Laura Cussonneau
email: laura.cussonneau@inrae.fr

Christian Boyer
email: christian.boyer@inrae.fr

Charlotte ; ; Brun
email: charlotte.brun@etu.unistra.fr

Christiane ; Deval
email: christiane.deval@inrae.fr

Emmanuelle Loizo
email: emmanuelle.loizon@inserm.fr

Emmanuelle Meugni
email: emmanuelle.meugnier@univ-lyon1.fr

Elise Gueret
email: elise.gueret@mgx.cnrs.fr

Emeric Dubois
email: emeric.dubois@mgx.cnrs.fr

Daniel Taillandier
email: daniel.taillandier@inrae.fr

Cécile Polge
email: cecile.polge@inrae.fr

Daniel Béchet
email: daniel.bechet@inrae.fr

Guillemette Gauquel-Koch

Ali L Evans
email: alina.evans@inn.no

Jon Arnemo
email: jon.arnemo@inn.no

Jon E Swens
email: jon.swenson@nmbu.no

Stéphane Blanc
email: stephane.blanc@iphc.cnrs.fr

Chantal Simon
email: chantal.simon@univ-lyon1.fr

Etienne Lefai
email: etienne.lefai@inrae.fr

Fabrice Bertile
email: fbertile@unistra.fr

Lydie Combaret
email: lydie.combaret@inrae.fr

E ; Loizon

Manuela Malatesta

Emerito Carlos Rodriguez-Merchan

Cécile Coudy-Gandilhon

Ghita Chaouki

Mehdi Djelloul-Mazouz

Yoann Delorme

Julien Hermet

Guillemette Gauquelinkoc
email: guillemette.gauquelinkoch@cnes.fr

Cécile Pol

Daniel Taillandi

Julien Averous

Alain Bruhat

Céline Jousse

Isabelle Papet

Pierre Fafournoux

Anne-Catherine Maurin

kinase B 10 wingless/int1-frizzled 11 AMP-activated protein kinase 12 nuclear factor-kappa B 13 janus kinases/signal transducers and activators of transcription Concurrent BMP Signaling Maintenance and TGF-β Signaling Inhibition Is a Hallmark of Natural Resistance to Muscle Atrophy in the Hibernating Bear

Keywords: congresses 2.3.1.1 Orals brown bear hibernation, mouse unloading, muscle atrophy, physical inactivity, RNA sequencing, TGF-β/BMP signaling

Aux membres du jury, le Pr Jean-Paul Thissen, la Dr Audrey Bergouignan, la Dr Jennifer Rieusset et le Dr Cédric Chaveroux

Je suis très honorée de vous compter parmi les membres de mon jury. Un immense merci pour votre

[…]

Sans la curiosité de l'esprit, que serions-nous ? Telle est bien la beauté et la noblesse de la science: désir sans fin de repousser les frontières du savoir, de traquer les secrets de la matière et de la vie sans idée préconçue des conséquences éventuelles.

Marie Skłodowska-Curie, 1938

A l'équipe Protéostasis Merci à tous les membres de l'équipe Protéostasis, on ne peut pas espérer un meilleur cadre de travail, joie et bonne humeur sont les maîtres mots de cette équipe. Je vous souhaite à tous le meilleur dans vos vies professionnelles et personnelles. J'aimerais remercier tout particulièrement certaines personnes. Merci à Pierre le chef d'équipe, alias Pr Dumbledore, pour tes conseils scientifiques toujours très avisés, et pour tes histoires de vie toujours très passionnantes. Merci à Christiane, pour ton aide technique, mais surtout pour ta générosité et ta gentillesse, j'ai beaucoup appris à tes côtés et je te souhaite la plus formidable des retraites. Merci à Cécile, ma partenaire de pilate et de stretching, pour ton aide en histologie mais aussi pour nos franches rigolades et confessions. Et merci à Lolo, pour ta bonne humeur et ton écoute, tu m'auras tellement fait rire, tu as été mon soleil du labo.

Aux animaliers de l'INRAE de Theix

Merci à toute l'équipe de l'animalerie de Theix qui fournit un travail formidable. Un merci tout spécial à Mehdi et Yoann, vous m'avez bien fait rire pendant ces trois ans, et également bien aidé avec mes souris suspendues, ce fût un réel plaisir de travailler avec vous. Vous me croyez maintenant, je n'étais pas une stagiaire !

A l'équipe du Brown Bear Project

Merci à toute l'équipe du BBP, sans qui ce projet de thèse n'aurait jamais existé. Cette équipe de vétérinaires, de rangers et de scientifiques experimentés permet à ce consortium de vivre et de faire naître de beaux projets scientifiques.

A Fabrice Bertile et Damien Freyssenet

Merci à Fabrice Bertile et Damien Freyssenet pour les échanges scientifiques de qualité que l'on a pu avoir lors de mes comités scientifiques de thèse. Merci à toi Fabrice également pour ton implication dans le BBP, et l'inoubliable expérience que tu m'as offerte avec Etienne lors de notre voyage en Suède.

A l'IUT Génie Biologique de Clermont-Ferrand

Merci à l'IUT Génie Biologique de Clermont-Ferrand, et spécialement à Mathilde Bonnet et Jérémy Denizot pour leur confiance en m'accordant un poste de vacataire pendant deux ans, ce fût une belle expérience qui a confirmé mon souhait de devenir enseignante chercheuse.

Aux étudiant.es de l'équipe de passage ou de plus longue durée

Merci aux doctorants qui ont partagé pendant trois ans cette incroyable expérience. Merci à Christian, même si tu râles beaucoup tu es très attachant, il faudrait te créer si tu n'existais pas. Merci à Aurore pour ta bienveillance et surtout tes conseils de maman chat quand je le suis devenue également. Merci à Ghita, pour ta présence et ton écoute. On aura partagé de jolis moments, je regrette déjà ta tajine somptueuse, tes appels avec ton franco-arabe qui me faisaient tant rigoler, tu vas beaucoup me manquer. Merci à Guillaume, mon petit frère ours, pour ces moments de franche rigolade au labo et en dehors, j'espère que tu t'occuperas bien de papa ours en mon absence. Merci à Maelle, ça a été un plaisir de travailler avec toi pendant quelques mois, et de partager de jolis moments de vie ensemble dans notre immeuble. Et merci à Maxime, alias Ludovic, Pierre, Enguerrand Raucroy, merci pour tous ces moments de rire, d'avoir relu mon manuscrit, de m'avoir consolée quand je doutais, et merci de m'avoir donné un peu plus confiance en moi.

A Dulce

Il était nécessaire de faire un paragraphe à part pour toi ma Dutz. Merci d'avoir été la première personne que j'ai rencontré dans cette ville, tu as transformé mon expérience Clermontoise et mon expérience de thèse comme je ne l'aurais jamais imaginé. Merci d'avoir partagé ces rires, ces doutes, ces questionnements et ces confessions pendant trois ans dans notre bureau, dans nos appartements ou pendant notre traversée de la Corse à pied. Je suis extrêmement chanceuse d'avoir croisé ton chemin. Te quiero mucho como la trucha y el trucho.

A mon fils

A mon petit chat Bibi, auvergnat d'origine, qui me rappellera à vie ce souvenir indélébile de l'Auvergne.

Merci pour tous ces câlins réconfortants et ta présence de tout instant.

Aux KAPAK

Ces trois ans n'auraient pas été les mêmes si je n'avais pas pu m'évader dans ma passion de toujours, la danse. Alors un immense merci à Yohann Sebileau, le meilleur professeur de danse contemporaine de la Terre, je pleure déjà la fin de tes cours, tu m'auras tellement fait progresser, merci pour tes chorées et ton engagement, merci pour ces moments sur scène gravés à tout jamais. Et merci à mes copines Tia et Claire pour ces jolis moments de danse partagés en cours, sur scène et en dehors.

A mes ami.es clermontois.es

Merci à Alexis, mon compère de soirées et de confidences, tes petits plats vont terriblement me manquer mais pas plus que toi. Merci à ma copine Melu, pour ton soutien, ton amitié, ces soirées à refaire le monde et à rêver ensemble. Et merci au groupe des Théseux pour la bonne humeur et les galères de thèse partagées ensemble.

A mes ami.es de longue date

A Chachou, Dédoule et Cass, mes meilleures amies depuis tant d'années maintenant, votre amour et soutien sont sans faille, vous êtes mes pilliers de vie, merci pour tout. Merci à Ele, ma précieuse amie, pour nos week-ends évasion et dégustation de vin. Merci à ma Caice pour ta joie de vivre et tes mots réconfortants. Merci à mes copains de master Juju, Roro, Fabi, Val et Mauricette pour nos week-ends découverte de la France et votre présence depuis Lyon. Et merci à mes copains bourguignons, à nos soirées au Galopin ou à la cocoloc, ces moments de franche rigolade m'ont permis de tenir le choc pendant trois ans.

A ma famille

Merci à ma famille, pour votre soutien et amour inconditionnel depuis 26 ans. A mon papa et ma maman, qui ont toujours les mots qu'il faut pour apaiser mes angoisses et doutes. A mes deux petites soeurs Marine et Ema, qui me comblent de bonheur. A ma tata Séverine, mon tonton Pascal et à ma mamie MC pour votre présence et votre amour, et à mon papi parti pendant ma thèse qui je sais aurait été très fier de moi.

List of figures

State of the art

Preface

Muscle wasting results from a wide range of pathophysiological conditions such as cancer and renal failure, but also microgravity, bed rest or inactivity. Muscle wasting is associated with adverse health effects such as a decline in independence and an increased morbidity and mortality. With increasing physical inactivity and improved life expectancy, muscle wasting is a major public health problem.

Muscle atrophy results from an imbalance between protein synthesis and degradation, and a variety of intracellular players are involved in this dysregulation, including the TGF-β superfamily and the ATF4 pathway. Their biological roles in muscle physiology are mainly described in humans or laboratory rodents. Despite the wealth of knowledge on this subject, an approved and readily available therapeutic or preventive treatment is still lacking. Hibernating brown bears are fascinating mammals because they naturally resist muscle atrophy although they remain completely inactive and starved for 5-7 months during the hibernation period. The main objective of this thesis was to find new underlying mechanisms that could become therapeutic targets to combat muscle atrophy in humans. To achieve this goal, we (1) used a comparative physiology approach in hibernating bears naturally resistant to atrophy, (2) investigated the role of the interaction between the ATF4 and the TGF-β/BMP pathways in unloaded mice susceptible to atrophy, and finally (3) initiated experiments in human muscle cells to validate hypotheses arising from the first two studies. This manuscript is therefore a compilation of all the work carried out during my 3-year thesis on the regulation of the TGF-β superfamily and ATF4 signalling pathways in the skeletal muscle of the hibernating brown bear and the unloaded mouse. It will be divided into 3 distinct parts. First, a stateof-the-art of (1) skeletal muscle physiology and muscle atrophy, (2) TGF-β superfamily and ATF4 signalling pathways, and their pivotal roles in muscle homeostasis, as well as (3) bear hibernation and the first clues to explain its resistance to muscle atrophy. In the second part, two studies will be presented. The first one is a published article on the transcriptomic analysis of muscles of brown bears during hibernation and of mice during unloading. The second study is a paper currently under review on the effect of ATF4 induction on skeletal muscle in both healthy and unloaded mice, and also in hibernating brown bears. A discussion of perspectives and questions arising from the data follows both studies. Finally, the last part consists of the presentation and discussion of the preliminary results of the effect of bear serum on human muscle cells. This thesis is written in english, and therefore a substantial abstract in french requested by the doctoral school is included in the Appendix, together with 2 articles of which I am co-author: one is an original article related to tissue adapatations (muscle, adipose tissue and serum) in the hibernating brown bear and the other is a review on ubiquitin ligases and their role in muscle atrophy. The word "muscle" was first used by Middle French speakers in the 14 th century, from the existing Latin words mus meaning "mouse" and musculus which translates to both "little mouse" and "muscle."

Ancient Romans thought that some muscles, especially the biceps, looked like little mice running under a person's skin. Our organism contains about 600 "little mice" accounting for approximately 40% of our total body weight, making skeletal muscle the most abundant tissue in the human body.

Generalities. Two types of muscles coexist: (1) the so-called smooth muscles located in walls of hollow organs (e.g intestine, stomach) under involuntary control, and (2) the striated muscles divided into two types, the cardiac muscles which also contract spontaneously, and the skeletal muscles which cover our skeleton and allow movements under voluntary control. Skeletal muscle is a very dynamic and plastic tissue. It is essential for movement, gesture and posture for functional autonomy. It also acts as the main tissue for energy metabolism, with heat production and absorption, use and storage of energy substrates (i.e. glucose, lipids and amino acids). Skeletal muscle is composed of water (75%), proteins (20%) and other constituents such as carbohydrates, lipids and minerals. It accounts for approximately 50-75% of body proteins and 30-50% of whole-body protein turnover [1,2].

Organisation. Skeletal muscle is a highly organised tissue containing several bundles of myofibers with each layer successively surrounded by the extracellular matrix (Figure 1). Myofibers are multinucleated and post-mitotic cells and contain adult stem cells called satellite cells that contribute to muscle growth and repair. Each myofiber contains thousands of myofibrils which are composed of the basic cellular unit of muscle, the sarcomere. The sarcomere itself is composed of billions of myofilaments, both thick (myosin) and thin (actin), which are essential for muscle contraction requiring high ATP consumption (Figure 1). Myofilaments represent the main protein content of muscle (i.e. 70-80% of the total protein content of a single fibre) [1,2]. The size of the muscle is primarily determined by the number and the size (i.e. cross-sectional area, CSA) of each myofiber, although fat and extracellular matrix infiltration can also influence its size [1,2].

Typology. Myofibers are classified into different types, with different characteristics such as the sarcomeric myosin heavy chain (MYH) gene expression, strength-velocity, response to neural inputs, or metabolic properties [3] (Figure 1). Since the first half of the 19 th century, scientists have distinguished skeletal muscles based on their colours and contractile properties: (1) red muscles composed of slow-twitch fibres (i.e. type 1) rich in mitochondria with oxidative metabolism and (2) white muscles composed of fast-twitch fibres (i.e. type 2) poor in mitochondria with glycolytic metabolism [4]. Over the past 40 years, this oversimplified schema has evolved with the notion of diversity of muscle fibre types, and four major fibre types have been identified in adult mammalian skeletal muscle (i.e. types 1, 2A, 2X, and 2B) (Figure 1). Humans, however, lack type 2B fibres, and the proportion of MYH within the same muscle may differ between mammals [3,5]. Skeletal muscle fibre type and mitochondrial function are sometimes uncoupled, for example for fast type 2A fibres with abundant mitochondrial content [2,3,6] (Figure 1). Based on differential MYH expression, a muscle may also consist of hybrid fibres (i.e., 1/2A, 2A/2X, 2X/2B), which allow muscles to utilise ATP in a nearly continuous gradient and thus be endowed with a fast type 2B to slow type 1 muscle contraction rate [2,3]. The heterogeneity of muscle fibres is the basis for the flexibility to use the same muscle for a variety of tasks, from fast and intense contraction (e.g. jumping) to slow and low-intensity activity (e.g. posture).

Mitochondria. Skeletal muscles are highly vascularised and innervated, and contain components of the metabolic machinery (e.g. mitochondria, sarcoplasmic reticulum), allowing efficient energy production. The precise coordination of activity between each of these components is essential to maintain muscle homeostasis and associated motor activity. The energy requirement during an intense contraction increases the normal ATP consumption in skeletal muscle by 100-fold [7]. To support this high energy demand, skeletal muscle relies in part on mitochondrial oxidative phosphorylation (OXPHOS) for ATP production. Adult myofibers exhibit specific subcellular localisation of distinct populations of mitochondria, namely subsarcolemmal (i.e. just below the plasma membrane) and intermyofibrillar. These two distinct populations of mitochondria are functionally highly interconnected but have a specific shape and exhibit differences in their biochemical and functional properties [8][9][10][11]. The morphology, arrangement, and connectivity of the mitochondrial network are adapted to the specific functional needs of each fibre type. For example, oxidative fibres have a gridlike organisation with elongated mitochondria oriented both parallel and perpendicular to the muscle contraction axis, in contrast to the mitochondrial network of glycolytic fibres, which is fragmented and oriented perpendicular to the muscle contraction axis [12]. Maintaining a functional mitochondrial network in skeletal muscle is fundamental to fulfilling the metabolic demands imposed by contraction, thereby regulating fuel utilisation, energy expenditure, and overall metabolism. Mitochondrial integrity and function are highly regulated by quality control systems (e.g., mitochondrial biogenesis, dynamics, and degradation) to maintain homeostasis [2,13]. Moreover, mitochondrial dysfunction has been linked to several human muscle diseases called mitochondrial myopathies [14][15][16][17][18][19].

Muscle-organ crosstalk

Myokinome. Over the past decades, skeletal muscle has been extensively studied for its role as an endocrine organ, producing and secreting hundreds of cytokines and other peptides, i.e. myokines, with autocrine, paracrine, or endocrine effects [20][21][22][23] (Figure 2). The first myokine described was myostatin [24] followed by . The latter is increased 100-fold in the bloodstream during exercise and shows multiple metabolic effects at the whole the body level [25,26]. Given the broad physiological and metabolic effects of physical activity throughout the body, it was clear that there was more than one myokine [27,28]. The biological name myokinome provided a new concept for understanding how muscles communicate with the rest of the body, and more than 650 myokines have been identified [20][21][22][23] (Figure 2). Myokines (e.g. myostatin,cathepsin B,irisin, are synthesised and released from myofibers during muscle contraction and provide communication between skeletal muscles and other organs, including the brain, adipose tissue, bone, liver, intestine, pancreas, endothelial cells, and skin, as well as communication within the muscle itself [20][21][22] (Figure 2, Figure 3). The biological roles of myokines include widespread body functions such as cognition, lipid and glucose metabolism, white fat browning, bone formation, endothelial cell function, hypertrophy, and skin structure (Figure 2, Figure 3). For muscle itself, myokines play a role in mitochondrial biogenesis, fat oxidation and glucose metabolism, and act as signals for muscle hypertrophy or atrophy [20,21,29] (Figure 3). It should be noted that most of the myokines are not yet sufficiently well characterised with respect to their biological functions [20][21][22][23]. Establishing proper crosstalk between body organs and muscles is essential for whole-body homeostasis [20][21][22][23].

Amino acid reservoir. Another major role of skeletal muscle is to be a reservoir of amino acids. Muscle amino acids can be mobilised in the absence of an adequate nutrient supply or in situations of increased need in other tissues to maintain their protein mass [30][31][32]. For example, obese individuals maintain normal plasma amino acid concentrations even after 60 days of total fasting [33]. Studies conducted by Jewish physicians in concentration camps during World War 2 suggested that death by starvation (i.e. uncomplicated by severe disease) occurred when amino acids mobilised from muscle proteins became insufficient to maintain the precursors necessary for gluconeogenesis. Indeed, amino 20 acids released from muscles serve as precursors for the maintenance of blood glucose levels through hepatic gluconeogenesis during starvation [34]. In the context of disease prevention and health maintenance, reduced muscle mass compromises the body's ability to respond to stress and chronic disease due to inappropriate crosstalk between muscles and organs. Therefore, loss of muscle mass is incompatible with life, and maintenance of muscle protein content through appropriate turnover is vital to maintain whole body homeostasis (see section 4.1.3).

Muscle protein turnover

During embryonic and early postnatal development, muscle growth occurs primarily through myogenesis and fusion of satellite cells [35,36]. In adult organisms, regulation of muscle mass results from growth within existing myofibers primarily via cellular pathways that control protein turnover [37,38] (see Appendix 10.4). Muscle proteins are constantly renewed, i.e. synthesised and degraded (Figure 4). The balance between the rates of muscle protein synthesis (MPS) and muscle protein breakdown (MPB), i.e. the net muscle protein balance, determines muscle protein content and homeostasis (Figure 4). MPS and MPB are sensitive to many factors, including nutritional status, hormonal balance, physical activity, injury or diseases. A decrease in muscle size in fully mature organisms, i.e. muscle atrophy (see section 4.1.3), results from a negative protein balance, whereas an increase in muscle size, i.e. hypertrophy, results from a positive protein balance. Muscle hypertrophy, in response to physical activity or a high-protein diet, is an interesting field of investigation that is of clinical interest in the search for treatments to limit or prevent muscle wasting.

Muscle protein synthesis

External stimuli. Amino acids (AA) provided by an appropriate diet act as extra-and intra-cellular anabolic molecules and are essential for inducing MPS [39,40] (Figure 4). High-protein diets do not enhance MPS as long as energy and protein requirements are met in the muscles and other organs. Amino acids bioavailability is strongly influenced by protein source, digestibility, and protein intake pattern, and is important for optimising MPS [40,41]. Mechanical cues are also considered anabolic stimuli, based on two basic lines of evidence: (1) muscles atrophy when mechanical load is reduced (e.g. bed rest) [42] and (2) muscle overload is sufficient for skeletal muscle hypertrophy [43,44]. Life on Earth has evolved in a 9.8m/s 2 environment that loads organisms. Therefore, cells have evolved with a plethora of sensors that detect mechanical stimuli. These mechanosensors help cells to adapt not only directly to the force produced by the contraction of a muscle fibre but also to more indirect mechanical signals, such as the stiffness of the extracellular matrix (ECM) that surrounds each cell [44,45]. However, these mechanical signals remain incompletely characterised. The increase in MPS after food intake is a systemic transient phenomenon, whereas physical activity stimulates a long-term AA: amino acid local adaptive response. Furthermore, adequate nutrition after physical activity can take advantage of anabolic pathways initiated by physical activity [41,46,47]. mTOR. One of the most recognized players in MPS is the mechanistic target of rapamycin (mTOR), which controls anabolic and catabolic signalling in skeletal muscle, resulting in the modulation of muscle hypertrophy and wasting [48,49] (Figure 5). mTOR inhibition by rapamycin or genetic invalidation respectively reduces the increase in MPS and/or muscle size after exercise in humans [50],

or results in severe myopathy leading to premature death in mice [51]. mTOR is a serine/threonine kinase that (1) senses a variety of environmental and intracellular changes, including nutrient availability, energy status and mechanical stimulation, and (2) coordinates a variety of cellular processes, including cell growth and survival, differentiation and autophagy [48,49,52]. There are two biochemically and functionally distinct mTOR complexes, namely mTORC1 and mTORC2 [48,52] (Figure 5). Both complexes share the mTOR catalytic subunit and are distinguished by their accessory proteins, and their unique substrates and functions [48,52] (Figure 5). On one hand, mTORC2 regulates cell survival and cytoskeleton organisation [48,52]. On the other hand, mTORC1 controls protein synthesis by activating S6K1 2 , which promotes ribosome biogenesis, and by inhibiting 4E-BP1 3 leading to protein translation (Figure 5). mTORC1 also promotes muscle hypertrophy by phosphorylating and suppressing ULK1 4 activity resulting in the inhibition of autophagy, one of the major protein degradation processes in muscle [48,52] (Figure 5).

Muscle protein breakdown

Physical activity, nutritional interventions, hormonal balance or inflammation also influence muscle protein breakdown (MPB) (Figure 4). The mechanisms are much less understood than for MPS, mainly because MPB measurement is technically more challenging than for MPS [53,54]. The main systems that contribute to MPB in skeletal muscle are the autophagy-lysosomal system (ALS) and the ubiquitinproteasomal system (UPS).

Autophagy-lysosomal system. The word autophagy is derived from two Greek words auto meaning "self", and phagy meaning "eating". Three different systems of autophagy have been described in mammals: macroautophagy, chaperone-mediated autophagy, and microautophagy. In this manuscript, ALS will refer to macro-autophagy, the most explored system in MPB. ALS involves the formation of a nascent membrane structure, i.e. the phagophore, surrounding bulk intracellular components, such as organelles, damaged proteins, or other target proteins (e.g. transporters, ion channels, receptors) (Figure 6). The origin of the membrane, i.e. endosomal, trans-golgi, nuclear or de novo synthesis, is unclear. After maturation of the autophagosome, it fuses with lysosomes to generate an autolysosome (Figure 6). Finally, activation of lysosomal proteases, i.e. cathepsins, or other enzymes such as DNAses or lipases, leads to the degradation of the autolysosome content and recycling of AAs [55] (Figure 6). It should be noted that ALS cannot degrade proteins in intact myofibrils; therefore, additional catabolic pathways are required [56,57]. Under normal conditions, ALS primarily prevents the accumulation of damaged organelles and misfolded proteins, but also degrades glycogen and lipid droplets, thus providing glucose, free fatty acids (FFAs), or AAs to the entire body to support basic cellular metabolism. In response to different stresses, such as starvation, ALS acts as a pro-survival mechanism in skeletal muscle, providing metabolic substrates [55]. Skeletal muscle is one of the organs with the highest rate of autophagy flux when nutrients are lacking [58]. In particular, basal autophagic flux is higher in glycolytic fibres than in oxidative fibres [59]. This is particularly important 2 ribosomal protein S6 kinase 1 3 eukaryotic translation initiation factor 4E binding protein 1 4 unc-51-like autophagy activating kinase because muscles may regulate ALS differently during specific stresses depending on their fibre type composition. Additionally, because muscle cells are highly sensitive to insulin [60], known to inhibit ALS [61], the autophagy flux fluctuates according to the food intake throughout the day and the level of physical activity [62,63]. While too much autophagy flux contributes to muscle wasting (see section 4.1.3), inhibition of ALS also leads to muscle atrophy [64]. In addition, inhibition of ALS leads to the accumulation of abnormal mitochondria, oxidative stress and protein aggregates, causing degeneration, weakness and premature death of myofibers [62][63][64][65]. Therefore, proper autophagic flux is required to maintain healthy muscle cells [62,63].

Mitophagy. In healthy skeletal muscles, damaged and depolarized mitochondria are selectively removed by the mitophagy process, which is a selective form of autophagy. The most studied mitophagy involves the ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1 5 [13,19,66].

Studies have demonstrated that mitochondrial dynamics and mitophagy are essential for skeletal muscle homeostasis [13,19,66]. There is a growing body of evidence that alterations in mitophagy or mitochondrial distribution and dynamics are present in muscles during wasting conditions (e.g. ageing, 5 PTEN induced kinase 1 AA: amino acids disuse or cancer cachexia) [66][67][68]. Importantly, enhancing mitophagy through genetic or nutritional approaches improves skeletal muscle function in aged rodents [13,19,66]. Thus, improving mitophagy in skeletal muscle appears to be a promising therapeutic target to prevent or even treat skeletal muscle dysfunction.

Ubiquitin-proteasomal system. The UPS is perhaps the best-known cellular proteolytic system and is responsible for degrading the majority of misfolded or defective proteins in all cell types. The UPS plays a fundamental role in normal muscle physiology, including degrading myofibrillar proteins [69,70].

Most proteins undergo degradation by being targeted to the 26S proteasome through the covalent attachment of a multi-ubiquitin chain (Figure 7). Protein ubiquitination involves the action of 3 enzymes: ubiquitin-activating enzymes E1, ubiquitin-conjugating enzyme E2, and ubiquitin-ligase E3 (Figure 7). The ubiquitin-tagged proteins are then recognized by the 26S proteasome, which initiates the ATP-dependent degradation process within the catalytic core (Figure 7). Through this mechanism, the UPS performs substrate-specific proteolysis [71]. Protein ubiquitination is both dynamic and reversible. Deubiquitinase or deubiquitinating enzymes (DUB) catalyze the removal of ubiquitin from target proteins and are also involved in ubiquitin maturation, recycling and editing. Several reports have demonstrated a relationship between UPS and lifespan, with proteasome activity decreasing with age in skeletal muscle, causing dysfunction [72]. Moreover, inhibition of proteasome activity in skeletal muscle is associated with a defect in muscle growth and shortened lifespan in rodent models [70,73]. UPS and ALS crosstalk. UPS and ALS have long been considered independent. However, emerging evidence suggests that there is a crosstalk between both pathways in skeletal muscle. Although ALS had been thought to be a non-specific degradation system, it has also been reported to degrade ubiquitinated proteins [74]. Studies also suggest that ALS and UPS are complementary because proteasome-deficient mice exhibit increased autophagic flux [70,73]. Therefore, the UPS and ALS are compensatory mechanisms, both essentials to sustain muscle homeostasis and integrity. However, since they are important for the health of skeletal muscle, dysfunctions in both systems lead to muscle pathological conditions.

Muscle atrophy

Causes and consequences

Causes. The loss of muscle mass and strength in the adult body is referred to as muscle atrophy. Muscle loss arises from inherited (congenital or genetic) or acquired conditions (pathological or physiological conditions) [75] (Figure 8). In addition, older adults exhibit age-induced muscle atrophy, primarily due to anabolic resistance, which may predispose this population to more pronounced muscle loss when exposed to periods of reduced physical activity [76]. Pathological conditions that cause muscle atrophy include cancer cachexia [77], chronic obstructive pulmonary disorders [78], diabetes and obesity [79], chronic kidney diseases [80], heart failure [81], sepsis [82], burns [83], and conditions associated with anorexia or malnutrition [84]. Physical inactivity also leads to muscle wasting, especially following leg fractures, immobilisation and bed rest [85][86][87][88][89][90] and even in those with sedentary lifestyles, as observed during COVID-19 home confinement [91]. It should be noted that 60-85% of people worldwide lead a sedentary lifestyle (World Health Organisation). Muscle atrophy results from an imbalance between MPS and MPB, with a negative balance in favour of protein breakdown [75] (Figure 8). Data suggest that both (1) decreased MPS and (2) increased MPB contribute to muscle loss and that the relative contribution of each process to muscle loss depends on the pathophysiological condition [92][93][94].

During this thesis, we were primarily interested in disuse-induced muscle atrophy.

Consequences and treatments.

The resulting muscle wasting is characterised by muscle alterations such as myofiber shrinkage, changes in fibre types or myosin isoforms, and net losses of cytoplasm, organelles, and total proteins. Loss of myofibers and/or a decrease in myofiber diameter are the most prominent histopathologic features of skeletal muscle atrophy (Figure 9). As mentioned above, skeletal muscle plays a central and major role in whole-body homeostasis. Lack of physical activity is associated with a wide network of diseases, including type 2 diabetes, cardiovascular diseases, cancer, dementia, and osteoporosis [22,95]. These adverse effects are likely, to some extent, mediated by a lack of release of myokines and/or resistance to their effects [20,23]. In addition, skeletal muscle is a major organ of insulin-induced glucose metabolism. Therefore, a loss of muscle mass is closely related to insulin resistance and metabolic syndrome [96]. Muscle atrophy limits daily activities, reduces quality AA: amino acid of life and lengthens recovery time after illness, while increasing morbidity and mortality (Figure 8).

Given its adverse consequences, increasing sedentary lifestyle and life expectancy worldwide, muscle wasting affects millions of people and remains a major economic and social burden. Currently, strategies for treating skeletal muscle atrophy include physical exercise, nutritional interventions and some medications [75]. In addition, natural products have a wide range of effects on muscle function.

However, their low bioavailability and low intestinal absorption limit their application [97]. To date, no drugs have been approved for clinical use, no effective remedies for muscle atrophy have been discovered, and exercise or nutritional interventions are strategies that are not suitable for all patients (e.g., immobilised or intensive care unit patients). Thus, although our understanding has improved considerably over the last two decades, mainly through the use of laboratory models inducing muscle atrophy, there is still a need to discover new targets and drugs to combat it.

An interconnected network of cellular actors

Signalling pathways. MPS and MPB are influenced by a wide range of external and internal molecular actors. External stimuli (1) include mechanical load, inflammatory factors such as cytokines (e.g. IL-6, TNF-α 6 ), endocrine factors such as growth factors (e.g. IGF-1 7 , insulin), catecholamines and angiotensin, and (2) activate various intracellular pathways (Figure 10). This interconnected network of intracellular actors contributes to the regulation of muscle protein balance by working in synergy or in antagonism to promote anabolism or catabolism [2,37,38,75] (see Appendix 10.4). In the context of muscle atrophy, dysregulation of one or more of these actors results in a blunting of anabolic signalling in favour of catabolism leading to either MPS inhibition, UPS and ALS overactivation, or both [2,37,38,75] (see Appendix 10.4) (Figure 10). In brief, anabolic pathways suppressed in many atrophy 6 tumor necrosis factor α 7 insulin growth factor-1 conditions include signalling from PI3K 8 -AKT 9 -mTORC1, ß2-adrenergic, WNT/FZD 10 , calcineurin, hippo and bone morphogenetic protein (BMP). In contrast, catabolic pathways overactivated in many cases of atrophy include signalling from transforming growth factor-β (TGF-β), AMPK 11 , NF-κB 12 , glucocorticoid receptors, angiotensin, IL-6-JAK/STAT 13 , kinin, sphingolipids, notch or activating transcription factor 4-endoplasmic reticulum stress (ATF4 and ER stress) [2,37,38,75] (see Appendix 10.4) (Figure 10). The precise interconnection and biological actions of these actors still need to be fully elucidated. A detailed description of their regulation is beyond the scope of this manuscript.

Nevertheless, a review of which I am a co-author is appended for more details (see Appendix 10.4).

Atrogenes. Atrogenes (i.e. atrophy-related genes) are referred to as a set of genes whose expression changes in different catabolic situations associated with muscle wasting. Regulation at the protein level is sometimes more complex to elucidate [98,99]. The atrogenes belong to different cellular pathways, mainly the UPS and ALS proteolytic systems, and include the E3-ubiquitin ligases containing tripartite motif 63 (TRIM63)/muscle ring finger-1(MuRF1) and F-Box protein 32 (FBXO32)/Atrogin-1, as well as some autophagy players such as cathepsin L and BCL2-interacting protein 3 (BNIP3) [99]. For example, TRIM63/MuRF1 targets myofibrillar proteins (i.e. thin and thick filaments), as well as sarcomere structural components such as telethonin, for UPS-dependent degradation (from Peris-Moreno et al., 2021, see Appendix 10.4 [38]).

In this thesis project, we focused on the pivotal role in muscle homeostasis of the TGF-β superfamily (see section below) and the Integrated Stress Response signalling (see section 4.3).

4.2 A pivotal role for the TGF-β superfamily in skeletal muscle homeostasis

Overview

The transforming growth factor-β (TGF-β) superfamily is an ubiquitious family that regulates a multiplicity of biological actions including proliferation, differentiation and apoptosis. This superfamily is divided into two signalling pathways, named TGF-β and bone morphogenetic protein (BMP).

Ligands. More than 30 secretable ligands belong to this family including activins A and B (INHBA and INHBB genes), myostatin (MSTN gene), TGF-β1-3, growth differentiation factor GDF1/10/11 for TGF-β signalling, and AMH 14 , BMP2-7, GDF5/7 for BMP pathway (Figure 11).

Signal transduction.

Ligands bind to a type 2 receptor, which subsequently recruits the type 1 receptor to form an heteromeric complex. These receptors pair up in different combinations to mediate the response of each ligand and the subsequent intracellular response [105][106][107]. Once the receptors are complexed, the adaptor protein SARA 15 recruits the receptor-regulated SMAD family member (R-SMAD), i.e. SMAD2 and 3 for the TGF-β signalling and SMAD1,5 and 8 for the BMP signalling (Figure 11).

Thereafter, the receptor complexes phosphorylate the R-SMAD making them recognizable by the common TGF-β and BMP mediator, SMAD4. Subsequently, SMAD4 forms an heteromeric complex with SMAD1/5/8 or SMAD2/3 that translocates into the nucleus and elicits a cell/environment/ligand specific transcriptional program [105-108] (Figure 11). 14 anti-müllerian hormone 15 SMAD anchor for receptor activation

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Muscle atrophy is a social and economic burden that results from an imbalance in muscle protein synthesis in favour of muscle protein breakdown. However, despite a better understanding of the anabolic and catabolic signalling pathways dysregulated during atrophy, there is still no proven therapeutic or preventive treatment suitable for all patients.

Regulation. Regarding the wide range of ubiquitous biological actions, the TGF-β superfamily is tightly regulated at multiple steps. First of all in the ECM, several mechanisms enable the activation of the secreted ligands from their latent inactive state. Second of all, the active ligands can be sequestrated by antagonists within the ECM, for instance by follistatin (FST gene) the best described for TGF-β signalling, and noggin (NOG gene) for BMP [109] (Figure 11). Moreover, each ligand can bind to several receptor subtypes which, themselves, can be post-translationally modified, adding layers of complexity [105][106][107].

In addition, the inhibitory SMADs (I-SMAD), SMAD6 and SMAD7 can antagonize the signal initiated by ligands by competing with R-SMAD for the binding to a given receptor. SMAD6 selectively inhibits BMP signalling whereas SMAD7 inhibits signalling for both TGF-β and BMP signalling (Figure 11). Moreover, both signalling are also tightly regulated by ubiquitination/deubiquitination processes from receptor and R-SMAD activation to induction of the transcriptional program [38,[105][106][107] (Figure 12). Finally,

TGF-β signalling: The master regulator of skeletal muscle atrophy

Pathophysiological conditions. TGF-β is a catabolic pathway of great interest within the field of skeletal muscle biology. In the late 1990's, the discovery that deletion of the myostatin gene (MSTN), one of its ligands, and inhibition of its receptor, caused a profound hyper-muscularity in mice, cattle, sheep, and dogs sparked the initial interest in its role in atrophy [24,[112][113][114]. Thereafter, MSTN was found to be elevated in muscles or blood in all type of catabolic situations characterised by muscle atrophy, such as in ageing subjects, in response to prolonged bed rest, in patients with acquired immune deficiency syndrome, renal failure or heart failure [115][116][117][118][119]. Serum levels of other TGF-β ligands such as activin A also rise in response to cancer, kidney failure and heart failure, all associated with muscle wasting [115,[120][121][122]. In addition, TGF-β1 is remarkably elevated in plasma of patients with muscular dystrophies [123]. Besides, microRNA positively controlling TGF-β signalling are increased in muscles of patients following 10 days of sustained bed rest or in patients in intensive care unit [124,125].

The binding of myostatin or activin A/B to activin receptor type-2B (i.e. to a lesser extent type 2A) ActR-2B/A (ACVR2A/2B genes) leads to the recruitment and phosphorylation of SMAD2/3 and is associated with muscle atrophy in a multiplicity of catabolic situations [110,114,118,[126][127][128].

TGF-β underlying mechanisms inducing muscle atrophy

Activation of MPB. TGF-β signalling is involved in the transcription of the atrogenes FBXO32/Atrogin-1 and TRIM63/MuRF1 known to be involved in ubiquitin-proteasome proteolysis. Mice or cultured mice myotubes treated with TGF-β ligands (i.e. activin A/B, myostatin) show muscle atrophy through the activation of SMAD2/3 resulting in overexpression of Fbxo32/Atrogin-1 and/or Trim63/MuRF1 atrogenes [127,129] (Figure 13). Similarly, exposing healthy mice to exogenous GDF11 ligand results in muscle wasting through the activation of the SMAD2-ubiquitin-proteasome pathway and autophagy axis [130] (Figure 13). Furthermore, overexpression of the transforming growth factor beta receptor 1 (Tgfbr1) in mice muscles also increases the expression of the atrogene Fbxo32/Atrogin-1 and induces muscle fibre atrophy via a SMAD2/3-dependent mechanism [131] (Figure 13). Conversely, mice with muscle specific deletion of Smad2 or 3 are resistant to muscle atrophy induced by Tgfbr1 surepexression or denervation [131,132]. Additionally, inhibition of TGF-β signalling through musclespecific KO type 1 receptors (i.e Tgfbr1 and Acvr1b) or follistatin administration, induces muscle hypertrophy in mice by reducing Fbxo32/Atrogin- 1 and Trim63/MuRF1 expression [133,134].

Mechanistically, overexpression of Smad3 in mice muscles is sufficient to induce Fbxo32/Atrogin-1 expression and ultimately induces muscle fibres atrophy [135]. In cultured mice myotubes, SMAD3 synergises with the transcription factor FOXO3 16 to induce the expression of Trim63/MuRF1 [136] (Figure 13). Finally, myostatin treatment also inhibits the expression of MyoD and Pax3 17 myogenic genes in cultured myotubes [129]. Altogether, these data showed that the canonical TGF-β signalling (i.e. SMAD2/3) is required for muscle damage. 16 forkhead box-O 3 17 paired box 3 Inhibition of MPS. TGF-β catabolic action also involves inhibition of protein synthesis. Myostatin or activin A administration are sufficient to inhibit protein synthesis in mice muscles through inhibition of the AKT/mTORC1 signalling [127,131,137,138]. The same phenotype is observed by overexpressing Smad3 [133,135] (Figure 13). Additionally, inhibition of TGF-β signalling through muscle-specific KO type 1 receptors (i.e Tgfbr1 and Acvr1b) or follistatin administration, induces muscle hypertrophy in mice by increasing AKT phosphorylation [133,134]. Moreover, the hypertrophic effect of myostatin blockade is reduced when mTORC1 is genetically or pharmacologically inhibited [131,133,137]. The mechanisms linking SMAD2/3 to AKT/mTORC1 signalling in muscle atrophy conditions remain unclear.

Treatment with insulin-like growth factor 1 (IGF-1) activates AKT and increases the interaction between AKT and SMAD3, leading to inhibition of TGF-β signalling in cultured myoblasts [139]. IGF-1/AKT The dotted lines correspond to the signalling impaired in numerous muscle wasting conditions, and the questions marks the unsolved questions.

signalling is altered in many catabolic situations in muscles [140], and impairement of IGF-1 receptor during muscle immobilisation contributes to SMAD2/3 protein accumulation [132]. Accordingly, TGFβ signalling might be further amplified in these situations due to compromised interaction between AKT and SMAD3. In turn, this may reinforce the vicious circle between activation of TGF-β signalling and impairment of AKT-mTORC1 signalling (Figure 13).

Other muscle detrimental actions.

A proteomic analysis of mice muscles overexpressing follistatin has uncovered changes in energy metabolism, fibres type, insulin and calcium signalling, providing insight into the intracellular modifications sensitive to TGF-β signalling [141]. In addition, TGF-β signalling in mice represses mitochondrial biogenesis [142,143] and is associated with mitochondrial disruption in cancer cachexia-induced muscle wasting in mice [144] (Figure 13). TGF-β-induced muscle wasting is also linked to reactive species oxygen (ROS). Injection of TGF-β1 ligand into mice muscles increases ROS content and induces atrophy, both being reversed by administration of an antioxidant treatment, suggesting that TGF-β1-induced muscle atrophy was . Finally, the TGF-β pathway is also known to play a major role in fibrosis, promoting muscle mechanical changes and muscle injury in many muscular dystrophies in mice and humans [146,147] (Figure 13).

TGF-β-targeted mediation in muscle atrophy: Panacea or smokescreen ?

The establishment of myostatin and activins as robust negative regulators of skeletal muscle has designated these ligands and partners as attractive therapeutic targets for various musculoskeletal disorders.

Promising results in vivo. Follistatin (FST gene) is a potent extracellular inhibitor of myostatin and of several other ligands of the TGF-β superfamily and its overexpression results in muscle hypertrophy in mice. This hypertrophy exceeded that observed in Mstn KO mice and was further exacerbated when overexpressing Fst in Mstn KO mice [133,148]. Inhibition of activin A by a specific antibody leads to muscle hypertrophy in mice and monkeys [128], and codelivery of specific activin A and myostatin inhibitors induces a synergistic response with an increase in muscle mass of up to 150% in mice [149].

Finally, the concomitant neutralization of both ACVR2A and ACVR2B receptors with BYM338 antibody results in a stronger skeletal muscle hypertrophy [150].

These observations led to the utilisation of such strategies during muscle atrophy situations: Pharmacological inhibition of myostatin alleviates muscle wasting in cachectic mice [151]. Inhibition of the receptor TGFBR1 by the LY364947 molecule abolishes diaphragm atrophy in rats undergoing sepsis [152]. Genetic or pharmacological blockade of the myostatin/activin A receptor ACVR2B improves muscle mass and function in mice models of cancer cachexia, spinal muscular atrophy, or microgravity [153][154][155]. Overexpression of Smad7, the intracellular TGF-β antagonist, prevents cancer-mediated muscle wasting in mice [156,157]. Other effective strategies using muscle-specific microRNAs have also been investigated. For instance, overexpression of miR-206 attenuates muscle atrophy during denervation in rat by inhibiting TGF-β-SMAD2/3 axis [158]. The system renin-angiotensin is involved in muscle loss. Interestingly, treatment with an angiotensin 2 inhibitor prevents muscle atrophy in mice through a blockade of the TGF-β-induced SMAD2 /3 activation [111,145]. Finally, the use of angiotensin 2 inhibitor, extracellular or receptor antagonists shows improvement in muscle function in different muscular dystrophies by inhibiting the TGF-β signalling and hence its consequences as a pro-fibrotic pathway [146].

Disillusion in human clinical trials.

Based on the pre-clinical studies, numerous TGF-β-inhibiting pharmacologic agents have progressed in human trials or are still currently under evaluation [119].

Treating elderly patients requiring hip replacement with an anti-myostatin has proven to be safe, although preservation of muscle mass following surgery was minimal [159]. Another anti-myostatin molecule showed promising results with amelioration of muscle locomotor function in spinal muscular dystrophy patients [119]. Other phase 2 clinical trials showed that the use of an antibody blocking the activin type 2 receptor was safe but with little or no functional benefit in patients with muscle wasting (i.e. hip fracture surgery, sporadic inclusion body myositis, sarcopenic elderly, cachexia, chronic obstructive pulmonary disease) [160][161][162][163].

Therefore, although these molecules were promising in rodents, they have shown only a minimal effect in humans or have demonstrated important side effects [119,164]. Indeed, most myostatin inhibitors also repress the activities of other closely related TGF-β family members including GDF11, activins, and BMPs, increasing the potential off-targets. Consequently, a careful distinction between targets is required to evaluate the use of these medications in human clinical practice [119,164].

BMP signalling: The silver bullet for muscle atrophy ?

The BMP signalling pathway was originally discovered for its ability to induce bone formation. BMP signalling is important in embryogenesis and development in all organ systems, and also in the maintenance of adult tissue homeostasis [165]. The role of BMP in the regulation of muscle mass was only discovered in 2013 [166,167] (Figure 14). For this reason, much less is known about this pathway and its underlying mechanistic in muscle homeostasis.

Fundamental in healthy adult muscle. BMP signalling controls the mass of healthy adult muscles, since increasing the expression of the ligand Bmp7 or a constitutively active BMP receptor type 1A (caBmpr1a, ALK3 protein) promotes a SMAD1/5-dependent hypertrophy phenotype in mice [166,167].

Furthermore, inhibition of the BMP pathway by using inhibitors of ligand-receptor interaction (i.e. LDN-193189 or noggin), or invalidation of Smad1 or 5, leads to muscle atrophy in healthy adult mice muscles [166]. In addition, the profound increase in muscle mass observed in Mstn KO mice is mediated by the activation of BMP signalling via SMAD1/5, whereas overexpressing the selective BMP inhibitor Smad6, significantly reduces this hypertrophic phenotype [149].

Regulation in catabolic conditions. SMAD1/5 phosphorylation increased in rodent muscles exhibiting atrophy associated with motor nerve degeneration, intensive care disuse or with amyotrophic lateral sclerosis [166,167]. In addition, the expression of BMP-related components, i.e. BMP ligands Gdf5 and Gdf6 and the BMP receptor type 1B (Bmpr1b, ALK6 protein), increase in denervated mice muscles.

Similarly, the DNA-binding protein inhibitor (ID1)-luciferase reporter, which mirrors BMP transcriptional activity also increases in this situation [166,167]. However, SMAD1/5/8 phosphorylation is down-regulated, whereas gene expression of the BMP inhibitor noggin is upregulated in muscles of tumour-bearing mice, and in muscles of pre-cachectic and cachectic patients [168]. Additionally, a decreased in gene expression of BMP-related components is also observed in the elderly with muscle atrophy following hip arthroplasty [169].

A central role to counteract muscle atrophy. Administration of tilorone, a molecule capable of inducing BMP signalling, restores BMP-mediated signalling in muscles, limits muscle wasting, and lengthens the survival of tumour-bearing mice [168]. These data showed the necessity of promoting/maintaining BMP signalling to limit cancer-induced muscle atrophy [168]. Overexpression of caBmpr1a/ALK3 or Bmp7 in mice blunts muscle atrophy induced by denervation or cancers [166,167], while Smad6 or Nog overexpression suppresses SMAD1/5 phosphorylation and exacerbates muscle atrophy during denervation and fasting [166,167]. Besides, the role of altered BMP signalling in muscle atrophy was confirmed by Sartori et al. who observed a severe aggravation of denervationinduced muscle atrophy in Gdf5 KO mice [166]. Moreover, Mstn KO mice, which are usually resistant to denervation-induced muscle atrophy, lose this ability when BMP signalling is concomitantly blunted [166]. Finally, a long non-coding RNA, Chronos, impairs muscle growth in ageing mice by repressing BMP signalling [170]. Altogether, these data strongly suggest that (1) activation of the BMP pathway in skeletal muscle during catabolic conditions is an adaptive response to counteract atrophy, and (2) a deficiency in this signalling plays a critical role in aggravation of muscle frailty [166,167].

Intracellular actions. In innervated mice muscles, hypertrophy induced by increased expression of

Bmp7 or caBmpr1a/ALK3 is associated with increased phosphorylation of AKT and of two mTORC1 substrates (i.e RPS6 and 4E-BP1), which is blunted by rapamycin treatment. These data provided the first demonstration that the mTORC1 pathway is indispensable in the regulation of BMP signallinginduced muscle growth [167] (Figure 14). In denervated mice muscles, overexpression of Nog significantly enhances the expression of the Fbxo30. This gene encodes a protein identified as MUSA1 for muscle ubiquitin ligase of SCF complex in atrophy-1 [166]. The authors proved that BMP signalling acts as a positive regulator of muscle mass by repressing the transcription of Fbxo30/MUSA1, whose induction is required for denervation-induced atrophy [166] (Figure 14). Similarly, increased expression of the BMP inhibitor Smad6 also results in increased Fbxo30/MUSA1 expression in denervated muscles [167]. Inhibition of the BMP signalling is also associated with increased expression of Trim63/MuRF1 and Fbxo32/Atrogin-1 during muscle atrophy associated with denervation [167] and cancer [168] in mice. The repression of the expression of these atrogenes by the BMP pathway is believed to be through the suppression of the HDAC4 18 -Myogenin axis, which is involved in the transcription of TRIM63/MuRF1, FBXO32/Atrogin-1, and FBXO30/MUSA1 [167] (Figure 14). 18 histone deacetylase 4 The dotted lines correspond to the signalling impaired when BMP signalling is activated.

A finely tuned balance between TGF-β and BMP signalling

SMAD4 shared custody

SMAD4 is the shared actor between the TGF-β and BMP signalling (Figure 15). Smad4 KO mice slightly lose muscle mass and are even more susceptible to muscle wasting during denervation or fasting [166].

Mstn KO mice exhibit a significant activation of the SMAD1/5/8 transcriptional activity with greater recruitment of SMAD4 on the promoter of BMP target genes. On the contrary, Gdf5 KO mice lead to an increased binding of the SMAD4-SMAD2/3 complex to the promoter of TGF-β target genes [166].

This has prompted the concept of a competition between SMAD 2/3 and SMAD 1/5/8 for SMAD4 recruitment. The authors speculated that inhibiting TGF-β signal would release SMAD4 from SMAD2/3 to be more available for SMAD1/5/8. In muscle wasting scenarios, TGF-β over activation is considered

B. A.

as a factor which reduces the availability of SMAD4 for BMP signalling (Figure 15). This article has strongly highlighted the need for a fine-tuning of the BMP/TGF-β balance to maintain muscle homeostasis [166] (Figure 15). This is consistent with the Myhre syndrome, a human rare autosomal dominant genetic condition characterised by muscle hypertrophy. This syndrome is explained by a missense mutation of SMAD4-leading to defects in ubiquitination, and hence Myhre syndrome patients have increased levels of SMAD4 protein rather than SMAD4 loss [171].

TGF-β/BMP non-canonical signalling and its dual role in muscle homeostasis

Non-SMAD signalling. In addition to the canonical SMAD-mediated TGF-β superfamily signal transduction, activated receptors also transduce signals through non-SMAD signalling [172] (Figure 16).

For example, the TGF-β-activated kinase 1 (TAK1) protein, originally identified as a member of the MAPK 19 family, is a major component of the non-canonical TGF-β superfamily signalling. TAK1 interacts with the TNF Receptor Associated Factor 6 (TRAF6), which is bound to a receptor type 1 of the canonical SMAD signalling [173,174] (Figure 16). Once the receptors type 1 and 2 are complexed , TRAF6 undergoes autoactivation and subsequently activates TAK1. Thereafter, TAK1 phosphorylates MAPK actors leading notably to the activation of the p38 MAPK [173,175] (Figure 16). In addition to the possible role of TAK1 in muscle atrophy, TAK1 has also been reported to be required for the maintenance of skeletal muscle mass in adult mice. Inducible skeletal muscle-specific Tak1-KO mice leads to severe muscle wasting which is accompanied by increased proteasome activity, elevated autophagy, redox imbalance and mitochondrial dysfunctions associated with decreased p38 19 mitogen-activated protein kinase phosphorylation [182,183]. Overexpression of Tak1 in mice induces muscle hypertrophy, increases protein synthesis, and attenuates denervation-induced muscle atrophy, while its genetic inactivation leads to neurogenic atrophy [184]. A very recent study has revealed promising findings. The authors identified a strong physical interaction between TAK1 and SMAD1 in denervated mice muscles. The authors assumed that TAK1 could regulate the spatial distribution of SMADs proteins by promoting (1) the nuclear localisation of SMAD1-SMAD4 to suppress FBXO30/MUSA1 transcription and ( 2) the cytosolic retention of the inhibitor SMAD6 in denervated muscles. The underlying mechanisms are however still completely unknown [184]. Whether such an interplay between TAK1 and SMADs also exists in other catabolic conditions has never been investigated (Figure 16). The red and green lines represent respectively the catabolic and anabolic signalling, and the questions marks the unsolved questions.

MEF2 a pro-maintenance actor. Myogenic enhancer factor 2 A-D (MEF2A-D) proteins are key transcriptional regulators of skeletal muscle development, sarcomeric gene expression, fibre type control and glucose uptake metabolism [185][186][187]. p38 MAPK directly phosphorylates MEF2, increases its transcriptional activity in myoblasts from mice or rats, and is required for a proper differentiation process [188,189]. In addition, Traf6 deletion in mice myotubes inhibits MEF2 transcription [190].

Furthermore, SMAD3 interacts with MEF2C resulting in reducing MEF2C transcriptional activity in mice myoblast cells and thus disrupt differentiation [191]. Finally, activin A treatment in human muscle cells reduces MEF2C expression and activity and leads to myotubes atrophy [192]. To our knowledge, no study has ever explored the TGF-β/BMP-TRAF6-TAK1-p38-MEF2 axis in skeletal muscle in vitro or in vivo (Figure 16).

The pivotal role for TRAF6-TAK1-p38 in muscle homeostasis beneath TGF-β and BMP signalling is full of unsolved questions. How does TAK1 act as a pro-atrophic actor through TGF-β-p38, and promotes muscle gain through BMP? How does TGF-β/BMP ligand-receptors pair to activate SMAD and non-SMAD signalling? How do these signals interact? These unsolved questions warrant further investigation (Figure 16).

The Integrated Stress Response signalling: Beneficial or harmful for skeletal muscle?

The Integrated Stress Response (ISR) signalling is another pathway involved in muscle homeostasis.

First of all, an overview of the signalling organisation will be presented and second of all, a focus will be made on the role of ISR in muscle homeostasis.

Overview

The ISR is a well-conserved signalling present in eukaryotic cells, which is activated in response to a range of physiological stresses [193,194]. Such stresses commonly include extracellular factors such as hypoxia, amino acids deprivation, glucose deprivation, heme deficiency, viral infection, and intracellular stresses such as ER stress. The core event of the ISR activation is the phosphorylation of

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A major conceptual insight emerging from these studies is that the balance between TGF-β and BMP signalling pathways plays a key role in determining skeletal muscle fate. Therapies targeting TGF-β induce challenging side effects due to its pleiotropic role. At present, extensive effort should be directed toward further a better understanding of the role of BMP signalling in muscle homeostasis.

the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α) on its serine 194] (Figure 17). To date, four kinases have been reported to phosphorylated eIF2α: PKR-like ER kinase (PERK), double-stranded RNA-dependent protein kinase (PKR), heme-regulated inhibitor (HRI), and general control nonderepressible 2 (GCN2) [193,194] (Figure 17). They all dimerise and autophosphorylate to be activated in response to distinct environmental stresses: amino acid deprivation for GCN2, ER stress for PERK, heme deficiency for HRI and viral infection for PKR [193,194] (Figure 17).

Phosphorylation of eIF2α leads to two consequences (1) a general inhibition of the translational machinery and ( 2) the translation of selected mRNA including ATF4.

Inhibition of protein translation.

In normal conditions, the GDP 20 -bound form of eIF2α is exchanged for a GTP 21 -bound form by the action of the guanine nucleotide exchange factor eIF2B (Figure 17). This 20 guanosine diphosphate 21 guanosine-5'-triphosphate event converts eIF2α to its active form which can recruit the translation initiation ternary complex and subsequently initiates the first step of protein translation. Upon stress conditions, p-eIF2α inhibits eIF2B action, which thus remains in its GDP-bound form, preventing the formation of the ternary complex and leading to the global inhibition of protein translation [193,194] (Figure 17).

Translation of specific mRNAs. In parallel, p-eIF2α results in the translation of specific mRNAs, including the activating transcription factor 4 (ATF4) (Figure 18). The mechanism is highly conserved, from yeasts to mammals. The ATF4 transcript is constitutively expressed in many cells and has several small upstream open reading frames (uORF) at its 5′ end, being out of frame with the main proteincoding sequence (CDS). These uORFs mediate basal repression of ATF4 translation (Figure 18). Upon normal conditions, when the ternary complex is abundant (i.e. eIF2α non-phosphorylated), ribosomes initiate scanning at uORF1 and re-initiate at uORF2 overlapping with ATF4 CDS, hence precluding ATF4 translation (Figure 18). Upon stresses conditions (i.e. p-eIF2α), a limited number of ternary complexes are formed. Ribosomes still initiate scanning at uORF1 but, due to low levels of the ternary complex, ribosomes take longer to re-initiate translation. Hence, they re-initiate scanning at the ATF4 CDS (Figure 18). Therefore, upon p-eIF2α, this results in approximately fivefold higher ATF4 protein expression [193][194][195]. Biological role of ATF4. ATF4 is a bZIP 22 transcription factor that belongs to the ATF/CREB 23 protein family [196]. ATF4 is a key determinant of cellular fate in response to ISR activation, mainly acting as a transcriptional activator of a cohort of genes involved in cellular stress adaptation (Figure 17). ATF4 has several dimerisation partners that influence its regulation of gene transcription and governs cellular outcome. ATF4 produces distinct tailored responses with the transcription of target genes being highly dependent on the cellular context/stresses [193,194]. For instance, upon nutritional stress, ATF4 stimulates the expression of genes involved in amino acid transport and biosynthesis, and in autophagy, to supply new amino acids for de novo protein synthesis [197][198][199] (see section 4.3.2).

Moreover, ATF4 induces the transcription of the protein phosphatase 1 regulatory subunit 15A (PPP1R15A, GADD34 24 protein), the main eIF2α phosphatase, acting as an important negative feedback loop to restore protein synthesis once the stress is overcome [193,194,200] (Figure 17). It has been proposed that the relative duration and intensity of the ISR signalling dictate the cellular outcome.

Therefore, ATF4 may also facilitate the execution of a cell death transcriptional program when cellular homeostasis cannot be restored, by activating the transcription of apoptotic genes [201][202][203][204] (Figure 17).

Other regulations of ATF4 translation and transcription. ATF4 translation can also be stimulated by anabolic hormones and growth factors, including insulin or IGF-1 (Figure 17), which activate mTORC1 to increase ATF4 translation [205][206][207][208]. mTORC1 activation enables ribosomes to bypass short uORF in the 5' of ATF4 mRNA in the same way as ISR activation does [195,[207][208][209]. In contrast to p-eIF2α, mTORC1 activity increases both ATF4 translation and general protein synthesis [205][206][207][208]. In this context, ATF4 heterodimers primarily induce the transcription of genes that promote amino acid uptake and synthesis to facilitate anabolism (Figure 17).

In addition to the translational regulation of ATF4, it can also be regulated at the transcript level. ATF4 mRNA levels are low under normal conditions but are induced in response to different stresses by different transcription factors (Figure 17). For example, ATF4 expression is induced in response to oxidative stress by NRF2 25 , chemotherapeutic drugs by CLOCK 26 , or ER stress and starvation by TFEB 27 and TFE3 28 [193,194]. Interestingly, in a positive feedback loop, ATF4 downstream gene targets, such as NUPR1 29 , can also elevate ATF4 mRNA levels [210] (Figure 17). 22 basic leucine zipper 23 cyclic AMP response element binding 24 growth arrest and DNA damage-inducible 34 25 NF-E2-related factor 2 26 clock circadian regulator 27 transcription factor EB 28 transcription factor binding to IGHM enhancer 3 29 nuclear Protein 1, transcriptional regulator How ATF4 facilitates such diverse cellular adaptations, ranging from anabolism to growth arrest, is an important and unsolved question. One possibility is that different ATF4 heterodimers or different combinations of ATF4 heterodimers mediate the different effects of signalling.

The ISR pathway involvement in autophagy and mitochondrial homeostasis

As written above, autophagy and mitochondrial quality control are cellular processes essential for muscle homeostasis, and deficiency in either is associated with muscle wasting [13,19,66]. The ISR is an important signalling involved in these two processes in a wide range of tissues and cells.

Role in autophagy.

During hypoxia, ER stress, amino acid deprivation, lipopolysaccharide treatment, or low protein diet, ATF4 binds to the specific promoter of genes involved in autophagy to promote (1) a pro-survival response in vitro or in the liver, heart and skeletal muscle of rodents [197,199,[211][212][213][214][215][216][217] or (2) a pro-lethal autophagy response in heart and kidney [218,219] (Figure 19). ER stress or hypoxia leads to the upregulation of certain autophagy actors in a PERK-dependent manner in vitro and in the heart of mice [197,211,212,214,216,218]. Of note, PERK regulates all stages of autophagy including induction, vesicle nucleation, phagophore elongation, and maturation [220]. GCN2 is also essential for the induction of autophagy-related genes upon amino acid starvation in vitro and in the mouse intestine, while a mutant form of eIF2α suppresses the autophagy process [197,[221][222][223]. Moreover, there are direct interactions between eIF2α subunits and core autophagy proteins, although it is not yet known whether these interactions are biologically significant [224,225].

Role in mitochondrial quality control. Over the past decades, growing evidence have placed the ISR signalling as essential in mitochondrial quality control, through the mitochondrial unfolded protein response (UPRmt). UPRmt is a mitochondria stress response induced by a loss of mitochondrial homeostasis. UPRmt activates a transcriptional program of mitochondrial chaperone proteins and proteases (i.e. encoded by nuclear DNA) to promote the recovery of mitochondrial proteostasis [226].

Nonetheless, if the UPRmt is unable to repair mitochondrial damages, it promotes the elimination of the entire mitochondrion by mitophagy. Finally, if the damages persist, cells undergo senescence and/or apoptosis [226]. UPRmt is evolutionarily conserved and there are three key regulatory proteins of UPRmt, including ATF4, which is often overexpressed upon mitochondrial damage [227]. Cells lacking a functional gene copy of ATF4 fail to upregulate several mitochondrial enzymes and exhibit a reduction in mitochondrial respiration. A global transcriptomic analysis has validated the presence of ATF4-binding motifs in many UPRmt genes [226][227][228]. In skeletal muscle, evidence of ATF4 activation by mitochondrial stresses is growing. For instance, ATF4 protein accumulation is linked to HRI or GCN2 activation following the loss of mitochondrial membrane potential in vitro [229,230], and is associated with PERK upon a genetic defect in mitochondrial fission in mice muscles [231,232] (Figure 19). In addition, mitochondrial stresses, induced by a genetic defiency in mitochondrial fusion or mitophagy in mice muscles, increase p-eIF2α, ATF4 protein levels and the expression of an ATF4 target gene, the fibroblast growth factor 21 (FGF21) [233]. Of note, although the biological effects of FGF21 are largely unknown, it is massively induced by a defect in mitochondrial homeostasis, and in turn, improves mitochondrial function [233]. There is evidence that the activation of UPRmt-ATF4 following mitochondrial disturbances in muscles can have protective or maladaptive effects. For instance, in a rare children mitochondrial myopathy (i.e. Reversible Infantile Respiratory Chain Deficiency), the induction of ATF4-FGF21 axis and the subsequent induction of mitochondrial biogenesis-related genes precede the complete disease recovery phase in humans [231]. On the contrary, genetic deletion of a mitochondrial fusion protein in muscles leads to an accelerated ageing phenotype with increased muscle atrophy and inflammation through an ATF4-FGF21-dependent mechanism [234].

Taken together, these studies highlight the intricate involvement of the ISR in autophagy and in mitochondrial quality control. These studies support a dual role for ATF4 in mediating survival and cell death responses, depending on the duration and type of stress, the cell type and the pathophysiological context. Moreover, much remain to be explored in understanding ISR-ATF4 involvement in autophagy and mitochondrial quality control in muscle homeostasis.

The ISR pathway implication in muscle atrophy

Some of the ISR members have been associated to muscle weakness and atrophy in different catabolic conditions and will be discussed in the following part (Figure 19).

The ISR kinases and eIF2α

PERK kinase. A ligand-activatable PERK kinase induces p-eIF2α, expression of ATF4 target genes, and leads to severe muscle atrophy within a few days after injection in mice muscles [235]. However, genetic ablation or pharmacological inhibition of PERK reduces skeletal muscle mass and strength and increases gene expression of UPS and ALS components in healthy mice muscles [236]. In addition, Perk is increased in a model of cancer cachexia-induced muscle atrophy in mice, but genetic ablation or pharmacological inhibition of PERK exacerbates muscle atrophy [237]. Of note, in this study, they observed that PERK increased (1) p-eIF2α and Atf4 expression and (2) gene expression of the Unfolded Protein Response (UPR) components, which is another PERK downstream signalling [237]. Therefore further studies are needed to decipher by which downstream pathways PERK acts as a negative or positive regulator of muscle mass (Figure 19).

GCN2 kinase. Gcn2 deficiency protects mice from denervation-induced muscle atrophy while forced

Gcn2 expression worsens denervation-induced atrophy [238]. The authors highlighted that GCN2 could promote FOXO3 nuclear accumulation, and the subsequent transcription of the atrogenes Trim63/MuRF1 and Fbxo32/Atrogin-1 [238] (Figure 19). Of note, whether the atrophic role of GCN2 is mediated by the downstream p-eIF2α-ATF4 signalling has not been yet demonstrated.

PKR kinase. Levels of phosphorylated PKR and eIF2α are increased in muscles of cancer cachectic patients [239] and mice [240]. In addition, the pharmacological inhibition of PKR attenuates muscle atrophy in cancer cachectic mice through a possible inhibition of NF-κB [240] (Figure 19). Increased levels of intracellular calcium might be the upstream stress activating PKR in skeletal muscle (Figure 19). Currently, as for GCN2 kinase, the atrophic role of PKR has not been demonstrated to be linked to the ATF4 downstream signalling. eIF2α. P-eIF2α is enhanced in muscles during cancer-cachexia and amyotrophic lateral sclerosis in mice or humans [237,239,241]. In addition, fasting-induced muscle atrophy is hampered when mice express a phosphorylation-resistant form of eIF2α [242]. However, p-eIF2α is decreased in atrophic mice muscles following food deprivation, while an upregulation of Atf4 and some of its target genes is observed [243]. In addition, p-eIF2α is also decreased in atrophying mice muscles during disuse and spinal cord isolation [244,245]. Therefore, p-eIF2α is definitely not a common feature of muscle atrophy, and its augmentation is likely more a consequence of various extracellular stresses during muscle wasting conditions (Figure 19).

The atrogene ATF4

Like other cell types, skeletal muscle fibres do not significantly express the ATF4 protein in the absence of cellular stress, especially as ATF4 is non-essential for the normal development or maintenance of skeletal muscle mass and function [246][247][248]. Mice with a lifelong absence of ATF4 expression in skeletal muscle fibres undergo normal skeletal muscle development and exhibit normal muscle mass and function until late in life, at which time they begin to exhibit protection from age-related muscle atrophy and weakness [246,247]. ATF4 is considered as an atrogene because its mRNA levels rise in muscle during many catabolic conditions that cause muscle atrophy in mice (i.e. denervation, spinal cord isolation, fasting, ageing, immobilisation, myopathies) [99,242,246,247,249]. In addition, a deletion of Atf4 in mice muscles limits muscle atrophy during starvation, immobilisation and ageing [242,[246][247][248]. Additionally, a transcriptionally inactive ATF4 does not lead to a reduction of myofiber size during fasting in mice. This suggests that the ATF4-mediated transcriptional program is required to induce atrophy [242]. When ATF4 is expressed in skeletal muscle fibres, it interacts with several different bZIP family members but only the heterodimerisation with C/EBPβ 30 is yet known as required for muscle atrophy caused by immobilisation [250] (Figure 19). The ATF4-C/EBPβ heterodimer induces the transcription of the growth arrest and DNA damage inducible alpha (GADD45A) gene in muscle fibres by binding a DNA sequence that is 100% conserved in all mammalian genomes [250] (Figure 19).

Of note, in most of the studies, the authors only measured ATF4 mRNA expression and expression of its target genes as evidence of its activity, because endogenous ATF4 protein cannot be reliably detected in skeletal muscle, presumably due to its low abundance, very short half-life, and lack of highquality antibodies. 30 CCAAT enhancer binding protein β The red and green lines represent respectively the proved pro-atrophic and pro-maintenance signalling, and the questions marks the unsolved questions.

At present, the mechanisms by which ATF4 is transcriptionally and translationally activated in catabolic conditions are unclear but may involve different mechanisms or combinations of mechanisms. For example, contrarily to anabolic conditions, mTORC1 activity is increased in muscle during advanced ageing and is thought to contribute to the pathogenesis of age-related skeletal muscle atrophy [207,251,252]. In this context, mTORC1 may be a driver of the ATF4 pathway in skeletal muscle. In contrast, many acute stress conditions repress mTORC1 activity in muscle fibres while inducing eIF2α kinase signalling [237,239,241,253]. Thus, in some situations, such as starvation, eIF2α signalling may be the driver of the ATF4 pathway in skeletal muscle fibres (Figure 19). Whether the canonical ISR signalling is always implicated as an atrophic inducer seems unlikely. Further investigation of these issues might uncover new unrelated signalling.

ATF4 target genes: the atrogenes GADD45A,CDKN1A and EIF4EBP1

The mechanism by which ATF4 promotes muscle atrophy does not lead to an increase of FBXO32/Atrogin-1 nor TRIM63/MuRF1 gene expression [242]. Much remains to be discovered, but it is currently known that ATF4 contributes to muscle atrophy by modulating the transcription of three genes considered as atrogenes: GADD45A, cyclin-dependent kinase inhibitor 1 (CDKN1A) and eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) [99] (Figure 19).

GADD45A.

GADD45A is a myonuclear protein that induces widespread transcriptional changes in muscles. It represses genes involved in anabolic signalling and energy production, and it induces proatrophic genes [246]. GADD45A transcript is weakly expressed in non-catabolic conditions in skeletal muscle fibres but strongly induced during muscle atrophy (e.g. ageing, amyotrophic lateral sclerosis, critically ill patients, fasting, immobilisation) in pigs, humans and mice [242,246,248,[254][255][256][257]. For example, GADD45A is increased by 22-fold in muscle biopsies from critically ill patients with severe generalized skeletal muscle atrophy compared to healthy controls [257]. Gadd45a is the earliest and most sustained gene shown to be increased in muscles after denervation in mice [258]. Forced expression of Gadd45a in mice muscles or cultured mice myotubes induces atrophy even in the absence of any catabolic stimuli. Additionally, ATF4 is necessary and sufficient to induce Gadd45a expression during fasting-and immobilisation-induced muscle atrophy in mice [242,246,259,260].

HDAC4 is required for increasing muscle atrophy induced by Gadd45a overexpression during denervation, and forced expression of Hdac4 is sufficient to induce muscle atrophy in healthy mice muscles [260]. GADD45A by forming a complex with MEKK4 31 in muscles, increases MEKK4 protein kinase activity, which leads to GADD45A-MEKK4-mediated skeletal muscle atrophy in healthy mice muscles [259]. (Figure 19). Despite the strong evidence that GADD45A is an atrogene, a recent study 31 mitogen-activated protein kinase kinase kinase 4 52 has shown that it is likely induced in the context of denervation for a protective effect because mice lacking Gadd45a show accelerated and exacerbated neurogenic muscle atrophy [258].

CDKN1A. Another important ATF4-C/EBPβ target gene in muscles is CDKN1A (P21 protein). CDKN1A gene expression is strongly associated with muscle atrophy in pigs, rodents and humans [242,246,248,[254][255][256][257]261,262]. Increased Cdkn1a expression in mice muscles is sufficient to induce muscle fibre atrophy and is required for ATF4-mediated muscle atrophy during immobilisation [248].

However, to date, the cellular mechanisms by which P21 induces muscle atrophy are not defined.

Although P21 protein is a well-known cell cycle inhibitor, its mechanistic role in muscle fibres seems likely to be different, essentially because muscle fibres have exited from the cell cycle. Its role in the control of skeletal muscle mass might involve the repression of the spermine oxidase (SMOX) gene expression, a gene suggested as anti-atrophic, even if the mechanisms remain completely unknown [262,263] (Figure 19).

EIF4EBP1. ATF4 heterodimers also induce the expression of the EIF4EBP1 gene, encoding for the wellestablished inhibitor of global protein synthesis 250,262] (Figure 5). EIF4EBP1 gene expression rises up in numerous catabolic conditions being as well considered as an atrogene [99,248,249,261,264]. Accordingly, Eif4ebp1 is often induced alongside Gadd45a and Cdkn1a during skeletal muscle atrophy in mice [246,247]. However, it remains unclear which ATF4 heterodimers regulate EIF4EBP1 gene expression [264] (Figure 19).

TRIB3.

Finally, another ATF4 target gene, the tribbles pseudokinase 3 (TRIB3), has been associated with muscle atrophy in numerous studies. Trib3 deficient mice show increased muscle mass and MPS rate while decreasing the expression of the atrogenes Trim63/MuRF1 and Fbxo32/Atrogin-1 in healthy muscles [265]. In addition, Trib3 deficient mice show attenuation of muscle fibre atrophy and fibrosis during ageing by increasing autophagy flux [266]. Finally, these mice are also partially protected from food deprivation-induced muscle atrophy [267] (Figure 19).

TAKE HOME MESSAGE

A major conceptual insight emerging from these studies is that the ISR pathway is involved (1) in the maintenance of muscle homeostasis likely through autophagy and mitochondrial quality control, but also (2) in the induction of muscle atrophy through ATF4 and its target genes. How such an interplay can occur with two different outcomes on skeletal muscle, remains to be elucidated.

A natural model of muscle atrophy resistance: the hibernating brown bear

The first three chapters of this review show (1) the need to preserve muscle mass in order to remain healthy, but also (2) that, despite the huge amount of data acquired on the multiple signalling pathways involved in atrophy, there is still no approved treatment that can be used in the clinic. Most, if not all, of the mechanisms have been elucidated using classical laboratory models in rodents and humans. In this thesis project, we have chosen to combine classical and biomimetic approaches.

Getting inspired by the oldest research laboratory: Nature

As noted earlier, global health is being challenged by an ageing population and epidemics of lifestyle diseases such as type 2 diabetes, obesity, atherosclerosis, osteoporosis, or muscle wasting. Rather than destroying and exploiting living beings, we should learn from and emulate ingenious evolutionary adaptations to solve current human challenges. Nature is the oldest of research and development laboratories, where failure becomes fossil and our environment is a secret of survival. Biomimicry or bio-inspiration is an approach that (1) seeks sustainable solutions to human challenges by mimicking nature's patterns and strategies and (2) has enabled significant human biomedical advances and progress [268]. For example, about one-third of the medicines we use today are derived from nature.

In addition, marine organisms have inspired polymers for medical adhesives [269] and microscopically small mosquito needles have inspired the development of small, flexible microprobes to be implanted in the brain [270]. A particular species of Namibian desert beetle has a system for collecting water by condensing fog into water droplets in its exoskeleton, and gradually channelling them to its head to drink. Inspired by this ingenious strategy, researchers have replicated this structure with glass and plastic, intending to cover existing objects to turn them into fog collectors, potentially ending the world's water shortage [271] (https://www.youtube.com/watch?v=IoflT3Uvels). The examples are vast and endless given the great diversity of the millions of species on Earth, which live in all types of environments from extreme temperatures to total hypoxia, to the driest places on the planet [268].

Therefore, the development of future drugs, technologies, or biomedical advances depends on humans preserving the diversity of nature. Hibernation is a perfect example of seasonal variability that holds clues to diverse solutions for human pathologies.

Hibernation: a bioinspired approach for human challenges

Hibernation comes from the word hibernare "the action of overwintering" and may date back 250 million years in the Antarctic Circle [272]. Hibernation is an adaptation used by some animals to cope with an episodic or seasonal lack of energy due to unfavourable environmental conditions (e.g., low food/water availability, high predation pressure) [273]. Torpor is at the heart of hibernation, it represents a period of metabolic suppression that can last from a few hours to several weeks.

Hibernation is a more elaborate behaviour, structured into several long periods of torpor often separated by brief periods of interbout arousals (IBA). IBA last approximately 24 hours and are present in nearly all small hibernators (i.e. <10kg) but not in hibernating bears (see section 4.4.3) [273]. The most typical hibernation season is the cold season (i.e. fall to spring) but is also found in mammals inhabiting temperate and tropical climates [273].

During hibernation, along with the sharp reduction in metabolic rate (MR), there is a strong reduction in respiratory and heart rates, followed by a decrease in body temperature (Tb) [274]. The decrease in Tb can be either extreme, as in small hibernators (e.g. Arctic ground squirrels) where Tb can drop below zero [275], or mild, as in bears, where Tb rarely drops below 30 °C [276-280]. Despite the multiplicity of phenotypes and regulations of hibernation onset, there is likely a single underlying mechanism that reduces MR, although it is still unknown. However, hypotheses are emerging about the role of the hypothalamus, in particular the dorsomedial hypothalamus and a recently discovered set of neurons in the preoptic area [281][282][283]. A complete understanding of the molecular mechanisms triggering torpor would be of great value in developing a process to induce a hibernation/torpor state in humans, for example for a manned deep space expedition [284].

In the next sections, we will discuss hibernation in bears and how understanding its characteristic could provide insights into human health challenges, particularly muscle atrophy.

Hibernation in bears

Hundred years of myths and legends

The English word "bear" reflects the long history between bears and humans. Around 500 BC, in Northern Europe, the brown bear was the undisputed predator. In Proto-Germanic, an ancient language spoken by the Nordic tribes, the bear was known by the harsh name of hrktos. Hunters were so scared of hrktos that they came to believe that the mere mention of the bear was a cause for trouble. Linguists believe that the word became so taboo that tribes began to use the euphemism bero "the brown one" instead. In other words, bears were the Voldemort of Northern Europe. As another example, among the Celtic islanders, the bear was more associated with power and sovereignty, as evidenced by the figure of King Arthur, the bear-king. The etymology of the name Arthur comes from the Celtic name of the bear, artos, which means both "bear" and "warrior". King Arthur's death occurs on All Saints' Day when the bears begin to hibernate. Like them, Arthur does not die, he goes into dormancy. According to the history books, it is at Candlemas that Arthur takes the sword Excalibur out of the rock, a symbolic day since it corresponds roughly to the end of the hibernation of bears.

Hundreds of myths and legends surround the bears. In recent decades, contemporaries have become interested in the bear for the characteristics of its hibernation characteristics and the opportunity that this represents for medicine.

Features of bear hibernation

Ursidae family. Bears are mammals in the diverse family Ursidae because (1) they comprise eight species in three subfamilies, (2) they are geographically widespread in North and South America, Europe and Asia and (3) they inhabit a wide range of ecological niches from Arctic ice to tropical rainforests [285]. Bears in warm climates, do not go into hibernation, nor do the giant panda or polar bears. In this manuscript, we will only discuss bears that hibernate, i.e. brown bears (Ursus arctos), American black bears (Ursus americanus) and Asiatic black bears (Ursus thibetanus).

Winter in the dens. Bears enter dens in October-November and remain there until late April or early May (Figure 20); both periods are highly dependent on weather conditions (i.e. snow levels).

Unfortunately, researchers have observed a decrease in hibernation duration due to global warming.

They estimate that for every 1°C increase in winter temperatures, bears hibernate six days less [286].

As mentioned before, hibernating bears do not exhibit IBA, and not only do they remain physically inactive inside their dens, but they also do not eat, defecate, drink or urinate [276][277][278][279][287][288][289]. isolation [279,290,291]. Furthermore, in hibernating bears, there is a significant decrease compared to active bears in average heart rate (i.e. from 50-80 to 10-30 beats per minute) [279,292,293] and respiratory rate (i.e. from 10-12 to 5-7 breaths per minute) [294] (Figure 21). At the renal level, the glomerular filtration rate decreases during bear hibernation compared to summer (i.e. from 117 ml to 37 ml per minute) resulting in the production of very small amounts of urine that are reabsorbed by the urothelium of the bladder [295]. MR and Tb are clearly linked. However, bears decrease their activity, heart rate and MR before decreasing their Tb before entering the den (Figure 21). Furthermore, Tb is the first physiological parameter to change before den exit, whereas bears maintain a reduced MR up to 3 weeks after den exit (Figure 21). Therefore, the pronounced reduction and delayed recovery of MR in hibernating bears suggest that the majority of metabolic suppression during hibernation is independent of Tb decline [278,279].

Energy storage. Fat storage is increased before hibernation (i.e. hyperphagia). For example, Swedish brown bears achieve this by eating lots of carbohydrate-rich berries [296]. During the fall, bears more than double their daily energy intake reaching a total weight gain of 40% [297]. During this period of hyperphagia, lipogenesis-related genes are upregulated in white adipose tissue (WAT) to promote fat storage [298]. Energy requirements in winter rely primarily on the mobilisation and oxidation of lipid fuels, with bears experiencing a loss of approximately 22-25% of their body mass during the hibernation season [299,300] and only a moderate loss of muscle protein (see section 4.4.4) [301,302].

The respiratory quotient 32 in bears decreases from 0.8 to nearly 0.7 in winter, reflecting pure fat burning [303,304]. In states of negative energy balance such as during hibernation or starvation, triglycerides (TG) stored in the WAT are converted to glycerol and FFA, which are used for gluconeogenesis 33 , ketogenesis 34 and β-oxidation 35 in the liver. Consistently, genes related to these 3 biological processes are upregulated in the liver of hibernating bears [305][306][307]. This is also consistent with the maintenance of blood glucose levels via gluconeogenesis, and the increase in circulating TG, FFA and ketone bodies during hibernation. Of note, a decrease in circulating glycerol is observed in winter, probably due to greater uptake by the liver and its reaction with ammonia to form amino acids (see section 4.4.4) [298,303,308-312]. Altogether, these data highlight the amazing metabolic flexibility 36 of hibernating bears [313,314]. 32 the ratio of the volume of carbon dioxide evolved to that of oxygen consumed by an organism or tissue in a given time 33 glucose production from non-carbohydrate carbon substrates 34 production of ketone bodies by fatty acid breakdown 35 fatty acid catabolism 36 capacity to switch among energy substrates to generate ATP depending on the physiological circumstances 57 Model for human pathologies. Hibernating bears appear to be insulin resistant compared to active bears. Insulin resistance is normally observed in hyperinsulinemic diabetic humans [312]. However and surprisingly, hibernating bears do not develop type 2 diabetes. Plasma cholesterol and TG levels are Evans et al., 2016 [279]). 58 twice as high in hibernating bears as in healthy humans, but they show no signs of developing atherosclerosis or cardiovascular damage [315]. In brief, bears readily emerge from their dens in spring and show no signs of organ damage [316] (Figure 22). Under similar conditions, humans would develop cardiovascular disease, obesity, muscle loss, osteoporosis and other deleterious health consequences.

Dozens of examples could be discussed regarding the extraordinary characteristics of bears during hibernation and the therapeutic possibilities it offers to treat human pathologies (Figure 22). The conservation of muscle mass during a long period of fasting and physical inactivity has attracted our attention and will be discussed in the next section.

Skeletal muscle features in hibernating bears

Over the six months of total physical inactivity and fasting, hibernating bears experience only a moderate loss of muscle protein content. Conversely, over a shorter period of time, this leads to a significant reduction in muscle mass and function in humans [87,90]. (2) decreases by 4-10% in the gastrocnemius and biceps femoris muscles [302,319], or (3) it decreases by 15% in the vastus lateralis muscle [301]. Interestingly, in the latter study, they found that the 15% muscle loss after 1 month of denning remained the same 4 months later [301] (Figure 23). Furthermore, the nitrogen content of the vastus lateralis muscle remained unchanged in winter compared to summer, indicating a moderate loss of proteins [301]. The limited decrease in muscle protein content is consistent with the slight increase in 3-methylhistidine observed in the serum of hibernating bears [309]. Some researchers have speculated that the small amount of atrophy exhibited by bears may simply be due to muscle dehydration [317]. However, other studies have not observed any change in muscle water composition during winter [302].

Muscle CSA. Remarkably, the number of muscle fibres as well as their CSA remain unchanged in hibernating bears in most studies [302,319,320]. A recent paper showed a 26% decrease in CSA in the sartorius muscle after 5 months of hibernation, but the authors considered that was a minimal loss compared to what would happen in humans [321]. Indeed, under a similar period of disuse, a muscle loss of about 150% would be expected in humans [322,323].

Fibre type composition.

It should be noted that the proportion of slow and fast muscle fibre types in active bears is roughly the same as in rodents or humans [320,324], and like humans, bears lack the type 2B fibre isoform [319,324]. Studies have reported conflicting changes in the proportion of fibre types between seasons with (1) an increase in type 1 fibre content and a decrease in type 2A fibre content [324,325], (2) a moderate shift towards more type 2 muscle fibres [302], or even (3) no change Lohuis et al. -2007 [301]). [302,319,320]. These discrepancies can be explained by the biochemical techniques used and the timing of the muscle sampling during hibernation.

Muscle strength and neuromuscular activity. The loss of muscle strength is about 29% after 110 days of anorexia and physical inactivity during hibernation in bears. This is about half of what is observed in humans confined to bed for 90 days, with a 54% loss in muscle strength while on a balanced diet [318,326]. Furthermore, hibernating bears show no or very limited changes in muscle contractile properties (e.g. contraction time, half relaxation time) [302,318,319,326]. Although bears do not exhibit as vigorous shivering thermogenesis as small hibernators, they do make occasional postural adjustments, wake up briefly and shiver. It has been suggested that this mild muscle activity may limit atrophy [278,318,327]. Finally, neural inputs cannot be considered as a mechanism to limit muscle atrophy. Indeed, the denervation-induced decrease in muscle mass in active bears is comparable to that observed in other mammals, whereas hibernating bears are partly resistant to denervationinduced muscle atrophy [328].

Regulation of metabolism and signalling pathways

Muscle protein sparing. Lohuis et al., showed that muscle protein synthesis and degradation were lower in bears during hibernation than during the active period [301]. Furthermore, they observed that both phenomena remained unchanged between the beginning and the end of hibernation, indicating that protein balance is maintained throughout the hibernation period (Figure 24). Furthermore, protein synthesis was greater than protein degradation in summer bear muscles, suggesting that they accumulate muscle protein during the season when food is available in abundance [301] (Figure 24). A comprehensive transcriptomic analysis in skeletal muscle also showed that bear hibernation results in (1) increased expression of genes involved in protein biosynthesis (translation) and ribosome biogenesis and (2) decreased expression of genes related to proteolysis in skeletal muscle [304,307,329] (Figure 25). Of note, whole-body protein sparing is also supported by transcriptional downregulation of genes related to amino acid catabolism in the liver of hibernating bears [305][306][307] (Figure 25).

Unchanged or decreased levels of circulating urea 37 and decreased aminotransferase activities reflect low muscle protein mobilisation in bears during winter [289,299,309,330,331]. This is consistent with the coordinated downregulation of genes involved in urea production in skeletal muscle, but also in the liver during hibernation in bears [304,307]. Furthermore, urea recycling is very efficient in hibernating bears, with 99.7% of the urea produced being recycled into protein, which probably limits muscle protein degradation [304,307]. The mechanisms remain to be clarified but urea recycling would 61 include a role for the gut microbiota in the hydrolysis of urea to ammonia, which would subsequently be used for the synthesis of amino acid, in particular glutamine [304,307] (Figure 25). Consistently, an increase in blood glutamine is observed in bears during hibernation [310].

Finally, hibernating bear muscles show an increase in a transcriptomic signalling mediated by MEF2A, contributing to a decrease of the expression of TRIM63/MuRF1 and FBXO32/Atrogin-1 [332] (Figure 25).

Muscle energy metabolism.

Glycolysis is preserved in the skeletal muscle of hibernating bears, as suggested by (1) an overall increase in protein abundance of all glycolytic enzymes, (2) an increase in muscle lactate dehydrogenase activity and (3) maintenance or reduction of circulating lactate levels [303,310,331] (Figure 25). Bear muscles still oxidise glucose and produce lactate during hibernation.

This could help maintain skeletal muscle functionality in unexpected situations, such as an emergency exit from the den that would require a rapid increase in ATP production [308,310]. Glycolysis could be fuelled by hepatic gluconeogenesis and mobilisation of muscle glycogen content, which is higher in bear muscles in winter compared to summer [310,320] (Figure 25). Together, these studies have led some authors to suggest that the Cori cycle 38 may be active in bears during hibernation thus contributing to muscle protein sparing [298,310] (Figure 25).

PDK4 39 is a switch that enables the use of lipid substrates and thus limits the entry of glycolytic intermediates into the tricarboxylic acid (TCA) cycle. PDK4 protein is increased in hibernating bears muscles [298,310,333], whereas proteins involved in the TCA cycle and β-oxidation are predominantly all downregulated in hibernating bear muscles [310,333] (Figure 25). Although lipids are the preferred 38 degradation of glucose into lactate in muscles, then transformation of lactate into glucose, and finally into glycogen in liver 39 pyruvate dehydrogenase kinase isoenzyme 4 Lohuis et al. -2007 [301]). fuels in winter, bear muscles metabolism is mainly characterised by reduced ATP turnover, so that the reduced β-oxidation in muscles during hibernation is due to and/or contributes to the depression of metabolic rate [307,310].

Hormonal/growth factor changes. Cortisol is a glucocorticoid hormone that reduces glucose uptake in peripheral tissues and stimulates lipolysis in adipose tissue and skeletal muscle. Elevated cortisol levels are observed during hibernation and are associated with reduced (1) protein levels of phosphorylated and total AMPK and ( 2) expression of PGC-1α/PPAR-α 40 in skeletal muscle and adipose tissue [308] (Figure 25). Low plasma levels of IGF-1 and IGF-2 are also recorded in hibernating bears, but the authors suggest that they would be present in a different spatial conformation than in summer and therefore more available to tissues such as skeletal muscle [334] (Figure 25). Serum from 40 peroxisome proliferator-activated receptor-gamma coactivator 1/ peroxisome proliferator-activator receptor The words in green, red and black represent respectively the functions/metabolites that are down-regulated, up-regulated or unchanged in winter compared to summer. Two-coloured words (e.g. urea, lactate) represent discrepancies found in the literature. Words in bold represent metabolic processes (e.g. glycolysis). Words in italics represent biological processes regulated at the genetic level. Dashed lines delineate altered intracellular mechanisms and pathways in skeletal muscle in winter versus summer.

hibernating bears is also enriched in some specific n-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA) [310,335] (see Appendix 10.5). DHA has previously been associated with increased muscle glycogen stores and subsequent prevention of muscle atrophy in fasted mice [336].

Interestingly, a 3-fold increase in muscle glycogen content is recorded in hibernating bears muscles compared to active summer bear muscles, while muscle protein is preserved [310] (Figure 25).

Antioxidant defences.

Most subunits of mitochondrial complexes are downregulated in hibernating bear muscles [310,337]. This may be due to reduced mitochondrial content in winter compared to summer in bear muscles. In addition, protein levels for UCP3 41 , which limits ROS production [338], are increased in bear muscles during hibernation [321,337]. Consistently, increased levels of proteins involved in cytosolic antioxidant systems, higher plasma antioxidant capacities and maintenance of the GSH/GSSG42 ratio are recorded in bear muscles during hibernation [337,339] (Figure 25). Overall, this suggests that bear muscles do not undergo significant oxidative damage in winter and/or that antioxidant defence systems remain effective [337].

Overall, metabolic adaptations in skeletal muscle (and other tissues such as liver and adipose tissue) promote ( 1) lipid catabolism during hibernation, (2) maintenance of glycolysis, and (3) regulation of intracellular pathways that contribute to the preservation of muscle proteins during this period of fasting and physical activity.

Circulating antiproteolytic compounds in hibernating bear serum

The preservation of vital functions of most organs in bears during hibernation, including skeletal muscle, has led researchers to speculate that active circulating factors may be responsible for these characteristics.

Rats muscles incubated ex vivo in the presence of hibernating bear serum exhibited a 40% decrease in net proteolytic rate compared to the muscles incubated with active bear serum. This inhibition of proteolysis was accompanied by a decrease in gene expression of the lysosomal (e.g. cathepsin B) and ubiquitin-dependent (e.g. ubiquitin) proteolytic systems. These results showed for the first time that a compound present in bear serum during hibernation had an anti-proteolytic property on skeletal muscle [340]. Subsequently, our team showed that cultivating primary human myotubes with hibernating bear serum favoured an increase in myotubes area compared to summer bear serum [341] (Figure 26). This was the first proof of concept that an active compound in bear serum was transmissible to human biological material. We showed that protein turnover in human myotubes was overall reduced when incubated with winter bear serum, with both a dramatic inhibition of proteolysis (i.e. UPS and ALS) and an average reduction in the rate of protein synthesis [341]. Therefore, winter bear serum was able to reproduce in human muscles cells the regulation of protein turnover already described in hibernating bears muscles (i.e. lower rate of protein synthesis and degradation). Recently, another team confirmed our results with an increase in total protein content in myotubes cultured with hibernating bear serum, although they did not observe any alteration in protein anabolism [342].

Overall, these few studies showed similar results, with hibernating circulating compounds being able to induce potent cross-species effects on human or rat muscle cells. It is therefore highly likely that the (2) myostatin and phosphorylated SMAD2 protein levels increase as squirrels emerge from torpor [343]. In the latter study, they also observed an increase in phosphorylated SMAD1/5 levels at the beginning of hibernation, which returned to normal levels when the squirrels emerged from torpor [343]. In the muscles of hibernating little brown bats (Myotis lucifugus), a decrease in myostatin Illustrative immunodetection and corresponding quantifcation of myosin heavy chain in cultured myotubes winter bear serum (WBS) or summer bear serum (SBS) treatment (from Chanon et al., 2018 [341]).

protein expression while an increase in the TGF-β inhibitor SMAD7 protein is observed compared to the active animal [344]. Similarly, a decrease in Mstn expression is also recorded in the muscles of hibernating ground squirrels (Spermophilus lateralis) [345]. To date, only one study has explored the regulation of Mstn expression in hibernating bear muscles and shown that it was lower in muscles at den exit compared to summer [321].

ATF4 signalling. In hibernating ground squirrels, ATF4 protein expression is strongly increased in skeletal muscle, and subcellular localisation studies have shown that ATF4 translocates into the nucleus during hibernation, as does its cofactor, the phosphorylated form of CREB-1 [346].

Furthermore, in the torpid muscles of the Daurian ground squirrels (Spermophilus dauricus), the levels of phosphorylated PERK and eIF2α, as well as ATF4 protein are increased during hibernation, and normalised during IBA and the active period [347].

Overall, these few data suggest that upregulation of ATF4 and downregulation of TGF-β signalling may play a role in the coordination of muscle maintenance in small hibernating mammals. However, the ability of hibernating bears to preserve skeletal muscle biochemical and performance characteristics during prolonged hibernation still needs to be explored with respect to these two signalling pathways.

TAKE HOME MESSAGE

(1) Biomimicry is an approach that seeks sustainable solutions to human challenges by mimicking nature's patterns and strategies and has enabled significant human biomedical advances and progress.

(2) Hibernation, particularly in bears, is of great interest for understanding the mechanisms that allow them to cope with prolonged fasting and physical inactivity without deleterious effects on the whole body. The hibernating bear, being resistant to muscle atrophy, is an interesting model for identifying new biomolecular actors that can possibly be translated to human pathophysiology where muscle atrophy is present.

(3) The regulation of TGF-β/BMP and ISR signalling pathway is important for muscle homeostasis in rodents and humans, and interesting clues have been found during hibernation in the muscles of small hibernators. However no studies have yet explored these signalling in the muscles of hibernating bears.

Objectives and strategies

The main objective of this thesis was to identify new molecular players and their mechanisms that could become therapeutic targets to combat muscle atrophy in humans. For this purpose, , we adopted a biomimetic approach using the brown bear, which is naturally resistant to muscle atrophy, and compared muscle adaptations to those observed in a classical model of sensitivity to atrophy. The project has been subdivided into three studies, as follows. The objectives were to (1) identify the underlying mechanisms implicated in the resistance of muscle atrophy although prolonged physical inactivity and (2) determine whether these mechanisms were oppositively regulated in a model of susceptibility to atrophy.

We performed a comparative transcriptomic analysis of the atrophy-resistant muscles of the hibernating brown bear and the atrophy-sensitive muscles of the hindlimb-suspended mouse.

Study 2 Induction of ATF4 atrogenes is uncoupled from disuse-induced muscle atrophy in halofuginone-treated mice and in hibernating brown bears (paper under review).

The objectif was to further explore the role of ATF4 signalling in skeletal muscle during basal and catabolic conditions.

KEY POINTS FROM THE LITERATURE:

(1) Muscle atrophy affects millions of people worldwide and despite intensive efforts using laboratory rodents models, there is still no easily used therapeuthic or preventive treatments.

(2) TGF-β signalling pathway have been extensively targeted for its pro-atrophic role. However, little is known about the BMP counterpart, which is promising for fighting muscle atrophy.

(3) ISR signalling has a dual and complex role in skeletal muscle homeostasis and needs to be further investigated.

(4) Hibernating bears resist muscle atrophy even when faced with long-term fasting and physical inactivity. Therefore, the hibernating bear is a promising model to find new avenues to fight muscle atrophy in humans.

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We first developed an experimental protocol for inducing the ATF4 signalling with the pharmacological molecule halofuginone (HF) in mice. We then (1) investigated the effect of ATF4 induction on mice muscles during basal and hindlimb suspension-induced atrophy conditions and ( 2) deciphered the HF molecular mechanisms in mice muscles. We also investigated the regulation of this pathway in the atrophy-resistant muscles of the hibernating brown bear.

Study 3

Winter bear serum induces similar characteristics in human muscle cells as those found naturally in hibernating bear muscles (preliminary results).

Our objective was to determine whether the molecular characteristics of the atrophy-resistant muscles of hibernating bears can be reproduced in human muscle cells.

We first analysed microarray data from human muscle cells cultivated with winter bear serum to assess whether there is a TGF-β/BMP signalling transcriptomic signature. We then measured TGF-β/BMP signalling transcriptional activity with luciferase reporter assay. We sought to compare at the genetic level, the changes occurring in muscles between a natural model of muscle atrophy resistance, the hibernating brown bear, and a model of susceptibility to muscle atrophy, the unloaded mouse. We thus performed muscle RNA sequencing and analysed common and different features in these two models to uncover unexplored intracellular mechanisms (Figure 27).

Experimental protocol

Muscle atrophy-sensitive model. We studied disuse atrophy in the hindlimb-suspended (HS) mice model [348]. HS is a method developed in the 1970s, used to mimic space flight and prolonged bed rest in humans. The mouse tail is attached to a device that elevates the hindlimb into an unloaded position (Figure 28). Unlike cast immobilisation-induced muscle atrophy in rodents, the HS procedure does not cause inflammation or fibrosis in muscles [349,350], making it an interesting model to study the role of disuse in the induction of muscle atrophy independently of any other parameters. Muscle atrophy-resistance model. The brown bear, remains resistant to muscle atrophy during hibernation, although confronted with two strong atrophy inducers (i.e. starvation and physical inactivity) (see section 4.4.4). Our laboratory belongs to an international consortium, the Scandinavian Brown Bear Research Project (http://bearproject.info/). Our team travels twice a year to Northern Sweden to collect biological samples (e.g. muscle, blood) from bears during the hibernation period (February) and the active period (June). A team of experienced veterinarians care for the bears under anaesthesia and monitor vital signs in the field (Figure 29). Bears are either male or female, from 2 to 3 years old, just before sexual maturity. The same bears are sampled twice a year using GPS collars. One of the disadvantages of using a wild animal is the difficulty of collecting biological samples due to the lack of proximity to their living area. Therefore, some periods during the hibernation or the active seasons remain unexplored in our analysis. Moreover, commercialized biological reagents (e.g. antibodies) do not necessarily react with the cellular components of the bear, and hence, the study of some signalling pathways, for example, is challenging. In the cages, there are two rails with wheels connected to the tails of the mice allow them to move. This system leaves the hind legs free to move without being able to grip. The mice can move with their front legs. Their food is underneath the grid.

Introduction

Muscle atrophy is defined as a loss of muscle mass and strength and is associated with adverse health outcomes, such as an autonomy decline and an increase in morbidity and mortality in many catabolic conditions (e.g., cancer cachexia, heart, and kidney failure, fasting, sepsis, injury, aging, or physical inactivity, etc.) [1][2][3][4][5]. Given the increase in sedentary behavior and improvement in life expectancy, and with to date still no proven therapeutic or preventive treatment to date, muscle atrophy remains a major public health issue (World Health Organization data) [6]. Skeletal muscle tissue represents an important reservoir of amino acids, which are mobilized during catabolic situations to preserve vital functions, resulting in an imbalance of contractile protein turnover (i.e., proteolysis exceeding protein synthesis) [7,8]. Catabolic stimuli (e.g., oxidative stress, endoplasmic reticulum disturbances, nutrient shortage, mitochondrial disruptions, etc.) activate a complex network of intracellular modulators, which in turn lead to the activation of the ubiquitin-proteasome system (UPS) and autophagy [9,10]. These two main proteolytic systems in muscle tissue involve a set of genes, i.e., atrogenes, whose expression at the mRNA levels is commonly altered during atrophy [11]. Cascades of events and players of muscle atrophy are well described and conserved in mammals [12,13] and include the transforming growth factor-β (TGF-β) superfamily, with TGF-β signaling acting as a negative regulator, and Bone Morphogenetic Proteins (BMP) signaling as a positive regulator of muscle mass [14]. The TGF-β pathway mediates muscle atrophy through cytoplasmic and nuclear signaling molecules SMAD2/3, mainly leading to the expression of the atrogenes TRIM63 (MuRF1) and FBXO32 (atrogin-1) [15]. Constitutive expression of SMAD3 triggers muscle wasting, and inhibition of SMAD2 and SMAD3 is sufficient to induce muscle growth in vivo [16][17][18][19]. Conversely, the BMP pathway mediates muscle mass maintenance through cytoplasmic and nuclear signaling molecules SMAD1/5/9, promoting a negative transcriptional regulation for a ubiquitin ligase required for muscle wasting, FBXO30 (MUSA1) [20], and increasing the expression of BMP receptors activity in muscles induced hypertrophy through Smad1/5mediated activation of mTOR signaling [21].

Hibernating bears (Ursidae family) are naturally resistant to muscle atrophy when facing the two major atrophic inducers, prolonged fasting and physical inactivity up to 5-7 months [22,23]. Conversely and during a shorter period, a loss of muscle mass and volume prevails in rodent models [24][25][26][27][28][29] and humans [4,5]. As in rodent models, the muscles of the active bear are sensitive to disuse after denervation, whereas the muscles of the hibernating brown bear (Ursus arctos) are resistant [30]. The hibernating brown bear, therefore, appears as a suitable model to study the underlying mechanisms of muscle mass maintenance [31]. How it withstands muscle loss in conditions where muscle atrophy is expected in non-hibernating mammals remains to be fully elucidated. However, several hypotheses can be raised; our recent analysis of muscle proteome in the hibernating brown bear revealed the maintenance of glycolysis and a turning down of ATP turnover [32]. In addition, we reported (i) a myogenic microRNA signature prone to promoting muscle regeneration and suppressing ubiquitin ligase expression in bear muscle during winter [33], as well as (ii) limited levels of oxidative stress [34]. To unravel the molecular basis of muscle maintenance at the mRNA level, the bear muscle transcriptome has already been explored using cDNA microarrays [35] or RNA sequencing [36,37]. These two transcriptomic studies suggested an overall reduction in energy and protein metabolism, consistent with metabolic suppression and lower energy demand in skeletal muscle during hibernation. Although these studies focused on the changes in bear muscle transcriptome during the hibernating versus active period, our study aimed to compare them with those that occur in the muscle transcriptome of disuse-induced atrophy in a mouse model. The rationale for such a comparative analysis of two contrasted situations of muscle atrophy or maintenance lies in the identification of potential new candidates, beyond already reported metabolic factors [35][36][37], that may help the hibernating bear resist atrophy, thereby providing new targets for fighting muscle atrophy in humans. Among the transcription factors involved in the regulation of the differentially expressed genes highlighted in bear muscle between the hibernation and active periods, eight were involved in the regulation of the TGF-β superfamily. We therefore subsequently focused on an in-depth analysis of the TGF-β and BMP intracellular pathways.

Materials and Methods

Animal Experiments

Bear Sample Collection

Biopsies from the vastus lateralis muscle were collected from 17 free-ranging brown bears, 2-3 years old (Ursus arctos; 11 females and 6 males), from Dalarna and Gävleborg counties, Sweden, from 2014 to 2019 (Table S6). The samples were immediately frozen on dry ice until storage at -80 • C. In a given year, the same bears were captured during winter hibernation (February) and recaptured during their active period (June). The study was approved by the Swedish Ethical Committee on Animal Experiment (applications Dnr C3/2016 and Dnr C18/2015), the Swedish Environmental Protection Agency (NV-0741-18), and the Swedish Board of Agriculture . All procedures complied with Swedish laws and regulations. Capture, anesthesia, and sampling were carried out according to an established biomedical protocol [38].

Mouse Model of Hindlimb Unloading

Our objective was to compare the muscle transcriptome of the hibernating bear with that in a rodent model of long-term physical inactivity. We chose the 10-day mouse model of unloading as an established disuse-atrophy model, with atrophic pathways still activated [28,39]. All experiments were conducted with the approval of the regional ethics committee (agreement n • D6334515) following the European Directive 2010/63/EU on the protection of vertebrate animals used for experimental and scientific purposes. This study was performed with 12 C57BL6/J adult male mice purchased from Janvier Labs (Le Genest-Saint-Isle, France). They were housed by pairs upon arrival in a polycarbonate cage in a controlled room (22 ± 2 • C, 60% ± 5% humidity, 12 h light/dark cycle, light period starting at 8 h), fed ad libitum, and given free access to water.

After 10 days of acclimatization, the mice were either kept unsuspended (Control, n = 6) or subjected to hindlimb unloading through tail suspension (Unloaded, n = 6) for 10 days. Custom tail suspension cages were adapted from previous studies [40,41]. The cages (43 × 29 × 24 cm) have an overhead frame to which two suspension systems are fixed in parallel on the width of the cage. These suspension systems are widely spaced in a cage so that mice can always be housed in pairs without touching each other. Unloaded mice had a metal ring attached near the base of their tails using surgical adhesive tape. This ring was then attached to a swivel that allowed a 360-degree rotation and was attached on a rail that covered the upper width of the cage. The height of the swivel has been adjusted to keep the mouse at an angle of about 30 • from the head so that the hindlimbs could not touch the ground or the walls. During the 10 days of unloading, mice showed only a very small body weight loss (<7%) that occurred within the first 3 days, with no change in food intake. At the end of the experiment, soleus muscles were rapidly dissected out and immediately frozen in liquid nitrogen and stored at -80 • C until analyses. As for the data used from the RNA sequencing of Zhang et al. [42], the soleus muscle atrophied by 37% (from 7.24 ± 0.27 mg in control mice to 4.58 ± 0.31 mg in unloaded mice, p < 0.001 according to the unpaired Student's t-test).

RNA Sequencing of Brown Bear Muscle

RNA Isolation

Total RNA from bear muscles was isolated as described [43]. Briefly, muscle RNA from six bears (paired samples collected in summer and winter in a given year for the same individual) was extracted using TRIzol reagent (Invitrogen, Courtaboeuf, France).

Illumina RNA Sequencing, Data Assembly, Statistical Analysis

We constructed RNA-Seq libraries with the Truseq stranded mRNA sample preparation kit from Illumina and sequenced them in two lanes on an Illumina HiSeq2500 (single-end, 50 bp, six libraries per lane). Image analyses and base calling were performed using the HiSeq Control Software (v2.2.70, Illumina, San Diego, CA, USA) and , Illumina, San Diego, CA, USA). Demultiplexing was performed using Illumina's conversion software (bcl2fastq 2.20). The quality of the raw data were assessed using FastQC from the Babraham Institute and the Illumina software SAV (Sequencing Analysis Viewer, Illumina, San Diego, CA, USA). A splice junction mapper, TopHat 2.1.1 [44] (using Bowtie 2.3.5.1 [45], Johns Hopkins University, MD, USA), was used to align the RNA-Seq reads to the Ursus arctos genome (GCA_003584765.1 ASM358476v1 assembly downloaded from NCBI) with a set of gene model annotations (GCF_003584765.1_ASM358476v1_genomic.gff downloaded on 17 June 2019, from NCBI). Final read alignments having more than three mismatches were discarded. Samtools (v1.9) (http://samtools.sourceforge.net) was used to sort the alignment files. Then, the gene quantification was performed with Featurecounts 1.6.2 (http://subread.sourceforge.net/) [46]. As the data were from a strand-specific assay, the read had to be mapped to the opposite strand of the gene (-s 2 option). Before statistical analysis, genes with less than 30 reads (cumulating over all the analyzed samples) were filtered out. Differentially expressed genes were identified using the Bioconductor(https://bioconductor.org/) [47] package DESeq2 1.26.0 [48] (R version 3.6.1 https://www.r-project.org/). Data were normalized using the DESeq2(https://bioconductor.org/packages/release/bioc/html/DESeq2.html, accessed on 16 June 2021) normalization method. Genes with an adjusted p-value below 5% (according to the Benjamini-Hochberg procedure that controls the FDR) were declared differentially expressed.

Functional and Pathway Enrichment Analysis

Hierarchical clustering of bear transcriptomic data (log-transformed) was performed using Cluster v3.0 software (University of Tokyo, Tokyo, Japan) from the 13531 reads [49]. Parameters were set as follows: median centering and normalization of genes for adjusting data and centroid linkage clustering for both genes and arrays. Dendrograms were generated and viewed using the Java Treeview v1.3.3 program (Alok Saldanha, Stanford University, Stanford, CA, USA) [50]. To identify the differentially expressed genes (DEGs), we selected a fold change (FC) Winter/Summer >|1.3| or <|0.77| and an adjusted p-value < |0.01| as cut-off standards, for the up-and down-regulated genes, respectively. Visualization of functional enrichment was performed using Metascape [51], a web-based portal for visualizing the inference of enriched biological pathways among the DEGs. For the given DEGs gene list, pathway and process enrichment analysis has been carried out with the following ontology sources: KEGG Pathway, GO Biological Processes, Reactome Gene Sets, Canonical Pathways, CORUM, TRRUST, DisGeNET, PaGenBase, Transcription Factor Targets, WikiPathways, PANTHER Pathway, and COVID. All genes in the genome have been used as the enrichment background. Terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor >1.5 (the enrichment factor is the ratio between the observed counts and the counts expected by chance) are collected and grouped into clusters based on their membership similarities. More specifically, p-values are calculated based on the accumulative hypergeometric distribution, and q-values are calculated using the Benjamini-Hochberg procedure to account for multiple testings. Kappa scores are used as the similarity metric when performing hierachical clustering on the enriched terms, and sub-trees with a similarity of >0.3 are considered a cluster. The most statistically significant term within a cluster is chosen to represent the cluster. The 10 top-score enrichment terms from that analysis are shown in Figure 1b,c S1). The color code for the functional cluster is indicated in the respective graphs, and the bold numbers in the different bars represent the numbers of DEGs found in the enriched terms. (d) Graph representing the 10 top-score of transcription factors involved in DEGs regulation from "Formation of a pool of free 40S subunits" and "Extracellular matrix organization" enriched terms. The bold TFs are involved in TGF-β superfamily regulation.

Transcriptomic Data Assembly and Statistical Analysis of Mouse Muscle

We used transcriptomic data from an already published study [42]. Briefly, in this study, C57BL6/J adult male mice were either kept unsuspended (Control, n = 4) or subjected to hindlimb unloading through tail suspension (Unloaded, n = 4) for 10 days. The fastq files of eight soleus muscles were downloaded from GEO (GSE102284). A splice junction mapper, TopHat 2.1.1(Johns Hopkins University, MD, USA) [44], was used to align the RNA-Seq reads to the mouse genome (UCSC mm10) with a set of gene model annotations (genes.gtf downloaded from UCSC on 29 October 2019; GeneIDs come from the NCBI gene2refseq file). Final read alignments having more than three mismatches were discarded. Samtools (v1.9, http://www.htslib.org/) was used to sort the alignment files. Then, the gene quantification was performed with Featurecounts 2.0.0 (http://subread.sourceforge.net/) [46]. As the data were from a strand-specific assay, the read had to be mapped to the opposite strand of the gene (-s 2 option). Before statistical analysis, genes with less than 20 reads (cumulating all the analyzed samples) were filtered out. Differentially expressed genes were identified using the Bioconductor [47] package DESeq2 1.26.0 [48] as previously described (cf. 2.2.2).

Western Blot

Vastus lateralis muscles from eleven bears (paired samples collected in summer and winter in a given year for the same individual; Table S6) and soleus muscles from 10-days control or unloaded mice (n = 6/group) (~30 mg) were used. Samples were homogenized using a polytron in 1 mL of an ice-cold buffer (10 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% Igepal CA630) containing inhibitors of proteases (Protease Inhibitor Cocktail) and phosphatases (1 mM Na 3 VO 3 , 10 mM NaF) (Sigma, Saint-Quentin-Fallavier, France). The homogenates were stirred for 1 h at 4 • C and then centrifuged at 10,000 g for 15 min at 4 • C. The resulting supernatants were then stored at -80 • C until use. The concentration of proteins was determined using the Bradford Protein Assay Kit (Biorad, Marnes-la-Coquette, France). Proteins were then diluted in Laemmli buffer and stored at -80 • C until use. Protein extracts were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using TGX™ FastCast™ 10% Acrylamide gels (Biorad, Marnes-la-Coquette, France) and transferred onto a PVDF membrane (Hybond P, Amersham, England). Blots were blocked for 1 h at room temperature with 5% bovine serum albumin in TBS buffer with 0.1% Tween-20 (TBS-T, pH = 7.8), then washed thrice in TBS-T and incubated (overnight, stirring, 4 • C) with appropriated primary antibodies against SMAD1/5 (PA5-80036, Thermo Fisher, Illkirch, France), SMAD2/3 (#8685, Cell Signaling Technology, Saint-Cyr-L'Ecole, France), SMAD4 (ab230815), CTGF (ab227180), and GDF5 (ab137698) (Abcam, Cambridge, United Kingdom). Blots were then washed and incubated for 1 h with an appropriate secondary horseradish peroxidase-conjugated antibody at room temperature. Signals were detected after incubation with Luminata Crescendo Western HRP substrate (Millipore, Burlington, MA, USA) and visualized using G: BOX ChemiXT4 (XL1) imaging system (Syngene, Frederick, MD, USA). Signals were then quantified using the GeneTools software (Syngene, Cambridge, UK) and normalized against the total amount of proteins determined by TGX signals to correct for uneven loading. Protein data were presented as individual values. The bilateral ratio paired Student's t-test was used to compare the muscles of bears during summer and winter (S and W, respectively). For muscles of control and unloaded mice (C and U, respectively), statistical significance was determined using the bilateral unpaired Student's t-test. Statistical analysis was performed using Prism 8 (GraphPad Prism 9, San Diego, CA, USA).

Results

Deep Changes in Brown Bear Muscle Transcriptome during Hibernation

The brown bear transcriptome data set revealed that, from 13531 transcripts commonly identified in all individuals, gene expression differed markedly between summer and winter (Figure 1a). We identified 4115 differentially expressed genes (DEGs) between muscles of the active and hibernating bear with mainly down-regulated genes (Table S1). The 10 top-score obtained from an annotation enrichment analysis performed from the down-and up-regulated DEGs highlighted several significant enriched terms regulated differentially in bear muscles between the two seasons (Figure 1b,c). For instance, the protein metabolism functional cluster was the most up-regulated in bear muscles in winter compared to summer, with "Formation of a pool of free 40S subunits", "Ribonucleoprotein complex biogenesis", or "Translation" enriched terms (Figure 1b). In addition, the tissue structure remodeling functional cluster was the most down-regulated in muscles of the hibernating versus active bear, with "Extracellular matrix organization", "Cell-substrate adhesion", or "Supramolecular fiber organization" enriched terms (Figure 1c). We then run a transcriptional regulatory network analysis to identify transcription factors (TFs) involved in the DEGs regulation from the two most differentially enriched terms between the two seasons (e.g., "Formation of a pool of free 40S subunits" and "Extracellular matrix organization") (Figure 1d). We found that SP1 was the TF the most involved in the DEGs regulation, as well as others TFs such as SP3, NFKB1, JUN, RELA, TFAP2A, ETS1, or SMAD4. Interestingly, these TFs cited above are all involved in the regulation or signal transduction of the TGF-β superfamily [52][53][54]. We therefore decided to focus on that superfamily, including (i) TGF-β signaling, which is a master regulator of the extracellular matrix organization and also involved in muscle mass loss, and (ii) BMP signaling, which has recently been discovered to be involved in muscle mass maintenance [20].

Hibernation Induces a Transcriptional Shift from the TGF-β to the BMP Pathway

From a thorough analysis of the literature [55][56][57], we have drawn up a list of the actors and regulators of the TGF-β superfamily. We then analyzed precisely how they were regulated at the mRNA level in the muscles of the hibernating bear between the winter and summer seasons.

The expression levels of two main TGF-β ligands, INHBA and MSTN, were dramatically lower (Fold change (FC) = 0.28 and 0.52, respectively) in winter, whereas INHBB expression was higher (FC = 1.85) (Figure 2, Tables S2 andS3). In BMP signaling, the main ligand described in muscle mass maintenance, GDF5, showed higher levels (FC = 2.5) in hibernating bear muscles compared to active muscles. Extracellular actors inhibiting (KCP, DCN, MGP, NOV, or CHRD) or promoting (CCN2 or BMPER) TGF-β and/or BMP signals were mainly down-regulated in winter compared to summer. Receptors from the TGF-β signaling were differentially expressed during hibernation, with ACVR1C and TGBBR2 levels being considerably lower (FC = 0.28 and 0.80, respectively) in winter compared to summer, whereas TGFBR1 and ACVR1B were higher (FC = 1.42 and 1.47, respectively). The GDF5 receptor, BMPR1B, was higher in winter compared to summer (FC = 1.37). The co-receptors that control intensity and specificity of the downstream TGF-β/BMP signaling were mainly down-regulated or unchanged in winter for both pathways, except for MUSK, an important BMP co-receptor in muscle cells, which was up-regulated (FC = 2.39). Overall, actors involved in the initiation of the TGF-β signal were mainly repressed while those driving the BMP signal were increased. S2 andS3). ECAg: Extracellular Agonist, ECAn: Extracellular Antagonist, L: Ligand, Co-R: Co-Receptor, RII: Receptor type II, RI: Receptor type I, TA: Transcriptional Activator, TR: Transcriptional Repressor, SBEs: SMAD Binding Element. Created with BioRender.com.

For TGF-β signaling, intracellular inhibitors such as ERBIN, LDLRAD4, EIF3I, STK17B, and PP2CA were up-regulated in winter bear muscles (FC = 1.26, 1.50, 1.37, 1.86, and 1.56, respectively), whereas some of the actors promoting the signal were lower expressed, i.e., DAB2 and TRAP1 (FC = 0.50 and 0.63, respectively). By contrast, for BMP signaling, intracellular inhibitors were mainly lower expressed in muscles of the hibernating bear, such as CTDNEP1, FKBP1A (FC = 0.70 and 0.67, respectively). Regarding the intracellular actors triggering TGF-β/BMP signaling, SMAD3 (TGF-β signaling; FC = 0.76) was less expressed, SMAD1 and SMAD5 (BMP signaling; FC = 1.99 and 1.30, respectively), and SMAD4 (common to TGF-β and BMP signaling; FC = 1.34) were more expressed in muscles of the hibernating bear compared to the active one. Thus, expression changes of the intracellular actors again suggest repression of TGF-β signaling but maintenance of the BMP signaling.

For nuclear components, transcriptional activators were either up-(FOXO3 and SP3, FC = 1.68 and 1.70) or down-regulated (e.g., KAT2B, ATF3 and ETS1, FC = 0.55, 0.46, and 0.54) for TGF-β, whereas mainly up-regulated in winter for BMP with YAP1, ZC-CHC12, and HOXC8 (FC = 1. 63, 2.21, and 2.46, respectively). Conversely, the transcriptional repressors were mainly up-regulated for the TGF-β pathway, i.e., TRIM33, YAP1, and SIRT1 (FC = 1.60, 1.63, and 1.82, respectively), whereas unchanged or lower expressed, i.e., TOB1 (FC = 0.78) for BMP during hibernation. Considering TGF-β target genes, an overall down-regulation was observed in winter compared to summer, as highlighted for the different collagen isoforms or the multiple metalloproteinases COL1A1/2 (FC = 0.04 and 0.07), COL3A1 (FC = 0.06), COL5A2 (FC = 0.28), COL6A1/3 (FC = 0.22 and 0.20), COL14A1 (FC = 0.14), and MMP2/14 (FC = 0.33 and 0.46) (Figure 2 and Table S2). For BMP target genes, it was less contrasted, with either an unchanged (e.g., RUNX2 or ID4), downregulated (e.g., ID1 or ID2, FC = 0.31 and 0.65), or up-regulated expression (RGS4 or KLF10, FC = 1.72 and 1.32) during hibernation, with RGS4 being a muscle-specific gene. Overall, this supports a general down-regulation of the transcriptional activity that drives TGF-β signaling, with possible maintenance of that for BMP signaling.

Finally, TGF-β and BMP signaling also use a shared SMAD-independent pathway, involving a branch of the MAPK (Mitogen-Activated Protein Kinase) pathway [58]. In this pathway, the expression of TRAF6 and its downstream actor MAP3K7 were higher in muscles of the hibernating bear compared to the active one (FC = 1.78 and 1.60, respectively). Some of the TRAF6 downstream actors, including MEF2A and MEF2C, which are key muscle transcription factors, were as well up-regulated (FC = 1.89 and 2.23) in winter compared to summer bear muscles (Figure 2).

TGF-β and BMP signaling pathways are tightly regulated by several UPS members, such as E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUB) [55,59]. TGF-β inhibitors localized from the receptors to the nucleus level were up-regulated in winter, such as for CUL1 (FC = 1.85), NEDD4L (FC = 1.63), and TRIM33 (FC = 1.60), the latter being also a positive regulator of BMP signal transduction (Figure 3 and Table S4). SMURF1, an E3 ligase inhibiting both TGF-β and BMP signaling, was also up-regulated (FC = 1.64) in muscles of the hibernating bear. For UPS activators of the TGF-β signaling pathway alone, several DUBs were up-regulated in winter, e.g., UCHL5, USP11, and USP4 (FC = 1.78, 1.77, and 1.37, respectively), the latter also promoting BMP signaling, and others were down-regulated such as for USP15 and TRAF4 (FC = 0.52 and 0.47). The TGF-β and BMP pathways regulate the transcription of some muscle-specific E3s (TRIM63, FBXO32, and FBXO30). None of them were differentially expressed in winter compared to summer (Figure 3 and Table S4).

Overall, this wide transcriptomic analysis highlighted a winter transcriptional pattern in muscles of the brown bear that was prone to shutting down TGF-β signaling while maintaining or even over-activating the BMP pathway.

Divergent Regulation of TGF-β and BMP Pathways in Atrophy-Resistant Muscles of the Hibernating Brown Bear versus Atrophied Muscles of the Unloaded Mouse

We compared the above-described brown bear muscle transcriptome to published transcriptomic data from a model of long-term physical inactivity in mice induced by 10 days of unloading, where the soleus muscle mass decreased by ~30% [42]. Comparing the two models, the muscle transcriptomic profiles appeared very different for selected gene expression related to TGF-β and BMP signaling pathways (Figure S1 and Tables S2-S4). Of note, gene expressions of TGF-β and BMP ligands were mainly differently regulated, as particularly evidenced for GDF5, which was up-regulated during bear hibernation, but down-regulated during unloading in the mouse model (FC = 0.18), and as well with MSTN strongly down-regulated during bear hibernation but unchanged in unloaded mouse muscles (FC = 1.57) (Figure 4a and Tables S2 andS3). Regarding receptor expressions, the two models responded quite similarly (Figure 4b and Tables S2 andS3), with three notable exceptions. Firstly, the gene expression of ACRV1C did not change in muscles of the unloaded mouse, unlike muscles of the hibernating bear, where a strong downregulation occurred (FC = 1.11 and 0.28, respectively), what we also observed with the gene expression of TGFBR2 (FC = 1.08 and 0.80, respectively). Secondly, the gene expression of BMPR1B, GDF5 receptor, was up-regulated in muscles of the hibernating bear but was down-regulated in muscles of the unloaded mouse (FC = 0.66) (Figure 4b). The intracellular actors SMAD3, SMAD4, and SMAD1 were regulated similarly by bear hibernation or mouse unloading, although not to the same extent (FC = 0.73, 1.10, and 1.50, respectively). However, SMAD2 was up-regulated (FC = 1.24), and SMAD5 and SMAD9 remained unchanged only in the muscles of the unloaded mouse (Figure 4c). S4). SBEs: SMAD Binding Elements. Created with BioRender.com. Regarding the E3s and DUB enzymes involved in the BMP pathway alone (Figure 5 upper panel), NEDD4 was similarly up-regulated in both models (FC = 1.63 and 1.28, respectively in bear and mouse), whereas FBXO30 (FC = 0.82) was down-regulated only in muscles of the unloaded mouse (Figure 5 and Table S4). The enzymes involved in the regulation of both TGF-β/BMP signaling were mainly up-or down-regulated in muscles of the hibernating bear but were mainly unaffected in unloaded mouse muscles (Figure 5 middle panel, Table S4). Finally, the E3s/DUB involved only in TGF-β signaling regulation were for half of them commonly unchanged or up-regulated, e.g., CUL1, UCHL5 or NEDD4L (FC = 1.17, 1.30 or 2.38, respectively), and for the other half oppositely regulated, e.g., SMURF2, CBLB, or TRIM62 (FC = 0.81, 1.50, and 1.97, respectively) in muscles of the unloaded mouse compared to muscles of the hibernating bear (Figure 5 lower panel and Table S4).

Overall, TGF-β and BMP signaling pathways were differentially regulated between atrophy-resistant muscles of the hibernating bear and atrophy-sensitive muscles of the unloaded mouse.

Hibernation Induces Changes in TGF-β and BMP Pathway Components at the Protein Level

To further compare these models of resistance and vulnerability to atrophy, we explored the protein levels of the SMAD intracellular actors. In the brown bear muscle, we observed a tendency to decrease for SMAD2 protein levels (p = 0.09) in winter compared to summer, whereas SMAD3 levels remained quite similar between the two seasons (Figure 6a,c,d). Protein levels of SMAD4 were higher in muscles of the hibernating bear compared to the active one (Figure 6a,e), but the converse was observed for SMAD1/5 (Figure 6a,f). As for mRNA, these SMAD proteins did not follow the same regulation pattern in muscles of the unloaded mouse, where none changed at the protein level (Figure 6b-f). The protein levels of CCN2, a TGF-β target gene that is also an extracellular activator of the TGF-β pathway and an inhibitor of the BMP pathway, were strongly lower in winter bear muscles but were unaffected in unloaded mouse muscles (Figure 6a,b,g). Finally, the protein levels for GDF5, a BMP ligand, remained unchanged in both muscles from the hibernating bear and the unloaded mouse (Figure 6a,b,h).

Discussion

Although basic knowledge regarding the underlying mechanisms of muscle atrophy is continuously growing, essentially from rodent models and clinical studies in humans, there are still no efficient therapeutic strategies for its prevention and treatment. To explore new avenues, we compared a model of muscle atrophy resistance, the hibernating brown bear [23], and a mouse model of disuse-induced muscle atrophy [42]. Therefore, we analyzed the bear muscle transcriptome and identified sweeping changes in gene expression between the summer-active period and the winter-hibernating period, and we compared them with transcriptomic data from muscles of the unloaded mouse.

Whereas the loss of muscle mass during inactivity is often associated with a decrease in muscle protein synthesis [42,[61][62][63], we reported here that genes implicated in protein metabolism were mainly up-regulated in muscles of the hibernating bear. This is consistent with previous studies that linked muscle atrophy resistance during hibernation to induction of protein translation [35,36,64]. Under activation of FOXOs transcription factors, atrogenes involved in both autophagy and UPS pathways are enhanced in rodents [65][66][67]. These atrogenes (e.g., MAP1LC3A, FBXO32, and ZFAND5) were indeed up-regulated or maintained in the unloaded mouse model but down-regulated or unchanged in muscles of the hibernating bear (Table S5). In agreement with previous studies, our data confirmed that proteolytic actors were up-regulated in disuse-induced muscle atrophy in rodents [11] but not in the bear model of muscle atrophy resistance [36]. Despite discrepancies between bear and mouse models (e.g., fed status, torpor vs. hindlimb muscle disuse), both display similar pathways controlling skeletal muscle mass and protein balance. For instance, the TGF-β signaling pathway has been reported to be evolutionarily conserved among several species, from Caenorhabditis elegans and Drosophila melanogaster to Mus musculus [68]. In addition, both models are responsive to denervation-induced atrophy when in active conditions [20,30]. We performed our analyses on the fast-twitch vastus lateralis muscle for the brown bear and the slow-twitch soleus muscle for the mouse. Thus, we cannot exclude the possibility that the differences recorded here between the models may partly have resulted from the specific nature of these muscles concerning their metabolic and contractile properties. However, it is noteworthy that, although fast-twitch muscles are not as sensitive to physical inactivity as slow-twitch muscles, both types of muscles atrophied in classical models of long-term physical inactivity in rodents [25,29,39,69,70] and humans [71][72][73]. Despite this difference in metabolic and contractile properties, a general down-regulation of genes involved in extracellular matrix (ECM) structure organization was observed in atrophied muscles from the unloaded mouse and atrophy-resistant muscles from the hibernating brown bear (Figure 1 and Table S2). This ECM structure remodeling is a common feature of other atrophic models reported during dystrophic diseases [74] or muscle disuse in rodents or humans [26,69,[75][76][77].

TGF-β is currently of major interest within the field of skeletal muscle biology. Indeed, the gene disruption of two of its ligands Myostatin (MSTN) or Activin A (INHBA), and the inhibition of their shared receptor ActRIIB (ACVR2B) promote a profound muscle hypertrophy phenotype in various conditions and species [78][79][80][81]. On the contrary, overexpression of MSTN or INHBA leads to the recruitment and phosphorylation of SMAD2-3, triggering an atrophic transcriptional program [16,17,[82][83][84]. Here, we reported that the expression levels of both ligands MSTN and INHBA were lower only in muscles of the hibernating bear. In addition, ACVR2B and SMAD2 mRNA levels were up-regulated in muscles of the unloaded mouse whereas maintained in muscles of the hibernating bear. At the protein level, SMAD2 was maintained in the muscles of the unloaded mouse and showed a tendency to decrease in muscles of the hibernating bear. However, despite extensive efforts and numerous antibodies tested, we could not characterize the SMADs phosphorylation status in bear muscles, which thus remains to be defined.

One TGF-β target gene, CCN2 (also known as CTGF), is an ECM protein associated with fibrotic activity that is up-regulated in several muscle chronic disorders (i.e., Duchenne muscular dystrophy or the amyotrophic lateral sclerosis) [85]. CCN2 is one of the main pro-fibrotic cytokines acting downstream of TGF-β signaling and can amplify its effects through enhancement of TGF-β ligand-receptor binding [85][86][87][88][89][90]. Inhibition of CCN2 gene expression reduced fibrosis and improved muscle and locomotor performance in a rodent model of amyotrophic lateral sclerosis [86]. During unloading, mRNA and protein levels of CCN2 were unchanged in the muscles of the unloaded mouse but were strongly lower in the muscles of the hibernating brown bear. Taken together, our data strongly suggest that TGF-β signaling is overall inhibited only in muscles resistant to atrophy during bear hibernation.

The BMP pathway is a potent inducer of bone and cartilage formation [91]. BMP signaling was also recently discovered as a regulator of muscle mass, as its inhibition abolished the hypertrophic phenotype of the MSTN-KO mouse [20], and an increase in its receptor activity induced important muscle hypertrophy [21]. Regarding BMP ligands, GDF5 is essential to muscle mass maintenance, binding preferentially to the type I receptor BMPR1B. GDF5 expression was strongly induced in denervated mouse muscles, and its inhibition worsened muscle atrophy, suggesting a role in counteracting denervation-induced atrophy [20]. We report here that the gene expression for both GDF5 and BMPR1B was strongly down-regulated in mouse muscles during unloading, whereas up-regulated in muscles of the hibernating bear. However, GDF5 protein levels were stable in the muscles of both the unloaded mouse and the hibernating bear. We recently demonstrated that circulating components of the hibernating bear serum were able to induce trans-species effects on human myotubes, notably inhibition of protein degradation. Therefore, we hypothesized that those components could be involved in the maintenance of muscle mass and strength in the hibernating bear [92]. Along with muscle mass maintenance, bear hibernation is also associated with bone mass maintenance [93], and thus GDF5 may constitute a possible target toward muscle and bone protection during long periods of physical inactivity and/or fasting. Unfortunately, the exploration of GDF5 concentration in bear serum was hampered by species cross-reactivity concerns with commercially available Elisa kits and thus remains to be addressed.

It has been proposed that the BMP and TGF-β common actor SMAD4 mainly engages with the TGF-β pathway, but switches to the BMP pathway when TGF-β transduction is reduced, and thus could be the limiting factor between these two signalings [20]. Moreover, denervation-induced muscle atrophy was exacerbated in the SMAD4 deficient mouse [20], and muscle mass was increased in humans with a mutation-associated gain of function in the SMAD4 gene [94]. We observed here higher mRNA and protein levels for SMAD4 in muscles of the hibernating bear, but only at mRNA levels in muscles of the unloaded mouse. This is concomitant with the overall down-regulation highlighted for the TGF-β signaling in the hibernating bear, which was not observed for the unloaded mouse. Thus, the inhibition of the TGF-β signaling in muscles of the hibernating bear may have released SMAD4 from TGF-β to BMP pathway to maintain muscle mass in a long period of disuse (Graphical abstract).

TGF-β and BMP share a SMAD-independent pathway that activates the E3 ubiquitin ligase TRAF6 [58]. In addition to its pro-atrophic role [95], TRAF6 is also required for myogenic differentiation and muscle regeneration via the MEF2 axis [96]. MEF2 is a conserved family of transcription factors involved in the control of muscle gene expression [97]. A recent muscle transcriptome analysis highlighted an inhibition of MEF2 transcription factors during human bed rest leading to skeletal muscle alterations [98]. Here, only MEF2A was up-regulated during unloading in mouse muscles, whereas TRAF6, MEF2A, and MEF2C were up-regulated in the hibernating bear muscles. We already observed a myogenic microRNA signature mediated by MEF2A signaling in the muscles of the hibernating bear, promoting mechanisms of muscle regeneration, suppression of ubiquitin ligases, and resistance to muscle atrophy [33]. Further studies are required to address whether this MEF2 signature could be under the control of the TGF-β and/or the BMP pathway through TRAF6.

Conclusions

Resistance to muscle atrophy in hibernating brown bears has so far been linked to a reduction in protein and energy metabolism. Here, we show for the first time that the TGF-β pathway is down-regulated whereas the BMP pathway is concomitantly sustained or even up-regulated in atrophy-resistant muscles of the hibernating brown bear. Thus, beyond strengthening the previous hypothesis of a hypometabolism enabling this natural resistance to muscle atrophy, our study provides new insights regarding the underlying mechanisms.

The originality of the current work lies in the choice to study the mechanisms involved in resistance to atrophy, and not solely, as in many studies, the mechanisms involved in the onset of atrophy. Our comparison of resistance and sensitivity to muscle atrophy animal models suggested the balance between the TGF-β and the BMP pathways as critical for preventing skeletal muscle atrophy over a long period of disuse. Many targeted therapies to counteract muscle atrophy already focus on TGF-β inhibition [99]. Our data open the way for further studies and clinical trials to test the effects of strategies to switch on (or sustain) the BMP pathway in combination with TGF-β inhibition to prevent disuse-induced muscle atrophy. CNRS

Discussion and perspectives

The main conclusion of our study is that the TGF-β/BMP balance appears to be crucial for the maintenance of muscle mass in hibernating brown bears (Figure 30). In addition to the points raised in the article, there are other issues worth discussing. First of all, we will discuss the transcriptomic regulation of genes related to protein synthesis in hibernating bears muscles. Second of all, we will address in 5 sub-sections the possible reasons why and how the TGF-β and BMP signalling pathways are differentially regulated in the hibernating bears muscles compared to summer. 24). This was associated with a lower protein degradation rate suggesting that overall muscle protein turnover was lowered in winter compared to summer in bears [301] (Figure 24).

Hibernation induces a transcriptomic reprogramming in genes related to protein synthesis and RNA metabolism in bear muscles

The decrease in the rate of protein degradation is consistent with the downregulation of genes related to protein degradation that we reported in the present transcriptomic study (see Paper 6.3) [351].

However, proteomic data from hibernating bears muscles revealed that proteins involved in the biological processes "protein biosynthesis" and "ribosome biogenesis" were not differentially regulated between winter and summer [310], whereas all were upregulated in winter bear muscles in our transcriptomic analysis (i.e. RPS2 43 , RPS4X, RPS5, RPS7 and RPSA) (see Paper 6.3) [351].

Induction of RNA metabolism-related genes. We identified RNA binding motif protein 3 (RBM3) as a gene that is highly up-regulated in winter compared to summer in bear muscles (see Paper 6.3) [351].

This appears to be a common feature of many hibernators in almost all tissues (e.g. brain, heart, liver muscle) [304,306,337,352,353]. RBM3 has been described as (1) facilitating the processing of RNA molecules in the cold and protecting mRNA transcripts from degradation in hibernating ground squirrels organs [352,354,355] and ( 2) playing a role in RNA metabolism and mRNA-related posttranscriptional processes (e.g. trafficking, stability, translation initiation) that ultimately affect protein synthesis [356]. This is consistent with other terms related to RNA metabolism that were found to be enriched in our transcriptomic study (e.g. mRNA 3'-end processing, mRNA splicing, regulation of mRNA stability) (Figure 31) (see Paper 6.3) [351].

What does the muscle translatome of the hibernating bear look like?

In many cell types, transcript levels do not always predict protein levels [357], hence the emergence of the term translatome. For example, synaptic plasticity requires relatively rapid de novo protein synthesis and specific mRNAs can be stored in neurons waiting for the precise moment to be translated [358]. To identify the level and type of mRNAs translated in hibernating versus active brown bears muscles, we could combine total RNA sequencing and ribosome profiling (ribosome sequencing). For the time being, many questions remain open, including what factors determine when and in which tissues these mRNAs should be translated? It is possible that elevated mRNAs levels of genes related to protein translation and ribosome biogenesis in winter bear muscles are waiting to be translated when needed. The transcriptional increase in protein biosynthetic-related genes detected in torpid squirrels facilitates the induction of translation in muscle during short bouts of arousal [354,355]. Bears, in contrast, do not undergo periods of arousal but maintain an alertness state during hibernation to potential dangers outside the den [294]. In agreement, we recently reported that hibernating bears have higher plasma levels of the endocannabinoid-like compound N-oleoylethanolamide, which has been described to have wakefulness-promoting effects in rodents (see Appendix 10.5) [335]. Therefore, in hibernating 99 bear muscles, the increase in protein translation-related mRNAs could serve as a rescue in case of unexpected exit from the den and thus a rapid reactivation of general protein synthesis.

Hibernation induces a transcriptomic reprogramming in TGF-β superfamilyrelated genes in bear muscles

The main message from our study is that the TGF-β pathway is down-regulated whereas the BMP pathway is concomitantly maintained or even up-regulated at the transcriptomic level in atrophyresistant muscles of the hibernating brown bear. Our data suggest that the balance between the TGFβ and the BMP pathways is crucial for preventing skeletal muscle atrophy during a long period of disuse

[351] (Figure 30). In the following subsections we will discuss (1) the expression of target genes of BMP Figure 31. Detailed enriched terms from the biological processes "Protein metabolism" and "RNA metabolism" from the differentially expressed genes in Cussonneau et al., 2021 [351].

Terms written in the same colour represent closely related biological terms.

signalling in skeletal muscle, (2) the regulation of the intracellular actor SMAD4, (3) the regulation of the extracellular actor CCN2, ( 4) the relationship between TGF-β/BMP signalling and changes in lipid membrane composition, (5) the relationship between TGF-β/BMP signalling and neuromuscular junction integrity and ( 6) muscle-organ crosstalk and connection with TGF-β/BMP signalling.

Identification of BMP target genes in skeletal muscle.

To our knowledge, no study has explored the transcriptomic signature of BMP signalling in skeletal muscle. Therefore, one of the final objectives of this thesis project was to draw a list of genes induced upon activation of BMP signalling. We first looked for easy-to-use tools for the preliminary experiments and therefore chose the immortalised human muscle cell line CCL136 (rhabdomyosarcoma cell line) as they are undifferentiated cells, which saved time and made the genetic manipulation more efficient. CCL136 were transfected with a dominant negative BMP type 1 receptor (BMPR1A/ALK3) where the kinase is inactivated (K261R) blocking the signal to be transduced [367], and treated with GDF5 ligand to activate BMP signalling (see Appendix 10.1). We observed a lower induction in total and phosphorylated SMAD1/5 and SMAD4 protein contents following GDF5 treatment in CCL136 expressing ALK3-KD compared to non-transfected cells (Figure 32).

The slight decrease in SMADs protein content could be explained by compensatory mechanisms set up by cells with other BMP receptors. Unfortunately, the transfection conditions were not optimal when tested with C2C12 myotubes and human primary myotubes. For these cell lines, the use of viral particles (e.g. lentivirus) to transduce the inactive receptor could improve the transfection efficiency.

In addition, other methods can be used to attenuate BMP signalling and overcome the compensatory mechanism of the receptors, for example, the use of siRNAs against SMAD1 and/or SMAD5 or treatment with the BMP inhibitor noggin. Once the optimisation is done, we will perform RNA and chromatin immunoprecipitation (ChIP) sequencing and obtain a list of BMP-dependent genes in muscle. Then, further analysis of the transcriptome of hibernating bear muscles will confirm whether the identified genes are indeed upregulated in this model of natural resistance to muscle atrophy. Immunoprecipitation protocols were optimised for SMAD4, SMAD1/5 and SMAD2/3. However, our conditions, unfortunately, did not enable the co-immunodetection of SMAD4 with either SMAD1/5 or SMAD2/3 (Figure 33).

Co-IP is highly dependent on protein-protein interactions. As bear muscles samples are necessarily frozen when sampling in the field, protein-protein interactions may have been disrupted by a freezethaw cycle, which may explain the lack of co-immunodetection. Another antibody against SMAD4 may be used to avoid a possible overlap of the epitope of the SMAD4 antibody with the protein-protein interaction site. Cross-linking the binding partners could also help to stabilize physiological interactions throughout extraction procedures involving mechanical and chemical stresses and thus enhance protein-protein interactions. However, for now, whether the proportion of SMAD4 recruited to TGF-β versus BMP signalling may change in winter versus summer in bear muscles remains unsolved.

SMAD4 stability could be different in bears vs. other mammals.

The analysis of the SMAD4 protein sequence revealed two highly conserved domains separated by a proline-rich linker, which is a substrate for kinases and phosphatases. This linker serves as a binding platform for cofactors and ubiquitin ligases, which tag SMAD4 protein for activation or degradation [368,369]. The SMAD4 protein sequence is highly conserved in metazoans. In mammals, the bear or mouse share 98% sequence 103 homology with the human SMAD4 protein. In the SMAD4 linker region, a Threonine is found at position 272 (Thr272) and is well conserved from drosophila to human (Figure 34). However, in all members of the Ursidae family (red box), this Threonine is replaced by a Serine (Figure 34). This characteristic is not shared by other small hibernators (black box) (Figure 34).

An in vitro study revealed a putative regulatory site consisting of four threonines in the linker region of SMAD4, including Thr272 [370,371]. The authors showed that activation of the MAPK pathway induces phosphorylation of Thr276, which initiates three sequential phosphorylations by GSK3 44 on Thr272, 268 and 264. This generated the recognition of SMAD4 by the E3 ubiquitin ligase β-TrCP leading to its proteasome-dependent degradation [370,371] (Figure 35). 44 glycogen synthase kinase-3 This sequential phosphorylation of SMAD4 may not be possible in the Ursidae due to the replacement of this Threonine by a Serine. This could thus stabilise SMAD4, explaining the slight increase in its protein content in hibernating bear muscles [351]. This change in sequence could also allow SMAD4 to interact differently with its partners. Whether this stabilisation and/or interaction with other proteins play a role in regulating the TGF-β/BMP balance in muscles remains to be explored. Myotube culture experiments could be performed by mutating Thr272 within SMAD4 to explore the consequences on TGF-β or BMP transcriptional activities. In addition, using Surface Plasmon Resonance, the real-time association and dissociation rates between wild-type or mutated SMAD4 and SMAD1/5 or SMAD2/3 could be accurately determined.

CCN2 is strongly reduced in hibernating bear muscles

We showed that the cellular communication network factor 2 (CCN2) mRNA and protein levels were strongly reduced in hibernating bear muscles compared to the summer counterpart (see Paper 6. has been reported to be induced by mechanical stretch in vitro in a TGF-β signalling-dependent manner Hibernating bear muscles do not experience fibrosis as shown by the downregulation of most of the extracellular matrix organisation-related genes (see Paper 6.3) [351]. Therefore, our data suggest that in addition to targeting CCN2 to reduce fibrosis in muscular dystrophies, this strategy could also be of interest in other muscle-related diseases without fibrosis likewise the hibernating bear muscles.

Is the regulation of TGF-β/BMP balance linked to the modification of the lipid membrane composition?

In hibernating bear serum, we and others published an increase in free circulating fatty acids and triglycerides arising from the lipolysis of the adipose tissue [298,303,309,310,331,335]. These NMJ in hibernating bear muscles. Amazingly, hibernating bears are partially resistant to denervationinduced muscle atrophy, whereas summer-active bears are susceptible to it like other mammals [328].

Whether the maintenance of BMP signalling in muscles is responsible for this resistance remains to be elucidated (Figure 36). No study has yet explored in detail the structure and characteristics of the NMJ in active versus hibernating bears. For that purpose, histological studies of NMJ markers will be soon initiated in our laboratory.

6.4.2.6

The muscle-organ crosstalk and connection with TGF-β/BMP signalling.

Is GDF5 synthesised and released by adipose tissue? GDF5 and its receptor BMPR1B are strongly upregulated in the hibernating bear muscles relative to its summer counterpart, whereas significantly downregulated in the unloaded mice muscles (see Paper 6.3) [351]. We wondered whether the GDF5 ligand was increased in winter bear serum and could explain the induction of BMP-related gene transcription. Unfortunately, serum GDF5 concentration could not be measured because the only commercially available ELISA 45 kit did not work in bears (see Appendix 10.1). GDF5 is primarily 108 synthesised and secreted by the salivary glands, which is probably not a dynamic tissue during hibernation since bears do not drink or eat for 6 months. GDF5 is also present in adipose tissue (Human Protein Atlas data https://www.proteinatlas.org/ENSG00000125965-GDF5/tissue). In mice, GDF5

promotes thermogenesis in subcutaneous white adipose tissue (sWAT) after cold exposure via non-SMAD p38 signalling [395]. In addition, GDF5 facilitates the development of brown fat-like cells in sWAT tissue via SMAD1/5 signalling in mice [396]. However, brown fat in hibernating brown bears has only been described in one study [397], which was subsequently refuted [398]. Shivering plays a role in active thermogenesis in muscles, but there is also non-shivering thermogenesis that occurs primarily through the metabolism of brown fat and, to a lesser degree, white fat [399]. If GDF5 is increased in winter bear serum, it would not only contribute to the maintenance of muscle mass induced by BMP signalling but could also play a role in non-shivering thermogenesis in white adipose tissue (Figure 37).

The concentration of GDF5 in bear serum could be assessed by mass spectrometry. Moreover, exploring the gene/protein expression of GDF5 and BMP-related components in adipose tissue could provide valuable information on whether GDF5 is synthesised and released from adipose tissue into the bloodstream during hibernation. In green and red, the genes down-and upregulated respectively in hibernating bear muscles in Cussonneau et al., 2021 [351].

TGF-β signalling and muscle-bone

In addition, myostatin, produced in muscle, stimulates the production and the differentiation of osteoclasts responsible for bone resorption through SMAD-dependent signalling [406][407][408][409] In this study, we aimed to explore the impacts of a controlled induction of ATF4 signalling on skeletal muscle. We selected the molecule halofuginone (HF), which (1) induces eIF2α-ATF4 signalling and ( 2)

is already used and well tolerated in mouse dystrophic models. We designed an experimental protocol, choosing (1) the dose of HF, ( 2) the mode and frequency of administration, and ( 3) the duration of treatment (data not shown). Once the protocol was validated, it was tested in mice either in basal conditions or when they were subsequently subjected to muscle atrophy induced by hindlimb suspension (HS) (Figure 38). We also took advantage of a model of muscle atrophy resistance that we previously explored (see Paper 6.3) [351] and studied the regulation of the ATF4 atrogenes in the hibernating brown bear muscles. We showed that induction of ATF4 signalling was not associated with atrophy in muscles from HF-treated mice or hibernating brown bears. We also investigated the molecular mechanisms of HF in skeletal muscle.

Introduction

Many unloading conditions (e.g., microgravity, bed rest, or physical inactivity) lead to a loss of muscle mass and strength. This muscle atrophy is associated with adverse health effects such as autonomy decline and increased morbidity and mortality [1][2][3]. Considering the lack of proven, easy-to-use therapeutic or preventive treatment, muscle atrophy remains a major public health issue (World Health Organisation data) [4].

The underlying molecular mechanisms of muscle atrophy involve the dysregulation of a complex network of intracellular pathways leading to an imbalance in protein turnover [5][6][7][8][9]. Activating transcription factor 4 (ATF4) is overexpressed in many conditions of muscle atrophy [10][11][12][13][14] and is, therefore, considered as an atrogene, i.e., one of the genes with expression at the mRNA level that is commonly altered during atrophy [14].

ATF4 belongs to the integrated stress response (ISR) pathway, a conserved intracellular network activated in response to various intrinsic and extrinsic stresses (e.g., amino acid (AA) depletion and endoplasmic reticulum (ER) stress) to restore cellular homeostasis [15]. Activation of the ISR involves phosphorylation of eukaryotic translation initiation factor 2 (eIF2α) by several kinases (i.e., general control nonderepressible 2 (GCN2), protein kinase RNA-like ER kinase (PERK), protein kinase R (PKR), heme-regulated inhibitor (HRI), and microtubule affinity-regulating kinase 2 (MARK2)), resulting in the global inhibition of protein synthesis but the increased translation of certain mRNAs, including ATF4 [15,16]. Inhibition of ATF4 in skeletal muscle limits starvation-, immobilisation-, and ageing-induced atrophy, whereas ATF4 induction results in muscle wasting [11][12][13]17]. ATF4 target genes include some atrogenes, such as GADD45A and CDKN1A, that are required for ATF4-mediated muscle atrophy [11][12][13]18], and TRIB3, which is involved in fasting-and ageing-induced muscle atrophy [19,20]. However, ATF4 targets also include genes that may be involved in the maintenance of muscle homeostasis. Indeed, ATF4 contributes to the transcription of autophagy-related genes [21][22][23][24] and is activated during mitochondrial perturbations (e.g., oxidative stress) to restore mitochondrial homeostasis [25,26]. In fact, activation of the eIF2α-ATF4 pathway by the pharmacological molecule halofuginone (HF), prior to stressful events (i.e., ischemia-reperfusion injury models) has shown positive effects on the preservation of kidney and liver function [27]. Moreover, the maintenance of autophagy and mitochondria homeostasis are essential for maintaining muscle mass [28][29][30]. Altogether, this led to the hypothesis that the ATF4 pathway may have a dual role in skeletal muscle.

Interestingly, halofuginone also improved muscle performance during dystrophies, mainly through its antifibrotic properties [31][32][33][34]. Whether these beneficial effects involve a regulation of the ATF4 pathway has never been investigated. However, they mainly involved the inhibition of the transforming growth factor-β (TGF-β) pathway [35][36][37]. The TGF-β signalling pathway acts as a negative regulator of muscle mass, notably through the transcriptional induction of the atrogenes TRIM63/MuRF1 and FBXO32/Atrogin-1 [38][39][40]. When inhibited, it promotes a profound muscle hypertrophy phenotype in various conditions and species [41,42]. Members of TGF-β signalling belong to the TGF-β superfamily [38,39], as do the bone morphogenetic protein (BMP) signalling members. The BMP signalling pathway [39,43] instead acts as a positive regulator of muscle mass through the transcriptional repression of the atrogene FBXO30/Musa1, which is required for denervation-induced muscle loss [44,45]. When inhibited, it profoundly exacerbates denervation-induced muscle atrophy [44,45].

This study aimed to explore the impacts of HF-induced ATF4 signalling on skeletal muscle under basal conditions and hindlimb suspension-induced atrophy in mice. We further deciphered the molecular mechanisms of HF by focusing on protein metabolism and TGF-β/BMP signalling. We also took advantage of a model of muscle atrophy resistance that we previously explored [46], and studied the regulation of the ATF4-induced atrogenes in hibernating brown bear muscle.

Results

Induction of ATF4-Regulated Atrogenes Does Not Affect Muscle Mass in Mice

We used halofuginone (HF) to induce ATF4 transcriptional activity in mouse muscles, and we investigated the effect on skeletal muscle mass. For that purpose, mice were treated with HF three times a week for up to 4 weeks. Six hours after the last HF administration at the end of each week, we measured the mRNA levels for some ATF4 target genes, involved in muscle atrophy, i.e., Trib3, Cdkn1a, Gadd45a, and Eif4ebp1 (Figure 1A-F). Except for Atf4, for which mRNA levels were elevated during the first 2 weeks of HF treatment, Trib3, Cdkn1a, and Gadd45a were all overexpressed in muscles after 2 weeks of HF treatment compared to H 2 O-treated mice. Of note, Eif4ebp1 mRNA levels increased in mouse muscles after 4 weeks of HF-treatment compared to H 2 O, with a noticeable trend after 3 weeks of treatment (Figure 1F). We also investigated the regulation of other ATF4 target genes and showed the overexpression of Asns over the 4 weeks of HF treatment as well as a trend for Ddit3 and Ppp1r15a (Supplementary Figure S1).

istration at the end of each week, we measured the mRNA levels for some ATF4 target genes, involved in muscle atrophy, i.e., Trib3, Cdkn1a, Gadd45a, and Eif4ebp1 (Figure 1A-F). Except for Atf4, for which mRNA levels were elevated during the first 2 weeks of HF treatment, Trib3, Cdkn1a, and Gadd45a were all overexpressed in muscles after 2 weeks of HF treatment compared to H2O-treated mice. Of note, Eif4ebp1 mRNA levels increased in mouse muscles after 4 weeks of HF-treatment compared to H2O, with a noticeable trend after 3 weeks of treatment (Figure 1F). We also investigated the regulation of other ATF4 target genes and showed the overexpression of Asns over the 4 weeks of HF treatment as well as a trend for Ddit3 and Ppp1r15a (Supplementary Figure S1). These data underline an activation of the ATF4 transcriptional activity. However, despite the overexpression of the ATF4-regulated atrogenes, the mass of the gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus (EDL) muscles remained unchanged during the 4 weeks of HF treatment (Figure 1G, Supplementary Figure S1). Altogether, these data suggest that a long-term halofuginone administration induced ATF4-regulated atrogenes without leading to muscle atrophy.

Overexpression of ATF4-Regulated Atrogenes during Hindlimb Suspension Is Uncoupled from Muscle Atrophy in HF-Treated Mice

We then investigated the effect of the induction of the ATF4 pathway by HF treatment during the muscle atrophy induced by hindlimb suspension (HS). Briefly, mice received HF three times a week for 3 weeks and were then, 3 days after the last HF administration, hindlimb-suspended or not for 3 or 7 days (Figure 2A). Measurements were thus performed at least 6 days after the last HF administration. HF induces ATF4 activation through the phosphorylation of eIF2α [47]. We observed that HF treatment resulted in overall higher phosphorylated and total eIF2α protein levels compared to H 2 O-treated mice (Figure 2B-D). In addition, HS led to an overall decrease in phosphorylated eIF2α protein levels compared to the controls (Ctrls) (Figure 2B,C). In addition, levels of the mRNA encoding the phosphatase GADD34 (Ppp1r15a) were higher at 3 days of hindlimb suspension (HS3) compared to the Ctrls in both H 2 O-and HF-treated mice (Supplementary Figure S2). The expression of ATF4-regulated genes was not different between Ctrl-HF and Ctrl-H 2 O groups (Figure 2 and Supplementary Figure S2). The mRNA levels of Atf4 and its target genes involved in muscle atrophy, i.e., Trib3, Cdkn1a, and Eif4ebp1, were higher during HS in both H 2 O-and HF-treated mice (Figure 2E-H), while the mRNA levels of the ATF4-regulated atrogene Gadd45a remained unchanged (Figure 2I). Of note, mRNA expression of other ATF4 target genes remained unchanged for Asns or slightly decreased upon HS for Ddit3. (Supplementary Figure S2). Altogether, Figures 1 and2 shows that (i) HF administration induced ATF4-regulated atrogenes after 6 h, but no more after 6 days, indicating that this effect was rapid and transient, and (ii) HS induced overexpression of these atrogenes.

We next investigated the outcomes on skeletal muscle. Gastrocnemius muscle had atrophied only in H 2 O-treated mice after 3 days of HS (H 2 O-HS3) and in both H 2 O-and HF-treated mice after 7 days of HS. Surprisingly, the average of the fibre cross-sectional area (CSA) did not change in H 2 O-HS3 mice compared to the Ctrls but was lower after HS7 regardless of the treatment (Figure 3B). We further analysed the distribution of gastrocnemius fibre CSA (Supplementary Figure S3A,B). We reported (i) a lower proportion of small fibres and (ii) a higher proportion of large fibres in HF-treated mice compared to the H 2 O group (Supplementary Figure S3A). This observation was restricted to fast-twitch fibres (i.e., 2X/2B) (Supplementary Figure S3B). Taken together, our data suggest that induction of ATF4-regulated atrogenes is not associated with muscle atrophy after 3 days of hindlimb suspension in HF-treated mice and, thus, that HF slightly preserves muscle mass during HS. We next investigated the outcomes on skeletal muscle. Gastrocnemius muscle had atrophied only in H2O-treated mice after 3 days of HS (H2O-HS3) and in both H2O-and HF-treated mice after 7 days of HS. Surprisingly, the average of the fibre cross-sectional area (CSA) did not change in H2O-HS3 mice compared to the Ctrls but was lower after HS7 regardless of the treatment (Figure 3B). We further analysed the distribution of gastrocnemius fibre CSA (Supplementary Figure S3A,B). We reported (i) a lower proportion of small fibres and (ii) a higher proportion of large fibres in HF-treated mice compared to the H2O group (Supplementary Figure S3A). This observation was restricted to fast-twitch fibres (i.e., 2X/2B) (Supplementary Figure S3B). Taken together, our data suggest that induction of ATF4-regulated atrogenes is not associated with muscle atrophy after 3 days of hindlimb suspension in HF-treated mice and, thus, that HF slightly preserves muscle mass during HS.

Halofuginone Treatment Inhibits TGF-β while Promoting BMP Signalling in Gastrocnemius Muscle

HF inhibits the TGF-β pathway [36,48]. We and others have recently reported that inhibition of the TGF- signalling is associated with the concomitant activation of BMP signalling [45,46]. Therefore, we investigated how HF treatment and subsequent HS treatment affected these pathways in skeletal muscle. The nuclear localisation of SMADs mirrors the upstream activation of the TGF-β or BMP pathway [49]. We, thus, measured protein levels for the transcription factors SMAD2/3 (TGF- signalling), SMAD1/5 (BMP signalling), and SMAD4 (TGF- and BMP signalling) in nuclear and cytosolic fractions (Figure 4A-D and Supplementary Figure S4A-F). The ratio of nuclear SMAD2/3 to total SMAD2/3 was very low in HF-treated mice compared to H2O-treated mice and decreased

Halofuginone Treatment Inhibits TGF-β While Promoting BMP Signalling in Gastrocnemius Muscle

HF inhibits the TGF-β pathway [36,48]. We and others have recently reported that inhibition of the TGF-β signalling is associated with the concomitant activation of BMP signalling [45,46]. Therefore, we investigated how HF treatment and subsequent HS treatment affected these pathways in skeletal muscle. The nuclear localisation of SMADs mirrors the upstream activation of the TGF-β or BMP pathway [49]. We, thus, measured protein levels for the transcription factors SMAD2/3 (TGF-β signalling), SMAD1/5 (BMP signalling), and SMAD4 (TGF-β and BMP signalling) in nuclear and cytosolic fractions (Figure 4A-D and Supplementary Figure S4A-F). The ratio of nuclear SMAD2/3 to total SMAD2/3 was very low in HF-treated mice compared to H 2 O-treated mice and decreased upon HS only in H 2 O-treated mice (Figure 4A,B). Consistently, the overall mRNA levels of several collagens, which are well-known target genes of TGF-β signalling, decreased upon HS (Supplementary Figure S5). Moreover, the ratio of nuclear SMAD1/5 to total SMAD1/5 was higher in HF-Ctrl mice than in H 2 O-Ctrl mice. This ratio was reduced at HS7 compared to the Ctrl in HF-treated mice, while it was increased in H 2 O-treated mice (Figure 4A,C). Finally, the ratio of nuclear SMAD4 to total SMAD4 was overall lower in HFvs. H 2 O-treated mice (Figure 4A-D), with a decrease in HF-treated mice at HS7 compared to the Ctrl.

TGF-β catabolic action involves the inhibition of protein synthesis [40,50,51], whereas the anabolic action of BMP involves its promotion [44]. The overall protein-synthesis rates measured by puromycin incorporation were reduced during hindlimb suspension. However, this decrease was only significant in H 2 O-HS7 compared to H 2 O-Ctrl mice (Figure 5A,B). TGF-β signalling acts also as a negative regulator of muscle mass through the induction of the atrogenes TRIM63/MurF1 and FBXO32/Atrogin-1 [38,39], while BMP signalling acts as a positive regulator with the transcriptional repression of the atrogene FBXO30/Musa1 [44,45]. Trim63 and Fbxo32 mRNA levels were upregulated only at HS3 in both H 2 O and HF-treated mice, while mRNA levels for the atrogene Fbxo30 remained unchanged (Figure 5C-E). Our data suggest that while HF inhibits TGF-β signalling, it also promotes BMP signalling in the control gastrocnemius muscles. We also showed that HF partially attenuates the drop in protein synthesis during hindlimb suspension.

ATF4-Regulated Atrogenes Are Overexpressed in Atrophy-Resistant Hibernating Brown Bear Muscle

Our data strongly suggest that the induction of ATF4 signalling is not always associated with muscle atrophy, either in basal conditions or in HS-induced muscle atrophy. We took advantage of a natural model, i.e., the hibernating brown bear, which experiences only a moderate loss of muscle protein content while remaining completely inactive for up to 6 months [52][53][54]. As with HF treatment, we recently reported a concomitant TGF-β pathway inhibition and BMP pathway activation in hibernating brown bear muscle [46]. We, thus, explored whether ATF4-regulated atrogenes may also be induced in this model. Interestingly, as shown in Figure 6, Atf4 was upregulated in hibernating brown bear muscle compared to the active counterpart. In addition, the two main ATF4-regulated atrogenes (Gadd45a and Cdkn1a) and Trib3 were also induced in hibernating brown bear muscle. Other ATF4 target genes were either down-(Eif4ebp1, Ppp1r15a, and Asns) or upregulated (Ddit3). These data show that ATF4-regulated atrogenes are induced in hibernating brown bear muscle, even if they are resistant to atrophy.

Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 19

Our data strongly suggest that the induction of ATF4 signalling is not always associated with muscle atrophy, either in basal conditions or in HS-induced muscle atrophy. We took advantage of a natural model, i.e., the hibernating brown bear, which experiences only a moderate loss of muscle protein content while remaining completely inactive for up to 6 months [52][53][54]. As with HF treatment, we recently reported a concomitant TGF- pathway inhibition and BMP pathway activation in hibernating brown bear muscle [46]. We, thus, explored whether ATF4-regulated atrogenes may also be induced in this model. Interestingly, as shown in Figure 6, Atf4 was upregulated in hibernating brown bear muscle compared to the active counterpart. In addition, the two main ATF4-regulated atrogenes (Gadd45a and Cdkn1a) and Trib3 were also induced in hibernating brown bear muscle. Other ATF4 target genes were either down-(Eif4ebp1, Ppp1r15a, and Asns) or upregulated (Ddit3). These data show that ATF4-regulated atrogenes are induced in hibernating brown bear muscle, even if they are resistant to atrophy.

Discussion

Several muscle-wasting conditions, including fasting or physical inactivity, are associated with eIF2 phosphorylation [55,56] and/or ATF4 overexpression, which trigger muscle atrophy [10][11][12][13]18,57]. Moreover, muscle atrophy is hampered during fasting or ageing in mice with reduced ATF4 expression or expressing a phosphorylation-resistant form of eIF2α [11][12][13]. Both CDKN1A and GADD45A are referred to as atrogenes and are required for ATF4-mediated muscle atrophy [11][12][13]18], and TRIB3 is another ATF4 target gene involved in fasting-and ageing-induced muscle atrophy [19,20]. We showed here that overexpression of ATF4-regulated atrogenes was dissociated from muscle wasting (1) in a basal condition, (2) during hindlimb suspension, and ( 3) in a natural model of muscle-atrophy resistance (Figure 7).

Discussion

Several muscle-wasting conditions, including fasting or physical inactivity, are associated with eIF2α phosphorylation [55,56] and/or ATF4 overexpression, which trigger muscle atrophy [10][11][12][13]18,57]. Moreover, muscle atrophy is hampered during fasting or ageing in mice with reduced ATF4 expression or expressing a phosphorylation-resistant form of eIF2α [11][12][13]. Both CDKN1A and GADD45A are referred to as atrogenes and are required for ATF4-mediated muscle atrophy [11][12][13]18], and TRIB3 is another ATF4 target gene involved in fasting-and ageing-induced muscle atrophy [19,20]. We showed here that overexpression of ATF4-regulated atrogenes was dissociated from muscle wasting (1) in a basal condition, (2) during hindlimb suspension, and ( 3) in a natural model of muscle-atrophy resistance (Figure 7). We first observed that the overexpression of the ATF4-regulated atrogenes Trib3, Cdkn1a Gadd45a, and Eif4ebp1, as well as Atf4 itself, induced by halofuginone treatment for up to 4 weeks, did not coincide with atrophy in all hindlimb muscles, including gastrocnemius. Subsequently, we then reported that pre-treatment with halofuginone mitigated the atrophy of the gastrocnemius muscle during hindlimb suspension. These positive effects of halofuginone treatment are consistent with reports showing that this dose (i.e., 0.25 μg/g) and frequency of administration (i) were very well-tolerated in mice for up to 3 months and (ii) improved muscle-cell survival, promoted membrane repair, and improved muscle performances in models of muscular dystrophies [31,33,[58][59][60]. However, none of these studies explored whether the potential effect of HF would involve the ATF4 pathway. Here, we showed that induction of ATF4-regulated atrogenes was uncoupled from muscle atrophy during hindlimb suspension. Indeed, although ATF4-regulated atrogenes were overexpressed during hindlimb suspension, halofuginone-treated mice displayed a partial preservation of gastrocnemius muscle mass and CSA. In addition, we took advantage of a natural model of resistance to muscle atrophy to examine the expression of ATF4-regulated atrogenes. The brown bear remains completely inactive during hibernation for up to 6 months but, surprisingly, is not sensitive to muscle atrophy [52][53][54], which provides an interesting model for finding new molecular mechanisms to fight muscle atrophy in humans. We showed that the atrogenes CDKN1A, GADD45A, ATF4 itself, and TRIB3 were upregulated in atrophy-resistant hibernating brown bear muscle compared to active bear muscle. Of note, ATF4 is mainly regulated at the translational level [15]. However, in most of the previous studies on the topic, the authors only measured Atf4 mRNA expression and expression of its target genes as evidence of its activity We first observed that the overexpression of the ATF4-regulated atrogenes Trib3, Cdkn1a Gadd45a, and Eif4ebp1, as well as Atf4 itself, induced by halofuginone treatment for up to 4 weeks, did not coincide with atrophy in all hindlimb muscles, including gastrocnemius. Subsequently, we then reported that pre-treatment with halofuginone mitigated the atrophy of the gastrocnemius muscle during hindlimb suspension. These positive effects of halofuginone treatment are consistent with reports showing that this dose (i.e., 0.25 µg/g) and frequency of administration (i) were very well-tolerated in mice for up to 3 months and (ii) improved muscle-cell survival, promoted membrane repair, and improved muscle performances in models of muscular dystrophies [31,33,[58][59][60]. However, none of these studies explored whether the potential effect of HF would involve the ATF4 pathway. Here, we showed that induction of ATF4-regulated atrogenes was uncoupled from muscle atrophy during hindlimb suspension. Indeed, although ATF4-regulated atrogenes were overexpressed during hindlimb suspension, halofuginone-treated mice displayed a partial preservation of gastrocnemius muscle mass and CSA. In addition, we took advantage of a natural model of resistance to muscle atrophy to examine the expression of ATF4regulated atrogenes. The brown bear remains completely inactive during hibernation for up to 6 months but, surprisingly, is not sensitive to muscle atrophy [52][53][54], which provides an interesting model for finding new molecular mechanisms to fight muscle atrophy in humans. We showed that the atrogenes CDKN1A, GADD45A, ATF4 itself, and TRIB3 were upregulated in atrophy-resistant hibernating brown bear muscle compared to active bear muscle. Of note, ATF4 is mainly regulated at the translational level [15]. However, in most of the previous studies on the topic, the authors only measured Atf4 mRNA expression and expression of its target genes as evidence of its activity in skeletal muscle. Indeed, endogenous ATF4 protein cannot be reliably detected in skeletal muscle, presumably due to its low abundance, very short half-life, and lack of a high quality antibody [11][12][13]17,18,61]. Altogether, these data strongly suggest that the induction of the ATF4 pathway can be dissociated from muscle atrophy. ATF4 target genes are highly dependent on the type and duration of stress stimuli [21,62], and the ability to restore homeostasis may be overwhelmed when the stress is too severe or sustained, resulting in cell death through the transcription of pro-apoptotic genes [63][64][65][66]. Therefore, to avoid chronic and acute activation, halofuginone was administrated periodically to activate the eIF2α-ATF4 pathway in mice. In our conditions, the ATF4 transcriptional program may, thus, (i) differ from the transcriptional program induced by a severe and sustained activation and (ii) include genes that might counteract the effect of ATF4-induced atrogenes.

Halofuginone is well-described to also target the TGF-β pathway [35,36]. The nuclear translocation of the TGF-β transcription factors SMAD2/3 requires the formation of a complex with SMAD4 [43]. Halofuginone-induced eIF2α phosphorylation has been reported to inhibit the nuclear translocation of this complex in intestinal porcine enterocyte cells in vitro [67]. Consistently, we reported here a concomitant overall (i) increase in phosphorylated eIF2α protein levels and (ii) reduction in SMAD2/3 and SMAD4 nuclear protein levels in HF-treated mice. Thus, this highlights in skeletal muscle, for the first time, the possible role of HF-induced eIF2α phosphorylation in TGF-β inhibition. Although, the concomitant collagen downregulation and decrease in SMAD2/3 nuclear protein levels during hindlimb suspension in H 2 O-treated mice suggest a decrease in TGF-β signalling, we cannot exclude that these events are disconnected. Indeed, the SMAD2/3 nuclear protein levels are consistently low in HF-treated mice and, thus, cannot explain the decreased collagen expression during hindlimb suspension. Much remains to be clarified about the mechanisms of action of HF. Indeed, HF is used for its antifibrotic properties mediated by TGF-β inhibition in situations already characterised by fibrosis [48]. This is, however, not the case in our study. In addition, the inhibition of TGF-β signalling in muscles of HF-treated mice could have led to transcriptional changes that remain to be explored. It is possible that TGF-β signalling is induced later during hindlimb suspension. In fact, the TGF-β signalling pathway has previously been reported to be either unchanged in skeletal muscle after 1-3 days or induced after 7-10 days of unloading [46,68,69]. We also reported an upregulation of Trim63 and Fbxo32 during hindlimb suspension. Although these atrogenes are targets of the TGF-β signalling activation, they are also regulated by other signalling pathways [5].

We and others reported that the balance between TGF-β and BMP signalling seems crucial for muscle-mass maintenance during catabolic situations [39,[44][45][46]70]. Indeed, using the hibernating bear model, we recently reported that TGF-β signalling, i.e., a negative regulator of muscle mass, was downregulated at the transcriptomic level in muscles that are resistant to atrophy, while BMP signalling, i.e., a positive regulator of muscle mass, was maintained [46]. Previous data suggested that TGF-β inhibition would release SMAD4, i.e., the common actor in TGF-β and BMP signalling, which could, thus, be recruited to BMP signalling and promote hypertrophy and/or counteract atrophy [45].

Here, we reported an increase in the SMAD1/5 nuclear protein levels in the halofuginonetreated control mice, suggesting there was concomitant BMP signalling activation and TGF-β inhibition. In addition, BMP activation was reported to increase during denervation, intensive care disuse, and amyotrophic lateral sclerosis and was described as essential to counteract excessive muscle wasting [44,45]. In agreement, we reported here that BMP transcription factors SMAD1/5 accumulated in the nucleus in H 2 O-treated mice but, surprisingly, declined in HF-treated mice after 7 days of hindlimb suspension. Whether the higher basal pools of nuclear SMAD1/5 and their maintenance after 3 days of hindlimb suspension in HF-treated mice contributed to attenuating skeletal muscle atrophy during hindlimb suspension remains to be explored. Of note, BMP signalling has been reported to promote protein synthesis in muscle [44]. Maintenance of the BMP pathway after 3 days of hindlimb suspension may have contributed to the partial preservation of protein synthesis and muscle mass in HF-treated mice. Nuclear translocation of SMAD1/5 represses the transcription of FBXO30/Musa1 [44,45]. However, we did not observe any change in Fbxo30 expression. Mechanisms by which BMP signalling controls muscle mass are still very poorly understood and will require further studies, particularly with a comprehensive characterisation of the BMP target genes in skeletal muscle. We can also speculate that HF-induced BMP activation has helped to limit muscle atrophy induced by ATF4-regulated atrogenes (Figure 7).

In conclusion, halofuginone treatment reproduced the muscle features of hibernating bears in gastrocnemius mice muscles with (i) the activation of ATF4-regulated atrogenes and (ii) the concurrent inhibition of TGF-β signalling and promotion of BMP signalling, without resulting in muscle atrophy (Figure 7). These characteristics were associated with mitigated muscle atrophy during physical inactivity. To date, clinical trials have all attempted to inhibit the TGF-β pathway, mostly with side effects or minimal efficiency [71].

Our study suggests halofuginone, as a well-tolerated chemical compound, already used in human clinical trials [36], was able to tune the TGF-β/BMP balance in vivo and likely sustained muscle mass. Moreover, our data open new ways to further decipher by which precise mechanisms ATF4 induces atrophy and how BMP activation can interfere.

Materials and Methods

Ethics, animals housing, and experimental design. All experiments were conducted with the approval of the regional ethics committee (agreement no. D6334515) following the European Directive 2010/63/EU on the protection of vertebrate animals used for experimental and scientific purposes. This study was performed with 12-week-old C57BL6/J male mice (25-30 g), purchased from Janvier Labs (Le Genest-Saint-Isle, France). They were housed individually upon arrival for 10 days of acclimatisation in a controlled room (22 ± 2 • C, 60 ± 5% humidity, 12 h light/dark cycle, and light period starting at 8 h), fed ad libitum a standard rodent diet (pellets A03 from Safe, Augy, France), and given free access to water. Two distinct animal experiments were performed. To evaluate the effects of a periodic halofuginone (HF) (#32481, Sigma, Saint-Quentin-Fallavier, France) administration, we performed a first protocol where mice received either HF (0.25 µg/g) or water (H 2 O) by gavage 3 times a week for 1 to 4 weeks (n = 6 animals per group). This dose was reported as well tolerated over longer periods [31,33,60]. Gastrocnemius muscle was sampled 6 h after the last HF/H 2 O administration at the end of each week. Subsequently, we performed a second protocol to test whether HF administration before hindlimb unloading had a positive effect on muscle mass and function. For that purpose, we performed two separate animal experiments. In each experiment, mice received either HF (0.25 µg/g) or H 2 O by gavage 3 times a week for 3 weeks and were afterwards subjected either to hindlimb unloading through tail suspension (HS) or kept unsuspended (Ctrl) for 3 or 7 days, as previously described [46] (n = 8-19 animals per group). We did not record any difference between Ctrl mice at 3 or 7 days for all the measurements reported in this manuscript. We, therefore, pooled the two groups of Ctrl mice for further analysis and data representation. Food intake and body weight were recorded throughout the different protocols. Unloading in control mice resulted in only a small body weight loss (<10%) that occurred within the first 3 days concomitantly with a decrease in food intake, whereas HF treatment did not modify food intake or body weight ( see Supplementary Figures S1 andS2).

Tissue collection. At the end of the experiments, mice were euthanised by cervical dislocation. The soleus, gastrocnemius, tibialis anterior, and extensor digitorum longus (EDL) muscles were carefully collected and weighed prior to immediate freezing in liquid nitrogen and storage at -80 • C until analyses.

Measurement of protein synthesis in gastrocnemius. At the end of protocol 2, mice received an intraperitoneal injection of 0.040 µmol/g puromycin (#P8833, Sigma, Saint-Quentin-Fallavier, France) dissolved in 100 µL of a saline solution before euthanasia, as described previously [72]. At exactly 30 min post-puromycin injection, gastrocnemius muscle was dissected and frozen in liquid nitrogen for Western blot analysis, as follows.

Histology and morphometric measurements. A part of the gastrocnemius muscle was collected at the end of protocol 2 and frozen in isopentane chilled with liquid nitrogen and stored at -80 • C until use. Serial muscle cross-sections (10 µm thick) were obtained using a cryostat (HM500M Microm International, Fisher Scientific, Illkirch, France) at -20 • C. Cross-sections were labelled with anti-laminin-α1 (L9393 Sigma, Saint-Quentin-Fallavier, France) to outline the fibre cross-sectional area (CSA) and BFF3 antibody (#AB_2266724, DSHB, Iowa City, IA, USA) to determine myosin heavy chain type 2B fibre. Both were subsequently hybridised with a corresponding secondary antibody conjugated to Alexa-Fluor (Invitrogen, Cergy-Pontoise, France). Image acquisitions were performed with a high-resolution ORCA-Flash4.0 LT+ Digital CMOS camera coupled to a IX-73 microscope (Olympus, Münster, Germany) and Cell-Sens dimension software (Olympus Soft Imaging Solutions, Münster, Germany). The CSA was determined for 1000-1500 fibres per animal, using ImageJ software 1.53f51 (http://rsb.info.nih.gov/ij/, accessed on 3 April 2018).

Protein isolation. Gastrocnemius muscles were pulverised in liquid nitrogen. ( 1) For all targets, ~30 mg of the resulting powders were homogenised using a polytron in 1 mL of an ice-cold buffer (10 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 0.5% Igepal CA630) containing inhibitors of proteases (Protease Inhibitor Cocktail) and phosphatases (1 mM Na3VO3 and 10 mM NaF) (Sigma, Saint-Quentin-Fallavier, France). The homogenates were stirred for 1h at 4 • C and then centrifuged at 10,000× g for 15 min at 4 • C. The resulting supernatants containing total soluble proteins were then stored at -80 • C until use. ( 2) For SMADs protein level analysis, subcellular fractionation was performed. For that purpose, ~50 mg of gastrocnemius powder samples were homogenised for 1 min on ice using a polytron in 500 µL of ice-cold extraction buffer (10 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM KCl, 0.1 mM EDTA, pH 8.0, and 0.1% Triton X-100) [73]. The resulting homogenates were subjected to sequential fractionation steps to separate soluble cytosolic and nuclear proteins as described [74]. Pellets containing nuclear proteins were solubilised in nuclear extraction buffer (20 mM HEPES, pH 7.9, 25% glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and pH 8.0) [73]. For all protein extracts, protein concentration was determined using the Bradford Protein Assay Kit (Biorad, Marnes-la-Coquette, France). Proteins were then diluted in Laemmli buffer and stored at -80 • C until use.

Western blots. Protein contents for (i) SMAD family members (anti-SMAD1-5, PA5-80036, Thermofisher, Illkirch, France; anti-SMAD2-3, #8685, Cell Signalling Technology, Saint-Cyr-L'Ecole, France; anti-SMAD4, ab230815, Abcam, Cambridge, UK), (ii) total and phosphorylated eukaryotic initiation factor 2 alpha (anti-eIF2α, #9722, Cell Signalling Technology; anti-p-Ser51eIF2α, ab32157, and Abcam), and (iii) incorporation of puromycin (anti-puromycin clone 12D10, MABE343, Millipore, Burlington, MA, USA) were assessed by immunoblotting. Briefly, 20-40 µg of protein extracts were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using TGX™ FastCast™ 10% Acrylamide gels (Biorad, Marnes-la-Coquette, France) and transferred onto a PVDF membrane (Hybond P, Amersham, England) using Trans-Blot ® Turbo™ Transfer System standard protocol (Biorad, Marnes-la-Coquette, France). Western blots were blocked for 1 h at room temperature in TBS (Tris-Buffered Saline) buffer with 0.1% Tween-20 (TBS-T, pH = 7.8) with 5% bovine serum albumin (BSA) for all the targets, in accordance with the instructions of the manufacturer. They were then washed thrice in TBS-T and incubated (overnight, stirring, 4 • C) with appropriate primary antibodies diluted at 1:1000, except for anti-puromycin diluted at 1:5000. Western blots were then washed and incubated for 1 h with an appropriate secondary antibody (HRP-conjugated anti-rabbit (#7074) or anti-mouse (#7076) IgGs) (Cell Signalling Technology, Saint-Cyr-L'Ecole, France). For anti-puromycin antibody, an anti-mouse IgG2Ak (115-035-206, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used. Signals were detected after incubation with Luminata Crescendo Western HRP substrate (Millipore, Burlington, MA, USA) and visualised using G: BOX ChemiXT4 (XL1) imaging system (Syngene, Frederick, MD, USA). Signals were then quantified using ImageJ 1.53f51 software. Two samples from each group were loaded on each gel. The signal recorded within each lane of one Western blot was normalised to the overall signal of that blot, and then signals were normalised to the total amount of proteins determined by the Biorad's stain-free system or ponceau S to correct for uneven loading. The normalised values were then averaged by group and expressed as the fold change from the mean of all H 2 O-ctrl samples.

RT-qPCR. Total RNA from gastrocnemius muscle samples was extracted with Macherey-Nagel™ NucleoSpin™ 96 RNA Kit and KingFisher™ Duo Prime Purification System, in accordance with the instructions of the manufacturer (Macherey-Nagel, Hoerdt Cedex, France). RNA was quantified by measuring the absorbance at 260 nm on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Then, 500 ng of RNA were treated with DNase I (Invitrogen, Cergy-Pontoise, France) prior to reverse transcription using random primers and SuperScript II (Invitrogen, Cergy-Pontoise, France), in accordance with the instructions of the manufacturer. Real-time PCR was carried out using the CFX96 Real-Time PCR detection system (Biorad, Marnes-la-Coquette, France). Primer sequences are provided in Supplementary Table S1. PCR reactions were performed using the IQ SYBR Green Supermix (Biorad, Marnes-la-Coquette, France), in accordance with the instructions of the manufacturer. The comparative threshold cycle (2∆∆CT) method was used to compare the relative mRNA expression between each group, using TBP (TATA binding protein) as a reference gene for muscle. The relative mRNA abundance was arbitrarily set to 1 for the H 2 O-Ctrl group.

Statistics. All data are means ± SEM and were analysed for normality of residuals using the Shapiro-Wilk test. No set of data was transformed for non-normality distribution. For protocol 1 (n = 6/group), we performed a multiple Welch t-test within each week. For protocol 2 (n = 8-19/group), we performed a two-way ANOVA with the factors "Hindlimb suspension" and "Halofuginone" and corrected the data for multiple comparisons using Tukey's test. These analyses were performed using Prism 9 (GraphPad Prism 9, San Diego, CA, USA).

Transcriptomic Data. We used transcriptomic data from already published studies [46]. The transcriptomic bear data supporting Figure 6 of this study are openly available in the GEO repository database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi, reference no. GSE144856, accessed on 1 September 2021). To identify the differentially expressed genes (DEGs) from this list, we selected a winter/summer (FC) > 1.0 with an adjusted p-value < 0.05 as cut-off for the up-regulated genes.

Supplementary Materials: The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms24010621/s1.

Institutional Review Board Statement:

The study was conducted according to the guidelines of the Declaration of Helsinki and of the European Directive 2010/63/EU and was approved by the institutional review board (or ethics committee) of ( 1) the Swedish Ethical Committee on Animal Experiment (application nos. Dnr C3/2016 and Dnr C18/2015), the Swedish Environmental Protection Agency (no. NV0741-18), and the Swedish Board of Agriculture (no. Dnr 5.2.18-3060/17) and ( 2) the C2E2A (Comité d'Ethique pour l'Expérimentation Animale Auvergne) (no. D6334515).

Data Availability Statement:

The analysed transcriptomic bear data that support the findings of Figure 6 are openly available in the GEO repository database at https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi, accessed on 16 June 2021, reference no. GSE144856. Other data that support the findings of this study are available in the supplementary materials of this article.

Asns

TACAACCACAAGGCGCTACA AAGGGCCTGACTCCATAGGT

Cdkn1a

GTCTTGCACTCTGGTGTC CTTGGAGTGATAGAAATCTG

Col1a1

CGTGGTGACAAGGGTGAGAC AACCAGGAGAACCAGGAGGA

Col3a1

CTGCTGGTCCTTCTGGTGCT AGCCACGTTCACCAGTTTCA

Col5a2

CCTGGTCCAAATGGTGAACA CCAGGGTTTCCTTCCTTTCC

Col6a1

CCACAACCAGATGCAAGAGC CACCAGCCATCCATTGTAGC

Ddit3

GCATGAAGGAGAAGGAGCAG CTTCCGGAGAGACAGACAGG

Eif4ebp1

CAGGCGGTGAAGAGTCACAA CCTTGGGGGACATAGAAGCA

Fbxo30

GTTGGGATTGCGTAGTGACC CCCTCATTAGCCGGGATACA

Fbxo32

AGTGAGGACCGGCTACTGTG GATCAAACGCTTGCGAATCT

Gadd45a

AGTCAACTTATTTGTTTTTGC GCAATTTGGTTCAGTTATTT

Ppp1r15a

GACTCAAGCCAGAGTCCCTG TAGAGGAATCTCGGGGTCCT

Tbp

TGGTGTGCACAGGAGCCAAG TTCACATCACAGCTCCCCAC

Trib3

CCAGAGATACTCAGCTCCCG GAGGAGACAGCGGATCAGAC

Trim63

ATGGAGAACCTGGAGAAGCA AACGACCTCCAGACATGGAC Atf4: activating transcription factor 4; Asns: asparagine synthetase; Cdkn1a: cyclin dependent kinase inhibitor 1a; Col1a1: collagen type I alpha 1; Col3a1: collagen type III alpha 1; Col5a2: collagen type V alpha 2; Col6a1: collagen type VI alpha 1; Ddit3: DNA damage inducible transcript 3; Eif4ebp1: eukaryotic translation initiation factor 4E binding protein 1; Fbxo 30/32: F-Box protein 30/32; Gadd45a: growth arrest and DNA damage inducible alpha; Ppp1r15a: protein phosphatase 1 regulatory subunit 15a; Tbp: tata binding protein; Trib3: tribbles pseudokinase 3; Trim63: tripartite motif containing 63

Discussion and perspectives

In our study, we found that the induction of ATF4 atrogenes in skeletal muscle was not associated with atrophy (1) in healthy and ( 2) catabolic conditions in halofuginone-treated mice, and also (3) in hibernating brown bears. Even more, we found benefits on gastrocnemius muscle mass in halofuginone-treated mice when subjected to hindlimb suspension (HS) compared to the untreated mice. Furthermore, we demonstrated that the molecular mechanisms of halofuginone involve the inhibition of TGF-β signalling while concomitantly promoting BMP signalling (Figure 39). In addition to the points discussed in the article, other points deserve to be discussed. First, we will discuss the halofuginone mechanisms of actions, its biological effects on muscle mass, and finally the dual role of ATF4 signalling in skeletal muscle.

Halofuginone mechanism of action in skeletal muscle

Halofuginone (HF) is a synthetic derivative of febrifugine that has been isolated from the roots and leaves of Dichroa febrifuga and also from Hydrangea [415] (Figure 40). Febrifugine, and subsequently HF, have been used in traditional Chinese medicine for many years for their therapeutic benefit against malaria, cancer, fibrosis and inflammatory diseases [416]. Currently, two modes of action of HF have been described: (1) inhibition of the prolyl-tRNA synthetase (ProRS) activity leading to ISR activation and ( 2) inhibition of type 1 collagen production via SMAD3 inhibition [416]. In addition, expression profiling of HF targets in epithelial cells revealed that this molecule can induce the expression of ATF4 target genes, including TRIB3, GADD45A and ATF4 itself [417]. Consistently, in our study, we found that Atf4 and its target genes were upregulated following HF treatment in mice muscles.

Does halofuginone induce GCN2 phosphorylation in skeletal muscle ?

HF binds and inhibits the activity of prolyl-tRNA synthetase (ProRS) [418]. This mimics the response to amino acid starvation by increasing GCN2 and subsequently eIF2α phosphorylation, and thus ATF4 translation [418]. Unfortunately, examining GCN2 phosphorylation in skeletal muscle is impossible given the commercially available antibodies. Therefore, it is unclear in our study whether the increased transcriptional activity of ATF4 following HF treatment was mediated by HF canonical activation of GCN2. ATF4 binds to specific CCAAT/enhancer binding protein (C/EBP)-ATF response elements (CAREs) located in the promoters of its target genes. Our team has developed a CARE-driven luciferase mouse model (CARE-LUC) that enables the study of the activity of the eIF2α-ATF4 pathway in the whole organism, and at tissue and cellular levels, by combining imaging, luciferase assays and immunochemistry [419]. We could use wild-type CARE-LUC mice and CARE-LUC Gcn2 KO mice available in our laboratory to compare the intensity of luciferase in muscles during HF treatment and subsequent hindlimb suspension. This will enable determination of whether ATF4 transcriptional activity induced by HF treatment is GCN2 dependent. Furthermore, we showed for the first time that TGF-β inhibition by HF was concomitant with a promotion of BMP signalling with nuclear translocation of SMAD1/5 (see Paper 7.2) [423]. We suggested that this promotion and the resulting transcriptional program, might be in favour of neutralising the atrophic actions of ATF4 in muscles subjected to HS but also in muscles of the hibernating bears. A study showed that BMP2 treatment induced an increase in ATF4 protein levels and its phosphorylation in chondrocyte cells [424]. Several post-translational modifications of ATF4, including phosphorylation, regulate its stability or enhance its transcriptional activity [194]. Whether BMP signalling in muscles can induce ATF4 phosphorylation and whether this post-translational modification can alter its stability and/or transcriptional activity has never been studied. As it is not possible to analyse ATF4 at the protein level in muscles in vivo, in vitro experiments will be needed to explore this hypothesis.

Biological effects of halofuginone on skeletal muscle

Duration of HF pre-treatment. In this study, we reported that 3-weeks of HF pre-treatment slightly preserved the mass and CSA of the gastrocnemius muscle during hindlimb suspension in mice (HS) (see Mice were treated with H2O or halofuginone (HF, 0.25µg/g) 3 times a week for 2 weeks and were then subjected to hindlimb suspension for 3 (HS3) days or kept unsuspended (Ctrl). Gastrocnemius muscle mass (mg) represented with means ± SEM. Two-way ANOVA * padj <0.05; ns= non-significant.

pre-treated with HF for 2 (Figure 43) or 3 weeks (data not shown). Consistently, the average CSA of soleus muscle fibres was similarly reduced in both untreated and HF-treated mice (Figure 43). We then analysed the distribution of fibre CSA in the soleus and compared it to the gastrocnemius muscle (Figure 43). Overall, HF reduced the proportion of small fibres and increase the proportion of large fibres in both the soleus and the gastrocnemius muscle in control mice (see Paper 7.2 and Figure 43) [423].

However, contrarily to the gastrocnemius muscle (see Paper 7.2) [423], this difference in CSA fibre distribution was not maintained during hindlimb suspension in the soleus muscle (Figure 43). This suggests that muscle fibre type may influence the effect of HF on skeletal muscle and could be specific to the pathophysiological condition and/or the nature of the muscle. It is therefore conceivable that HF treatment is even more successful in preserving muscle mass in glycolytic muscles under catabolic conditions where glycolytic fibres are more likely to atrophy, such as ageing, cancer or glucocorticoid treatment.

A. B.

C. Finally, to examine whether this slight preservation of gastrocnemius mass and CSA might have had functional muscle benefits, we performed locomotor experiments using the Rotarod and Catwalk devices. The Rotarod test is widely used to assess the effects of drugs on motor coordination and balance, while the Catwalk is used for the quantitative assessment of stepping and motor performance in rodents. These functional measures did not show much difference between control and HS mice with or without HF treatment. Other functional measures could be considered, such as electromyography.

The dual role of ATF4 signalling in skeletal muscle

As mentioned in the state of the art (see section 4.3.2), ATF4 target genes comprise atrogenes, but also genes that may be involved in muscle homeostasis including autophagy. In our study, we showed that the induction of ATF4 atrogenes was not associated with muscle atrophy during disuse in halofuginone-treated mice and in hibernating bears. For these reasons, we hypothesised that ATF4 may play a dual role in skeletal muscle, either pro-atrophic or pro-homeostatic, and that this may depend on the frequency and duration of its activation.

Does halofuginone-induced ATF4 signalling lead to the expression of autophagy-related genes?

ATF4 is a transcription factor involved in the transcription of autophagy-related genes in response to various stresses (e.g. amino acid starvation, ER stress) [197,[211][212][213][214]216,217]. We thus examined the expression of some autophagy-related genes known to be targets of ATF4 [217]. We observed no change in the muscle expression of Atg5, Atg12 nor Atg16, whether the mice were treated with HF or hindlimb suspended (Figure 44) (see Appendix 10.2). However, mRNA and/or protein levels for microtubule associated protein 1 light chain 3 alpha (LC3) and BCL2-interacting protein 3 (BNIP3), involved in autophagosome formation were increased during HS in both H2O-and HF-treated mice [426,427] (Figure 44). Therefore, the uncoupling of ATF4 from atrophy observed in our study in halofuginone-treated mice does not seem to be dependent on autophagy induction. Interestingly, BNIP3, with the help of LC3, can sequester ATF4 into mitophagosomes leading to ATF4 degradation by mitophagy in response to nutrient deprivation in cancer cells [428]. It would therefore be very interesting to study the cellular localisation of ATF4 upon HF treatment. In addition, RNA sequencing analysis of muscles from HF-treated mice would provide insight into the signalling pathways that may explain the uncoupling between ATF4 atrogenes induction and muscle atrophy. (A) A list of 614 ATF4-related genes has been drawn up from the Genecards web-based portal. Their expression has been analysed in a model of atrophy resistance (the hibernating brown bear) as described before (Cussonneau et al., 2021 [351]). Biological processes represents the protein-protein enrichment analysis performed on Metascape from the respective down-(green) or up-(red) regulated genes. (B-C) Vastus lateralis relative protein levels were assessed by Western blotting and the ratio phosphorylated/total form for eIF2α was calculated after quantification and normalisation using TGX signal for uneven loading. A representative western blot is shown (see Appendix 10.2). Data are presented as individuals values with mean bars (n=12 bears/season, the same individuals were sampled and analysed in summer and winter). S: summer; W: winter.

Enrichment analysis revealed that ATF4-related genes in the biological processes such as heme deficiency response, oxidative stress, endoplasmic reticulum stress and amino acid deficiency were upregulated in atrophy-resistant muscles of the hibernating bear (Figure 45). In contrast, ATF4-related genes in the biological processes of the aerobic electron transport chain and amino acid biosynthesis were predominantly downregulated (Figure 45). These data suggest that the ATF4 transcription factor is transcriptionally active in hibernating bear muscles. We also observed that the ratio of phosphorylated eIF2α to total eIF2α remains unchanged between hibernating and active bear muscles (Figure 45) (see Appendix 10.2). ATF4 target genes are highly dependent on the intensity and duration of the stress [197,204,429]. When the stress is too severe and sustained, eIF2α is phosphorylated leading to the induction by ATF4 of a pro-apoptotic transcriptional program [202][203][204]. The maintenance of a low level of phosphorylated eIF2α may avoid any death-like transcriptional response in bear muscles during hibernation, but also suggest an uncoupling between eIF2α phosphorylation and ATF4 transcriptional activity. This is consistent with the decrease in phosphorylated eIF2α protein levels that occurs alongside an increase in the expression of ATF4 target genes during HS in mice (see Paper 7.2) [423]. Therefore, the transcriptional activity of ATF4 may be independent of the level of phosphorylated eIF2 and may depend on other signals in certain situations such as disuse.

Halofuginone-like compound enriched in bear food

As mentioned above, HF is a synthetic derivative of febrifugine that has been isolated from the roots and leaves of Dichroa febrifuga and also from Hydrangea [415]. Dichroa febrifuga is mainly found in Asia. However, a certain type of Hydrangea, named Hydrangea macrophylla occurs in northern and southern Europe, as well as in southern China. Thus, the geographical distribution of this plant overlaps with some of the brown bear habitat areas (Figure 46). Interestingly, the fruiting of this plant occurs from April to September, and hyperphagia of the brown bear occurs before den entry in late October [296]. Regarding the biological similarity between the muscles of HF-treated mice and hibernating bears (i.e. ATF4 and BMP signalling induction and TGF-β inhibition), it could be envisaged that a halofuginone-like molecule is present in the brown bear food before it enters hibernation. This molecule/compound could be stored in the adipose tissue and released into the bloodstream during the hibernation period. This hypothesis is part of a larger hypothesis that active circulating compounds may be present in the serum of the hibernating bear, which could explain the general hypometabolism present during hibernation and the consequent preservation of organ functions such as skeletal muscle.

Analysis of microarrays in human muscle cells cultivated with WBS

Prior to the start of this thesis project, human primary myotubes (HM) were cultured with SBS or WBS and a microarray sequencing experiment was performed. We analysed these data focusing on the TGFβ/BMP-related genes. Interestingly, we observed common features with hibernating bear muscles, with down-regulation of CCN2, ID1,3 and 4, and up-regulation of MEF2A expression when HM were treated with WBS compared to SBS (Figure 49). Furthermore, we found that NOG (noggin protein), a well-known antagonist of BMP signalling, was also downregulated in HM after WBS treatment (Figure 49), although this was not the case in the hibernating bear muscles (see Paper 6.3) [351]. The downregulation of NOG and CCN2 are key features of TGF-β inhibition and promotion of BMP signalling. These data indicate that WBS can induce transcriptional changes in the TGF-β/BMP balance in HM similar to those occurring in hibernating bear muscles, supporting the existence of circulating active compounds in the winter bear serum.

Optimisation of tools for the screening active compounds in WBS

We used luciferase reporters to visualise the induction/inhibition of BMP or TGF-β transcriptional activity in muscle cells cultivated with bear serum (see Appendix 10.3). To minimise the need for bear serum, we miniaturised the protocol using the immortalised CCL136 human muscle cell line (rhabdomyosarcoma cell line).

BMP signalling. We first used a BMP response element (BRE) luciferase reporter containing the mouse ID1 promoter responsive region for BMP [430]. We first validated that this pathway is active and that the machinery to transduce the BMP signal from the ligand to the target genes is operational in CCL136 cells (data not shown). Thereafter, we observed a decrease in luciferase intensity when CCL136 were treated with WBS for 6 to 24h compared to SBS (Figure 50). This was confirmed on HM after 24h WBS treatment (Figure 50). These data suggest that the downregulation of ID1 observed in hibernating bear muscles in vivo is the result of a compound present in WBS. Furthermore, this effect of WBS could be direct since it occurred after only 6h of treatment (Figure 50).

TGF-β signalling.

We also used a SMAD binding element (SBE) luciferase reporter containing four copies of the SMAD binding site GTCTAGAC corresponding to the main TGF-β responsive element the cells treated with SBS (Figure 50). This preliminary result is very promising as this is the second proof of concept that a compound in the WBS can mimic what happens naturally in winter bear muscles in vivo. Further studies are needed to determine whether this MEF2 signature observed in hibernating bear muscle, in vivo and in human muscle cells cultured with WBS, could be under the control of the TGF-β and/or BMP pathway via a circulating compound.

Perspective on identifying the circulating active compound in WBS

The identification of compounds in hibernating bear serum that have the potential to control the TGFβ/BMP balance is the core of a project recently granted by the ANR 46 with a PhD student just starting.

The prospects are promising. They will include confirmation of the results with the MEF2-Luc reporter in HM, and repetition with more individuals for the ID1-Luc reporter.

Is ID1 disconnected from BMP signalling in skeletal muscle?

The decrease in the luminescence of ID1-Luc reporter observed in HM cultivated with WBS is coherent with what we showed in hibernating bear muscles (see Paper 6.3) [351]. However, ID1 is a well-known BMP target gene in bone and cartilage (see Discussion 6.4.2.1), therefore its reduction raises questions about the disconnection of ID1 transcription and BMP transcriptional acitivity in skeletal muscle. Moreover, ID1 expression is under the control of other signalling pathways, including 363]. Whether the decrease in ID1 expression in hibernating bear muscles or HM cultivated with WBS reflects inhibition of TGF-β signalling remains to be explored. Therefore, we will examine the SMADs nuclear translocation, as performed in study 2, in HM cultivated with either WBS or SBS. Then, once the genetic signature of BMP signalling in muscle cells will be identified (see Discussion 6.4.2.1), we will design a BMP-Luc reporter with a BMP-dependent gene in muscle to address whether a circulating compound can activate the transcriptional activity of BMP signalling when HM are cultivated with WBS.

Is BMP canonical signalling involved? Subsequently, we will decipher the molecular mechanisms capable of inducing BMP signalling when human muscle cells are exposed to winter bear serum. For example, we can examine whether BMP inhibition with silencing RNAs (e.g. siSMAD1) or pharmacological treatments (e.g. noggin) abolish/limit ID1 down-regulation or MEF2A up-regulation in cells treated with SBS or WBS. This will allow us to find out whether the biological effect observed on muscle cells treated with WBS is mediated by BMP canonical signalling.

What is the nature of the active compound in WBS?

The project also includes serum fractionation processes (e.g. delipidation, dealbumination, heat denaturation) to determine the nature of the compounds that may be involved in the regulation of the TGF-β/BMP balance in muscle cells. Serum 155 fractionation is already performed by one of our collaborators, Dr Bertile Fabrice at the IPHC in Strasbourg. This is also why we have miniaturised the reporter assay experiments in 96-well plates using CCL136 cells to test the different fractions and easily read the luminescence of the TGF-β/BMP reporters. Therefore, we will be able to investigate the nature of the compound (e.g. lipid, protein, …) leading to transcriptional modifications of the TGF-β /BMP signalling in vitro.

Finally, the aim is also to reproduce in vitro and then in vivo the atrophy resistance phenotype of hibernating bear muscles, using active fractions and/or compounds of bear serum to prevent or reverse atrophy under catabolic conditions (e.g. dexamethasone-induced atrophy on myotubes in vitro).

General conclusion

Some therapies to combat muscle wasting have been developed, including exercise, nutritional interventions and certain medications. However, no effective treatment has been found to completely and safely prevent muscle wasting. Furthermore, despite all its preclinical success, modulation of TGFβ signalling has not translated into the desired effects in humans. The promotion of BMP signalling has received very little attention to date, and the concept that simultaneous fine-tuning of the BMP and TGF-β pathways might be of interest against the development of muscle atrophy has been mentioned in very few papers. The use of a model of natural resistance to muscle atrophy, the hibernating brown bear, has great potential for the discovery of new therapeutic targets for the human clinic. In addition, our comparative physiology strategy between an induced atrophy model and an atrophy-resistant model unveiled new and promising avenues for new future treatments. In this thesis project, we (1) performed a transcriptomic analysis comparing hibernating versus active bear muscles to unloaded versus control mouse muscles, ( 2 The signal was measured for 12 seconds. The firefly values were then normalised by the renilla luciferase values.

Introduction

Cachexia is a multifactorial syndrome leading to serious clinical complications with high mortality rates and is present in almost all chronic diseases [1]. Besides inflammation and metabolic modifications, skeletal muscle loss is an important factor of cachexia and limiting muscle wasting is a major challenge for maintaining well-being of patients, the capacity of the organism to fight against diseases and the tolerance of the patients towards challenging therapies like cancer chemotherapies [2].

Muscle homeostasis is mainly driven by the ubiquitin-proteasome system (UPS) that controls signaling pathways, contractile structure, cellular architecture, energy metabolism, protein translation, etc., thus allowing a fine-tuning of skeletal muscle metabolism [3][4][5][6]. The UPS is composed by hundreds of proteins and controls protein fate by ubiquitination, a post-translational modification carried out by the E1, E2, E3 enzymatic cascade (see [7] for a review). Ubiquitin (Ub) is covalently attached to the target proteins thanks to the interactions between Ub conjugating E2 enzymes (35)(36)(37)(38)(39)(40) members according to species) and E3 Ub ligases (>600 in human). Another complexity of the UPS resides in the multitude of Ub signals that can be synthesized on the target proteins, from mono-Ub, multiple mono-Ub, or poly-Ub chains with at least eight different topologies. Each type of Ub modification is dedicated to a specific fate for the target protein, the role of some Ub linkages being still obscure. This Ub code can send the target protein for either proteasome or autophagy degradation or for non-proteolytic purposes (addressing, stabilization, activation, etc.) [7]. Furthermore, the multiple possible combinations between a given E3 and several E2s (and vice versa) further increase the potential of the UPS for controlling cellular metabolism.

E3 ligases can be either monomeric or multi-protein complexes and are classified into three families according to their structure and mode of action (recently reviewed [8]). The first class contains 28 members that contain a C-terminal Homologous to E6-Associated Protein C Terminus (HECT) domain that is necessary and sufficient to accept Ub from an E2 enzyme and to transfer it to the substrate, HECT E3 ligases having their own catalytic activity. Their N-terminal domain is involved in the recognition of the substrate. The second class comprises ≈90% of the E3 Ub ligases and are known as Really Interesting New Gene-finger (RING) type. RING domains are defined by eight cysteine and/or histidine residues coordinating four zinc atoms that allow interaction with E2 enzymes. RING-type E3s do not bind Ub, but they serve as a platform for the E2 and the substrate and promote the Ub transfer from the E2 to the substrate. Within multi-protein RING-E3 complexes, also named cullin-containing RING Ligase E3s (CRLs), several families of proteins with motifs involved in protein-protein interactions (e.g., F-box pattern) are responsible for substrate recognition [9]. The third class of E3 ubiquitin ligases are the RING-in-Between-RING (RBR)-type that combine properties of RING-and HECT-type E3s. They utilize an E2-binding RING domain and a second domain (called RING2) that binds Ub before transferring it to substrate [10,11].

Within muscle atrophy, numerous ubiquitinating enzymes are now identified for their involvement in the regulation of both anabolic and catabolic pathways during the atrophy process, notably by being responsible for the degradation of the contractile proteins [12]. The E3 Ub ligases appear to be at the heart of these regulations and some of them may prove to be efficient therapeutic drug strategies with roughly two main approaches: (i) indirect modulation of an E3 ligase by targeting the signals involved in its regulation [13][14][15][16] or (ii) direct inhibition of the E3 ligase [17][18][19]. However, the intertwinement between anabolic and catabolic processes (including the signaling pathways) often renders difficult an indirect modulation of E3 ligases, while direct inhibition strategies is limited by the somehow limited data available on E3 ligases.

This review summarizes the signaling pathways implicated in muscle homeostasis, and highlights the E3 ligases playing a role in the regulation of skeletal muscle mass and function, excluding the muscle regeneration process where numerous E3 Ub ligases are also involved. We more specifically focus on the strategies that have already been used for modulating E3 ligase activity, including pharmaceutical drugs or natural compound-based approaches.

Signaling Pathways Regulating Skeletal Muscle Mass and Function

Skeletal muscle homeostasis is controlled by numerous signaling pathways (Figure 1) that act either as anabolic or catabolic factors. Depicting in detail their regulation is beyond the scope of this review and we just briefly summarize their implication in muscle mass control. Skeletal muscle hypertrophy via the PI3K/AKT (phosphatidylinositol 3-kinase/protein kinase B) pathway can be induced by nutrients (amino acids, glucose and fatty acids) [20], hormones (insulin) [20,21] growth factors (Insulin Growth Factor-1 (IGF-1)) [22,23], and mechanical stimuli (e.g., exercise) [24]. Upon ligand binding, the PI3K/AKT pathway activates mTORC1 that phosphorylates numerous substrates [25,26], which regulate the activation of translation, transcription, ribosome biogenesis, and autophagy [27,28]. AKT also phosphorylates and inactivates GSK3β (a negative regulator of protein translation) [29] and the pro-catabolic FOXOs transcription factors (TF), the latter being crucial inducers of muscle loss upon catabolic situations via the expression of numerous atrophy-related genes [30][31][32][33]. Moreover, mTORC1 also inhibits the autophagy induction complex [34]. Intriguingly, mTORC1 can also exhibit adverse effects on skeletal muscle homeostasis upon denervation [35] or ageing [36,37]. In these situations, a negative feedback loop from mTORC1 to AKT was involved, thus favoring FOXOs activation and the subsequent expression of proteolytic genes like the atrophy-related E3 ligases MuRF1/TRIM63 and MAFbx/Atrogin-1.

G Protein-Coupled Receptors (GPCRs) and cAMP Signaling 1. ß2-Adrenergic Receptors Signaling Pathway

Upon stimulation by endogenous catecholamines or synthetic agonists, ß2-Adrenergic Receptors (ß2-ARs) lead to skeletal muscle hypertrophy (Figure 1) through: (i) PKAmediated expression of genes containing cAMP response elements (follistatin, NR4A3, calpastatin) via CREB [38] (ii) PKA-mediated inhibition of FOXO activity in vivo [39] or (iii) the activation of PI3K/AKT/mTORC1 [40,41], or both AKT and CaMKII/HDAC4 signaling [42].

WNT/FZD Signaling Pathway

The Wingless-type mouse mammary tumor virus integration site (Wnt) family of proteins induce hypertrophy via Wnt/ß-catenin and PI3K/AKT/mTORC1 cascades [43,44] (Figure 1). The former one controls the transcriptional regulation of growth-related genes (e.g., C-myc and Cyclin 1) via ß-catenin and T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors [45,46] whereas the latter regulates the protein synthesis process. The PI3K/AKT/mTORC1 pathway is induced via the specific interaction of WNT7a (ligand) and FZD7 (receptor) proteins [47][48][49][50]. Under mechanical stimulation, WNT is the only pathway able to stabilize ß-catenin and therefore to promote growthrelated gene expression [51,52]. Accordingly, therapeutic stimulation of WNT7a/FZD7 by injection of recombinant Wnt7a resulted in a significant increase in muscle strength and a reduce contractile damages in mdx mice (Duchenne Muscular Dystrophy (DMD) model) [49]. By contrast, in dystrophic muscles WNT7a increased fibrosis by inducing transforming growth factor-β2 (TGFβ2) [53], and Wnt activation enhanced the fibrotic response in aged mice [54]. These data suggest WNT7a to have a context-dependent effect in skeletal muscle, thus complicating future therapeutic strategies.

Calcineurin Signaling Pathway

Different downstream effectors have been proposed for calcineurin (Cn) during skeletal muscle hypertrophy, such as NFAT [55], GAT-2 [55] and MEF-2 [56], which seem to be activated during skeletal muscle hypertrophy in a fiber-specific manner [57]. Cn can modulate these TFs and downstream effectors (including the E3 ligases MuRF1/TRIM63 and MAFbx/atrogin-1) upon several conditions (dexamethasone [58], diabetes [56], exercise [59] or starvation [60] (Figure 1).

Hippo Signaling Pathway

The Hippo signaling pathway consists of a cascade of kinases that inhibits the transcriptional co-activators YAP and TAZ (Figure 1) (for a review, see [61]). Upon exercise and myostatin/activin inhibition in mdx mice [62], mechanical overloading [63] and following injury or degeneration of motor nerves [64], the expression and phosphorylation of YAP increased [62,63] along with those of other pro-hypertrophy proteins [40]. Furthermore, YAP negatively regulated the myostatin/activins signaling pathway by inhibiting SMAD2/3 transduction and consequently blunted the SMAD-mediated MuRF1/TRIM63 E3-ligase expression [63].

Transforming Growth Factor (TGFs), Pro-Anabolic and Pro-Catabolic Pathways

The transforming growth factor (TGF) multifunctional cytokine family is divided in two subfamilies with opposite outcomes on muscle mass: myostatin/activin/TGF-β are negative regulators of muscle mass and BMPs (Bone Morphogenic Proteins)/GDF (Growth and Differentiation Factors) are positive regulators [65]. Myostatin/activin/TGF-β activate the pro-catabolic SMADs 2-3 whereas BMP ligands recruit pro-anabolic Smads 1-5-8 and elicit an anabolic transcriptional program (Figure 1). SMAD4 is shared by both pro-anabolic and pro-catabolic SMADs and can be a limiting factor for SMADs downstream effects [45].

Upon myostatin binding, Mafbx/Atrogin-1 and genes involved in the degradation of several anabolic factors (ribosomal proteins, translation initiation factors, MyoD, desmin and vimentin) are up-regulated [49,66] and the AKT/mTORC1 pathway is inhibited [67]. TGF-ß signaling also regulates MuRF1/Trim63 expression through the synergistic action of FOXO3a and SMAD3 [68,69] (see [12] for a recent review). Similarly, Activin A ligand negatively regulates muscle mass by binding to the same receptor than myostatin and by activating the same intracellular pathway [70][71][72]. Interestingly, the non-canonical TGF-ß pathway involving TAK1-p38 MAP kinase can also be activated under Activin A treatment in cellulo and in vivo, with MAFbx-mediated myotube atrophy [73]. Moreover, TGF-ß induces skeletal muscle atrophy through a mechanism dependent on NOX-derived ROS production, in vivo [69]. The TGF-ß pathway is also known for its master role in fibrosis, which promotes muscle mechanical constraints and injuries [74,75]. Recent reports showed that the canonical NF-κB and angiotensin pathways mediate the TGF-ß effects in cellulo and in vivo [76].

Conversely, the BMP pathway regulates hypertrophy by repressing the E3 ligases MUSA1/Fbxo30 [77] MAFbx/Atrogin-1, MuRF1/Trim63 [78,79] and through the positive modulation of mTORC1 and consequently protein synthesis [80]. Additionally, the long non-coding RNAs Myoparr and Chronos negatively modulate the BMP pathway (and muscle mass) by repressing Gdf5 [81] and Bmp7 [82] respectively. Altogether, a major conceptual idea is that a net balance between TGF-ß/BMP pathways plays a major role in determining skeletal muscle mass.

Catabolic Pathways 2.3.1. AMPK Signaling Pathway

The adenosine 5 -monophosphate-activated (AMP)-activated protein kinase (AMPK) is an energy sensor that preserves energy by turning on catabolic pathways and turning off ATP-consuming anabolic pathways [83][84][85]. In skeletal muscle, AMPK inhibits protein synthesis through the reduction of the mTORC1 signaling and favors contractile protein breakdown via the activation of FOXO1 and FOXO3a TFs (Figure 1) [86]. Consequently, MuRF1/TRIM63 and MAFbx/Atrogin-1 E3 ligases target different proteins involved in muscle contraction and protein synthesis initiation for UPS-dependent degradation [86,87]. Additionally, AMPK also promotes skeletal muscle autophagy [88].

The NF-κB Signaling Pathway

NF-κB, a major pro-inflammatory transcription factor, is considered one of the main effectors of muscle atrophy via the regulation of UPS-related proteins expression [89][90][91][92][93][94][95][96].

Indeed, the NF-κB pathway is consistently upregulated upon catabolic conditions in both mouse models [89,97,98] and patients suffering from chronic obstructive pulmonary disease (COPD) [99] or chronic heart failure (CHF) [100] patients. A hypertrophic response is also observed in myotubes when blunting NF-κB activation upon catabolic TNFα exposure [93]. In addition to TNFα induction of NF-κB signaling, other proinflammatory cytokines (such as IL6 and TWEAK), bacterial products, growth factors, ROS, genotoxic stress, and viruses can activate this pathway [101]. Interestingly, for controlling the proper signaling, the NF-κB pathway comprizes several E3 ubiquitin ligases, TRAF6 [95,102,103], cIAP1 [19,104], LUBAC [95,105], SCF β-TRCP [105,106] that represent several opportunities for future potential therapies (Figure 2). NF-κB, a major pro-inflammatory transcription factor, is considered one of the main effectors of muscle atrophy via the regulation of UPS-related proteins expression [89][90][91][92][93][94][95][96]. Indeed, the NF-κB pathway is consistently upregulated upon catabolic conditions in both mouse models [89,97,98] and patients suffering from chronic obstructive pulmonary disease (COPD) [99] or chronic heart failure (CHF) [100] patients. A hypertrophic response is also observed in myotubes when blunting NF-κB activation upon catabolic TNFα exposure [93]. In addition to TNFα induction of NF-κB signaling, other proinflammatory cytokines (such as IL6 and TWEAK), bacterial products, growth factors, ROS, genotoxic stress, and viruses can activate this pathway [101]. Interestingly, for controlling the proper signaling, the NF-κB pathway comprizes several E3 ubiquitin ligases, TRAF6 [95,102,103], cIAP1 [19,104], LUBAC [95,105], SCF β-TRCP [105,106] that represent several opportunities for future potential therapies (Figure 2).

Glucocorticoid Receptor Signaling Pathway

Glucocorticoids (GCs) are endogenous stress hormones involved in modulating inflammation [107]. GCs are well known for their catabolic effects on skeletal muscle [108] and can exert their action via different mechanisms (Figure 1). In skeletal muscles, GCs mainly operate through the glucocorticoid receptor (GR), that interacts with specific DNA sequences, DNA-bound TFs as well as transcriptional co-regulatory proteins, which modulate the transcription of numerous genes [108-110] like MuRF1/Trim63, MAFbx/Atrogin-1, Foxo transcription factors, the myokine Gdf8, Klf15,Redd1 and Sesn1 [110]. Intriguingly, the effect of GCs on muscle mass is dependent on the type of GC, fiber type composition, muscle type, sex and dose, but also on the type of catabolic situation (e.g., starvation, diabetes, sepsis, cancer cachexia, etc.) (for details, refer to [110,111]). Recent works at least partly explained these differential effects by the capacity of GCs to use different signaling pathways, such as IGF-1/PI3K/AKT, MEK/ERK, Myostatin [112], , NOTCH [114] or to depend on co-factors such as connexin-based hemichannels [115], high-fat diet [116], oxidative stress [111] or mechanical load [51,52,117].

Angiotensin Signaling Pathway

Angiotensin (Ang) is a peptide hormone that upon enzymatic processing [118] renders different variants like Ang-II and Ang-(1-7) (Figure 1) that can either be linked to catabolic conditions (Ang-II) [118][119][120][121][122][123] or counteract muscle atrophy (Ang-(1-7)) [124][125][126][127][128]. However, Ang-II can also exhibit anticatabolic properties, but only in some circumstances [127,129]). High levels of Ang-II have been associated with skeletal muscle atrophy in CHF, CKD, and SARS-CoV-2 pathologies [120,130]. Ang-II induced atrophy was also linked to increased proteasome activity [131], elevated polyubiquitinated protein conjugates [132], and early and transient accumulation of MuRF1/Trim63 and MAFbx/Atrogin-1 mRNA [119,123]. Therefore, the differential modulation of the enzymes processing Ang may be a promising approach for improving skeletal muscle atrophy.

Glucocorticoid Receptor Signaling Pathway

Glucocorticoids (GCs) are endogenous stress hormones involved in modulating inflammation [107]. GCs are well known for their catabolic effects on skeletal muscle [108] and can exert their action via different mechanisms (Figure 1). In skeletal muscles, GCs mainly operate through the glucocorticoid receptor (GR), that interacts with specific DNA sequences, DNA-bound TFs as well as transcriptional co-regulatory proteins, which modulate the transcription of numerous genes [108-110] like MuRF1/Trim63, MAFbx/Atrogin-1, Foxo transcription factors, the myokine Gdf8, Klf15,Redd1 and Sesn1 [110]. Intriguingly, the effect of GCs on muscle mass is dependent on the type of GC, fiber type composition, muscle type, sex and dose, but also on the type of catabolic situation (e.g., starvation, diabetes, sepsis, cancer cachexia, etc.) (for details, refer to [110,111]). Recent works at least partly explained these differential effects by the capacity of GCs to use different signaling pathways, such as IGF-1/PI3K/AKT, MEK/ERK, Myostatin [112], , NOTCH [114] or to depend on co-factors such as connexin-based hemichannels [115], high-fat diet [116], oxidative stress [111] or mechanical load [51,52,117].

Angiotensin Signaling Pathway

Angiotensin (Ang) is a peptide hormone that upon enzymatic processing [118] renders different variants like Ang-II and Ang-(1-7) (Figure 1) that can either be linked to catabolic conditions (Ang-II) [118][119][120][121][122][123] or counteract muscle atrophy (Ang-(1-7)) [124][125][126][127][128]. However, Ang-II can also exhibit anticatabolic properties, but only in some circumstances [127,129]). High levels of Ang-II have been associated with skeletal muscle atrophy in CHF, CKD, and SARS-CoV-2 pathologies [120,130]. Ang-II induced atrophy was also linked to increased proteasome activity [131], elevated polyubiquitinated protein conjugates [132], and early and transient accumulation of MuRF1/Trim63 and MAFbx/Atrogin-1 mRNA [119,123]. Therefore, the differential modulation of the enzymes processing Ang may be a promising approach for improving skeletal muscle atrophy.

JAK/STAT Signaling Pathway

In skeletal muscle, the Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) pathway has been reported to be essential for transducing signals from growth factors and IL-6 among others (For a recent review, see [133,134]). STAT3, one of its effectors (Figure 1), is particularly implicated in skeletal muscle atrophy upon disease [recently reviewed elsewhere [135], notably through the development of skeletal muscle insulin resistance in Type 2 diabetes mellitus [136,137], the induction of myostatin [138], caspase -3 [139] and UPS [14], and increased mitochondrial ROS [140].

Kinin Signaling Pathway

Kinins are a group of peptides that act via inducible (B1) or constitutive (B2) receptors [141]. Using B1 receptors, kinins participate to muscle atrophy by blunting the PI3K/AKT/mTORC1 axis and by stimulating the IKK/NF-κB pathway (Figure 1) [142]. Both genetic or pharmacologic ablation of B1 receptor protect skeletal muscles from atrophy in androgen-sensitive mice, mainly by blunting MuRF1/Trim63 expression [142]. The role of kinin B2 receptors is more controversial as they may either be pro-catabolic via activation of myostatin signaling [143] or pro-anabolic [144]. Therefore, kinin receptors may regulate muscle mass but more studies are clearly needed before they become potential targets to modulate muscle atrophy.

Sphingolipids Signaling Pathway

The sphingomyelin pathway plays a role in skeletal muscle mass through the hydrolysis of plasma membrane sphingomyelin (SM) and the subsequent formation of ceramide and sphingosine-1-phosphate (S1P) (Figure 1). Ceramide, is linked to muscle atrophy through (i) the reduction of protein synthesis [145][146][147][148] the Notch Intracellular Domain (NICD) translocates to the nucleus (Figure 1) and binds directly to the MuRF1/Trim63 promoter to activate its transcription, thereby establishing NOTCH signaling as a proteolysis inducer [161].

Oxidative Stress Is an Inducer of Skeletal Muscle Atrophy

Oxidative stress is characterized by increased levels of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) and is a well-known mechanism of atrophy induction in skeletal muscle under several conditions and proteolytic mechanisms (reviewed elsewhere [162,163]) (Figure 1). Both ROS and RNS negatively impact muscle mass during COPD [164,165]. ROS induce a FOXO1-dependent MuRF1/Trim63 and MAFbx/Atrogin-1 overexpression in COPD peripheral muscle cells in cellulo [166]. NOS activation was suggested to occur through inflammation and hypoxia in COPD patients with low body weight via an activation of NF-κB and iNOS-generated RNS [99]. Besides increased protein breakdown, a decrease in protein synthesis via AKT/mTORC1 also contributes to muscle mass loss by ROS [162]. Importantly, depending on the type, duration and intensity of the imposed stress, specific signaling mechanisms are activated [162,163,[166][167][168][169] indicating that the underlying mechanisms by which oxidative stress contributes to muscle wasting is context-dependent. One strategy to fight against atrophy may be to stimulate the anabolic pathways leading to skeletal muscle hypertrophy. Insulin-like growth factor 1 (IGF1) induces skeletal muscle hypertrophy by activating the IGF1R/PI3K/AKT pathway, a critical mediator and checkpoint being IRS1. Indeed, the effect of IGF1 is time-limited by the phosphorylation of IRS1 by IGF1R and its subsequent ubiquitination and proteasome-mediated degradation.

E3 Ligases Involved in the

Different E3 ligases can target IRS1 in different tissues. For example, in embryonic fibroblasts, the CUL7 E3 ligase, containing FBXW8, has been shown to target IRS1 for ubiquitin-dependent degradation [170]. In skeletal muscle, Casitas B-lineage lymphoma-b (CBL-B), a RING E3 ligase, targets IRS1 for degradation and thus impairs muscular trophic signals in response to unloading conditions [171][172][173], which inhibits downstream IGF1 signaling [173] (Figure 2 and Table 1). Accordingly, mice deficient for CBL-B were partly resistant to unloading-induced skeletal muscle atrophy and dysfunction [173]. These results highlight the importance of CBL-B in the process of muscle atrophy in response to unloading.

FBXO40 is a muscle-specific F-box protein [174], component of an SCF (Skp1-Cullin1-F-box protein) E3 ligase complex. Following IRS1 activation, IGF1R phosphorylates IRS1 leading to its ubiquitination by FBXO40 and its degradation by the 26S proteasome, in cultured myotube and in mice [22,175]. FBXO40 expression is decreased in muscles from Limb-girdle muscular dystrophy (LGMD) patients, and up-regulated in mice skeletal muscle following denervation and in chronic kidney disease (CKD) mice model, but not during starvation [174,175]. Accordingly, the knock-down of Fbxo40 resulted in thicker myotubes (20% to 50% increase in diameter) [22] and its deletion in mice also induced muscle hypertrophy during the growth phase, a phase associated with high IGF1 levels [22] (Figure 2 and Table 1). IRS1 is thus an important checkpoint of the IGF1/PI3K/AKT pathway controlled by at least 2 E3 ligases (CBL-B and FBXO40). Although being an attractive target for fighting against muscle atrophy, the multiple ways for degrading IRS1 may complicate the development of drugs.

3.1.2. NEDD4-1 E3 Ubiquitin Ligase, Friend or Foe?

In muscles undergoing atrophy, NEDD4-1 mRNA levels are elevated upon severe sepsis [191], denervation or unloading [178,192,193]. On the one hand, NEDD4-1 E3 Ub ligase targets phosphatase and tensin homologue (PTEN). PTEN is a redox sensitive phosphatase that negatively regulates the PI3K-AKT signaling pathway, thereby affecting metabolic and cell survival processes. The deletion of PTEN improves muscle mass and function in a mouse model of Duchenne muscular dystrophy [194]. PTEN inhibition may thus also represent a potential therapeutic strategy to maintain muscle function during catabolic situations. The over-expression of NEDD4-1 is sufficient for activating the PI3K/AKT signaling in cardiac muscle, following myocardial ischemia/reperfusion (I/R) [176]. However, the negative regulation of PTEN by NEDD4-1 remains to be confirmed in skeletal muscle, especially since NEDD4-1 has also been shown to promote skeletal muscle atrophy in a denervation model. Indeed, NEDD4-1-KO mice exhibited increased weights and type II muscle fiber cross-sectional areas in denervated gastrocnemius muscle [178]. Moreover, NEDD4-1 also negatively regulates the hypertrophic BMP signaling (Figures 1 and2). Indeed, NEDD4-1 ubiquitinates phosphorylated-SMAD1 leading to its proteasomal degradation, thereby silencing BMP signaling in C2C12 myoblasts, and conversely the knock-down of Nedd4-1 potentiates BMP signal through upregulation of phospho-SMAD1 [195]. Altogether, the exact function of NEDD4-1 in skeletal muscle is still obscure and needs more work. Among the E3s involved in the regulation of the NF-κB pathway, two promising candidates may be manipulated to limit muscle atrophy, namely cIAP and TRAF6 (Figures 1 and2). cIAP1 is up-regulated in denervated gastrocnemius muscle, paralleling the upregulation of MAFbx/atrogin-1 and MuRF1/Trim63 mRNA [19]. Mice with genetic ablation of cIAP1 (cIAP1-KO mice) displayed limited denervation-induced atrophy in TA, gastrocnemius and EDL muscles. This was correlated with the blunting of the denervation-induced upregulation of MAFbx/Atrogin-1 and MuRF1/Trim63 [19]. The authors further demonstrated that cIAP1 induced atrophy through the up-regulation of the canonical NF-κB signaling. Conversely, cIAP1 overexpression in myotubes induced atrophy and the strong up-regulation of MAFbx/Atrogin-1 and MuRF1/Trim63 protein expression [19]. The E3 Ub ligase cIAP1 represents thus a potential therapeutic target at least for fighting against denervation-induced muscle atrophy.

E3 Ubiquitin Ligases

TRAF6 is a RING-type Ub ligase that plays an important role during skeletal muscle atrophy. TRAF6 expression is enhanced during starvation or within aged-induced muscle atrophy [179,196,197]. Traf6-KO mice are resistant to skeletal muscle loss (rescue of myofibril degradation, preservation of myofiber size and strength) induced by denervation, cancer cachexia, starvation or Dex and a concomitant suppression of the expression of key regulators of muscle atrophy was observed, including MAFBx/Atrogin-1, MuRF1/TRIM63, p62, Lc3b,Beclin1,Atg12,and Fn14 [179,180,[196][197][198]. Moreover, inhibition of Traf6 expression through miR-351 administration in C2C12 myotubes or in denervated mice attenuated Dexinduced muscle atrophy and concomitantly decreased the expression of MAFBx/Atrogin-1 and MuRF1/Trim63 [199,200]. Overexpression of miR-125b targeted Traf6 for degradation and protected skeletal muscle samples from atrophy in starved myotubes or in denervated rat tibialis muscle [201]. The implicated mechanisms involved both direct and indirect effects of TRAF6 on protein breakdown with TRAF6-mediated ubiquitination being re-quired for the optimal activation of JNK, AMPK, FOXO3, and NF-κB catabolic pathway in muscle [202].

In human, gastric cancer patients suffering from cachexia exhibited an upregulation of TRAF6 associated with an upregulation of ubiquitination in the rectus abdominis muscle [203]. Altogether, this highlights the importance for targeting TRAF6 inhibition to counteract muscle atrophy.

WWP1 in the Regulation of Muscle Atrophy

WWP1 is a HECT E3 ligase that is involved in chicken muscular dystrophy. Indeed, a missense mutation in the gene coding WWP1 was identified as the most promising candidate responsible for chicken muscular dystrophy (MD), potentially affecting the E3 function of WWP1 protein [204]. WWP1 was also shown to target the transcription factor KLF15 [181]. In response to glucocorticoids, KLF15 is up-regulated at the mRNA levels [205]. This induction leads to the up-regulation of the E3 ligases MAFbx/Atrogin-1 and MuRF1/Trim63 expression, likely in cooperation with a FOXO transcription factor, while inhibiting the anabolic mTORC1 [205]. Likewise, exogenous KLF15 expression in myotubes and in TA muscle leads to myofiber atrophy [205]. It has recently been shown that KLF15 protein expression was upregulated in skeletal muscle of diabetic mice, without any change in its mRNA expression [181]. This increase correlated with an increase in MAFbx/Atrogin-1, Murf1/Trim63 and Foxo3 genes expression and accordingly, the muscle-specific deletion of Klf15 in this model prevented from diabetes-induced muscle atrophy [181]. The authors identified WWP1 as an E3 ligase targeting KLF15 and showed that knocking-down WWP1 in both C2C12 myotubes and in tibialis anterior muscles increased MuRF1/Trim63 and MAFbx/Atrogin-1 expression and induced atrophy [181] (Figure 2). WWP1 E3 ligase is indeed induced by high glucose conditions in myotubes [206]. Conversely, in high glucose conditions, WWP1 has also been implicated in the down-regulation of AMPKα2 protein levels [206]. The authors have shown that WWP1 interacted with AMPKα2 leading to a proteasome-dependent decrease of AMPKα2 in myotubes; however, direct ubiquitination was not addressed [206]. WWP1 may thus control muscle mass through a direct action on AMPK, a known modulator of FOXO3a, MuRF1/TRIM63 and MAFbx/Atrogin-1 [88].

TRIM32 in the Regulation of Autophagy

TRIM32 is a RING E3 Ub ligase whose mutation is responsible for the development of limb girdle dystrophy 2H (LGMD2H) [207]. Several substrates have been identified for TRIM32 in non-muscle cells, including cell cycle regulators (c-Myc, MYCN, p53), the cell growth and transformation factor ABI2 and PIASY (a SUMO E3 ligase). TRIM32 is also involved in the targeting of factors influencing myogenesis (NDRG2 and TRIM72) that regulate muscle satellite cells renewal and differentiation [208]. While initially postulated to promote muscle atrophy, TRIM32 is in fact a master regulator of myogenesis during recovery situations [208]. Indeed, the dystrophic phenotype of TRIM32 mutations appeared to be largely due to impaired myogenesis [208][209][210].

More recently, TRIM32 was implicated in the early events leading to autophagy. Indeed, TRIM32 targets ULK1, a Ser/Thr protein kinase (Figures 1 and2). ULK1 is an upstream regulator of autophagy rapidly activated to ensure a rapid response to stress conditions [211]. The authors showed that TRIM32 deficiency was directly responsible for autophagy defects both in cultured cells and in mice treated with Dex. The mechanisms by which TRIM32 controls the activation of autophagy through ULK1 involves its binding to AMBRA1, a positive regulator of autophagy [211]. AMBRA1 is a pivotal factor able to bind several E3 ligases during the course of the autophagy process. In presence of AMBRA1, TRIM32 binds to ULK1, synthesizes unanchored K63 Ub chains that activate ULK1 kinase activity, thus promoting autophagy. The role of TRIM32 during the autophagy process is not limited to ULK1 as p62, an important autophagy receptor [212], is also a TRIM32 substrate. p62 activity is modulated by multi mono-Ub catalyzed by TRIM32 and loss of function of TRIM32 largely abolished autophagy [213]. Altogether, TRIM32 appears as a master regulator of muscle renewal through the initiation of autophagy.

FOXO Transcription Factors Are Regulated by MDM2 and SKP2 E3 Ubiquitin Ligases

Alternatively to phosphorylation, FOXO can be regulated by acetylation/deacetylation, methylation and ubiquitination to modulate its activity, localization as well as degradation [214][215][216].

Ubiquitination modulate FOXO activity by either mono-or polyubiquitination through MDM2 and SKP2 E3 Ub ligases (Figures 1 and2). MDM2 is the enzyme responsible of a single addition of an ubiquitin moiety to FOXOs, specifically to FOXO4, thus allowing its nuclear localization and transcriptional activation [217,218]. Mono-Ub of FOXO4 is observed under oxidative stress conditions and can be counteracted by deubiquitinating enzymes such as ubiquitin-specific protease (USP7). Importantly, ubiquitination mediated by MDM2 is context specific and upon growth factor stimulation can induce FOXO1 and 3 degradation [217]. In addition, interaction between FOXOs and SKP2, a subunit of the SKP/cullin 1/F-box protein E3 ligase leads to proteasomal degradation of FOXO1 in the cytosol [218].

Combined with the other posttranslational modifications, ubiquitination allows FOXOs to integrate information arising from insulin, growth factors, cytokines, and oxidative stress and to control downstream signaling. Interestingly, FOXO TFs have systematically been envisioned as crucial drivers of catabolic pathways during muscle wasting. Nonetheless, recent work showed that Muscle-specific RING finger protein 1 (MuRF1), also named TRIM63, is a RING-type E3 ligase and a founding member of the so-called "atrogenes" (see [6] for a recent review). MuRF1/TRIM63 is a master regulator of skeletal muscle atrophy development occurring in numerous catabolic conditions and MuRF1/Trim63 mRNA appeared to be upregulated in more than 25 atrophying situations [6] (Figures 1 and2). Mice deleted for MuRF1/TRIM63 (MuRF1-KO mice) were partially resistant (preservation of muscle mass and structure) to skeletal muscle atrophy induced by denervation [4], hindlimb suspension [4,223], glucocorticoid [224], amino acid deprivation [225], and acute lung injury [226]. MuRF1/TRIM63 is responsible for the coordinated breakdown of both thick and thin filaments occurring during catabolic states in skeletal muscle, targeting to degradation the main proteins of the contractile apparatus: myosin heavy chains (MHC) [227], alpha-actin [228], troponin I [229], TCAP/telethonin [230]. During denervation and starvation, MuRF1/TRIM63 has also been involved in the degradation of acetylcholine receptor (CHRN), the major postsynaptic ion channel of the neuromuscular junction. This degradation is mediated by the activation of selective autophagy and degradation of CHRN, likely via the degradation of BIF-1 (Bax interacting factor 1)/EndoB1 (EndophilinB1) and/or SQTM1/p62 (sequestosome-1) [231,232].

While numerous studies have promoted a major role of MuRF1/TRIM63 in the development of skeletal muscle atrophy during catabolic states, in the heart, the analyses of MuRF1 mutants have highlighted a beneficial cardioprotective role [233]. These opposites roles in both kind of muscles imply the development of skeletal muscle-specific drugs to inhibit MuRF1/TRIM63. Moreover, one should also take into account that MuRF1/TRIM63 has two homologs, MuRF2 and MuRF3 that share some redundant functions and could replace its role [12].

MAFbx/Atrogin-1/FBXO32

The multimeric E3 ligase MAFbx/atrogin-1/FBXO32 is another founding member of the atrogene family ([6] for a recent review) crucial for the development of muscle atrophy. Interestingly, nearly all catabolic situations induce an overexpression of both MAFbx/Atrogin-1 and MuRF1/TRIM63, which are controlled by the same TFs (FOXO1/FOXO3a, NF-κB, C/EBP β, Smad 3, etc.) and the same signaling pathways [234] (Figures 1 and2).

In contrast with MuRF1/TRIM63 that targets directly the contractile proteins for their degradation MHC,, MAFbx appeared to target pro-anabolic factors like MyoD, myogenin or eIF3f [235][236][237]. MyoD is a muscle-specific transcription factor that plays crucial roles during cell cycle and muscle differentiation [238]. The eukaryotic initiation factor 3 subunit f (eIF3f) is a pivotal element of protein synthesis and its control by MAFbx allows the latter to master the anabolic processes [235]. While a putative role of MAFbx/Atrogin-1 on sarcomeric proteins was hypothesized using an indirect approach, this has never been confirmed [239]. By contrast, the authors found that desmin, a main component of the intermediate filaments, physically interacted with MAFbx and was degraded in myostatin-treated cultured C2C12 myotubes.

As MAFbx/Atrogin-1 and MuRF1/TRIM63 are controlled by similar signaling pathways, the strategies for the upstream control of MuRF1/Trim63 expression are generally also valid for MAFbx/Atrogin-1 (Table 2). By contrast with MuRF1/TRIM63, no direct inhibitor of MAFbx/Atrogin-1 has been described so far but general strategies, like targeting the interface responsible for substrate recognition or impeding the assembly of the F-box (i.e., the subunit recognizing the substrates) into the SCF complex, may prove to be efficient.

Altogether, controlling concomitantly MAFbx/Atrogin-1 and MuRF1/TRIM63 E3 ligases allows skeletal muscle cells to both increase the degradation of the contractile apparatus and to depress the protein synthesis machinery, which allows a tight regulation of protein homeostasis.

PARKIN Controls Muscle Mass through the Maintenance of Mitochondrial Homeostasis

PARKIN is an E3 ubiquitin ligase implicated in the regulation of mitophagy, a quality control process in which defective mitochondria are degraded. Mitochondrial quality control through both mitochondria turnover and dynamic plays an essential role in the maintenance of muscle mass (see [240] for a review). During mitophagy, PARKIN ubiquitinates several outer mitochondrial membrane proteins leading to subsequent autophagosomal engulfment and lysosomal degradation (Figures 1 and2). This role of PARKIN has been emphasized in rodent models or in humans where a deregulation of PARKIN mRNA and/or protein expression prevailed in response to catabolic or anabolic situations. An accumulation of PARKIN protein prevailed during: (i) muscle wasting situations such as chronic kidney disease [241], chronic obstructive pulmonary disease (COPD) [242], physical inactivity [243,244] and (ii) upon exercise training [245,246]. Conversely, PARKIN mRNA or protein levels decreases in skeletal muscles from some elderly populations, perhaps related to the loss of muscle mass and poor physical function, e.g., physically inactive frail older women [247,248] or gastric cancer patients with cachexia [249].

In the last two years many studies using loss/gain of function models have provided insight on the role of PARKIN in skeletal muscle. Loss of function mouse models pointed out the essential role of PARKIN in basal conditions for the maintenance of (i) mitochondrial function [250,251] and (ii) skeletal muscle mass and normal contractile function [184,251]. Such studies also reported that PARKIN helps to resist to some drug-induced muscle damages [252] and is required for exercise-induced mitophagy flux and for the accumulation of functional mitochondria following muscle adaptations to training [250]. In addition, these loss-of-function studies also highlighted that PARKIN-mediated mitochondrial clearance contributes to proteasome activation during denervation in atrophied slow-twitch mus-cles [253]. On the flip side, gain-of-function studies showed that PARKIN overexpression in mice: (i) attenuates the ageing-related and the sepsis-induced muscle wasting and causes hypertrophy in adult skeletal muscle, (ii) increases mitochondrial content and enzymatic activities and (iii) protects from ageing-related increases of oxidative stress markers, fibrosis and apoptosis [185,186]. It is very likely that this role of PARKIN in controlling muscle mass has been evolutionary conserved. Indeed, similar observations were also reported in the fruit fly model: Parkin deficiency in Drosophila leads to severe degeneration of the flight muscles with accumulation of swollen mitochondria [254] whereas Parkin overexpression promotes mitophagy in older muscles and extend lifespan.

Together, these studies clearly indicate that PARKIN is an important player in the control of muscle mass through its role in the maintenance of mitochondrial homeostasis. This makes it a potential therapeutic target of interest for preserving muscle mass or fighting against atrophy. Nevertheless, the regulation of PARKIN can be very different according to the physiological or pathological situation or during ageing. Further investigations should enable defining how this actor could be a target of interest according to the population considered.

MUSA1/FBXO30

FBXO30, also called muscle ubiquitin ligase of the SCF complex in atrophy-1 (MUSA1), is a FBOX protein forming an SCF complex with SKP1, Cullin1 and ROC1 [77]. Proteins targeted by MUSA1 remain undefined, but its inhibition in denervated muscles reduces remarkably muscle atrophy, and reverts almost completely the strong atrophic phenotype of Smad4-KO mice [77] (Figures 1 and2). In muscle, Musa1 expression is upregulated in atrophic mice muscle undergoing CKD [255] or sepsis [256].

FBXL21

Very recently, a new E3 ubiquitin ligase involved in muscle function control has emerged, FBXL21 [188]. FBXL21 forms an SCF E3 ligase complex and was first identified as clock-controlled E3 ligase modulating circadian periodicity via subcellular cryptochrome degradation [257]. Accordingly, in mice, the Psttm mutation, corresponding to a hypomorphic mutation of FBXL21 with reduced FBXL21 activity, caused circadian period shortening [257]. Further studies of these mice revealed that they also displayed skeletal muscle deficiencies with a decrease in fiber CSA (gastrocnemius) and impaired exercise tolerance and grip strength for both forelimbs and hindlimbs [188]. The authors nicely demonstrated the circadian degradation of the cytosolic TCAP/Telethonin by FBXL21 (Figure 2), under the control of GSK-3β. They reported that GSK-3β phosphorylated both FBXL21 and TCAP leading to FBXL21-CULLIN1 complex formation and phosphodegron-dependent TCAP turnover.

Ubiquitin Ring-Type E3 Ligases (UBR)

Ubiquitin Ring-type (UBR, also referred to as E3α) proteins are RING finger E3 ligases that compose a 7-member family and that mainly recognize their substrate through the N-end rule pathway [258]. A first member, UBR2/E3alpha-II, has been shown to be significantly induced in skeletal muscle, in two different animal models of cancer cachexia, at the onset and during the progression of muscle wasting [259]. However, its exact function and importance in skeletal muscle maintain during catabolic states have not been further studied. UBR4 is overexpressed in the skeletal from fasted mice and genetic ablation of UBR4 preserves muscle mass in tumor-bearing mice [189] (Table 1). Intriguingly, the protection of UBR4 knockout against tumor-induced atrophy was limited to type IIA fibers. In contrast, UBR5 has been implicated in muscle hypertrophy [260] and reported to be at least partially associated to the proteasome [261]. Recently several members of the UPS have been described as UBR5 substrates, which included an E2 (UBE2B, an abundant muscle E2), several E3 ligases, proteins involved in chromatin remodeling, etc. [189]. As the main UBR4 targets are positive regulators of muscle growth, the authors concluded that UBR4 acts as a negative regulator of muscle hypertrophy. 3.3.7. FBXO21/SMART FBXO21/SMART forms an SCF complex with Skp1, Cullin1 and Roc1, in skeletal muscle and has been shown to promote atrophy during denervation [187]. Indeed, the authors showed that FBXO21/SMART upregulation was required for atrophy while, knockdown in TA muscle protected denervated muscles from atrophy (Table 1), probably due to a global reduction of protein ubiquitination [187]. FBXO21/SMART might therefore be a new critical E3 to target to limit skeletal muscle atrophy. Further work should determine whether this E3 is crucial for the development of atrophy in other catabolic conditions and what are the mechanisms involved.

Promising E3 Ubiquitin Ligases Regulating Muscle Mass and Function

Other E3 ubiquitin ligases are also promising putative targets for maintaining muscle mass and function, if we rely on what has been published in other organs or organisms. For example, the SIAH-1 RING E3 ligase has been identified in the same RNAi screen that UBR4, performed to identify ubiquitin-related enzymes that regulate myofiber size, using the fruit fly Drosophila [189]. In Drosophila, SIAH1 knock-down led to muscular hypertrophy while its overexpression led to atrophy [189]. It is noteworthy that, in space flown rats, SIAH1 mRNA expression has been shown upregulated suggesting also a putative role during this process in mammals [172]. However, in mammals two isoforms, SIAH1 and SIAH2, are expressed in muscle and could share redundant functions [189].

SMURF1, an HECT ubiquitin ligase interacts with SMAD1 and SMAD5 (BMP pathway) and SMAD4 in a certain context, leading them all to proteasomal degradation in vitro [262]. Moreover, it can degrade the main TGF-β receptor through an indirect recruitment to the receptor by SMAD7, leading to the receptor degradation [263]. In, COPD leading to muscle atrophy, TGF-β signaling is abnormally up-regulated and this, is negatively correlated to SMURF1 expression. This highlights that the inhibitory effect of SMURF1 over TGF-β is needed for muscle homeostasis [264].

The C terminus of Hsc70-interacting protein (STUB1/CHIP) serves as an E3 ubiquitin ligase. This E3 plays a dual role in BMP/TGF signaling. Overexpression of CHIP inhibits TGF-β luciferase reporter through the ubiquitination and degradation of SMAD3, and conversely silencing it leads to increase the signal transduction in HEK293T cells [265]. In cellulo experiments showed that CHIP mediates as well SMAD1-5 poly-ubiquitination, and subsequent degradation to terminate BMP signaling [266]. In muscle, CHIP is highly expressed. For instance, Chip-/mice at 6 months shows muscle morphological changes consistent with increased sarcoplasmic reticulum compartments in quadriceps muscle and gastrocnemius, resulting in damages and fiber switch composition [267]. From our knowledge, no studies have shown the implication of CHIP in TGF/BMP signaling-mediated muscle atrophy.

TRIM62 belongs to the TRIM/RBCC family. This enzyme acts as a negative regulator of TGF-β signaling by binding to SMAD3 and promoting its ubiquitination and degradation, resulting in a decrease of TGF-β/SMAD3 target genes in HEK and human mammary epithelial cells [268]. TRIM62 is increased in the skeletal muscle of ICUAW patients (Intensive care unit-acquired weakness), a devastating illness characterized by loss of muscle mass [269]. In this context, the authors proposed TRIM62 contribution in inflammation-induced muscle atrophy through IL-6 pathway. Indeed, Trim62-KD inhibited LPS-induced IL-6 expression in C2C12 cells [269].

TRIM72/MG53 is a muscle-specific E3 ligase, also called mitsugumin 53, specifically expressed in the plasma membrane of skeletal muscle, and has a critical role in membrane repair. Membrane repair deficiency causes muscle cell death, injury, and dystrophy. Accordingly, the overexpression of human TRIM72 in a hamster model of genetic muscular dystrophy protects skeletal muscle damage through enhancement of membrane repair [270]. Similarly, short-term TRIM72 injection ameliorates the underlying defects in dysferlin-deficient muscle by increasing sarcolemma membrane integrity [271] while Trim72 -/-mice develop significant skeletal muscle myopathy and cardiovascular defects due to defective sarcolemma repair [272].

Current Treatments/Potential Modes of Action

The importance of maintaining muscle mass together with the discovery of several E3 ligases implicated in muscle homeostasis has rapidly end up with multiple approaches to chemically alter the expression of these enzymes. This includes chemical drugs but also several natural molecules that have been tested for their ability to modulate the UPS and more particularly the E3 ligases (Table 2). As E3 ligases are controlled by several signaling pathways, one possibility that was first addressed was to block these signals. The PI3K-AKT-mTORC1 axis is known to control muscle mass by directly acting on FOXO transcription factors, the latter being master regulators of several E3 ligases, like MAFbx/Atrogin-1, MuRF1/TRIM63, MUSA1, SMART and FBXO31, during several atrophy situations [187]. As such, clenbuterol (Table 2 and Figure 2), an activator of the AKT-mTORC1 pathway, is able to decrease MuRF1/Trim63 and MAFbx/Atrogin-1 expression in denervated or hindlimb suspend rats and to partially preserve muscle mass [273].

Glucocorticoids

Glucocorticoids are potent manipulators of muscle mass and the glucocorticoid receptor antagonist RU486 proved to be efficient in rats for blocking dexamethasone (Dex)induced induction of MuRF1/Trim63 of MAFbx/Atrogin-1, the main regulators of muscle mass [13] (Table 2 andFigure 2). Similarly, the authors demonstrated that blocking TNFα by the TNF-binding protein (TNFBP) was efficient for blunting LPS-induced expression of MuRF1/Trim63 and MAFbx/Atrogin-1. However, when sepsis was induced by cecal ligation and puncture, neither RU486 nor TNFBP were able to counteract the overexpression of MuRF1/Trim63 and MAFbx/Atrogin-1, indicating that multiple signals were activated by sepsis. This points out the difficulty of treating complex catabolic signals in vivo. Infliximab is an anti-TNF-α agent able to lower the downstream NF-κB signaling. In patient's suffering from Crohn disease, treatment with infliximab was able to ameliorate muscle atrophy but, although hypothesized by the authors, the expression of MuRF1/Trim63 or any other E3 ligase was not addressed [274]. Il-6 is another inflammatory cytokine that can be implicated during muscle wasting conditions like muscle disuse [275]. Increased IL-6 in tail-suspended mice paralleled skeletal muscle atrophy and was accompanied by increased levels of MuRF1/Trim63 and MAFbx/Atrogin-1. The inhibition of the IL-6 receptor by hydroxymethyl butyrate (HMB, a metabolite of leucine) or vitamin D tended to decrease IL-6 levels and when combined, HMB and vitamin D exhibited better efficiency for blunting IL-6 production [275] (Table 2 and Figure 2). By contrast, each molecule was sufficient for decreasing MuRF1/TRIM63 and MAFbx/atrogin-1 levels and to attenuate muscle atrophy. While the authors attributed the beneficial effects of HMB and vitamin D on IL-6 receptor, using a monoclonal antibody directed against IL-6 receptor (MR16-1) proved to be inefficient as only MuRF1/Trim63 expression was decreased with no amelioration on muscle mass. As for the TNF-α, this work underscores the multiplicity of signaling during atrophy situations and the difficulty of blunting efficiently receptor-linked signaling. STAT-3 is a downstream effector of IL-6 signaling and a specific inhibitor of was investigated for its capacity to block muscle atrophy in a model of mice deficient for the vitamin D receptor (VDR) [14].

In these conditions, VDR -/-mice exhibited exacerbated MuRF1/Trim63 expression and increased muscle atrophy. While C188-9 was able to partially preserve muscle mass, its efficacy against MuRF1/TRIM63 was not addressed.

NF-κB

Inhibition of the NF-κB signaling pathway was also efficiently performed using high doses of salicylate (Table 2 andFigure 2), which allowed the reversion of MuRF1-induced muscle atrophy in tumor bearing or denervated mice [89]. However, the high doses used for achieving a potent inhibitor would be toxic when administered to humans. β2-AR agonists can exert both anabolic and anti-catabolic effects on skeletal muscles either by decreasing catabolic signals or by promoting anabolic ones or both. Formoterol (Table 2 andFigure 2 β2-AR reversion of E3 ligases expression and muscle sparing was also observed in a rat rheumatoid arthritis model and was attributed to modulation of both the AKT and the NF-κB pathways [293]. Other 2-AR agonists like espindolol have also been shown to ameliorate muscle loss and to blunt E3 ligase expression in aged rats. The authors found that both NF-κB and myostatin expression was reduced with no effect on AKT and FOXO3a [292]. Altogether, this strongly suggests that the positive effects of 2-AR agonists on muscle mass are mediated through the modulation of different signaling pathways depending on the catabolic stimuli, which complicates future therapeutical strategies. p38α MAPK is known to play an important role in the development of muscle atrophy [295]. Inhibition of the p38α MAPK receptor by the selective inhibitor VX-745 (Table 2 and Figure 2) partially improved muscle weight in hindlimb suspended rats with a modest inhibition of MuRF1 expression but no modification of MAFbx [292].

NOTCH

The NOTCH pathway is mainly known for its implication in muscle development and regeneration upon injury. However, it has been also implicated in muscle atrophy linked to either cancer or amyotrophic lateral sclerosis (ALS) mice models [161]. Using a tocopherol derivative (AGT251) (Table 2 andFigure 2), the authors found that this antioxidant molecule was protective against muscle atrophy and MuRF1/Trim63 expression, and that the effects may be mediated through NOTCH1 and 3 expression.

Ion Channels

Electrical stimulation is an important signal that controls muscle mass and ion exchange through specific channels, e.g., K + -channels, [296]. Following nerve injury, improvement of muscle mass was observed by blocking K+ channels with 4-aminopyridine (4-AP) [278]. 4-AP (Table 2) was able to partially restore muscle fiber diameter with a concomitant decrease of MuRF1/Trim63 expression accompanied by decreased Foxo1 and Foxo3a expression. 4.1.9. Acute-Phase Protein Serum Amyloid A1 (SAA1) Skeletal muscle loss in intensive care unit patients has been at least partially attributed to the acute-phase protein serum amyloid A1 (SAA1) [256] (Table 2). Recent work performed in cultured C2C12 myotubes and septic mice showed that SAA1 effects were mediated through TLR-dependent IL-6 expression and recruitment of the NF-κB pathway. This leads with muscle atrophy and an overactivation of MuRF1/TRIM63, MAFbx/Atrogin-1 and MUSA1 E3 ligases. Using BMS-345541, an inhibitor of the IκB kinase, the authors found that the expression of the E3 ligases returned to basal levels and muscle sparing was observed, indicating that blocking the NF-κB pathway may be an efficient way for indirectly modulating E3 ligases [266]. 4.1.10. TGF-β TGF-β family ligands, including myostatin and activin, are potential effectors of muscle atrophy in several situations of muscle atrophy like cancer [16]. The injection of a truncated form (aa 7-100) of the TGF-β ligands ActRIIB (Table 2 andFigure 2) in mice subjected to several models of cancer cachexia was sufficient for blocking MuRF1/Trim63 and MAFbx/Atrogin-1 expression together with complete sparing of both skeletal muscle and heart mass [16]. 4.1.11. Reactive oxygen species (ROS) ROS are downstream modulators of muscle wasting and may be also potential levers for preserving muscle mass [162]. Several molecules have been tested for their potency to modulate E3 ligase expression and thus to preserve muscle mass. Dehydroepiandrosterone (DHEA) (Table 2 andFigure 2), a multifunctional steroid with antioxidant properties was shown to decrease MuRF1/Trim63 expression (but not MAFbx/Atrogin-1) in tumor-bearing rats, which helped moderately preserving muscle mass [168]. Transforming growth factor type beta 1 (TGF-β1) regulates the function and pathological status of skeletal muscle and was found to modulate muscle mass by increasing the activity of NADPH oxidase (NOX), a major ROS producer [69]. This was accompanied by an increased expression of MuRF1. Interestingly, N-acetylcysteine (NAC, a clinically used anti-oxidant) and apocynin (NOX inhibitor) were able to reverse both MuRF1 overexpression and muscle mass loss in cultured myotubes treated with TGF-β1. Similarly, NAC or pyrroloquinoline quinone (PQQ, a naturally occurring antioxidant) were able to decrease MuRF1/Trim63 and MAFbx/Atrogin-1 expression and to preserve muscle mass in denervated mice or in starved cultured myotubes [285]. SS-31 is a cell-permeable mitochondria-targeted antioxidant tetrapeptide undergoing clinical trials [297]. This peptide is efficient for lowering ROS production, improving muscle atrophy and decreasing MuRF1/Trim63 and MAFbx/Atrogin-1 expression [287]. While ROS modulation seems to be efficient for protecting muscle mass, the mechanisms involved in the decrease of E3 ligases expression is far from being understood. Vitamin E is another antioxidant that has been used in a rat model of muscle disuse (hindlimb suspension) [291]. Vitamin E supplementation was able to largely prevent the overexpression of several proteolytic enzymes including MuRF1/TRIM63 and MAFbx/atrogin-1 but the impact on muscle mass fiber cross section was moderate. Interestingly, the authors attributed the protective role of vitamin E to a direct action on gene expression and not to its antioxidant properties [291].

4.1.12. Leucine and Its Derivative ß-Hydroxy-ß-Methylbutyrate (HMB)

The essential amino acid leucine and its derivative HMB were described as modulators of protein synthesis through an action on the mTORC1 pathway [298,299]. The efficiency of HMB and Leucine on MuRF1/Trim63 expression was addressed in Dex-treated rats [282] (Table 2). However, while HMB and leucine ameliorated muscle function and decreased MuRF1 expression, no effect of both HMB and leucine was observed on muscle weight. This might be due to partial effect of the treatment on muscles. Interestingly, the modulation of FOXO1 nuclear translocation was the putative mechanism for MuRF1/Trim63 downregulation. Leucine was also implicated in the modulation of both MuRF1/TRIM63 and MAFbx/atrogin-1 with an improvement of myotube diameter in Dex-treated primary muscle cells [283,300]. The authors found that the effect of leucine on E3 ligase expression was mediated by FOXO3a cytoplasmic sequestration and concomitant vacuolar protein sorting 34 (VPS34) nuclear accumulation. Alternatively, a supplementation with Vital01 (composed by high levels of BCAAs, increased ratio of whey and casein proteins, vitamin D, and ursolic acid) in calorically restricted mouse model of muscle atrophy preserved muscle mass both during and after the atrophic conditions were stablished. The catabolic phenotype was ameliorated by Vital01, notably through the modulation of the UPS (decreased expression of MuRF1/Trim63 and MAFbx/Atrogin-1) and the autophagy-lysosome pathways, [301]. However, Leu and HMB exhibit no effect on E3 ligase expression (MuRF1/Trim63 and MAFbx/Atrogin-1) in human during fasting [210] and the beneficial muscle sparing was attributed to a stimulation of the mTORC1 pathway [298]. On the whole, the potential beneficial effect of Leu and HMB is still controversial for both its action on E3 ligases and for muscle preservation effect.

Plant Derivatives

Plant derivatives were also tested for their potency to protect skeletal muscle atrophy. Ursolic acid (Table 2), was able to partially decrease muscle atrophy in mice subjected to chronic kidney disease and a moderate effect on MuRF1/TRIM63, MAFbx/Atrogin-1 and MUSA1 expression was observed, that was attributed to decreased expression of myostatin and inflammatory cytokines [255]. However, ursolic acid was unable to modify E3 ligases expression in cultured myotubes treated with Dex, and ursolic acid was able to directly induce the expression of MuRF1/Trim63 and MAFbx/Atrogin-1 in C2C12 myotubes. More investigation is clearly needed before concluding of any potential therapy using ursolic acid. A polyphenol from green tea, epigallocatechin-3-gallate (EGCG), was also used as a countermeasure for fighting against cancer cachexia [279]. EGCG was able to reduce NF-κB expression and the downstream E3 ligases MuRF1/TRIM63 and MAFbx/Atrogin-1 (only a trend for MuRF1/TRIM63). However, the decrease of the tumor volume renders difficult the interpretation of the effect of ECGC as its protective role on muscles might be indirect. Teaghrelin, an analog of the human ghrelin, was efficient for decreasing the catabolic effect of Dex in cultured C2C12 myotubes, with depressed expression of MuRF1/Trim63 and MAFbx/Atrogin-1 [289]. The authors suggested that increased myogenin expression might be implicated in the beneficial effect of teaghrelin. In rats submitted to thermal injury, ghrelin blunted the expression of MuRF1/Trim63 and MAFbx/Atrogin-1 [302]. While the exact mechanism was not addressed, the authors found that TNFα and IL-6 mRNA levels were normalized upon ghrelin infusion. Interestingly, mice knocked out for ghrelin exhibit an increased expression of MuRF1/Trim63 and are less protected from fasting atrophy [290]. Sabinene is a terpene present in plant essential oil and was found to decrease muscle atrophy in starved rats through reversal of the increased MuRF1/Trim63 overexpression that is commonly observed upon fasting [286]. The mechanism proposed by the authors was the repression of ROS-mediated activation of ERK and p38 MAKP.

Matrine (Table 2 and Figure 2) is a natural compound used in traditional medicine and approved for cancer therapy in China [284]. The authors demonstrated that this compound was able to partially reverse muscle atrophy in mice subjected to Colon 26 adenocarcinoma with a concomitant decrease of MuRF1/Trim63 and MAFbx/Atrogin-1 expression. Using cultured C2C12 myotubes, the authors found that the effect of matrine was mainly driven by the AKT/mTORC1/FOXO3a signaling pathway with both a repression of the catalytic axis and an up regulation of the anabolic one.

E3 Ligases Inhibitors

The main E3 ligase that has been investigated so far for the design of inhibitors is MuRF1/TRIM63. This can be explained by the fact it is also the only E3 ligase known to target contractile proteins from both the thin and the thick filament [227,228,230,303].

In a first attempt, the screening of a small molecule library for finding MuRF1/TRIM63 inhibitors identified a compound (P013222) (Table 2 andFigure 2) that was able to decrease MuRF1/TRIM63 autoubiquitylation [294]. The selectivity was within the µM range with a 10 times preference for MuRF1/TRIM63 compared to other E3 ligases and P013222 was able to inhibit the degradation of MHC in Dex-treated C2C12 myotubes.

More recently, the screening of a library identified another small molecule compound (ID#704946/MyoMed-946) able to alter MuRF1-titin interaction (IC50 around 25 µM), thus targeting the coiled-coil region of MuRF1/TRIM63 [293]. Compound ID#704946/MyoMed-946 was able to decrease in vitro MuRF1/TRIM63 self-ubiquitination and surprisingly was also able to decrease the mRNA levels of MuRF1/Trim63 in catabolic C2C12 myotubes [293]. This suggests that this compound may be interfering on several mechanisms modulating MuRF1/TRIM63 action. This compound was at least partially effective for preserving muscle mass in catabolic mice. The mechanism by which compound ID#704946/MyoMed-946 preserve muscle function needs further investigations as the same laboratory found that it was also able to modulate MuRF2 expression [17,18].

The cellular inhibitor of apoptosis 1 (cIAP1) E3 ligase is a negative regulator of muscle mass by acting on TNFα-mediated NF-κB signaling. cIAP1 is in fact an E3 ligase whose role is to blunt the non-canonical NF-κB signaling and its genetical ablation was reported to improve muscle mass in mdx mice [91]. Recently, an inhibitor of cIAP1 (LCL161) was addressed for its capacity to improve skeletal muscle mass in denervated mice [19]. While genetic ablation of cIAP1 was able to preserve muscle mass in denervated mice, its inhibition by LC161 was only moderately efficient as only the EDL muscle was preserved, indicating either a poor inhibition efficiency of LCL161 or a compensation by other E3 ligases and/or signaling pathways.

CBL-B is an E3 ligase involved in the targeting of the Insulin Receptor Substrate 1 (IRS1) that mediates IGF1 signaling, notably by activating the AKT-mTORC1 pathway. CBL-B is involved in spaceflight-induced muscle atrophy and genetic ablation of CBL-B protects skeletal muscle from disuse atrophy [171]. CBL-B can be inhibited by a small pentapeptide mimetic of tyrosine608-phosphorylated IRS-1 that restores IGF1 signaling and protects from atrophy. Interestingly, IGF1 signaling restoration induced a concomitant decrease of MAFbx expression while no variation on MuRF1/Trim63 mRNA levels was observed [171]. Another peptide, called cblin, was also reported to exhibit some protective action on skeletal muscle through the inhibition of .

Conclusions and Future Directions

The discovery of molecules able to lower muscle loss during catabolic situations is a promising field of investigation and numerous possibilities can be envisaged, from directly blunting the signals arriving at the cellular membrane levels to more specifically inhibiting the E3 ligase(s) involved in the degradation of the muscle contractile apparatus. Each strategy has advantages and disadvantages. The first approaches are not specific and alter numerous metabolic pathways, which may end up with side effects both at the short-and long-term levels. For example, suppressing the general protein breakdown by acting on the PI3K/AKT/FOXO pathway might be deleterious by accumulating misfolded proteins. On the other side, receptor or metabolic pathways have been studied for decades and several inhibitors have been well characterized, which allows more straightforward investigations dedicated to muscle atrophy.

The drugs targeting directly the E3 ligases, so far mostly focused on MuRF1/TRIM63, have the advantage of being more selective and should prove to be better tolerated by the muscle cells and the whole organism. Indeed, MuRF1/TRIM63 (and some other ligases) is muscle-specific, which means that drugs will only affect muscles. This is an important advantage over metabolic pathways that are shared by several organs. More investigations are clearly needed for ameliorating the first generation of molecules or for finding new ones, which includes new strategies for modulating E3 ligases activity.

Background

To deal with seasonal cold and food shortage during winter, hibernating mammals show a combination of behavioral and physiological changes. To save energy during hibernation, hibernating animals use periods of torpor characterized by decreased metabolic rate and body temperature, reduction in respiratory and heart rates, and physical inactivity [1,2]. Brown bears (Ursus arctos) exhibit unique features, as they hibernate at mild hypothermia (32-35 °C) and can stay inside their dens for up to 7 months, without drinking, eating, defecating or urinating, and with no arousal episodes [3][4][5][6]. While denning, they reduce their metabolic rate by about 75% [7], and rely primarily on mobilization of fat stores, which is reflected by increased circulating fatty acid concentration and body fat store depletion during winter [8][9][10].

Beyond energy substrates, lipids also have pleiotropic actions in the regulation of metabolism, and changes in membrane fatty acid composition have already been described in hibernating animals [11][12][13][14], including the brown bear [9]. Membrane phospholipids can also provide long-chain fatty acids for the synthesis of bioactive lipid mediators, such as endocannabinoids [15][16][17]. The endocannabinoid system (ECS) was originally described as being composed of Gprotein coupled receptors (CB1 and CB2) and their endogenous ligands, of which the main ones are derived from arachidonic acid 20:4n-6 (AA) esterified into phospholipids, and called 2-arachidonoyl glycerol (2-AG) and anandamide (AEA) [15][16][17][18][19][20]. These two well-characterized compounds clearly show varying affinity for CB1 and CB2 receptors. Indeed, AEA is considered as a high affinity CB1-partial agonist (and weak CB2 agonist), whereas 2-AG is described as a low-to-moderate affinity CB1 and CB2 full agonist [21,22]. 2-AG and AEA belong to the large family of 2-acylglycerols (2-AcGs) and N-acylethanolamines (NAEs), respectively [17,19]. N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPEPLD) and sn-1-specific diacylglycerol lipase-α and β (DAGLA and DAGLB) are the main enzymes involved in the biosynthesis of NAEs and 2-AcGs, respectively [17,19]. Fatty acid amide hydrolase (FAAH) is responsible for NAEs catabolism (and to a lesser extend for 2-AG) [23], and monoacylglycerol lipase (MGLL) specifically catabolizes 2-AcGs [17,19]. eCBs can also be metabolized by lipoxygenases (LOXs) and by cyclooxygenase-2 (COX-2), an alternative pathway for eCBs catabolism [17].

The ECS includes structurally related compounds like Noleoylethanolamine (OEA), called «endocannabinoids-like compounds» (eCBs-like). The latter are metabolized by the same biosynthetic and catabolic enzymes as eCBs [17]. Although eCBs-like compounds are not able to bind to CB1 and CB2 receptors, they can bind to other G-protein coupled receptors (e.g. GPR119 and GPR55) or nuclear receptors, like peroxisome proliferator-activated receptor α (PPARA) [17].

Endogenous cannabinoids are involved in the regulation of many physiological processes, including neuronal signaling [24], stress response [25], metabolism [25][26][27], feeding behavior and energy storage [25,28]. Evidences support the fact that the ECS could be involved in sleep cycles [29], circadian and potentially circannual rhythms [30]. At the central level (e.g. hypothalamus), CB1 is able to promote food intake and reduce energy expenditure [25,31]. In addition, CB1 activation in adipose tissue leads to fatty acid and glucose uptake, and to upregulation of lipogenesis [25]. In liver, CB1 signaling leads to increased expression of genes involved in the synthesis of fatty acids [32], and in skeletal muscle tissue, CB1 activation triggers a decrease in glucose uptake and insulin sensitivity [25]. The CB2 receptor is well known to be widespread over immune cells and to have numerous immunomodulatory roles [33]. CB2 has also been detected in metabolic tissues, like adipose tissue and skeletal muscle [34,35] and CB2 pharmacological or genetic inactivation in murine obesity models promote insulin-mediated glucose uptake in skeletal muscles, reduce adipose tissue inflammation, and thus improves insulin sensitivity [36,37]. Finally, the eCB-like OEA promotes lipolysis, fatty acid oxidation in skeletal muscle and liver, and triggers an anorexigenic signal, notably through the nuclear receptor PPARA [38,39]. Considering the pleiotropic roles of ECS in neuronal signaling, regulation of feeding behavior, energy metabolism and circannual rhythms, important changes are expected during hibernation. Several ECS circulating compounds have been quantified in hibernating black bears, during and around the torpor phase [40],with no major changes observed except a slight increase in 2-AG in the period of metabolic drop before torpor. Although a decrease in ECS tone has been observed in hibernating marmots (Marmota monax and flaviventris) and ground squirrels (Spermophilus richardsonii) [30,41,42], we hypothesize that a similar decrease should occur in hibernating bears, not excluding specific changes due to their unique features during hibernation (mild hypothermia, no periodic arousal, and maintenance of alertness). Therefore, we explored here seasonal variations in fatty acid composition and ECS tone, in both circulating compartment and in muscle and adipose tissues, in winter-hibernating and summer-active brown bears.

Results

Seasonal differences in serum lipids

We explored the fatty acid (FA) composition of winterhibernating (WBS) and summer-active (SBS) bear serum (see supplementary Table S1). From the lipidomic data, we compared both the summer and winter concentrations and proportions of fatty acids (see supplementary Table S2 and S3 for detailed lipidomic results). As shown in Fig. 1a, the total concentration of FAs was about twofold higher in WBS relative to SBS (28.82 ± 1.71 vs. 15.99 ± 1.09 mmol/L). All but two quantified lipid species were higher in concentration in hibernating bears, i.e. saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and n-6 polyunsaturated fatty acids (PUFAs) (Supplementary Table S2). Only concentrations of alpha-linolenic acid C18:3 n-3 (ALA) (0.49 -fold, non-significant) and eicosapentaenoic acid C20:5 n-3 (EPA) (0.26-fold) were lower in WBS (Supplementary Table S2).

Meanwhile, the molar percent of total n-6 species were found to be lower in WBS compared to SBS (Fig. 1b). Lipid species with the highest molar percent are presented in Fig. 1c (see Supplementary Table S3). Among SFAs, palmitic acid C16:0 (PA) was found in higher proportion, whereas stearic acid C18:0 (SA) was in lower proportion in winter serum. Similar proportions of oleic acid 18:1n-9 (OA), belonging to the n-9 MUFAs, were found in winter and summer bear serum. Concerning n-6 PUFAs, the proportion of arachidonic acid C20:4 n-6 (AA) was lower during winter, whereas proportion of linoleic acid C18:2 n-6 (LA) remained unchanged (Fig. 1c). For individual species of the n-3 family (Fig. 1d and Supplementary Table S3), docosapentaenoic acid C22:5 n-3 (DPA, 1.5-fold) and docosahexaenoic acid C22:6 n-3 (DHA, 2.2-fold) were found in higher proportions.

The proportion of C20:5 n-3 (EPA) was found much lower (0.15-fold) in winter serum, as well as the alphalinolenic acid C18:3 n-3 (ALA, 0.27-fold), a precursor of the EPA, DPA and DHA species.

From molar percent values, the DHA/AA ratio was 3.2-fold higher in winter (Fig. 1e).

Changes in plasma endocannabinoids and endocannabinoids-like compounds

We next assessed circulating eCBs and eCBs-like in bear plasma. Paired samples were collected in winter and in summer from eight bears (Supplementary Table S1) and quantification of AEA, 2-AG and OEA are presented in Fig. 2 and supplementary Table S5. Lower concentrations were observed for AEA (0.63-fold) in winter compared to summer, whereas the reverse was observed for OEA (3.3-fold). No difference was found for 2-AG plasma concentration.

Changes in endocannabinoid concentrations in muscle and adipose tissues

Quantification of endocannabinoids was then performed in bear muscle and adipose tissues. Paired tissues samples were collected from bears in winter and in summer (Supplementary Table S1) and quantification of AEA, 2-AG and OEA are presented in Fig. 3 and supplementary Table S5. AEA concentration was lower in both muscle and adipose tissues during winter versus summer, close to the statistical threshold (p = 0.064 and p = 0.069, respectively). 2-AG concentration was significantly lower in muscle and adipose tissues samples during winter, by about 1.6-and 9-fold, respectively. By contrast, no seasonal changes were found in OEA concentrations in both muscle and adipose tissues.

Changes in endocannabinoid pathway-related gene expressions in muscle and adipose tissues

To explore tissue metabolism of endocannabinoids, we quantified gene expression in muscle and adipose tissue of the eCBs membrane receptors CB1 and CB2, and several enzymes involed in the synthesis and catabolism of eCBs. For muscle tissue, paired samples were from 8 bears at the two time points, while for adipose tissue, data are coming from 5 bears in summer and 13 bears (See figure on previous page.) Fig. 1 Lipidomic from summer and winter brown bear serum. The winter and summer bear serum mixes were prepared as described (Supplementary Table S1). a: Total fatty acid (FA) concentration. b: Total n-6 and n-3 FA relative proportions of total lipids. c: Highest molar percent lipid species D: Molar percent of the n-3 family lipid species. e: Molar ratios of DHA/AA in summer and winter serum. Detailed lipidomic results are presented in Supplementary Tables S2 andS3. Data are expressed in mmol/L for total FAs concentration, or molar percentage of total lipids and are represented as mean ± SEM of separate extractions and quantifications from the twelve mixes (six summer and six winter serum mixes, except for EPA with data from only three summer and three winter mixes). Paired Student t-test were used to compare wummer and winter data and Benjamini-Hochberg correction was applied for multiple comparisons. * indicates BH adjusted p value < 0.05 when comparing seasons, ** for p < 0.01, *** for p < 0.001, NS: non significant. AA:arachidonic acid, ALA: alpha-linolenic acid, DHA: docosahexaenoic acid, DPA: docosapentaenoic acid, EPA: eicosapentaenoic acid, LA: linoleic acid, OA: oleic acid, PA: palmitic acid, SA: stearic acid, SBS: summer bear serum, WBS: winter bear serum Fig. 2 Circulating endocannabinoids concentration in brown bear plasma. Concentration of three major endocannabinoids compounds in bear plasma. Plasma were collected from bears at both winter-hibernating and summer-active time points (Supplementary Table S1). Data are expressed in ng/mL and are represented as mean ± SEM of separate extractions and quantifications (n = 8). Paired Student t-test were used to compare wummer and winter data. * indicates p value < 0.05 when comparing seasons, *** for p < 0.001, NS: non significant. AEA: anandamide, 2-AG: 2-arachidonoylglycérol, eCBs: endocannabinoids, OEA: N-oleoylethanolamine, SBP: summer bear plasma, WBP: winter bear plasma in winter (Supplementary Table S1). Data are presented in Fig. 4 and Supplementary Table S5.

For genes that encode the membrane receptors CB1 and CB2 in muscle tissue, CNR1 mRNA level, but not CNR2, was decreased (0.63-fold) in winter (Fig. 4). Concerning enzymes that catabolize AEA and 2-AG, mRNA level of FAAH was induced (2.3-fold) in winter, but MGLL gene expression did not change. For genes encoding enzymes of the biosynthetic pathway, DAGLA mRNA level was strongly reduced in muscle tissue during winter (0.40-fold), whereas DAGLB mRNA level was increased (1.53-fold). Finally, gene expression of NAPEPLD did not change in muscle (Fig. 4).

Conversely, in adipose tissue (Fig. 5), no significant changes in CNR1 gene expression were reported whereas CNR2 expression was strongly decreased in winter (0.42fold). For gene expression of catabolic enzymes (FAAH and MGLL), did not change in adipose tissue between seasons. Finally, for genes encoding biosynthetic enzymes, mRNA levels of DAGLB and NAPELD were respectively found higher (1.44-fold) and lower (0.75-fold) in winter.

Discussion

Thanks to repeated capture sessions, we were able to gather samples of serum, plasma and tissues from high number of free-living brown bears (Ursus arctos). From the 28 bears included in this study, samples were collected both in February during winter hibernation and in June during summer active period. Due to limited amount of available biological material, the analyses were performed on samples coming from different subsets of the 28 bears. In all but adipose tissue, analyses were performed on winter and summer paired samples (Supplementary Table S1). We examined circulating lipid and ECS compounds in both summer-active and winter-hibernating brown bears to explore the extent to which regulation of the ECS reflects bear hibernation peculiarities, including survival due to lipid oxidation, maintenance of muscle glycolysis, and maintained alertness during dormancy. The seasonal shift we highlighted in serum FAs composition, together with a decrease in tissue AEA and 2-AG, and a three-fold increase in circulating OEA during winter, could contribute to the behavioral and metabolic changes that occur in hibernating bears.

Hibernators experience extended periods of food shortage during hibernation and primarily rely on mobilization of fat stores from white adipose tissue [1]. Accordingly, we found that the concentration of total circulating fatty acids was elevated in hibernating bears, a finding in line with previous studies [5,43]. Considering both the amount and Fig. 3 Endocannabinoids concentration in brown bear muscle and adipose tissue. Concentration of three major endocannabinoids compounds in bear muscle and adipose tissue. Tissues were collected from bears at both winter-hibernating and summer-active time points (Supplementary Table S1). Data are expressed in pg/mg and are represented as mean ± SEM of separate extractions and quantifications (n = 5 for muscle tissue and n = 6 for adipose tissue). Paired Student t-test were used to compare wummer and winter data. *** indicates p value < 0.0001 when comparing seasons, NS: non significant. AEA: anandamide, 2-AG: 2-arachidonoylglycérol, OEA: N-oleoylethanolamine, SBA: summer bear adipose tissue, SBM: summer bear muscle, WBA: winter bear adipose tissue, WBM: winter bear muscle relative proportions of circulating lipids, our results are consistent with changes in serum and plasma lipid profiles during hibernation that have been previously published [5,9,10], notably an enrichment in DHA C22:6 n-3 and depletions in ALA C18:3 n-3 and EPA C20:5 n-3, during winter compared to summer. Whether the depletion in the ALA and EPA precursor species could be directly linked to the observed DHA increase remains to be elucidated.

Here, the DHA serum enrichment that we observed in hibernating bears is actually not coming from dietary FAs intake but rather due to lipid stores mobilization. The health benefits that have been attributed to n-3 PUFAs (e.g. DHA), essentially triggered by DHA dietary intervention studies, could potentially be transposed in the context of hibernation. Indeed, it has already been hypothesized that DHA could be involved in the bear's resistance to muscle atrophy during hibernation [10]. DHA appears to prevent muscle atrophy in fasting mice, and increases muscle glycogen stores [44]. Strikingly, in parallel to DHA serum enrichment, hibernating bears have more than a 3-fold higher glycogen muscle content compared to summer-active animals [10]. In addition to its anti-inflammatory effects, DHA is also known to exert a positive effect on protein balance by decreasing expression of factors involved in protein breakdown [45] and enhancing protein synthesis, notably by promoting mammalian Target Of Rapamycin (mTOR) activation [46].

Concomitantly to serum DHA enrichment, we observed a drop in AA proportion, thus leading to a sharp increase in the DHA/AA ratio. Omega-3/omega-6 ratio is known to have an impact on global health [47], and the balance of this ratio could also impact the endocannabinoid system [48], notably because AA is a precursor of the two main eCBs 2-AG and AEA. Indeed, n-6 PUFAs-enriched diets have been shown to increase the level of 2-AG or AEA in the brain, plasma, and peripheral tissues in nonhibernating animal models [49][50][51][52]. It is noteworthy to mention that, in response to DHA supplementation, an enrichment of this fatty acid in phospholipids of cell membranes occurs in parallel with a decrease in AA content [38,49,53,54]. By remodeling the amount of AAcontaining phospholipids, DHA is able to reduce the synthesis of AEA and 2-AG [49,54]. Further studies on bears, focusing on fatty acid membrane composition in tissues at different time points, will be helpful to characterize the Fig. 4 Fold change in gene expression of target genes involved in endocannabinoids biosynthesis and catabolism in brown bear muscle tissue. Muscle tissues were collected from bears at both winter-hibernating and summer-active time points (Supplementary Table S1), total RNA was extracted and expression levels were measured by RT-qPCR. Data are normalized against TBP mRNA levels and expressed as a fold change relative to the summer condition, represented as mean ± SEM of separate extractions and quantifications (n = 8). Paired Student t-test were used to compare wummer and winter data. * indicates p value < 0.05 when comparing seasons, NS: non significant. CNR1: cannabinoid receptor 1, CNR2: cannabinoid receptor 2, DAGLA: diacylglycerol lipase alpha, DAGLB: diacylglycerol lipase beta, FAAH: fatty acid amide hydrolase, MGLL: monoacylglycerol lipase, NAPEPLD: N-acyl phosphatidylethanolamine phospholipase D, SBM: summer bear muscle, WBM: winter bear muscle remodeling of membrane lipids that could affect the availability of FAs precursors for eCBs biosynthesis. Data on eCBs compounds from experimental short fasting in nonhibernating mammals are very divergent, depending on the tissue considered (e.g. brain or peripheral tissues) and the duration of food deprivation, but tissue levels of eCBs are mainly regulated by the availability of their membrane phospholipid precursors and by the activity of biosynthetic and catabolic enzymes [28,49,55,56].

We hypothesized that drastic reduction in metabolic activity, lack of intake of dietary PUFAs, significant increase in the serum DHA/AA ratio, and perhaps reduction in tissue AA-phospholipids concentration, could lead to a global reduction in ECS tone during the hibernation period. The reduction in ECS tone has already been documented in hibernating marmots [30,41], but not confirmed in large-bodied hibernators.

Comparing active and hibernation states in brown bears, we reported here a decrease in plasma concentration of AEA, and an unexpected 3-fold increase in OEA circulating levels in hibernating bears. In both muscle and adipose tissues, 2-AG and AEA (close to statistical threshold) were found lower in winter, while OEA did not change. Quantification of winter serum eCBs was previously reported in black bears during and around the topor phase, but summer active bears were not investigated [40]. Nutritional status of the captured animals and diet were not specified. These elements strongly limit comparison between the two studies.

Taken together, our data allowed us to make several hypotheses about possible mechanisms by which ECS could contribute to the metabolic and behavioral changes that occur in bears during hibernation. First, considering that AEA and 2-AG CB1 agonists favor food intake and stimulate lipogenesis [25], CB1 signaling is expected to be upregulated during the active summer period in order to promote energy storage, and downregulated during winter hibernation to stimulate lipolysis and FAs oxidation. The tissue concentration drops in 2-AG and AEA observed during winter could be due to a decrease in tissue AAphospholipids concentration, as we hypothesized above. The degradation of AEA could also be increased in muscle tissue during hibernation, as reflected in the higher mRNA levels of FAAH, the main hydrolase that degrades AEA [19,23]. In adipose tissue, lower NAPEPLD mRNA level content during hibernation may support a decrease in AEA synthesis, and ultimately content. The tissue content in 2-AG is decreased in winter with no changes in mRNA levels Fig. 5 Fold change in gene expression of target genes involved in endocannabinoids biosynthesis and catabolism in brown bear adipose tissue. Adipose tissues were collected from bears at both winter-hibernating and summer-active time points (Supplementary Table S1), total RNA was extracted and expression levels were measured by RT-qPCR. Data are normalized against TBP mRNA levels and expressed as a fold change relative to the summer condition, represented as mean ± SEM of separate extractions and quantifications (n = 5 for summer and n = 13 for winter samples). Unpaired Student ttest were used to compare wummer and winter data.* indicates p value < 0.05 when comparing seasons. CNR1: cannabinoid receptor 1, CNR2: cannabinoid receptor 2, DAGLA: diacylglycerol lipase alpha, DAGLB: diacylglycerol lipase beta, FAAH: fatty acid amide hydrolase, MGLL: monoacylglycerol lipase, NAPEPLD: N-acyl phosphatidylethanolamine phospholipase D, SBA: summer bear adipose tissue, WBA: winter bear adipose tissue of the catabolic enzyme MGLL. Furthermore, opposite changes in DAGLA and DAGLB gene expression do not allow to speculate on the biosynthetic/degradation balance. One limitation of our study is that gene expression could not reflect biological activity. Moreover, we only focused on main biosynthetic and catabolic enzymes involved in eCBs metabolism, and investigation on alternative degradation route as endocannabinoid oxygenation by cyclooxygenases and lipoxygenases would bring new insights.

During hibernation, lower 2-AG (and AEA close to statistical threshold) tissue content and the reduction of CNR1 and CNR2 mRNA levels in muscle and adipose tissue, respectively, strongly support reduced ECS tone in both tissues. In non-hibernating mammals, pharmacological inhibition of CB1 leads to a decrease in PDK4 expression [25,57]. PDK4 is a major negative regulator of PDH activity, that in turn regulates the whole body oxidative carbohydrate metabolism. In hibernating bear muscle, recent studies have shown that PDK4 is upregulated compared to summer active state [10,58] and expression of PDK4 during hibernation appear thus to be disconnected from direct regulation by CB1. CB1 receptor antagonism also leads to an increased uptake of glucose in muscle via PI3K signaling [59], and glycolysis appears preserved in bear skeletal muscle during hibernation, as suggested by an overall increase in the protein abundance of all glycolytic enzymes [10]. As proposed by Chazarin et al. and Vella et al., bears still oxidize glucose and produce lactate in skeletal muscle during hibernation [10,60].

Overactivation of the ECS is a hallmark of obesity [61,62], and 2-AG is predominantly found in higher concentration in tissues of obese people [61,63]. Interestingly, in murine models of obesity, gain of adipose tissue often leads to increased fat inflammation [36,37]. Genetic or pharmacological inactivation of CB2 receptor contribute to reduce adipose tissue inflammation, increase insulin sensitivity and skeletal muscle glucose uptake [36,37]. Strikingly, insulin resistance has been described in hibernating bears adipocytes [64]. As bears don't experience health consequences of circannual high body fat storage [65], a reduced CB2 signaling in adipose tissue could dampen adipose tissue inflammation. Lower amounts of 2-AG and AEA could also reduced CB1 signaling in adipose tissue, thus limiting lipogenesis and promoting lipolysis during hibernation in bears, as also suggested for hibernating marmots [30].

OEA is a high-affinity agonist for peroxisome proliferator-activated receptor α (PPARA), regulating food intake and stimulating fat catabolism [38,39,53,66,67]. The eCB-like OEA is generally synthesized in response to dietary oleic acid intake by enterocytes of the small intestine [49,54], and inhibits food intake. It has already been shown in rodents that food deprivation inhibits OEA synthesis in the small intestine, but stimulates its synthesis in liver [38,53,68,69]. Therefore, during bear hibernation, circulating OEA could originate from tissue synthesis (probably hepatic) and be released in the blood flow. The high OEA level that we found in hibernating bears, not triggered by food intake, could participate in a sustained anorexigenic signal during the hibernation state.

Consequences of high levels of circulating OEA have been studied in non-hibernating rodents. Intraperitoneal OEA administration in rats notably impairs locomotor activity, which is supported by a decrease in ambulation, an increase of the time spent in inactivity, and the presence of signs of catalepsy [66,70]. We thus can hypothesize that a higher amount of plasma OEA during bear hibernation can participate in the maintenance of prolonged physical inactivity. It has also been shown that intracerebroventricular injections of OEA promote alertness, with the observation of enhanced dopamine and c-Fos expressions in wakerelated brain areas [71]. Bears are known to stay sensitive to disturbance during hibernation [72][73][74]. High circulating amounts of OEA might thus participate in alertness to external stimuli from the environment in hibernating bears. OEA during winter possibly also favors body fat mobilization for energy needs, with stimulation of FA and glycerol release from adipocytes [38,39]. Finally, a potential role for OEA in the promotion of fasting-induced ketogenesis during hibernation could also be considered, as OEA has been demonstrated to increase 3-hydroxybutyrate production in in vivo rodent models [38,39].

Conclusions

In conclusion, our results show a reduction in ECS tone in hibernating bears and suggest a coordinated downregulation of CB1 and CB2 signaling in skeletal muscle and adipose tissue. As summarized in Fig. 6, these features could favor energy mobilization through lipolysis, and optimization of glucose uptake by skeletal muscles. Despite high fat stores in winter, bears do not exhibit features of ECS overactivation, and decrease in CB2 signaling could dampen adipose tissue inflammation. The observed increase in circulating OEA level may participate in the behavioral and physiological adaptations during bear hibernation state, like maintenance of an anorexigenic signaling pathway, and promotion of lipolysis and fatty acid β-oxidation. We also speculated about OEA involvement in torpor maintenance and in motor activity reduction, as well as a role in conservation of alertness at the level of central nervous system.

Methods

Bear sample collection

A total of 28 free ranging subadult brown bears (Ursus arctos) from Dalarna and Gävleborg counties, Sweden, were included in this study, including 4 bears captured two consecutive years. All samples and data were collected under protocols approved by the Swedish Ethical Committee on Animal Experiment (applications Dnr C3/2016 and Dnr C18/2015), the Swedish Environmental Protection Agency (NV-00741-18), and the Swedish Board of Agriculture . All procedures complied with Swedish laws and regulations.

As described previously [10,75], blood, subcutaneous adipose tissue, and muscle tissue (vastus lateralis) samples were collected at two time points, in February during winter hibernation (W) and in June during summer-active period (S). Blood samples were collected from the jugular vein into 8 ml dry tubes for serum (Vacuette® Z serum Sep Clot Activator, Greiner Bio-One GmbH, Kremsmünster, Austria) or into 10 ml EDTA-coated tubes (BD Vacutai-ner®, FisherScientific, Illkirch, France) for plasma.

The analyses were performed on samples coming from different subsets of bears as described in Supplementary Table S1.

Lipid extraction and analysis

To perform serum lipidomic analysis, serum mixes were prepared as followed: for a given year, 50 μl of summer serum from each bear of the year was pooled to obtain the summer mix. In parallel, 50 μl of winter serum from the same bears was pooled to obtain the winter mix. A total of 6 summer and winter paired mixes were obtained (Supplementary Table S1). Lipids were extracted and analyzed as previously described [76]. After addition of an internal standard (tri-17:0 triacylglycerol), total lipids were extracted twice from bear serum mixes with ethanol/chloroform (1:2, v/v). The organic phases were dried under nitrogen and lipids were transmethylated. Briefly, samples were treated with toluene-methanol (1:1, v/v) and boron trifluoride in methanol (14%). Transmethylation was carried out at 100 °C for 90 min in screw-capped tubes. Then 1.5 mL K 2 CO 3 in 10% water was added and the resulting fatty acid methyl esters were extracted by 2 mL of isooctane and analyzed by gas chromatography (GC) with a HP6890 instrument equipped with a fused silica capillary BPX70 SGE column (60 × 0.25 mm). The vector gas was hydrogen. Temperatures of the Ross injector and the flame ionization detector were set to 230 °C and 250 °C, respectively. Data were expressed in mmol/L for total or individual fatty acids (FAs) concentration or molar percentage of total lipids for individual FAs. Detailed lipidomic results are presented in supplementary Table S2 (serum fatty acid concentrations) and S3 (serum fatty acid relative proportions).

Endocannabinoid quantification

For quantification of circulating endocannabinoids, analysis was performed on 500 μl of plasma collected at the two time points (S and W) from 8 individual animals (see supplementary Table S1). Standard endocannabinoids (eCBs), i.e.-PEA, PEA-d5, OEA, OEA-d4, AEA, AEA-d4, 2AG, and 2AG-d5, were purchased from Cayman (Bertin BioReagent, Saint-Quentin en Yvelines, France). Mass spectrometry quality grade solvents were purchased from Fischer Scientific (Illkirch, France). Tissue samples (adipose and muscle tissues); c.a 100 mg) were crushed in an Omni Bead Ruptor 24 apparatus (Omni International, Kennesaw, USA) with circa twenty 1.4 mm OD zirconium oxide beads (S = 6.95 m/s, T = 30s, C = 3; D = 10s) and 900 μl of methanol/Tris-buffer (50 mM, pH = 8) 1/1 containing 20 ng of PEA-d5, 2 ng OEA-d4, 10 ng AEA-d4, and 20 ng 2AG-d5. Then, each homogenate was added with 2 mL of CHCl 3 / MeOH (1:1, v/v) and 500 μL of Tris (50 mM, pH = 8), vortexed and centrifuged 10 min at 3000 g. The organic layer was recovered and the upper aqueous phase was extracted twice with chloroform (1 mL). Finally, organic phases were pooled and evaporated under vacuum.

Plasma (500 μL) were mixed with 500 μL cold methanol containing 11 ng AEA. After protein precipitation at -20 °C for 2 h, endocannabinoids were extracted with methanol/chloroform (1:1, v/v) (5 ml) and saline (1.25 mL). The organic phase was recovered and the aqueous phase was extracted twice with chloroform (3 mL). Organic phases were finally pooled and evaporated under vacuum.

Dried extracts were solubilized with methanol (200 μL) and centrifuged for 5 min at 20,000 g. Four microliters of the supernatant were injected into a 1200 LC system coupled to a 6460-QqQ MS/MS system equipped with an ESI source (Agilent technologies). Separation was achieved on Zorbax SB-C18 2.1 × 50 mm, 1.8 μm column (Agilent technologies) at a flow rate of 0.4 mL/min, 40 °C, with a linear gradient of (solvent A) water containing 0.1% formic acid and (solvent B) methanol containing 0,1% formic acid as follows: 10% of B for 1 min, up to 85% of B in 8 min, and then 100% B for 4.5 min. Acquisition was performed in positive Selected Reaction Monitoring (SRM) mode (source temperature: 350 °C, nebulizer gas flow rate: 10 L/ min, 40 psi, sheath gas flow 10 L/min, sheath gas temperature 350 °C, capillary 4000 V, nozzle 1000 V).

Transitions used were: 2AG-d5 384.3 → 91.1 (frag 120 V, CE 62 V), 2AG 379.1 → 91 (frag 120 V, CE 62 V), AEA-d4 352.2 → 66.1 (frag 115 V, CE 14 V), AEA 348.2 → 62 (frag 120 V, CE 14 V), OEA-d4 330.2 → 66.1 (frag 120 V, CE 14 V), OEA 326.2 → 62 (frag 115 V, CE 14 V), PEA-d5 305.2 → 62 (frag 124 V, CE 14 V), and PEA 300.2 → 62 (frag 124 V, CE 14 V).

Endocannabinoids quantification in tissues was performed on tissue samples collected at the two time points (S and W) from 5 (muscle tissue) and 6 (adipose tissue) bears (Supplementary Table S1). eCBs from tissues were quantitated according to the isotope dilution method. Results are expressed as pg per mg of wet weight of tissue. eCBs from plasma were quantitated using calibration curves obtained with authentic standards extracted by the same method used for plasma samples. Linear regression was applied for calculations. Results are expressed as ng of endocannabinoid per mL of plasma.

Quantification of mRNAs by real-time RT-PCR

For mRNA quantification using RT-qPCR, total RNAs were obtained from muscle and adipose tissues collected at the two time points (S and W). For the muscle tissue, RNAs were extracted from 8 bears in summer and winter, while for adipose tissue, RNAs were extracted from 5 bears in summer and 13 bears in winter (Supplementary Table S1).

Muscle and adipose tissue total RNA was isolated using the TRIzol reagent (Invitrogen, Courtaboeuf, France) according to the manufacturer's instructions. First-strand cDNAs were synthesized from 1 μg of total RNA using the PrimeScript RT kit (Ozyme, saint quentin en Yveline, France) with a mixture of random hexamers and oligo(dT) primers, and treated with 60 units of RnaseH (Ozyme). Real-time PCR assays were performed with Rotor-Gene 6000 (Qiagen, Courtaboeuf, France). The primers and real-time PCR assay conditions are listed in supplementary Table S4. The results were normalized by using TBP (TATA box binding protein) mRNA concentration, measured as reference gene in each sample.

Statistical analysis

Statistical analysis was performed using the R software environment v3.0.2 [77]. For each set of values, distribution of the data was tested using the Shapiro-Wilk normality test, and using the p = 0.01 threshold normal distribution was considered in all cases. Differences between summer and winter data were tested using paired Student t-test for lipidomic, endocannabinoid quantification in plasma and tissues, and mRNA level in muscle tissue. For mRNA level in adipose tissue, differences between summer and winter data were tested using unpaired Student t-test. For multiple comparison (lipidomic data), the Benjamini-Hochberg correction using the p. mitochondries sont présentes dans les myofibres, subsarcolemmales et les intermyofibrillaires [10]. Le maintien d'un réseau mitochondrial fonctionnel dans le muscle est fondamental pour soutenir les demandes métaboliques imposées par la contraction. L'intégrité et la fonction mitochondriale sont hautement régulées par des systèmes de contrôle qualité (la biogenèse, la dynamique et la dégradation mitochondriales) afin de maintenir l'homéostasie. Cependant, un dysfonctionnement mitochondrial peut résulter en plusieurs maladies musculaires humaines appelées myopathies mitochondriales [19].

Réservoir d'acide aminés.

Un des rôles majeurs du muscle squelettique est d'être le principal réservoir d'acides aminés (AA) du corps. Les AA musculaires peuvent être mobilisés en l'absence d'un apport nutritionnel adéquat pour assurer de nombreuses fonctions au niveau corps entier [30]. Par exemple, les AA libérés par les muscles servent de précurseurs pour le maintien du niveau de glucose sanguin grâce à la gluconéogenèse hépatique pendant le jeûne. Cependant, la réduction de la masse musculaire compromet la capacité de l'organisme à répondre à différents stresses en raison d'une altération des interactions entre les muscles et les organes.

Balance protéique musculaire. Chez les organismes adultes, la régulation de la masse musculaire résulte de la croissance des myofibres existantes par le biais de signalisations intracellulaires qui contrôlent la balance protéique [38]. L'équilibre entre les taux de synthèse des protéines musculaires (SPM) et de dégradation des protéines musculaires (DPM) détermine le contenu en protéines et donc l'homéostasie musculaire. La SPM et la DPM sont sensibles à de nombreux facteurs, notamment l'état nutritionnel, l'équilibre hormonal, l'activité physique ou les maladies. La diminution de la taille des muscles chez l'adulte, c'est-à-dire l'atrophie musculaire, résulte d'une balance protéique négative, tandis que l'augmentation de la taille des muscles, c'est-à-dire l'hypertrophie, résulte d'une balance positive.

Les AA apportés par une alimentation adaptée agissent comme des substrats et des signaux et sont essentiels pour induire la SPM. L'un des acteurs les plus reconnus de la SPM est le complexe mTORC1 qui joue un rôle central dans la régulation de la synthèse des protéines et de la biogenèse ribosomale [48]. Par exemple, la délétion spécifique de mTOR (la kinase du complexe) dans les muscles de souris induit une myopathie sévère entraînant une mort prématurée.

Concernant la DPM, les principaux systèmes qui y contribuent sont les systèmes autophagie-lysosome (ALS) et ubiquitine-protéasome (UPS). L'ALS implique la formation du phagophore, qui englobe des composés intracellulaires environnants, tels que des protéines endommagées, et qui fusionne avec les lysosomes pour entraîner la dégradation du contenu protéique et le recyclage des AA [55]. Dans des conditions normales, l'ALS empêche principalement l'accumulation d'organites endommagés et de protéines mal repliées. En réponse au stress, comme le jeûne, l'ALS agit principalement comme un mécanisme pro-survie dans le muscle, fournissant des substrats métaboliques [55]. Alors qu'un flux d'autophagie trop important contribue à l'atrophie musculaire, l'inhibition de l'ALS entraîne également une atrophie musculaire [62]. Dans les muscles sains, les mitochondries endommagées et dépolarisées sont sélectivement éliminées par le processus de mitophagie, une forme spécifique d'autophagie. Des études ont démontré que la mitophagie est essentielle au maintien de l'homéostasie du muscle squelettique [10]. Il existe de nombreuses preuves que des altérations de la mitophagie sont présentes dans le muscle au cours de nombreuses conditions cataboliques conduisant à l'atrophie musculaire [10]. Le système UPS joue également un rôle fondamental dans la physiologie du muscle, notamment en dégradant les protéines myofibrillaires [70]. La plupart des protéines subissent une dégradation en étant ciblées par le protéasome 26S grâce à la fixation covalente d'une chaîne d'ubiquitine. Ces protéines marquées sont ensuite reconnues par le protéasome 26S, qui initie un processus de dégradation dépendant de l'ATP. Grâce à ce mécanisme, l'UPS dégrade spécifiquement le substrat. L'inhibition de l'activité du protéasome dans le muscle est associée à un défaut de croissance musculaire et à une diminution de la durée de vie chez les rongeurs [70]. 10.6.1.2 L'atrophie musculaire Causes. La perte de masse et de force musculaire est appelée atrophie musculaire. Les causes peuvent être diverses, par exemple congénitales ou génétiques, ou acquises suite à certaines conditions physiopathologiques [75] (Figure 51). Les conditions pathologiques qui conduisent à l'atrophie musculaire comprennent la cachexie cancéreuse, les troubles pulmonaires obstructifs chroniques, le diabète et l'obésité, ou encore des conditions associées à l'anorexie ou à la malnutrition [75] (Figure 51). L'inactivité physique entraîne également une fonte musculaire, comme dans le cas de fractures, d'immobilisation et d'alitement prolongé et même chez les personnes ayant un mode de vie sédentaire, comme observé lors du confinement de la COVID-19 [75] (Figure 51). L'atrophie musculaire résulte d'un déséquilibre entre la SPM et la DPM, en faveur de la DPM [75] (Figure 51). Au cours de ma thèse, je me suis principalement intéressée à l'atrophie musculaire induite par l'inactivité physique.

Conséquences. Le muscle est un organe majeur du métabolisme du glucose, de fait l'atrophie musculaire est étroitement liée à la résistance à l'insuline et au syndrome métabolique. De plus, l'atrophie musculaire limite les activités quotidiennes, réduit la qualité de vie et prolonge les temps de convalescence après une maladie tout en augmentant la morbidité et la mortalité (Figure 51). Compte tenu de ses conséquences néfastes, de l'augmentation de la sédentarité et de l'allongement de l'espérance de vie dans le monde, la fonte musculaire touche des millions de personnes et reste un fardeau social et économique majeur. Actuellement, les stratégies thérapeutiques pour limiter l'atrophie musculaire comprennent essentiellement l'exercice physique et des stratégies nutritionnelles, qui ne sont cependant pas applicables à tous (par exemple aux patients immobilisés ou en unité de soins intensifs) [75]. A ce jour, aucun médicament n'a été approuvé pour un usage clinique et aucun remède efficace contre l'atrophie musculaire n'a été découvert. Il est donc nécessaire de mieux comprendre les mécanismes sous-jacents et de découvrir de nouvelles cibles thérapeutiques potentielles. Notre compréhension s'est considérablement améliorée au cours des dernières décennies, principalement grâce à l'utilisation de modèles de rongeurs de laboratoire.

Les acteurs moléculaires. La SPM et la DPM sont influencés par un large éventail d'acteurs moléculaires extra et intracellulaires. Les stimuli extracellulaires comprennent des facteurs inflammatoires tels que des cytokines ou des facteurs endocriniens tels que des facteurs de croissance, qui activent diverses voies intracellulaires. Ces acteurs intracellulaires interconnectés contribuent à la régulation de l'équilibre protéique musculaire en travaillant en synergie ou en opposition, favorisant soit l'anabolisme, soit le catabolisme [38]. Dans le contexte de l'atrophie musculaire, le dérèglement d'un ou de plusieurs de ces acteurs entraîne une atténuation de la signalisation anabolique en faveur du catabolisme, ce qui conduit soit à l'inhibition de la SPM, soit à la suractivation des systèmes UPS et ALS, soit aux deux [38]. Les atrogènes sont désignés comme un ensemble de gènes dont l'expression La manière dont ATF4 facilite des adaptations cellulaires aussi diverses, allant de l'anabolisme à l'arrêt de la croissance, est une question importante et non résolue. Une des possibilités pourrait venir des différents hétérodimères d'ATF4 ou différentes combinaisons d'hétérodimères qui médieraient les différents effets de la signalisation.

Le role de l'ISR dans l'autophagie et l'homéostasie mitochondriale. Comme indiqué plus haut, l'autophagie et le contrôle de la qualité mitochondriale sont des processus cellulaires essentiels à l'homéostasie musculaire, et une déficience de l'un ou l'autre est associée à la l'atrophie musculaire [13]. L'ISR est impliquée dans ces deux processus dans un large éventail de tissus et de cellules. Lors de divers stress, ATF4 se lie au promoteur spécifique de gènes impliqués dans l'autophagie pour promouvoir une réponse pro-survie ou une réponse pro-létale, de manière PERK-ou GCN2dépendante (Figure 55). La signalisation de l'ISR est également essentielle au contrôle de la qualité des mitochondries, par le biais de UPRmt 60 Métabolisme énergétique musculaire. La glycolyse est préservée dans les muscles des ours hibernants [310]. Cela pourrait aider à maintenir la fonctionnalité des muscles dans des situations inattendues, comme une sortie urgente de la tanière qui nécessiterait une augmentation rapide de la production

Résumé

L'atrophie musculaire impacte des millions de personnes à travers le monde, inlcuant des personnes âgées, des personnes atteintes de maladies ou encore des personnes immobilisées pendant de longues périodes. La perte de masse musculaire conduit à un déclin de l'autonomie, favorise l'apparition de maladies, augmente la résistance aux traitements mis en place, et est associée à une augmentation de la mortalité. De ce fait, l'atrophie musculaire constitue un problème majeur de santé publique. Enormément de mécanismes biomoléculaires ont été documentés pouvant expliquer l'apparition de l'atrophie musculaire, principalement grâce à l'utilisation de modèle de rongeurs de laboratoire. Pourtant, aucun traitement n'est réellement efficace et/ou adaptable pour tous aujourd'hui. L'objectif principal de cette thèse était de trouver de nouveaux mécanismes sous-jacents qui pourraient devenir des cibles thérapeutiques pour combattre l'atrophie musculaire chez l'Homme. Nous avons choisi une approche basée sur le biomimétisme. Notre stratégie a été (1) de réaliser une étude de physiologie comparée entre le modèle de l'ours brun naturellement résistant à l'atrophie pendant hibernation et la souris suspendue sensible à l'atrophie musculaire, (2) d'étudier le rôle des voies de signalisations ATF4 et TGF-β/BMP dans ces deux modèles, et enfin (3) d'initier des études sur des cellules musculaires humaines pour valider les hypothèses issues des deux premières études. Dans notre première étude, la stratégie a été d'identifier les gènes différentiellement régulés dans les muscles de l'ours brun entre la période d'hibernation et la période d'activité. Ensuite, nous les avons comparés à ceux différentiellement régulés dans les muscles de la souris suspendue par rapport à la souris contrôle. Nous avons montré que la concomitance de l'inhibition de la signalisation TGF- et de l'induction de la signalisation BMP semblait être cruciale pour le maintien de la masse musculaire en condition d'inactivité physique prolongée. Dans notre deuxième étude, nous avons montré que l'induction de la voie de signalisation d'ATF4 dans le muscle était découplée de l'atrophie musculaire chez la souris saine ou soumise à une situation d'inactivité physique lorsqu'elles étaient préalablement traitées par la molécule d'halofuginone, et également chez l'ours hibernant. Dans ces 3 situations, le maintien de la masse musculaire était associé à la fois à l'induction de la signalisation d'ATF4 et de BMP et à l'inhibition de TGF-β. Enfin, des résultats préliminaires obtenus en cultivant des cellules musculaires humaines avec du sérum d'ours brun hibernant suggèrent la présence d'un composé actif circulant pouvant reproduire certaines caractéristiques observées dans le muscle de l'ours brun hibernant résistant à l'atrophie. En conclusion, ces travaux ouvrent de nombreuses perspectives dans la modulation de la balance des voies de signalisation TGF- et BMP dans des situations d'inactivité physique prolongée. De plus, ils ouvrent de nouvelles recherches sur l'identification de composés actifs dans le sérum de l'ours pouvant être utilisables en clinique humaine afin de limiter ou prévenir l'apparition d'atrophie musculaire lors de l'immobilisation ou dans d'autres conditions physiopathologiques.

Figure 1 .

 1 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 2 .

 2 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 3 .

 3 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 4 .

 4 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 5 .

 5 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 6 .

 6 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 7 .

 7 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 8 .Figure 9 .

 89 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 10 .

 10 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 11 .

 11 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 12 .

 12 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 13 .

 13 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 14 .

 14 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 15 .

 15 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 16 .

 16 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 17 .

 17 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 18 .

 18 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 19 .

 19 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 20 .

 20 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 21 .Figure 22 .

 2122 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 23 .Figure 24 .

 2324 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ... Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. ....................................

Figure 26 . 1 Figure 27 .

 26127 Figure 1. Skeletal muscle organisation and contractile apparatus structure* 1 . ...................................Figure 2. Myokinome overview of muscle-organ crosstalk*. ...............................................................Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk*. .........................................Figure 4. Muscle protein balance in physiological conditions*. ...........................................................Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects*. ...................Figure 6. Autophagy-lysosomal system*...............................................................................................Figure 7. Ubiquitin-proteasomal system*. ............................................................................................Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy*. ...Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA)*. ............................................................................... Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis. ..........................................................................................................................................Figure 11. TGF-β superfamily organisation and signal transduction*. .................................................Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes*. .........Figure 13. TGF-β signalling involvement in muscle atrophy*. ..............................................................Figure 14. BMP signalling involvement in muscle hypertrophy*. .........................................................Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions*. ..................Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis*. .......Figure 17. The Integrated Stress Response pathway organisation and signal transduction*. .............Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition.* ...............................................................................................................................................................Figure 19. The Integrated Stress Response involvement in muscle homeostasis*. .............................Figure 20. Pictures of hibernating brown bear dens in North Sweden. ...............................................Figure 21. Main physiological characteristics of hibernating bears. ..................................................... Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation*. ..............................................................................................................................Figure 23. Protein content in bear muscles in summer, early denning and late denning. ................... Figure 24. Protein turnover in bear muscles in summer, early denning and late denning. ................. Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation*. .......................................................................................Figure 26. Winter bear serum promotes hypertrophy in human muscle cells. .................................... Study 1 Figure 27. Schema of the experimental strategy of study 1*. ..............................................................Figure 28. Hindlimb suspension model in laboratory mice. .................................................................Figure 29. Pictures from the collection of free-ranging bears samples in the forest of Northern Sweden as part of the Brown Bear Research Project. ........................................................................................Figure 30. Graphical abstract of the study 1*. ......................................................................................Figure 31. Detailed enriched terms from the biological processes "Protein metabolism" and "RNA metabolism" from the differentially expressed genes in Cussonneau et al., 2021. ............................. Figure 32. Western blots of total and phosphorylated SMAD1/5, and SMAD4 in CCL136 human muscle cells expressing an inactive BMP receptor. ......................................................................................... 101

Figure 28 .

 28 Figure 27. Schema of the experimental strategy of study 1*. .............................................................. Figure 28. Hindlimb suspension model in laboratory mice. .................................................................Figure 29. Pictures from the collection of free-ranging bears samples in the forest of Northern Sweden as part of the Brown Bear Research Project. ........................................................................................ Figure 30. Graphical abstract of the study 1*. ......................................................................................Figure 31. Detailed enriched terms from the biological processes "Protein metabolism" and "RNA metabolism" from the differentially expressed genes in Cussonneau et al., 2021. ............................. Figure 32. Western blots of total and phosphorylated SMAD1/5, and SMAD4 in CCL136 human muscle cells expressing an inactive BMP receptor. ......................................................................................... 101

Figure 29 .

 29 Figure 27. Schema of the experimental strategy of study 1*. .............................................................. Figure 28. Hindlimb suspension model in laboratory mice. .................................................................Figure 29. Pictures from the collection of free-ranging bears samples in the forest of Northern Sweden as part of the Brown Bear Research Project. ........................................................................................ Figure 30. Graphical abstract of the study 1*. ......................................................................................Figure 31. Detailed enriched terms from the biological processes "Protein metabolism" and "RNA metabolism" from the differentially expressed genes in Cussonneau et al., 2021. ............................. Figure 32. Western blots of total and phosphorylated SMAD1/5, and SMAD4 in CCL136 human muscle cells expressing an inactive BMP receptor. ......................................................................................... 101

Figure 30 .

 30 Figure 27. Schema of the experimental strategy of study 1*. .............................................................. Figure 28. Hindlimb suspension model in laboratory mice. .................................................................Figure 29. Pictures from the collection of free-ranging bears samples in the forest of Northern Sweden as part of the Brown Bear Research Project. ........................................................................................ Figure 30. Graphical abstract of the study 1*. ......................................................................................Figure 31. Detailed enriched terms from the biological processes "Protein metabolism" and "RNA metabolism" from the differentially expressed genes in Cussonneau et al., 2021. ............................. Figure 32. Western blots of total and phosphorylated SMAD1/5, and SMAD4 in CCL136 human muscle cells expressing an inactive BMP receptor. ......................................................................................... 101

Figure 31 .Figure 32 .Figure 33 .Figure 34 .Figure 35 .

 3132333435 Figure 27. Schema of the experimental strategy of study 1*. .............................................................. Figure 28. Hindlimb suspension model in laboratory mice. .................................................................Figure 29. Pictures from the collection of free-ranging bears samples in the forest of Northern Sweden as part of the Brown Bear Research Project. ........................................................................................ Figure 30. Graphical abstract of the study 1*. ......................................................................................Figure 31. Detailed enriched terms from the biological processes "Protein metabolism" and "RNA metabolism" from the differentially expressed genes in Cussonneau et al., 2021. ............................. Figure 32. Western blots of total and phosphorylated SMAD1/5, and SMAD4 in CCL136 human muscle cells expressing an inactive BMP receptor. ......................................................................................... 101

Figure 36 .Figure 37 . 2 Figure 38 .Figure 39 .Figure 40 .Figure 41 .Figure 42 .

 363723839404142 Figure 33. Co-immunoprecipitation of SMAD4 with SMAD1/5 or SMAD2/3 in bear muscles. .......... Figure 34. SMAD4 linker protein sequences in different species. ...................................................... Figure 35. SMAD4 degradation involving MAPK, GSK3 and β-TrCP proteins*. ..................................Figure 36. Hypothetical schema to explain the denervation-induced muscle atrophy resistance in the hibernating brown bears muscles*. .................................................................................................... Figure 37. Hypothetical schema of the origin of muscle transcriptomic changes of TGF-β superfamily*. ............................................................................................................................................................. Study 2 Figure 38. Schema of the experimental strategy of study 2*. ............................................................ Figure 39. Graphical abstract of the study 2*. .................................................................................... Figure 40. Chemical structures of febrifugine and halofuginone. ...................................................... Figure 41. Effect of halofuginone treatment prior to hindlimb suspension on extracellular matrix components expression in gastrocnemius muscle in mice. ................................................................ Figure 42. Halofuginone treatment for 2 weeks prior to hindlimb suspension mitigates atrophy in gastrocnemius muscle in mice. ........................................................................................................... Figure 43. Effect of halofuginone treatment prior to hindlimb suspension in soleus muscle. ...........Figure 44. Effect of halofuginone treatment prior to hindlimb suspension on autophagy-lysosomal system in gastrocnemius muscle. ........................................................................................................Figure 45. ATF4 signalling regulation in atrophy-resistant muscle of the hibernating bear...............Figure 46. Worldwide geographic repartition of (A) Hydrangea macrophylla and (B) brown bear living area (Ursus arctos). .............................................................................................................................Figure 47. Hypothetical halofuginone and halofuginone-like molecular mechanisms in skeletal muscle*. ..............................................................................................................................................

Figure 43 .

 43 Figure 38. Schema of the experimental strategy of study 2*. ............................................................ Figure 39. Graphical abstract of the study 2*. .................................................................................... Figure 40. Chemical structures of febrifugine and halofuginone. ......................................................Figure 41. Effect of halofuginone treatment prior to hindlimb suspension on extracellular matrix components expression in gastrocnemius muscle in mice. ................................................................ Figure 42. Halofuginone treatment for 2 weeks prior to hindlimb suspension mitigates atrophy in gastrocnemius muscle in mice. ........................................................................................................... Figure 43. Effect of halofuginone treatment prior to hindlimb suspension in soleus muscle. ...........Figure 44. Effect of halofuginone treatment prior to hindlimb suspension on autophagy-lysosomal system in gastrocnemius muscle. ........................................................................................................ Figure 45. ATF4 signalling regulation in atrophy-resistant muscle of the hibernating bear............... Figure 46. Worldwide geographic repartition of (A) Hydrangea macrophylla and (B) brown bear living area (Ursus arctos). .............................................................................................................................Figure 47. Hypothetical halofuginone and halofuginone-like molecular mechanisms in skeletal muscle*. ..............................................................................................................................................

Figure 44 .

 44 Figure 38. Schema of the experimental strategy of study 2*. ............................................................ Figure 39. Graphical abstract of the study 2*. .................................................................................... Figure 40. Chemical structures of febrifugine and halofuginone. ......................................................Figure 41. Effect of halofuginone treatment prior to hindlimb suspension on extracellular matrix components expression in gastrocnemius muscle in mice. ................................................................ Figure 42. Halofuginone treatment for 2 weeks prior to hindlimb suspension mitigates atrophy in gastrocnemius muscle in mice. ........................................................................................................... Figure 43. Effect of halofuginone treatment prior to hindlimb suspension in soleus muscle. ...........Figure 44. Effect of halofuginone treatment prior to hindlimb suspension on autophagy-lysosomal system in gastrocnemius muscle. ........................................................................................................ Figure 45. ATF4 signalling regulation in atrophy-resistant muscle of the hibernating bear............... Figure 46. Worldwide geographic repartition of (A) Hydrangea macrophylla and (B) brown bear living area (Ursus arctos). .............................................................................................................................Figure 47. Hypothetical halofuginone and halofuginone-like molecular mechanisms in skeletal muscle*. ..............................................................................................................................................

Figure 45 .Figure 46 .

 4546 Figure 38. Schema of the experimental strategy of study 2*. ............................................................ Figure 39. Graphical abstract of the study 2*. .................................................................................... Figure 40. Chemical structures of febrifugine and halofuginone. ......................................................Figure 41. Effect of halofuginone treatment prior to hindlimb suspension on extracellular matrix components expression in gastrocnemius muscle in mice. ................................................................ Figure 42. Halofuginone treatment for 2 weeks prior to hindlimb suspension mitigates atrophy in gastrocnemius muscle in mice. ........................................................................................................... Figure 43. Effect of halofuginone treatment prior to hindlimb suspension in soleus muscle. ...........Figure 44. Effect of halofuginone treatment prior to hindlimb suspension on autophagy-lysosomal system in gastrocnemius muscle. ........................................................................................................ Figure 45. ATF4 signalling regulation in atrophy-resistant muscle of the hibernating bear............... Figure 46. Worldwide geographic repartition of (A) Hydrangea macrophylla and (B) brown bear living area (Ursus arctos). .............................................................................................................................Figure 47. Hypothetical halofuginone and halofuginone-like molecular mechanisms in skeletal muscle*. ..............................................................................................................................................

Figure 47 . 3 Figure 48 .Figure 49 .Figure 50 .

 473484950 Figure 38. Schema of the experimental strategy of study 2*. ............................................................ Figure 39. Graphical abstract of the study 2*. .................................................................................... Figure 40. Chemical structures of febrifugine and halofuginone. ...................................................... Figure 41. Effect of halofuginone treatment prior to hindlimb suspension on extracellular matrix components expression in gastrocnemius muscle in mice. ................................................................ Figure 42. Halofuginone treatment for 2 weeks prior to hindlimb suspension mitigates atrophy in gastrocnemius muscle in mice. ...........................................................................................................Figure 43. Effect of halofuginone treatment prior to hindlimb suspension in soleus muscle. ...........Figure 44. Effect of halofuginone treatment prior to hindlimb suspension on autophagy-lysosomal system in gastrocnemius muscle. ........................................................................................................ Figure 45. ATF4 signalling regulation in atrophy-resistant muscle of the hibernating bear............... Figure 46. Worldwide geographic repartition of (A) Hydrangea macrophylla and (B) brown bear living area (Ursus arctos). .............................................................................................................................Figure 47. Hypothetical halofuginone and halofuginone-like molecular mechanisms in skeletal muscle*. .............................................................................................................................................. Study 3 Figure 48. Schema of the experimental strategy of study 3*. ............................................................Figure 49. Human myotubes cultivated with winter bear serum induces similar transcriptional changes than those occurring in hibernating bears muscles. ........................................................................... Figure 50. Winter bear serum mimics in human muscle cells what occurs naturally in the muscles of hibernating bears. ...............................................................................................................................

Figure 1 .

 1 Figure 1. Skeletal muscle organisation and contractile apparatus structure.

Figure 2 .

 2 Figure 2. Myokinome overview of muscle-organ crosstalk.

Figure 3 .

 3 Figure 3. Myokinome overview of muscle autocrine/paracrine crosstalk.

Figure 4 .

 4 Figure 4. Muscle protein balance in physiological conditions.

Figure 5 .

 5 Figure 5. mTORC1 and mTORC2 intracellular organisation and their biological effects.

Figure 6 .

 6 Figure 6. Autophagy-lysosomal system.

Figure 7 .

 7 Figure 7. Ubiquitin-proteasomal system.

Figure 8 .

 8 Figure 8. Muscle protein imbalance in pathophysiological conditions leading to muscle atrophy.

Figure 9 .

 9 Figure 9. Histopathological characteristics of healthy muscle (A) versus atrophied muscle (B) with a decrease of myofiber cross sectional area (CSA).

  [100]. FBXO32/Atrogin-1 is involved in the degradation of ribosomal proteins and translation initiation factors, as well as several other proteins such as myoblast determination protein (MyoD), desmin, and vimentin (i.e. the intermediate filament in muscle). Thus, overexpression of FBXO32/Atrogin-1 in the context of muscle atrophy may reduce MPS and regeneration and thus leads to muscle wasting [101]. Atrogenes are markers of atrophy, but their involvement as active inducers of atrophy remains an open question. Furthermore, whether rodent atrogenes are shared with humans remains to be established for most of them. Drugs targeting. Several actors in this interconnected network have been proven effective when targeted to limit or counteract skeletal muscle atrophy in rodent models. For instance, targeting myostatin ligand or its activin receptor type 2B (TGF-β signalling) has shown beneficial effects in preserving muscle mass in different catabolic conditions (see section below 4.2.2.2). Moreover, the stimulation of β2-adrenoceptors prevents or even reverses muscle wasting and weakness in several 30 catabolic conditions, including cancer cachexia [102], ageing [103] and muscular dystrophies [104]. Yet, so far, no effective drug has been used in the clinical practice.

Figure 10 .

 10 Figure 10. Overview of anabolic and catabolic signalling pathways involved in muscle protein homeostasis (from Peris-Moreno et al., 2021, seeAppendix 10.4 [38]).

Figure 11 .

 11 Figure 11. TGF-β superfamily organisation and signal transduction.

  ribosylation and linker-domain phosphorylation, all of which changing the fate of the intracellular response [105]. TGF-β superfamily is a master regulator of adult muscle mass with (1) TGF-β signalling as a negative regulator and (2) BMP signalling as a positive regulator [110,111].

Figure 12 .

 12 Figure 12. TGF-β superfamily regulation by E3-ubiquitin ligase and deubiquitinase enzymes. Green and blue circles respectively represent deubiquitinase and E3-ubiquitin ligase enzymes. The schema is adapted from Cussonneau et al., 2021. Full and dotted lines respectively represent the SMAD and non-SMAD signalling. SBE: SMAD Binding Element.

Figure 13 .

 13 Figure 13. TGF-β signalling involvement in muscle atrophy.

Figure 14 .

 14 Figure 14. BMP signalling involvement in muscle hypertrophy.

Figure 15 .

 15 Figure 15. SMAD4 recruitment in muscle in basal (A) or muscle wasting (B) conditions.

  TRAF6 a pro-atrophic actor. TRAF6 mediates the activation of p38 and induces the expression of the atrogenes Trim63/MuRF1 and Fbxo32/Atrogin-1, as well as autophagic-related actors in atrophying muscles during denervation and starvation in mice [176-178] In addition, TRAF6 protein levels increase in muscles of gastric cancer patients [179]. Conversely, Traf6 deletion suppresses the increase expression of Fbxo32/Atrogin-1 and Trim63/MuRF1, improves AKT phosphorylation, and limits muscle atrophy in mice during ageing, starvation, denervation, cancer cachexia or dexamethasone treatment [176-179]. A dual role for TAK1. TAK1-p38 signalling is activated under activin A treatment in mice myotubes and in vivo ending up by up-regulation of Fbxo32/Atrogin-1 and muscle atrophy. Interestingly, the catabolic effect of activin A was abolished by p38 inhibitor administration [180]. Moreover, muscles damages were alleviated through pharmacologic inhibition of TGF-β1-TAK1 axis by the neuroprotective molecule catalpol in a model of Duchenne muscular dystrophy [181].

Figure 16 .

 16 Figure 16. Non-SMAD TGF-β/BMP signalling and its dual involvement in muscle homeostasis.

Figure 17 .

 17 Figure 17. The Integrated Stress Response pathway organisation and signal transduction.

Figure 18 .

 18 Figure 18. ATF4 mRNA sequence and its translation upon (A) basal condition or (B) stress condition. uORF: upstream open reading frame; CDS: coding sequence.

Figure 19 .

 19 Figure 19. The Integrated Stress Response involvement in muscle homeostasis.

  MR and Tb. Hibernating bears show a 75-85% decline in MR and their Tb only decreases by a few degrees Celsius compared to the values of the active summer season, remaining around 32-33°C [276-280] (Figure 21). Thermoregulatory mechanisms could explain the maintenance of a relatively high Tb, but also body insulation due to high fur coverage, accumulation of subcutaneous fat, and also den

Figure 20 .

 20 Figure 20. Pictures of hibernating brown bear dens in North Sweden.

Figure 21 .

 21 Figure 21. Main physiological characteristics of hibernating bears. Average of the daily mean values for ambient temperature (A), bear body temperature (B), heart rate (C) and activity level in accelerometery units (D) for 14 individual free-ranging brown bears in central Sweden collected over 3 years. The X-axis indicates the time of year. Green vertical bars indicate the den entry and exit periods (fromEvans et al., 2016 [279]).

Figure 22 .

 22 Figure 22. Overview of the spectacular characteristics of bears resistance to physiological damage during hibernation.

Figure 23 .

 23 Figure 23. Protein content in bear muscles in summer, early denning and late denning (fromLohuis et al. -2007 [301]).

Figure 24 .

 24 Figure 24. Protein turnover in bear muscles in summer, early denning and late denning (fromLohuis et al. -2007 [301]).

Figure 25 .

 25 Figure 25. A complex and non-exhaustive overview of the mechanisms identified in bears to save muscle protein content during hibernation.

  maintenance of muscle mass during hibernation in bears involves one or more circulating factors. The identification of these factors will undoubtedly open up a new field of study that will lead to new solutions for preventing and/or reversing muscle atrophy in humans.

4. 4 . 6

 46 The ISR and TGF-β superfamily signalling regulation in hibernating mammal muscles Very few papers have explored the regulation of ISR or TGF-β/BMP signalling in the skeletal muscle of hibernating mammals and only one in hibernating bears. TGF/BMP signalling. Studies have shown that (1) myostatin protein expression does not change during early or late torpor in muscles of thirteen-lined ground squirrels (Spermophilus tridecemlineatus), but

Figure 26 .

 26 Figure 26. Winter bear serum promotes hypertrophy in human muscle cells.

Study 1

 1 Concurrent BMP Signaling Maintenance and TGF-β Signaling Inhibition Is a Hallmark of Natural Resistance to Muscle Atrophy in the Hibernating Bear (published paper)

6 .

 6 Study 1: Concurrent BMP Signaling Maintenance and TGF-β Signaling Inhibition Is a Hallmark of Natural Resistance to Muscle Atrophy in the Hibernating Bear 6.1 Objective and strategy

Figure 27 .

 27 Figure 27. Schema of the experimental strategy of study 1.

Figure 28 .

 28 Figure 28. Hindlimb suspension model in laboratory mice.

Figure 29 .

 29 Figure 29. Pictures from the collection of free-ranging bears samples in the forest of Northern Sweden as part of the Brown Bear Research Project.

  , and the 10 top-score enrichment transcription factors regulating the DEGs are shown in Figure1d. The heat map representing the expression changes of the TGF-β/BMP gene sets in bear versus mouse muscles was made using the Pheatmap package (R 1.4.1106, University of Tartu, Tartu, Estonia). Briefly, the gene hierarchical clustering is based on Euclidean distance calculated from the log2FC value (Winter/Summer and Unloaded/Control for bear and mouse muscles, respectively).

Figure 1 .

 1 Figure 1. Deep changes in brown bear muscle transcriptome during hibernation. (a) Heatmap from brown bear muscle (vastus lateralis) transcripts (n = 6 bears/season, the same individuals were sampled and analyzed in summer and winter); red indicates high and green indicates low expression level of the 13531 genes. (b) Graph representing the 10 top-score of significantly enriched

Figure 2 .

 2 Figure 2. Deep transcriptomic reprogramming of TGF-β and BMP pathways in muscle during brown bear hibernation. Scheme showing brown bear vastus lateralis muscle transcripts involved in TGF-β and BMP signaling and depicting (i) their relationships [55-57] and (ii) the difference in their expression levels between hibernation and activity periods. Red and green boxes indicate, respectively up-and down-regulated genes during hibernation compared to the summer season, and white boxes indicate unchanged genes. Target genes of the TGF and BMP pathways are indicated in italic and are in green when down-regulated, in red when up-regulated, and in black when unchanged. Dashed lines show the SMAD-independent pathway and full lines the canonical signaling pathway. Arrows indicate activation, and ⊥ bars indicate inhibition. (n = 6 bears/season, the same individuals were sampled and analyzed in summer and winter, padj < |0.05|; TablesS2 and S3). ECAg: Extracellular Agonist, ECAn: Extracellular Antagonist, L: Ligand, Co-R: Co-Receptor, RII: Receptor type II, RI: Receptor type I, TA: Transcriptional Activator, TR: Transcriptional Repressor, SBEs: SMAD Binding Element. Created with BioRender.com.

Figure 3 .

 3 Figure 3. A transcriptomic reprogramming of UPS components involved in TGF-β and BMP regulation prevails in muscle during brown bear hibernation. Scheme showing the E3s/DUBs enzymes regulation of brown bear vastus lateralis muscle transcripts involved in TGF-β and BMP signaling pathways and depicting (i) their relationships [12,60] and (ii) the difference in their expression levels between hibernation and activity periods. Red and green boxes indicate, respectively up-downregulated genes during hibernation compared to the summer season, and white boxes indicate unchanged genes. Dashed lines show the SMAD-independent pathway and full lines the canonical signaling. Arrows indicate activation and bars inhibition. (n = 6 bears/season, the same individuals were sampled and analyzed in summer and winter, padj < |0.05|; TableS4). SBEs: SMAD Binding Elements. Created with BioRender.com.

Figure 4 .

 4 Figure 4. The gene expression pattern of TGF-β and BMP components is different in brown bear muscle resistant to atrophy during hibernation compared to atrophied muscles of the unloaded mouse. Genes expression level in vastus lateralis muscle of

Figure 5 .

 5 Figure 5. The gene expression pattern of muscle E3s/DUB enzymes involved in TGF-β and BMP pathway regulation differed in the atrophy-resistant brown bear muscle during hibernation compared to the atrophied muscle of the unloaded mouse. Genes expression level (a) in vastus lateralis muscle of active and hibernating brown bears (n = 6 bears/season, the same individuals were sampled and analyzed in summer and winter, log2FC Winter/Summer, dotted white bars), and (b) in soleus muscle of control and unloaded mice (n = 4 mice per condition, log2FC Unloaded/Control, gray bars). Data are expressed as log2FC ± lfcSE. Statistical significance is shown (* padj < |0.05|; ** padj < |0.01|; *** padj < |0.001|).

Figure 6 .

 6 Figure 6. Cont.

Figure 6 .

 6 Figure 6. Hibernation induces changes in TGF-β and BMP components at protein level. Protein levels for total SMAD2/3, SMAD1/5, SMAD4, CCN2, and GDF5 were assessed by Western blots (a) in the vastus lateralis muscle of brown bears during summer (S) and winter (W) and (b) in the soleus muscle of control (C) and unloaded mice (U). Representative western blots are shown for three couples of bears and mice. (c-h). Data are presented as individuals' values with mean bars (n = 11 bears/season, the same individuals were sampled and analyzed in summer and winter, and n = 6 mice per condition). Gray and black dots are for muscles of bears, in summer and winter, respectively, and gray and black triangles are for control and unloaded muscle of mice, respectively.

1 Supplementary Figure 1 .

 11 The gene expression pattern of TGF-β and BMP components is different in brown bear muscle resistant to atrophy during hibernation compared to atrophied muscles of the unloaded mouse. Heatmap from vastus lateralis muscle of active and hibernating brown bears (n=6 bears/season, the same individuals were sampled and analyzed in summer and winter, log2FCWinter/Summer), and soleus muscle of control and unloaded mice (n=4 mice per condition, log2FC Unloaded/Control) of 171 TGF-β/BMP related genes. The green and red colours indicate that the gene expression decreased or increased, respectively, and each line represents one gene.

Figure 30 .

 30 Figure 30. Graphical abstract of the study 1.

Figure 32 .

 32 Figure 32. Western blots of total and phosphorylated SMAD1/5, and SMAD4 in CCL136 human muscle cells expressing an inactive BMP receptor. CCL136 human muscle cells were transfected with the plasmid ALK3-KD (kinase dead) receptor, followed by 6h treatment with GDF5 ligand. BMP intracellular actors SMAD1/5 and SMAD4 were measured by Western blotting (see Appendix 10.1).

Figure 33 .

 33 Figure 33. Co-immunoprecipitation of SMAD4 with SMAD1/5 or SMAD2/3 in bear muscles. SMAD4 immunoprecipitation (IP) in vastus lateralis muscle from hibernating versus active brown bears was followed by immunodetection of SMAD1/5, SMAD4 and SMAD2/3 by Western blotting (IB). IgGs have been used as negative control. W: Winter; S: Summer (see Appendix 10.1).

Figure 34 .

 34 Figure 34. SMAD4 linker protein sequences in different species. The T bold in black represents the Threonine 272, while the S bold in blue represents the Serine replaced at position 272. The red box represents the Ursidae family members, while the black box represents examples of small hibernators.

3 )[

 3 351]. CCN2 is a secreted matricellular protein predominantly expressed during development in almost all tissues, during numerous pathological conditions that involve enhanced fibrogenesis, and during several cancers [372]. CCN2 and TGF-β signalling. CCN2 expression is regulated by growth factors, cytokines and hormones, including TGF-β1 [372]. TGF-β1 induces CCN2 gene expression and plays an important role in fibrosis, especially during dystrophies [372-375]. In turn, once secreted, CCN2 directly interacts with TGF-β ligands and thereby facilitates the signal to transduce [373,376]. Interestingly, CCN2 gene expression

Figure 35 .

 35 Figure 35. SMAD4 degradation involving MAPK, GSK3 and β-TrCP proteins.

[

  377]. The strong reduction in CCN2 mRNA and protein expression in winter bear muscles is consistent with the very limited mechanical demand in hibernating bear muscles, and the general downregulation of the TGF-β signalling[351]. CCN2 and TGF-β1 proteins are significantly overexpressed in muscles from Duchenne muscular dystrophy patients and both are positively correlated with the degree of pathology and clinical severity[378]. Furthermore, TGF-β signalling also induces the gene expression of other CCN family members with common biological actions, such as CCN4 (also known as WISP1)[379], which is strongly downregulated in hibernating bear muscles compared to summer in our study (seePaper 6.3) [351]. It remains to be determined whether the downregulation of CCN2 at the transcriptomic and proteomic level in the hibernating bear muscles is a cause and/or a consequence of the TGF-β signalling inhibition. However, CCN2 inhibition is likely to be a consequence of reduced TGF-β signalling given the large number of downregulated TGF-β target genes in winter bear muscles (seePaper 6.3) [351].CCN2 and BMP signalling. In addition, CCN2 can antagonize the activity of BMP4 and BMP7 ligands by preventing their binding to BMP receptors in Xenopus embryos and mice kidneys respectively, resulting in reduced SMAD1/5 signal transduction[376,380]. Moreover, surface plasmon resonance spectroscopy shows that CCN2 and GDF5 protein interacts[381]. Whether CCN2 inhibits the transduction of BMP signalling in muscles remains an open question. However, our data show that CCN2 downregulation is correlated with the maintenance of BMP signalling in winter bear muscles. From a clinical perspective. CCN2 is considered a therapeutic target in combating fibrosis and related disorders in a variety of organs and tissues. Muscle function is improved by anti-CCN2 antibody in a mouse model of Duchenne muscular dystrophy [382]. In addition, clinical trials in phase 2 and phase 3 are currently testing another fully human monoclonal antibody that interferes with the action of CCN2 during Duchenne muscular dystrophy (ClinicalTrials.gov Identifier: NCT02606136 and NCT04632940).

  profound changes in circulating lipids may have altered the composition of membrane lipids and therefore the membrane fluidity of organs, including muscle. The plasma membrane is a critical hub for signalling proteins. Membrane lipids are organised into different microdomains rich in specific lipid species, which attract different types of proteins[383,384]. A change in membrane lipid composition may have altered the heterodimerisation of TGF-β superfamily receptors known to be a dynamic process[107]. TGF-β receptors are distributed in both lipid rafts/caveolae and non-raft membrane microdomains (i.e. clathrin-coated pits). The internalisation of TGF-β receptors via clathrin-coated pits enhances TGF-β signalling, whereas lipid rafts-mediated endocytosis of TGF-β receptors facilitates receptor degradation and thus the turnoff of signalling[385][386][387]. Cholesterol has been suggested to inhibit SMAD2 activation, promote TGF-β receptor degradation, and therefore inhibit TGF-β signalling.This effect may result from the shifted localisation of TGF-β receptors from non-raft to lipid-raft microdomains in the plasma membrane[386][387][388]. On the contrary, BMP receptors have been suggested to be distributed in lipid rafts-mediated endocytosis, and a decrease in cholesterol level specifically blocks the BMP receptors-mediated intracellular signalling[389]. To test the dynamics of TGF-β superfamily receptors, we could treat myotubes with winter or summer bear serum and analyse the localisation of the receptors by confocal microscopy. We could also perform lipidomic analysis of membrane phospholipids in myotubes or biopsied bear muscles by functional two-photon microscopy.6.4.2.5 Is the resistance to denervation-induced muscle atrophy in hibernating bearsrelated to BMP signalling? BMP signalling in NMJ organisation. BMP signalling is important for the conservation of muscle mass when the neuromuscular junction (NMJ) is compromised, as reported in a model of denervationinduced muscle atrophy in mice [166-168]. In addition, disruption of presynaptic architecture and NMJ degeneration concomitantly to BMP signalling perturbation are observed in muscles of tumourbearing mice before muscle loss occurred [168]. The same feature was also observed in muscles from pre-cachectic cancer patients [168]. Promoting BMP signalling using genetic or drug-based interventions (tilorone) preserves NMJ function during the development of cachexia and therefore counteract muscle atrophy [168]. On the contrary, overexpression of the BMP inhibitor noggin in muscles of healthy mice induced muscle atrophy, mimicked the loss of presynaptic motor neuron terminals and increased the presence of denervation markers [168]. The BMP pathway regulates peripheral synaptic development and plasticity in Drosophila [390,391] and is essential for proper axon elongation in motor neurons during development in mice [392]. However, the role of BMP in controlling postnatal NMJ remodelling in adult mammals, particularly in pathological contexts, remains largely unexplored. TAK1 is involved in non-SMAD TGF-β/BMP signalling (see section 4.2.4.2) and the

Figure 36 .

 36 Figure 36. Hypothetical schema to explain the denervation-induced muscle atrophy resistance in the hibernating brown bears muscles.

  communication. A new concept has emerged that bone also acts as an endocrine tissue targeting other organs such as muscle. Therefore, muscle and bone communicate via soluble factors [400]. Both bone and muscle volumes are sensitive to mechanical loading, which regulates many of their secreted factors. Therefore, muscle and bone mass are both reduced during immobilisation [401]. Bone resorption releases TGF-β1 into the bloodstream in pediatric burn patients and tumour-bearing mice [402,403]. The use of antiresorptive drugs protects bone and muscle mass, demonstrating that a factor released by bones contributes to muscle wasting in these conditions. TGF-β1 released from bone suppresses activation of the AKT/mTOR anabolic pathway and promotes expression of UPS players in myoblasts in vitro [403]. TGF-β ligands are produced by osteoblasts, stored in the extracellular matrix of bone and released by osteoclastic proteolysis during bone resorption [404,405].

Figure 37 .

 37 Figure 37. Hypothetical schema of the origin of muscle transcriptomic changes of TGF-β superfamily.

7. Study 2 :

 2 Induction of ATF4 atrogenes is uncoupled from disuseinduced muscle atrophy in halofuginone-treated mice and in hibernating brown bear 7.1 Objective and strategy

Figure 38 .

 38 Figure 38. Schema of the experimental strategy of study 2.

Figure 1 .

 1 Figure 1. Halofuginone activates the expression of ATF4-regulated atrogenes in muscle without leading to atrophy. (A) Schematic representation of the experimental protocol, where mice received H 2 O (white bars) or HF (0.25 µg/g, grey bars) 3 times a week for up to 4 weeks (WK). Muscles were collected 6 h after the last HF administration at the end of each week (dotted arrows). (B-F) Relative mRNA levels in gastrocnemius for Atf4, Trib3, Cdkn1a, Gadd45a, and Eif4ebp1 were measured by RT-qPCR. Data were normalised using Tbp. Data are expressed as fold change vs. H 2 O within each week and are presented as individual values with mean bars ± SEM. (G) Gastrocnemius muscle mass per gram of body weight (BW). Data are expressed as a percentage from H 2 O within each week and presented as individual values with mean bars ± SEM. Statistics are described in Section 4. * p adj < 0.05; ** p adj < 0.01; *** p adj < 0.001; **** p adj < 0.0001.

Figure 2 .

 2 Figure 2. Hindlimb suspension induces ATF4 pathway. (A) Schematic representation of the imental protocol, where mice received H2O or halofuginone (HF) oral administration (0.25 μ

Figure 2 .

 2 Figure 2. Hindlimb suspension induces ATF4 pathway. (A) Schematic representation of the experimental protocol, where mice received H 2 O or halofuginone (HF) oral administration (0.25 µg/g) 3 times a week for 3 weeks (WK) (black arrows) and were then subjected to hindlimb suspension

Figure 3 .

 3 Figure 3. Halofuginone treatment prior to hindlimb suspension mitigates atrophy in gastrocnemius muscle. Mice were treated with H2O or halofuginone (HF, 0.25 μg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 (HS3, light grey bars) or 7 (HS7, white bars) days or kept unsuspended (Ctrl, dark grey bars), as described in Figure 2A. (A) Gastrocnemius muscle mass per gram of body weight (BW). Data are expressed as a percentage from H2O-Ctrl and presented as individual values with mean bars ± SEM. (B) Mean fibre cross-sectional area in gastrocnemius muscle. Data are presented as individual values with mean bars ± SEM. Statistics are described in Methods section. ** padj < 0.01; *** padj < 0.001; **** padj < 0.0001; ns = non-significant.

Figure 3 .

 3 Figure 3. Halofuginone treatment prior to hindlimb suspension mitigates atrophy in gastrocnemius muscle. Mice were treated with H 2 O or halofuginone (HF, 0.25 µg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 (HS3, light grey bars) or 7 (HS7, white bars) days or kept unsuspended (Ctrl, dark grey bars), as described in Figure 2A. (A) Gastrocnemius muscle mass per gram of body weight (BW). Data are expressed as a percentage from H 2 O-Ctrl and presented as individual values with mean bars ± SEM. (B) Mean fibre cross-sectional area in gastrocnemius muscle. Data are presented as individual values with mean bars ± SEM. Statistics are described in Section 4. ** p adj < 0.01; *** p adj < 0.001; **** p adj < 0.0001; ns = non-significant.

  Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 19

Figure 4 .

 4 Figure 4. Halofuginone treatment inhibits TGF-β while promoting BMP signalling in gastrocnemius muscle. Mice were treated with H2O or halofuginone (HF, 0.25 μg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 (HS3, light grey bars) or 7 (HS7, white bars) days or kept unsuspended (Ctrl, dark grey bars), as described in Figure 2A. (A-D) The ratio of protein levels in gastrocnemius for the transcription factors SMAD2/3 (TGF-β signalling), SMAD1/5 (BMP signalling), and SMAD4 (TGF- and BMP signalling) have been assessed in the nuclear and cytosolic subcellular fractions, quantified, and normalised to the total protein content. Representative Western blots are shown. The ratio of nuclear SMAD contents on the total (cytosolic and nuclear) SMAD content was calculated. Data are expressed as fold change vs. H2O-Ctrl and presented as individual values with mean bars ± SEM. Statistics are described in Methods section. * padj < 0.05; **** padj < 0.0001.

Figure 4 .

 4 Figure 4. Halofuginone treatment inhibits TGF-β while promoting BMP signalling in gastrocnemius muscle. Mice were treated with H 2 O or halofuginone (HF, 0.25 µg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 (HS3, light grey bars) or 7 (HS7, white bars) days or kept unsuspended (Ctrl, dark grey bars), as described in Figure 2A. (A-D) The ratio of protein levels in gastrocnemius for the transcription factors SMAD2/3 (TGF-β signalling), SMAD1/5 (BMP signalling), and SMAD4 (TGF-β and BMP signalling) have been assessed in the nuclear and cytosolic subcellular fractions, quantified, and normalised to the total protein content. Representative Western blots are shown. The ratio of nuclear SMAD contents on the total (cytosolic and nuclear) SMAD content was calculated. Data are expressed as fold change vs. H 2 O-Ctrl and presented as individual values with mean bars ± SEM. Statistics are described in Section 4. * p adj < 0.05; **** p adj < 0.0001.

Figure 5 .

 5 Figure 5. Halofuginone treatment prior to hindlimb suspension partially prevents the decrease in protein synthesis in gastrocnemius muscle. Mice were treated with H2O or halofuginone (HF, 0.25 μg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 (HS3, light grey bars) or 7 (HS7, white bars) days or kept unsuspended (Ctrl, dark grey bars), as described in Figure 2A. (A,B) Relative puromycin incorporation into gastrocnemius muscle was assessed by Western blotting, quantified, and normalised to the total protein content. A representative Western blot is shown. (C-E) Relative mRNA levels in gastrocnemius for Trim63, Fbxo32, and Fbxo30 were measured by RT-qPCR. Data were normalised using Tbp. Data are expressed as fold change vs. H2O-Ctrl and presented as individual values with mean bars ± SEM. Statistics are described in Methods section. * padj < 0.05; **** padj < 0.0001, or ns = non-significant.

Figure 5 .

 5 Figure 5. Halofuginone treatment prior to hindlimb suspension partially prevents the decrease in protein synthesis in gastrocnemius muscle. Mice were treated with H 2 O or halofuginone (HF, 0.25 µg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 (HS3, light grey bars) or 7 (HS7, white bars) days or kept unsuspended (Ctrl, dark grey bars), as described in Figure 2A. (A,B) Relative puromycin incorporation into gastrocnemius muscle was assessed by Western blotting, quantified, and normalised to the total protein content. A representative Western blot is shown. (C-E) Relative mRNA levels in gastrocnemius for Trim63, Fbxo32, and Fbxo30 were measured by RT-qPCR. Data were normalised using Tbp. Data are expressed as fold change vs. H 2 O-Ctrl and presented as individual values with mean bars ± SEM. Statistics are described in Section 4. * p adj < 0.05; **** p adj < 0.0001, or ns = non-significant.

Figure 6 .

 6 Figure 6. ATF4-regulated atrogenes are induced in atrophy-resistant hibernating brown bear muscles. Gene expression levels for ATF4, GADD45A, CDKN1A, TRIB3, EIF4EBP1, PPP1R15A, ASNS, and DDIT3 in vastus lateralis muscle of active and hibernating brown bears (n = 6 bears/season, the same individuals were sampled and analysed in summer and winter, log2FC winter/summer). Data are presented as individual values as log2FC with mean bars ± lfcSE (log2 fold change standard error). Statistics are described in [46]. * padj < 0.05; ** padj < 0.01; **** padj < 0.0001. FC: fold change; W: winter (hibernating season); S: summer (active season)

Figure 6 .

 6 Figure 6. ATF4-regulated atrogenes are induced in atrophy-resistant hibernating brown bear muscles. Gene expression levels for ATF4, GADD45A, CDKN1A, TRIB3, EIF4EBP1, PPP1R15A, ASNS, and DDIT3 in vastus lateralis muscle of active and hibernating brown bears (n = 6 bears/season, the same individuals were sampled and analysed in summer and winter, log2FC winter/summer). Data are presented as individual values as log2FC with mean bars ± lfcSE (log2 fold change standard error). Statistics are described in [46]. * p adj < 0.05; ** p adj < 0.01; **** p adj < 0.0001. FC: fold change; W: winter (hibernating season); S: summer (active season).

Figure 7 .

 7 Figure 7. Graphical abstract. The red and green lines represent catabolic and anabolic effects, respectively. Dotted lines represent hypothetical connections. The arrows/T bars above the ATF4 atrogenes, SMAD2/3, and SMAD1/5 boxes represent the induction/inhibition by halofuginone or by an as-yet-unknown mechanism in mouse or bear muscle, respectively. Created with BioRender.com.

Figure 7 .

 7 Figure 7. Graphical abstract. The red and green lines represent catabolic and anabolic effects, respectively. Dotted lines represent hypothetical connections. The arrows/T bars above the ATF4 atrogenes, SMAD2/3, and SMAD1/5 boxes represent the induction/inhibition by halofuginone or by an as-yet-unknown mechanism in mouse or bear muscle, respectively. Created with BioRender.com.

Author

  Contributions: Conceptualisation, L.C. (Laura Cussonneau), A.-C.M. and L.C. (Lydie Combaret); methodology, L.C. (Laura Cussonneau), L.C. (Lydie Combaret), C.D., C.C.-G., G.C., J.H., M.D.-M. and Y.D.; analysis, L.C. (Laura Cussonneau), L.C. (Lydie Combaret), C.D. and C.C.-G.; writing-original draft preparation, L.C. (Laura Cussonneau) and L.C. (Laura Cussonneau); writingreview and editing, all authors; project administration, L.C. (Laura Cussonneau); funding acquisition, E.L., F.B. and L.C. (Laura Cussonneau). All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Center National d'Etudes Spatiales (CNES, #865, #974, #1006, and #1905), the iSITE Challenge 3 Mobility program (Université Clermont Auvergne), and the Agence Nationale de la Recherche (B-STRONG ANR-22-CE14-0018). L.C. (Laura Cussonneau) was supported by a grant from the Institut National de la Recherche Agronomique et Environnement and Clermont Métropole. The long-term funding of the Scandinavian Brown Bear Research Project (SBBRP) comes primarily from the Swedish Environmental Protection Agency, the Norwegian Environment Agency, the Austrian Science Fund, and the Swedish Association for Hunting and Wildlife Management.

Figure 39 .

 39 Figure 39. Graphical abstract of the study 2.

Figure 40 .

 40 Figure 40. Chemical structures of febrifugine and halofuginone (from Pines et Spector 2015 [416]).

Figure 41 .

 41 Figure 41. Effect of halofuginone treatment prior to hindlimb suspension on extracellular matrix components expression in gastrocnemius muscle in mice. Mice were treated with H2O or halofuginone (HF, 0.25µg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 (HS3) or 7 (HS7) days or kept unsuspended (Ctrl). (A-D) Gastrocnemius relative mRNA levels for Col1a1, Col3a1, Col5a2 and Col6a1 by RT-qPCR (see Appendix 10.2). Data were normalised using Tbp gene. Data are means ± SEM (expressed as fold change vs. H2O-Ctrl). Twoway ANOVA: * padj < 0.05; ** padj <0.01; *** padj <0.001; **** padj < 0.0001.

Paper 7 . 2 )

 72 [423]. We also recorded slight preservation of muscle mass during HS in mice that were pretreated with HF for a shorter period (i.e. 2 weeks) (Figure42). This slight preservation of muscle mass occurred even if ATF4 atrogenes were overexpressed during HS both in mice pre-treated for 2 or 3 weeks with HF (see Paper 7.2 and data not shown) [423]. The effect of HF may depend on the nature of the muscle. The slight preservation of gastrocnemius CSA observed in HF-treated mice subjected to HS was mainly observed in glycolytic fibres (i.e. type 2X/2B) (see Paper 7.2) [423]. Oxidative fibres (i.e. type 1/2A) are well-documented to be more susceptible to disuse-induced atrophy than glycolytic fibres [425]. Unlike the glycolytic gastrocnemius muscle, the mass of the oxidative soleus muscle was not protected after 3 days of HS when mice were

Figure 42 .

 42 Figure 42. Halofuginone treatment for 2 weeks prior to hindlimb suspension mitigates atrophy in gastrocnemius muscle in mice.

Figure 43 .

 43 Figure 43. Effect of halofuginone treatment prior to hindlimb suspension in soleus muscle. Mice were treated with H2O or halofuginone (HF, 0.25µg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 (HS3) or 7 (HS7) days or kept unsuspended (Ctrl). (A) Soleus muscle mass (mg) represented with means ± SEM. (B) Mean fibre cross-sectional area (CSA) of soleus muscle in Ctrl, HS3 and HS7. Data are means ± SEM. Two-way ANOVA. * padj<0.05; ** padj <0.01; **** padj <0.0001. (C) Frequency distribution proportion of fibres CSA of soleus muscle in Ctrl, HS3 or HS7 of mice treated with H2O or HF, for all fibres type. Data are means ± SEM.

7. 3 . 3 . 2

 332 ATF4 signalling in hibernating bear musclesInduction of ATF4 atrogenes is associated with moderate muscle atrophy in hindlimb suspended mice when treated with HF or in hibernating brown bears muscles (seePaper 7.2) [423]. We further explored the transcriptome of the muscle of the hibernating brown bear from study1 [351] to analyse the ATF4 gene signature. Based on an extensive literature review and the use of databases (i.e. GeneCards), we have established a list of ATF4-related genes (seeAppendix 10.2). Using the same strategy as in study 1, we performed an enrichment analysis from the down-(161) and up-regulated (105) ATF4-related genes.

Figure 45 .

 45 Figure 45. ATF4 signalling regulation in atrophy-resistant muscle of the hibernating bear.

8. Study 3 :

 3 Winter bear serum induces similar characteristics in human muscle cells as those found naturally in hibernating bear muscle 8.1 Objective and strategyThis last part contains preliminary results. We sought to replicate the molecular characteristics of atrophy-resistant muscles of hibernating bears in human muscle cells. Our team previously reported an increase in total protein content in human myotubes (HM) cultured with hibernating bear serum.This result proved for the first time that a circulating compound in bear serum could transfer biological properties to human muscle cells[341]. In this thesis project, we showed concurrent TGF-β inhibition and BMP activation in atrophy-resistant muscles of the hibernating brown bear (seePaper 6.3) [351] that we replicated in muscles of hindlimb-suspended mice treated with HF (see Paper 7.2)[423]. We aimed at determining whether a compound in bear serum during hibernation could reproduce these changes in TGF-β/BMP balance in human muscle cells. Our strategy was first to analyse microarray data from human muscle cells cultivated with winter bear serum (WBS) or summer bear serum (SBS) to assess whether there is a transcriptomic signature of TGF-β/BMP signalling. Subsequently, we optimised tools to measure TGF-β/BMP signalling transcriptional activity through their canonical or non-canonical signalling, using luciferase reporter assays in human muscle cells cultivated with SBS or WBS (Figure48).

Figure 48 .

 48 Figure 48. Schema of the experimental strategy of study 3.

Figure 49 .

 49 Figure 49. Human myotubes cultivated with winter bear serum induces similar transcriptional changes than those occurring in hibernating bears muscles. Human myotubes cultured upon winter bear serum (W) or summer bear serum (S) treatment for 48 hours. Gene expression assessed by DNA microarrays was analysed focusing on TGF-β and BMP signalling components. Data are expressed as log2FoldChange (FC) W/S ± lfcSE of 3 independent experiments (different cell preparations and bear serum mixes). Statistical significance is shown * padj < 0.05.

Figure 50 .

 50 Figure 50. Winter bear serum mimics in human muscle cells what occurs naturally in the muscles of hibernating bears. CCL136 cells were transfected with (A) ID1-Luc, (C) SBE-Luc and (D) MEF2-Luc and cultivated with summer bear serum (SBS) or winter bear serum (WBS) for (A) 6, 12 or 24 hours or (B and C) 24 hours. Cells were then lysed and luciferase activity was measured. Data are presented as individuals values with mean bars (n = 8-12 bear serum/season the same individuals were sampled and analysed in summer and winter). (B) Human primary myotubes (HM) were transfected with ID1-Luc, cultivated with SBS or WBS for 24 hours, and then lysed before measuring luciferase activity (see Appendix 10.3). Data are presented as individuals values with mean bars (n= 4 bear serum/season). Statistical significance is shown (Ratio paired t test) * pvalue<0.05; ** pvalue<0.01; ns: non-significant.

  ) studied the impact of controlled ATF4 induction by halofuginone in mice subjected to hind limb suspension and (3) explored the effect of winter bear serum on human muscle cells. This work(1) demonstrated that the BMP/TGF-β balance is important in the muscle atrophy resistance phenotype and (2) suggested that it could be replicated in human muscle cells by the presence of circulating compounds in hibernating bear serum. The identification of new relevant targets within the BMP and TGF-β pathways that could be modulated by compounds in the hibernating bear serum and the identification of these compounds would therefore allow the development of innovative strategies against muscle atrophy. The continuation of this project is therefore the first step in the future development of new therapeutic solutions to confer resistance to muscle atrophy in humans.

1 5 (

 15 Identification of BMP target genes in skeletal muscleCell culture. The CCL136 human rhabdomyosarcoma cell line (ATCC, USA) was maintained in dulbecco's modified eagle medium (DMEM) 4,5g/L glucose medium (Gibco, USA) containing 1% penicillinstreptomycin (Gibco, USA) and 10% fetal bovine serum (FBS) (Gibco, USA). The cells were seeded in 12-wells plates at a density of 1.5 × 10 5 cells/well, cultured at 37 °C under 5% CO2, and transfected the day after. Plasmid. Plasmid cloning (pc)DNA3-ALK3 K261R was a gift from Aristidis Moustakas (Addgene plasmid # 80875; http://n2t.net/addgene:80875; RRID: Addgene_80875) [367]. This pcDNA3 back bone contains the DNA sequence of the BMP receptor BMPR1A/ALK3 carrying a K261R mutation leading to an inactive kinase. Transfection. CCL136 cells were transfected using the ViaFect Transfection Reagent (Promega, E4981). The ratio of ViaFect™ Transfection Reagent volume (µL) to DNA amount (µg) was optimised with green fluorescent protein (GFP) transfection assays prior to the actual experiments. We chose the 3:1 ratio (3 µl reagent: 1 µg DNA) as increasing the quantity of DNA or reagent did not increase the efficacy of the transfection. On the day of transfection, DNA and Viafect transfection reagent were mixed at the 1:3 ratio described above in a serum-free medium and incubated for 15 minutes at room temperature to form the ViaFect™ Transfection Reagent:DNA complex. Cells were transfected with 1 µg of DNA/well by adding 100 µl of the transfection mixture to the wells. GDF5 treatment. After one night of transfection, the media have been replaced by fresh media. Half of the wells were treated with recombinant human GDF5 protein (Accession # P43026, BioLegend) at 0.1 µg /mL for 6 hours. Cells were then washed with phosphate-buffered saline (PBS), harvested for protein extraction in the same protein extraction buffer as described in study 2 (see Paper 7.2) [423], and then stored at -80 until use. Western blots experiments have been performed as described in study 2 (see Paper 7.2) [423] with the same SMAD4 and SMAD1/5 antibodies, and Phospho-SMAD1/Ser463/465) (#9516, Cell Signalling Technology, Saint-Cyr-L'Ecole, France) diluted 1:1000 in 5% bovine serum albumin (BSA).

10. 1 . 2

 12 Is SMAD4 recruited more by TGF-β or BMP signalling: Coimmunoprecipitation Protein extraction. 5 mg of bear muscle powder were lysed on ice using a polytron with 500 µL NP-40 buffer (10mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.0% Nonidet P-40, 20mM betaglycerophosphate) containing inhibitors of proteases (Protease Inhibitor Cocktail) and phosphatases (1 mM Na3VO3, 10 mM NaF) (Sigma, Saint-Quentin-Fallavier, France). The homogenates were then centrifuged at 10,000 g for 10min at 4°C and the concentration was determined using the Bradford Protein Assay Kit (Biorad, Marnes-la-Coquette, France). An aliquot was taken for protein expression analyses.Immunoprecipitation. The remaining lysates, containing equivalent amounts of 1 mg of total protein, were pre-cleaned for 1 h with 40 µL of Protein A-Agarose beads(sc-2001, Santa Cruz Biotechnology) and 1 µg of IgG isotype control antibody (sc-2027, Santa Cruz Biotechnology, Nanterra, France) with gentle rotation at 4°C. The samples were then centrifuged at 3200g for 30 seconds at 4°C, and the supernatants were stored. Immunoprecipitation was performed by the addition of 1.8 µg of SMAD4 antibody (ab230815, Abcam, Cambridge, UK) and protein A-Agarose, followed by incubation at 4°C overnight with gentle rotation. The immune complex was isolated by centrifugation at 3500g for 5 minutes. The resulting pellet was then washed with 350 µl of wash buffer (PBS pH 7.4, 5mM EDTA, 10mM NaF) and centrifuged at 3500g for 5 minutes without vortexing. This last step was repeated twice.Immunodetection. The resulting pellet was eluted in 80 µL Laemmli 1X. Proteins were then denatured at 95°C for 5min, and 25 µL of the eluate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using tris-glycine eXtended (TGX)™ FastCast™ 7,5% Acrylamide gels (Biorad, Marnes-la-Coquette, France) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Hybond P, Amersham, England) using Trans-Blot® Turbo™ Transfer System standard protocol (Biorad, Marnes-la-Coquette, France). Blots were blocked for 1 h at room temperature in Tris-Buffered Saline (TBS) buffer with 0.1% Tween-20 (TBS-T, pH = 7.8) and 5% BSA for all the targets according to the manufacturer's instructions. They were then washed thrice in TBS-T and incubated (overnight, stirring, 4°C) with appropriate primary antibodies: (1) for validating the immunoprecipitation, SMAD4 (ab230815, Abcam, Cambridge, United Kingdom) antibody was used diluted 1:1000 in 5% BSA and (2) for checking the protein partners, membranes were hybridised with either SMAD1/5 (PA5-80036, Thermo Fisher, Illkirch, France) or SMAD2/3 (#8685, Cell Signalling Technology, Saint-Cyr-L'Ecole, France) both diluted 1:1000 in 5% BSA overnight at 4°C. Blots were then washed and incubated for 1 h at room temperature with VeriBlot for IP Detection Reagent (HRP) (ab131366, Abcam) diluted 1:2000 in 5% non-fat dried milk. Signals were detected after incubation with Luminata Crescendo Western HRP substrate (Millipore, Burlington, MA, USA) and visualized using G: BOX ChemiXT4 (XL1) imaging system (Syngene, Frederick, MD, USA). 10.1.3 Is GDF5 synthesised and released by adipose tissue: GDF5 ELISA We used the only commercially available GDF5 ELISA kit (Catalog Number: DY853-05 and DuoSet Ancillary Reagent Kit2 Catalog number: DY008; R&D Systems Europe) and performed GDF5 immunodetection in serum from hibernating and active bears following the supplier protocol.

Figure 1 .

 1 Figure 1. Signaling pathways regulating skeletal muscle mass and function. Myofiber representation of the different signaling pathways controlling skeletal muscle mass and function during atrophy conditions. Ligands and arrows (both with head or perpendicular line) in green denote those signaling pathways and interactions with an anabolic effect whereas the red ones represent catabolic signaling. Orange ligands and arrows stand for pathways with a dual role (context-dependent). ß2-AR: ß-2 Adrenergic Receptor; 𝛾-sec: 𝛾-secretase; Ang: Angiotensin; AT1R: Angiotensin II Type 1 Receptor; AT2R: Angiotensin II Type 2 Receptor; BCAAs: Branched-chain amino acids; BMP R: Bone Morphogenetic Receptor; Calp: Calpain; CSL: CBF1, Suppressor of Hairless, Lag-1; Dsh: Dishevelled; Fzd: Frizzled; GR: Glucocorticoid Receptor; IL-6: Interleukin-6; NCID: Notch Intracellular domain; NOX: NADPH oxidase activator; P: Phosphorylation; S1P: Sphingosine-1phosphate; SLC T: Solute Carrier Transporter; STAPs: Signal Transducing Adaptor Proteins; TF: Transcription Factors; TGF-ß R: Transforming Growth Factor ß Receptor; TKR: Tyrosine-protein Kinase Receptor; TLR: Toll-like Receptor; TNF R: Tumor Necrosis Factor Receptor; Transl. Fact.: Translational Factors.

Figure 1 .

 1 Figure 1. Signaling pathways regulating skeletal muscle mass and function. Myofiber representation of the different signaling pathways controlling skeletal muscle mass and function during atrophy conditions. Ligands and arrows (both with head or perpendicular line) in green denote those signaling pathways and interactions with an anabolic effect whereas the red ones represent catabolic signaling. Orange ligands and arrows stand for pathways with a dual role (contextdependent). ß2-AR: ß-2 Adrenergic Receptor; γ-sec: γ-secretase; Ang: Angiotensin; AT1R: Angiotensin II Type 1 Receptor; AT2R: Angiotensin II Type 2 Receptor; BCAAs: Branched-chain amino acids; BMP R: Bone Morphogenetic Receptor; Calp: Calpain; CSL: CBF1, Suppressor of Hairless, Lag-1; Dsh: Dishevelled; Fzd: Frizzled; GR: Glucocorticoid Receptor; IL-6: Interleukin-6; NCID: Notch Intracellular domain; NOX: NADPH oxidase activator; P: Phosphorylation; S1P: Sphingosine-1-phosphate; SLC T: Solute Carrier Transporter; STAPs: Signal Transducing Adaptor Proteins; TF: Transcription Factors; TGF-ß R: Transforming Growth Factor ß Receptor; TKR: Tyrosine-protein Kinase Receptor; TLR: Toll-like Receptor; TNF R: Tumor Necrosis Factor Receptor; Transl. Fact.: Translational Factors.

Figure 2 .

 2 Figure 2. Cont.

Figure 2 .

 2 Figure 2. E3 ubiquitin ligases regulating skeletal muscle mass and molecules developed to modulate their activity and expression. Myofiber representation of the different E3-ligases and molecules targeting the signaling pathways controlling skeletal muscle mass and function during atrophy conditions. Ligands and arrows (both with head or perpendicular line) in green denote those signaling pathways and interactions with an anabolic effect whereas the red ones indicate catabolic signaling. ß2-AR: ß-2 Adrenergic Receptor; BCAAs: Branched-chain amino acids; BMP R: Bone Morphogenetic Receptor; Calp: Calpain; CSL: CBF1, Suppressor of Hairless, Lag-1; GR: Glucocorticoid Receptor; IL-6: Interleukin-6; NCID: Notch Intracellular domain; NOX: NADPH oxidase activator; P: Phosphorylation; STAPs: Signal Transducing Adaptor Proteins; TF: Transcription Factors; TGF-ß R: Transforming Growth Factor ß Receptor; TKR: Tyrosine-protein Kinase Receptor; TLR: Toll-like Receptor; TNF R: Tumor Necrosis Factor Receptor; Transl. Fact.: Translational Factors.

Figure 2 .

 2 Figure 2. E3 ubiquitin ligases regulating skeletal muscle mass and molecules developed to modulate their activity and expression. Myofiber representation of the different E3-ligases and molecules targeting the signaling pathways controlling skeletal muscle mass and function during atrophy conditions. Ligands and arrows (both with head or perpendicular line) in green denote those signaling pathways and interactions with an anabolic effect whereas the red ones indicate catabolic signaling. ß2-AR: ß-2 Adrenergic Receptor; BCAAs: Branched-chain amino acids; BMP R: Bone Morphogenetic Receptor; Calp: Calpain; CSL: CBF1, Suppressor of Hairless, Lag-1; GR: Glucocorticoid Receptor; IL-6: Interleukin-6; NCID: Notch Intracellular domain; NOX: NADPH oxidase activator; P: Phosphorylation; STAPs: Signal Transducing Adaptor Proteins; TF: Transcription Factors; TGF-ß R: Transforming Growth Factor ß Receptor; TKR: Tyrosine-protein Kinase Receptor; TLR: Toll-like Receptor; TNF R: Tumor Necrosis Factor Receptor; Transl. Fact.: Translational Factors.

  and (ii) the activation of NF-κB [149-151]. Oppositely, S1P can promote skeletal muscle mass in denervated mice [152] although the downstream signaling depends on the context and the S1P-receptor type [153]. 2.3.8. NOTCH Signaling Pathway Hyperactivation of NOTCH leads to atrophy during cancer cachexia [154], denervation [155-157], chronic alcohol consumption [158], hypovitaminosis D [159], and glucocorticoid treatment [114]. Upon cleavage of the NOTCH receptor by secretases [160],

  Regulation of Muscle Atrophy 3.1. E3 Ligases Involved in the Regulation of Anabolic Pathways 3.1.1. The CBL-B and FBXO40 E3 Ubiquitin Ligases Target IRS1 to Degradation in Skeletal Muscle

  Involved in the Regulation of Catabolic Pathways 3.2.1. Regulating the Canonical NF-κB Pathway via the Manipulation of cIAP and TRAF6 E3 Ligases

  FOXO1 and 3a participate to skeletal muscle adaptation upon exercise thus adding a new of FOXOs in the control of muscle cell homeostasis [219-222].

3. 3 .

 3 E3 Ubiquitin Ligases Involved in the Regulation of Muscle Mass and Function 3.3.1. MuRF1/TRIM63

4. 1 .

 1 Indirect Action on E3 Ligases 4.1.1. PI3K-AKT-mTORC1

  ), a β2-AR agonist, was shown to reverse MuRF1/Trim63 and MAFbx/Atrogin-1 overexpression with a concomitant muscle sparing in tumor-bearing mice[276]. Intriguingly, neither a repression of FOXO1 and FOXO3a transcription factors nor an activation of AKT-mTORC1 pathway explained the positive effect of formoterol. By contrast, formoterol was able to blunt MuRF1/Trim63 and MAFbx/Atrogin-1 expression in LPS-induced muscle atrophy through restoration of the AKT-mTORC1 pathway and reversal of P-FOXO/FOXO1 ratio[277].

Fig. 1 (

 1 Fig. 1 (See legend on next page.)

Fig. 6

 6 Fig. 6 Hypothetical consequences of changes in circulating lipids and endocannabinoid system tone during hibernation in brown bear. Black arrows represent possible behavior and metabolic outcomes

  adjust function (Package stats version 4.0.0 of R studio) was applied. Data are presented as means ± SEM and individual values are plotted as grey and black dots for respectively summer and winter values. Means, SEM, fold change and associated p-values are reported in supplementary TablesS2 to S5. Statistical significance was considered with p values or adjusted p values lower than 0.05.

  TableS5: Endocannabinoids (eCBs) and mRNA quantification in plasma and tissues in winter hibernating (W) and summer active (S) bears. 10.6 Résumé de la thèse en français 10.6.1 Introduction bibliographique 10.6.1.1 Le muscle squelettique Physiologie. Il existe 3 différents types de muscle, et dans ce projet de thèse nous nous sommes intéressés au muscle squelettique. Les muscles squelettiques recouvrent notre squelette et son essentiellement responsables des mouvements volontaires et de la posture. Il s'agit également d'un tissu très dynamique et plastique qui agit comme principal tissu du métabolisme énergétique avec la production de chaleur, l'absorption, l'utilisation et le stockage de substrats énergétiques tels que le glucose et les acides aminés (AA). Le muscle est essentiellement composé d'eau (75%) et de protéines (20%) [1]. C'est un tissu hautement organisé contenant plusieurs faisceaux de myofibres dont chaque couche est successivement encapsulée par la matrice extracellulaire. Les myofibres sont des cellules multinucléées et post-mitotiques. Chaque myofibre contient des milliers de myofibrilles qui sont composées de l'unité cellulaire de base du muscle, le sarcomère. Le sarcomère lui-même est composé de milliards de myofilaments, à la fois épais (myosine) et fins (actine) qui sont essentiels à la contraction musculaire nécessitant une forte consommation d'ATP. Les myofilaments représentent le principal contenu protéique du muscle (c'est-à-dire 70-80% du contenu protéique total d'une seule fibre) [1]. Mitochondries. Les muscles sont hautement vascularisés et innervés. Les besoins énergétiques pendant une contraction multiplient par 100 la consommation normale d'ATP dans le muscle. Pour répondre à cette demande énergétique élevée, le muscle dépend en partie de la phosphorylation oxydative mitochondriale (OXPHOS) pour la production d'ATP. Deux populations distinctes de

Figure 51 .

 51 Figure 51. Déséquilibre dans la balance protéique dans des conditions physiopathologiques conduisant à l'atrophie musculaire. AA : acides aminé

Figure 52 .

 52 Figure 52. Organisation et transduction du signal de la superfamille du TGF-β.

  SMAD4 le facteur contrôlant la balance protéique. SMAD4 est l'acteur commun entre la signalisation du TGF-β et du BMP (Figure 53). Les muscles des souris déficients pour Smad4 s'atrophient fortement et présentent une protéolyse excessive après un mois de dénervation chez la souris. Les souris déficientes pour la Mstn présentent un recrutement plus important de SMAD4 sur le promoteur des gènes cibles de la signalisation BMP. Au contraire, les souris déficientes pour Gdf5 présentent une augmentation de la liaison du complexe SMAD4-SMAD2/3 sur le promoteur des gènes cibles du TGFβ. De ces résultats a émergé le concept d'une compétition entre SMAD2/3 et SMAD 1/5/8 pour le recrutement de SMAD4. L'inhibition du signal TGF-β libérerait SMAD4 de SMAD2/3 pour être plus disponible pour SMAD1/5/8. Ainsi, la suractivation du TGF-β lors de situations cataboliques est considérée comme un facteur qui réduit la disponibilité de SMAD4 pour la signalisation BMP. Il y a donc une vraie nécessité d'un équilibre finement régulé de la balance BMP/TGF-β afin de maintenir l'homéostasie musculaire [110] (Figure 53).

  . L'UPRmt est une réponse à divers stress mitochondriaux. Il active un programme transcriptionnel codé par l'ADN nucléaire pour favoriser le retour à une homéostasie mitochondriale [226]. Néanmoins, si l'UPRmt est incapable de réparer les dommages mitochondriaux, l'élimination de la mitochondrie entière par mitophagie est favorisée. Il existe trois protéines régulatrices clés de l'UPRmt, dont la protéine ATF4 qui est souvent surexprimée suite à des dommages mitochondriaux. Une analyse transcriptomique globale a validé la présence de motifs de liaison d'ATF4 dans de nombreux gènes UPRmt [226] (Figure 55). Dans les muscles de souris, des stress mitochondriaux causés par une mauvaise fusion mitochondriale ou mitophagie, augmentent p-eIF2α et les niveaux protéiques d'ATF4. Cependant, des preuves montrent que l'activation de l'UPRmt-ATF4 suite à des perturbations mitochondriales dans les muscles peuvent avoir à la fois des effets protecteurs ou néfastes pour le muscle [226]. Ainsi, ces études mettent en évidence l'implication complexe de l'ISR dans l'autophagie et dans le contrôle de la qualité mitochondriale. En outre, il reste beaucoup à explorer pour comprendre l'implication de l'ISR-ATF4 dans l'autophagie et le contrôle de la qualité mitochondriale dans l'homéostasie musculaire. 227 Le role de l'ISR dans l'atrophie musculaire. Certains membres de l'ISR ont été associés de près ou de loin à l'atrophie musculaire dans différentes conditions cataboliques. Pour la kinase PERK et p-eIF2α, certaines études les associent à l'atrophie musculaire, pendant que d'autres leurs confèrent une action anti-atrophique. Pour les kinases GCN2 et PKR, il semblerait que leurs activations soient associées à l'atrophie musculaire, bien qu'aucune connection avec ATF4 n'ait été faite. Le gène ATF4 est quant à lui considéré comme un atrogène car ses niveaux d'ARNm augmentent dans de nombreuses conditions cataboliques induisant l'atrophie musculaire [99,207]. Les souris déficientes pour Atf4 dans les fibres musculaires se développent normalement et présentent une masse et une fonction musculaire normale jusqu'à un âge avancé. Par la suite, elles commencent à présenter une protection contre l'atrophie musculaire liée à l'âge [207]. Une délétion d'Atf4 dans les muscles de souris limite l'atrophie musculaire induite par le jeûne, l'immobilisation ou encore le vieillissement [207]. Le rôle catabolique d'ATF4 passe par la transcription des atrogènes Gadd45a 61 , Cdkn1a 62 and Eif4ebp1 63 dans les muscles 61 growth arrest and DNA damage inducible alpha 62 cyclin-dependent kinase inhibitor 1 63 eukaryotic translation initiation factor 4E binding protein 1

Figure 55 .

 55 Figure 55. Le rôle double de l'ISR dans l'homéostasie musculaire. Les flèches vertes représentent des conséquences cellulaires anaboliques, les flèches rouges des effets cataboliques, et noires des conséquences encore mal comprises.

Figure 56 .

 56 Figure 56. Caractéristiques physiologiques fascinantes de l'ours brun hibernant.

Figure 58 .Etude 3

 583 Figure 58. Schéma expérimental de l'étude 2.

Figure 59 .

 59 Figure 59. Schéma expérimental de l'étude 3. Les cellules musculaires humaines sont transfectées avec un plasmide rapporteur luciférase pour le gène ID1 avant d'être traitées par du sérum d'ours d'été (SBS) ou d'hiver (WBS), avant de lire la luminescence.

Figure 61 .

 61 Figure 61. Expression des gènes associés aux signalisations TGF-β (à gauche) et BMP (à droite) dans les muscles de l'ours hibernants.

Figure

  Figure simplifiée extraite de la publication Cussonneau et al., 2021 [351]. Schéma montrant les transcriptions des gènes impliqués dans les signalisations TGF-β et BMP du muscle vastus lateralis de l'ours brun et décrivant (1) leurs relations et (2) la différence de leurs niveaux d'expression entre les périodes d'hibernation et d'activité. Les cases rouges et vertes indiquent, respectivement, les gènes régulés à la hausse et à la baisse pendant l'hibernation par rapport à la saison d'été, et les cases blanches indiquent les gènes inchangés. Les gènes cibles des signalisations TGF-β et BMP sont indiqués en italique et sont en vert lorsqu'ils sont régulés à la baisse, en rouge lorsqu'ils sont régulés à la hausse, et en noir lorsqu'ils sont inchangés. Les flèches indiquent l'activation, et les barres ⊥ l'inhibition (n = 6 ours/saison, les mêmes individus ont été échantillonnés et analysés en été et en hiver). SBEs : SMAD Binding Element. Créé avec BioRender.com.

Figure 60 .

 60 Figure 60. Les niveaux protéiques de SMAD4 sont augmentés dans le muscle de l'ours brun hibernant. Les niveaux de la protéine SMAD4 ont été évalués par Western blots dans le muscle vastus latéralis des ours bruns pendant l'été (S) et l'hiver (W), et des Westerns blots représentatifs sont présentés pour trois couples d'ours. Les données sont représentées sous forme de valeurs individuelles avec des barres moyennes (n=11 ours/saison, les mêmes individus ont été échantillonnés et analysés en été et en hiver). Les points gris et bleus représentent les muscles des ours en été et en hiver respectivement.

Figure 63 .

 63 Figure 63. Le traitement à l'halofuginone inhibe la signalisation du TGF-β tout en favorisant la signalisation du BMP dans le muscle gastrocnémien chez les souris. Le même protocole expérimental qu'expliqué dans la légende de la figure 63 a été réalisé. Les niveaux protéiques relatifs du gastrocnémien pour les facteurs de transcription (B) SMAD2-3 (TGF-β), (A) SMAD1-5 (BMP) et (C) SMAD4 (TGF-/BMP) ont été évalués dans la fraction subcellulaire nucléaire, quantifiés et normalisés en utilisant le signal TGX. Des Western blots représentatifs pour les fractions subcellulaires nucléaires et cytosoliques sont présentés. Les données sont des moyennes ± SEM (exprimées par rapport aux souris H2O-Ctrl). Test statistique d'ANOVA à deux facteurs : ** padj <0,01 ; **** padj <0,0001. # padj (effet de l'HF) <0,05.

Figure 62 .

 62 Figure 62. Le traitement à l'halofuginone avant la suspension par le train arrière atténu l'atrophie du muscle gastrocnémien chez les souris.Les souris ont été traitées avec de l'H2O ou de l'halofuginone (HF, 0.25µg/g) 3 fois par semaine pendant 3 semaines et ont ensuite été soumises à une suspension du train arrière pendant 3 (HS3, barres hachurées) ou 7 (HS7, barres noires pointillées) jours ou non suspendues (Ctrl, barres grises). Masse du muscle gastrocnémien par gramme de poids corporel, les données sont des moyennes ± SEM (exprimées par rapport aux souris H2O-Ctrl). Test statistique d'ANOVA à deux facteurs : ** padj <0.01; **** padj <0.0001; ns= non-significatif ; # padj (effet de l'HF) <0.05.

Figure 64 .

 64 Figure 64. Le sérum d'ours d'hiver réprime la transcription du gène ID1 dans les cellules musculaires humaines. Des cellules CCL136 ont été transfectées avec le plasmide ID1-Luc et cultivées avec du sérum d'ours d'été (SBS, points gris) ou du sérum d'ours d'hiver (WBS, points noirs) pendant 6, 12 ou 24 heures, puis les cellules ont été lysées et l'activité de la luciférase a été mesurée. Les données sont présentées sous forme de valeurs individuelles avec des barres moyennes (n = 12 sérum d'ours/saison, les mêmes individus ont été échantillonnés et analysés en été et en hiver). La signification statistique est indiquée (test t apparié de ratio) * pvalue<0,05 ; ** pvalue<0,01 ; ns : non significatif.

  and Strasbourg University (H2E project; MyoBears project of the PEPS ExoMod program). MGX acknowledges financial support from France Génomique National infrastructure, funded as part of "Investissement d'Avenir" program managed by the Agence Nationale pour la Recherche (contract ANR-10-INBS-09). L.C. (Laura Cussonneau) was supported by a grant from the Institut National de la Recherche Agronomique et Environnement and Clermont Métropole. C.B. (Christian Boyerand) and C.B. (Charlotte Brun) were supported by grants from the Ministère Français de l'Enseignement Supérieur, de la Recherche et de l'Innovation. The long-term funding of Scandinavian Brown Bear Research Project (SBBRP) has come primarily from the Swedish Environmental Protection Agency, the Norwegian Environment Agency, the Austrian Science Fund, and the Swedish Association for Hunting and Wildlife Management. The study was conducted according to the guidelines of the Declaration of Helsinki and of the European Directive 2010/63/EU and approved by the Institutional Other data that support the findings of this study are available in the supplementary material of this article.

	(GSE144856).
	Institutional Review Board Statement: Review Board (or Ethics Committee) of (1) the Swedish Ethical Committee on Animal Experiment
	(applications Dnr C3/2016 and Dnr C18/2015), the Swedish Environmental Protection Agency (NV-
	0741-18), and the Swedish Board of Agriculture (Dnr 5.2.18-3060/17), and (2) the C2E2A (Comité
	d'Ethique pour l'Expérimentation Animale Auvergne) (D6334515-08/2018).
	Informed Consent Statement: Not applicable.

Table 6 . Bears features

 6

	Supplementary ID_number	Year of collection	Age (year)	Gender	Experiments
	w1305		2	F
	w1316	2014	2	M
	w1317		2	M	RNA
	w1509		2	F	sequencing
	w1511	2016	2	F
	w1512		2	F
	w1509 w1610	2017	3 2	F M
	w1709 w1710	2018	2 2	F F
	w1707		3	F
	w1802		2	M	Western Blot
	w1803		2	F
	w1806	2019	2	F
	w1812		2	M
	w1813		2	F
	w1814		2	M
	N = 17		2.117647059	Sex ratio : 11F/6M

  . Inhibition of myostatin in muscles can result in either increased bone formation under physiological conditions or decreased bone resorption under pathological conditions[410][411][412]. Interestingly, TGFB1 and MSTN are downregulated in hibernating bear muscles in our study (seePaper 6.3) [351] and previous studies have clearly shown that bears do not suffer from osteoporosis despite long-term inactivity, lack of food, and cold exposure during hibernation[413]. Furthermore, peripheral blood mononuclear cells collected from hibernating Japanese black bears and cultured with hibernating bear serum do not differentiate into osteoclasts, unlike cells cultured with active bear serum[414]. This study implies the presence of circulating compounds during hibernation that protect bone from resorption. Altogether, these observations raise questions about whether or not soluble bone and muscle factors are secreted during hibernation in bears. Because TGF-β/BMP pathways are ubiquitous throughout the body, their regulation in one place is likely to imply consequences in another place. Is it because bone resorption does not occur in bears during winter that muscle atrophy does not occur either, or is it the other way around (Figure37)?

Transcriptomic Data and Functional Pathway Enrichment Analysis for ATF4 pathway.

  Transcriptomicdata from the already published study 1 (seePaper 6.3) [351] are openly available in the GEO repository databases (bear: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi, reference number Plasmid. pGL3 BRE Luciferase was a gift from Martine Roussel & Peter ten Dijke (Addgene plasmid # 45126; http://n2t.net/addgene:45126; RRID: Addgene_45126) [430]. Two copies of the SMAD binding element present in the ID1 promoter are cloned into pGL3-MLP-luc minimal promoter vector.Transfection. CCL136 were transfected with 50 ng of DNA/well the day after seeding and HM cells with 1 µg of DNA/well at the end of the differentiation process, by adding respectively 5 or 100 µl of the transfection mixture described above (seeAppendix 10.1). We added a renilla luciferase plasmid (i.e.

	Pools	10.2.2 ATF4 signalling in hibernating bear muscles Bear ID_number Year of collection Age (year) Gender	Experiments
			1813	2020	3	F
	P1	1509 1701	2017 2018	2 2	F F
	SBE4-Luc was a gift from Bert Vogelstein (Addgene plasmid # 16495; http://n2t.net/addgene:16495; 1814 2019 2 M (GSE144856)). Genecards web-based portal was used to draw up an ATF4 gene set list, selecting genes with a score > 1.3. To identify the differentially expressed genes (DEGs) from this list, we selected a Winter/Summer fold change (FC) >|1.0| or <|1.0| with an adjusted p-value < |0.05| as cut-off RRID: Addgene_16495) [431]. This vector contains four copies of the SMAD binding site (GTCTAGAC) which are cloned into pBV-Luc. 1604 2018 3 F HM cell line, BRE plasmid transfection 1610 2017 2 M P2 1710 2018 2 F
	3XMEF2-luc was a gift from Ron Prywes (Addgene plasmid # 32967 ; http://n2t.net/addgene:32967 ; 1803 2019 2 F standards, for the up-and down-regulated genes, respectively. Visualization of functional enrichment was performed using Metascape [432], a web-based portal for visualizing the inference of enriched biological pathways and protein-protein interaction among the DEGs as described in study 1 (see Paper 1707 2019 3 F RRID:Addgene_32967). This plasmid was cloned from pFOS WT-GL3 (Addgene #11983), where the human c-fos promoter was removed and replaced with 4 MEF2 sites. 1608 2017 2 F P4 1806 2019 2 F
	10.2 Materials and Methods discussion 2 6.3) [351]. 1812 2019 2	M
	10.2.1 Complementary RT-qPCR and Western blots of study 2 Immunodetection of total and phosphorylated eIF2α protein. The total and phosphorylated eIF2α
	protein content has been assessed by Western blots in vastus lateralis muscle of bears, as previously
	described in study 1 (see Paper 6.3) [351] using eIF2α (#9722S, Cell Signalling) and p-Ser51eIF2α
	(#ab32157) antibodies diluted 1:1000 in 5% BSA.
			Gene			Forward	Reverse
	Atg5 Atg12	TCAACCGGAAACTCATGGAA CGGAACAGCTTCTGGATGAA TAAACTGGTGGCCTCGGAAC CCATCACTGCCAAAACACTCA 10.3 Materials and Methods discussion 3
	Atg16		TCCCGTGATGACCTGCTAAA CAGTCAGAGCCGCATTTGAA 10.3.1 Optimisation of tools for screening active compounds in WBS
	Map1lc3a Cell culture. CCL136 cells were cultured as described in Materials and Methods discussion 1, and GAGCGAGTTGGTCAAGATCA GGAGGCGTAGACCATGTAG
	Bnip3 seeded in 96-wells plates at a density of 2.0 × 10 4 cells/well. TCACTGTGACAGCCCACCTC GCTGTTTTTCTCGCCAAAGC
	Col1a1 Human myotubes (HM) were derived from vastus lateralis muscle biopsies obtained from healthy CGTGGTGACAAGGGTGAGAC AACCAGGAGAACCAGGAGGA
	Col3a1 Col5a2 control donors (Diomede experimental protocol). All procedures were approved by the French Ethical CTGCTGGTCCTTCTGGTGCT AGCCACGTTCACCAGTTTCA CCTGGTCCAAATGGTGAACA CCAGGGTTTCCTTCCTTTCC Committee SUD EST IV (Agreement #12/111A 13-02) and performed according to French legislation Bear ID_number Year of collection Age (year) Gender Experiments
	Col6a1 (Huriet's law). All patients gave their written consent after being informed of the nature, purpose, and CCACAACCAGATGCAAGAGC CACCAGCCATCCATTGTAGC w1601 2 M 2017 w1610 2 M
	possible risks of the study. The myoblasts were thawed from liquid nitrogen, directly platted into 6-w1604 3 F 2018 w1701 2 F well plates coated with collagen (Corning® BioCoat® Collagen I 6-well Clear Flat Bottom TC-treated Immunodetection of LC3 protein. The lipidation of the LC3 protein has been assessed by Western blots w1707 3 F
	in gastrocnemius muscle, as previously described in study 2 (see Paper 7.2) [423]. As LC3 protein has a pHi > 8.0, a CAPS (3-(cyclohexylamino)-1-propanesulfonic acid)-ethanol buffer (10 mM CAPS, 10% Multiwell Plate, Product Number 356400), and maintained in medium Ham's F10 (1g/L glucose, Dutscher) containing 1% penicillin-streptomycin (Gibco, USA) and 10% FBS (Gibco, USA). The cells were CCL136 cell line, BRE, SBE w1709 3 F or MEF2 plasmid transfection w1803 2 F 2019 w1806 2 F seeded in 12-wells plates at a density of 4 × 10 4 cells/well and cultured at 37 °C under 5% CO2. After ethanol, pH = 11) was used to optimise protein transfer. Blots were blocked for 1 h at room w1812 2 M
	w1814		2	M
	w1813 w1909	2020	3 2	F F

RT-qPCR.

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) of gastrocnemius muscle have been performed as previously described in study 2 (see

Paper 7.2) [423]

and the primers used are described in the following table

:

temperature in TBS buffer with 0.1% Tween-20 (TBS-T, pH = 7.8) with 5% non-fat dried milk according to the manufacturer's instructions. They were then washed thrice in TBS-T and incubated (overnight, stirring, 4°C) with LC3 antibody (#ab48394, Abcam) diluted 1:1000 with 5% non-fat dried milk. reaching 80% confluence, differentiation was triggered by replacing the previous medium with DMEM 1g/L glucose containing 2% FBS for 5 days. The differentiation media were changed every other day. 0,25 ng for CCL136 and 5 ng for HM) in the transfection mixture for intra-well luminescence normalisation Bear serum treatment. After one night of transfection, the media have been replaced by fresh media containing 5% winter or summer bear serum instead of 5% FBS for 6, 12 or 24 hours. CCL136 cells were treated with the serum of 12 individuals bears for each season. HM cells were treated with 3 mixes of bear serum for each season. Bears characteristics for individuals sera or for pools of sera are described in the table below. Cells were then washed with PBS and lysed with the passive lysis buffer from Dual-Luciferase® Reporter Assay System 100 assays E1910 (Promega, Charbonnières-les-Bains, France) by adding 20 µl for CCL136 or 250 µl for HM cells. Luminescence reading. In the Dual-Luciferase® Reporter (DLR™) Assay System, the activities of firefly (from BRE-Luc, SBE-Luc or MEF2-Luc plasmids) and renilla luciferases are measured sequentially from a single sample. 10 µl of the lysed cells were added in each well of a 96-well flat-white plate. Thereafter, the plate was placed into the plate-reading Synergy™ 2 luminometer (Biotek, Colmar, France) equipped with two reagent injectors. The firefly luciferase reporter was measured first by adding 50 µl of Luciferase Assay Reagent II (LAR II) in each well. After quantifying the firefly luminescence for 12 seconds, this reaction was quenched, and the renilla luciferase reaction was simultaneously initiated by adding 50 µl of Stop & Glo® Reagent to the same well. The Stop & Glo® Reagent also produces a stabilized signal from the renilla luciferase, which decays slowly over the course of the measurement.

Table 1 .

 1 Phenotypes of transgenic mice for genes encoding ubiquitin ligases involved in the control of muscle mass and function.

	Gene Product	E3 Family	Mouse Model	Phenotype	References
			E3 ligases regulating the anabolic pathways
	CBL-B	RING	KO	Protection from unloading-induced muscle atrophy and dysfunction	[171]
	FBXO40	RING	KD KO	Myofibers hypertrophy Muscle hypertrophy	[22]
	NEDD4-1	HECT	OX	Myocardial activation of AKT during I/R	[176,177]
			KO	Partially resistant to denervation-induced skeletal muscle atrophy	[178]
			E3 ligases regulating the catabolic pathways
	TRAF6	RING	m.KO m.KO	Resistance to starvation induced muscle atrophy Resistance to denervation-induced loss of muscle mass and function	[179] [180]
	cIAP1	RING	KO	Limitation of denervation-induced muscle atrophy	[19]
			OX	Myotube atrophy
	WWP1	HECT	KD	Muscle fiber atrophy	[181]
	TRIM32	RING	KO DN	Muscular dystrophy Muscular dystrophy	[182] [183]
			Other E3 ligases involved in the control of muscle mass and function
	MuRF1	RING	KO	Resistance to catabolic-induced muscle atrophy	[4]
	MAFbx	RING	KO	Resistance to catabolic-induced muscle atrophy	[4]
	PARKIN	RBR	KO	Impaired mitochondrial function and muscle atrophy	[184]
			OX	Increased muscle mass and function in young and old mice	[185]
			OX	Prevention of sepsis-induced muscle atrophy	[186]
	SMART/FBXO21	RING	KD	Resistance to denervation-induced muscle atrophy	[187]
	MUSA1/FBXO30	RING	KD	Resistance to denervation-induced muscle atrophy	[77]
	FBXL21	RING	HM	Impaired muscle functions	[188]
	UBR4	HECT	KD	Muscle hypertrophy	[189]
	UBR5	HECT	KD	Muscle atrophy	[190]

DN, Dominant Negative mutation; HM, Hypomorphic Mutation; I/R, Ischemia/Reperfusion; KD, knock-down mutant; KO, Knock-out mutant; m.KO, skeletal muscle-specific KO mice; OX, overexpressing mutant; PTEN, Phosphatase and tensin homologue.

Table 2 .

 2 Treatments influencing E3 ligases expression and/or activity.

	E3 Ligases Inhibited	Molecule	Mode of Inhibition	Signal inhib-ited/Activated	Efficiency on E3 Ligases	Efficiency on Muscle Mass	References
			Indirect inhibition of E3 ligases
	MuRF1/MAFbx Expression	4-aminopyridine (4-AP)	K+-channels blockade	K+-channels blocking	Yes	Yes	[278]
			Notch1, Notch3
	MuRF1 Expression	AGT251	expression	NOTCH	Yes	Yes	[161]
			inhibition
	MuRF1/MAFbx/ MuSA1 Expression	Anti-TLR2	IKK2 (NF-κB)	TLRs Serum Amyloib A1	Yes	Yes	[256]
	MuRF1 Expression	Anti-TLR4	IKK2 (NF-κB)	TLRs Serum Amyloib A1	Yes	Yes	[256]
	MuRF1/MAFbx/ MuSA1 Expression	BMS-345541	IKK2 (NF-κB)	TLRs Serum Amyloib A1	Yes	Yes	[256]
	MuRF1 expression	C188-9	STAT3 inhibition	STAT3 signaling	ND	Partially	[14]
	MuRF1/MAFbx Expression	Clenbuterol	AKT-FOXO axis	Activation of PI3K-AKT	Yes	Yes	[15]
	MuRF1 not MAFbx	Dehydroepiandros-terone (DHEA)	ND	ND	Yes	Yes	[168]
	MuRF1 Expression	Epigallocatechin-3-gallate/EGCG	ND	NF-κB	Yes	Yes	[279]
	MuRF1 Expression	Espindolol	ND	Myostatin and NF-κB	Yes	Yes	[280]
	MuRF1/MAFbx Expression	Formoterol	ND	ND	Yes	Yes	[276]
	MuRF1/MAFbx Expression	Formoterol	AKT/mTORC1/ FOXO1	ß2 Adrenergic receptor?	Yes	Yes	[277]
	MuRF1/MAFbx Expression	Formoterol	ND	AKT and NF-κB	Yes	Yes	[281]
	MuRF1/MAFbx Expression	HMB	IL-6 receptor inhibition	NF-κB	Yes	Partially	[275]
	MuRF1 expression	HMB or Leucine	FOXO1 nuclear translocation	Glucocorticoid	Yes	No	[282]

Table 2 .

 2 Cont.

	E3 Ligases Inhibited	Molecule	Mode of Inhibition	Signal inhib-ited/Activated	Efficiency on E3 Ligases	Efficiency on Muscle Mass	References
	Cbl-b activity	IRS1 peptide mimetic	Cbl-b targeting	Activation of PI3K-AKT	Yes	Yes	[171]
	MuRF1/MAFbx Expression	Leucine	ND	FOXO3a and VPS34 nuclear translocation	Yes	Yes, myotube diameter	[283]
	MuRF1/MAFbx Expression	Matrine	AKT/mTORC1/ FOXO3α	FOXO3a and translocation VPS34 nuclear	Yes	Yes	[284]
	MuRF1 expression	MR16-1	Anti-IL-6 receptor	NF-κB	Mitigated	No	[275]
	MuRF1 expression	N-acetyl cysteine	ROS	TGF-ß	Yes	Yes	[69]
	MuRF1/MAFbx Expression	Pyrroloquinoline quinone (PQQ)	ROS	ND	Yes	Yes	[285]
	MuRF1/MAFbx Expression	RU486	GR	Glucocorticoid	Yes	ND	[13]
	MuRF1 Expression	Sabinene	ROS	ERK, p38 MAPK	Yes	Yes	[286]
	MuRF1/MAFbx Expression	sActRIIB	ActRIIB antagonist	SMADs	Yes	Yes	[16]
	MuRF1/MAFbx/ MuSA1 Expression	Salicylate	IKK2 (NF-κB)	NF-κB	Yes	Yes but toxic	[89]
	MuRF1/MAFbx Expression	SS-31	ROS	No	Yes	Yes	[287]
	MuRF1/MAFbx Expression	Teaghrelin	ND	Myogenin	Yes	Moderate	[288-290]
	MuRF1/MAFbx Expression	TNF-BP	TNF binding	TNF	Yes	ND	[13]
	MuRF1/MAFbx/ MuSA1 Expression	Ursolic acid	ND	Myostatin and cytokines inflammatory	Yes	Moderate	[255]
	MuRF1/MAFbx Expression	Vitamin E	ND but seems ROS independent	Unknown	Yes	Moderate	[291]
	MuRF1/MAFbx Expression	Vitamin-D	IL-6 receptor inhibition	NF-κB	Yes	Partially	[275]
	MuRF1 expression	VX-745/Neflamapimod	p38α MAPK	p38α MAPK	Partially	Moderate	[292]
			Direct inhibition of E3 ligases
	MuRF1 expression	ID#704946/MyoMed-946	ND	MuRF1 Expression	Yes	Partially	[293]
	MuRF1 Expression	ID#704946/MyoMed-946	ND	MuRF1 and Expression MuRF2	Yes	Partially	[17,18]
	MuRF1 and MuRF2 Expression	MyoMed-205	ND	MuRF1 expression			[17]
	MuRF1 activity	P013222	MuRF1 targeting	-	Yes	ND	[294]
	cIAP1 (activity??)	LCL161	cIAP1	NF-κB	Yes	Very moderate	[19]

* schemas created with BioRender.com

glutathione/oxidized glutathione ratio

Int. J. Mol. Sci. 2023, 24, 621. https://doi.org/10.3390/ijms24010621 https://www.mdpi.com/journal/ijms

Molecules 2021, 26, 407. https://doi.org/10.3390/molecules26020407 https://www.mdpi.com/journal/molecules

71 6.3 Paper Acknowledgments: The authors thank the field capture team (D. Ahlqvist, A. Friebe, H. Nordin, H. Blomgren, and S. Persson) and the IEN (INRAE Clermont-Ferrand-Theix, France) for excellent assistance with mouse care (A. Cissoire, Y. Delorme, and M. Djelloul-Mazouz), and L. Parry for help during animal slaughtering. The authors are also grateful to T. Brioche (Unité Mixte de Recherche INRAe, Dynamique Musculaire et Métabolisme, Université de Montpellier, France) for his help in implementing the unloading model in mouse and F. Rocher for helpful discussions. This is paper N • 317 of the Scandinavian Brown Bear Research Project. Acknowledgments: The authors thank the IEN (INRA Clermont-Ferrand-Theix, France) for the technical assistance with animal care. We are also grateful to Bogdan Vulpescu (Laboratoire de Physique de Clermont, UMR6533) for the helpful discussions regarding the statistical treatment of fibre CSA distribution. Acknowledgments: This work was supported by the French Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE). Laura Cussonneau is supported by Clermont-Auvergne-Métropole and Dulce Peris-Moreno by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 813599.

Acknowledgments

The authors wish to thank the field capture team (D Ahlqvist, A Friebe, H Nordin, H Blomgren, S Persson), and are grateful to Hélène Choubley and Victoria Bergas from the lipidomic platform of the university of Bourgogne-Franche-Comté for their valuable technical assistance. This is scientific paper no. 296 from the SBBRP.

Funding: This work was supported by the Center National d'Etudes Spatiales (CNES, #865, #974, #1006, #1905), the iSITE Challenge 3 Mobility program (Université Clermont Auvergne), and the Funding This work was supported by the French Space Agency (CNES), iSITE Challenge 3 Mobility program (UCA), CNRS and Strasbourg University (H2E project; MyoBears project of the PEPS ExoMod program), French Proteomic Infrastructure (ProFI; ANR-10-INSB-08-03, and MetaHUB (French infrastructure in metabolomics & fluxomics; ANR-11-INBS-0010). CBo was supported by a grant from the MESRI, LCu by grants from the INRAE and Clermont Metropole and CBr by a grant from French space agency (CNES). The long-term funding of Scandinavian Brown Bear Research Project (SBBRP) has come primarily from the Swedish Environmental Protection Agency, the Norwegian Environment Agency, the Austrian Science Fund, and the Swedish Association for Hunting and Wildlife Management.

Data Availability Statement:

The analyzed transcriptomic mouse data that support the findings of this study are openly available in the GEO repository database at https://doi.org/10.1093/gerona/ gly051, accessed on 16 June 2021, reference number (GSE102284). The generated transcriptomic bear data that support the findings of this study are openly available in the GEO repository database at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi, accessed on 16 June 2021, reference number Cells 2021, 10, 1873 Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Availability of data and materials

The datasets generated during and/or analyzed during the current study available from the corresponding author on reasonable request.

Supplementary Materials:

The following are available online at https://www.mdpi.com/article/10 .3390/cells10081873/s1, Figure S1, Tables S1-S6, and Blots quantification.

Conflicts of Interest:

The authors declare no conflict of interest.

Paper

Conflicts of Interest: The authors declare no conflict of interest.

Supplementary Figure S1

Supplementary Figure S1. Effect of halofuginone treatment on muscle mass. Mice were treated with H2O (white bars) or HF (0.25 µg/g, grey bars) 3 times a week up to 4 weeks (WK) as described in

Supplementary Figure S2

Supplementary Figure S2. ATF4-regulated alternative target genes expression in muscle during hindlimb suspension. Mice were treated with H2O or halofuginone (HF) oral administration (0.25µg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 or 7 days (HS3 and HS7, light grey and white bars, respectively) or kept unsuspended (Ctrl, dark grey bars).

Supplementary Figure S3

Supplementary Figure S3. Effects of halofuginone treatment prior to hindlimb suspension on skeletal muscle. Mice were treated with H2O or halofuginone (HF, 0.25µg/g) 3 times a week for 3 weeks and were then subjected to hindlimb suspension for 3 or 7 days (HS3 and HS7, respectively) or kept unsuspended (Ctrl) as described in Figure 2A

Supplementary Figure S5

Supplementary Table S1: Primers Gene Forward Reverse Atf4

E.

A.

B.

C. D.

F.

G.

TGX

A. B.

Figure 46. Worldwide geographic repartition of (A) Hydrangea macrophylla and (B) brown bear living area (Ursus arctos).

(A) Map of Hydrangea macrophylla geographic repartition found on https://identify.plantnet.org/fr/the-plant-list/species.(B) Map of brown bear living area repartition found on https://databayou.com/bear/habitat.html.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the writing of the manuscript.

Abbreviations

Paper

Supplementary Information

The online version contains supplementary material available at https://doi. org/10.1186/s12983-020-00380-y.

Additional file 1 Table S1. Characteristics of brown bears included in the study. Table S2. Serum fatty acid concentrations (mmol/L) in winter hibernating (WBS) and summer active (SBS) bears. Table S3. Serum fatty acid relative proportions (mol %) in winter hibernating (WBS) and summer active (SBS) bears. Table S4. List of primers used for RT-qPCR.

Ethics approval

All samples and data were collected under protocols approved by the Swedish Ethical Committee on Animal Experiment (applications Dnr C3/2016 and Dnr C18/2015), the Swedish Environmental Protection Agency (NV-00741-18), and the Swedish Board of Agriculture . All procedures complied with Swedish laws and regulations.

Competing interests

The authors declare no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. [193] (Figure 54). A ce jour, quatre kinases sont connues pour phosphoryler eIF2α, à savoir PERK 54 , 53 integrated stress response 54 PKR-like ER kinase

B. A.

Figure 53. Compétition pour le recrutement de SMAD4 en conditions physiologiques (A) ou cataboliques (B).

DPM : dégradation protéique musculaire ; SPM : synthèse protéique musculaire.

225

PKR 55 , HRI 56 , et GCN2 57 [193] (Figure 54). P-eIF2α conduit à deux conséquences : (1) une inhibition générale de la machinerie traductionnelle et (2) la traduction d'ARNm spécifiques dont ATF4 58 [193] (Figure 54). ATF4 est un facteur de transcription qui agit principalement comme un activateur transcriptionnel d'une cohorte de gènes impliqués dans l'adaptation au stress cellulaire en réponse à l'activation de l'ISR [193] (Figure 54). ATF4 produit des réponses distinctes en fonction du stress cellulaire. Par exemple, lors d'un stress nutritionnel, ATF4 stimule l'expression de gènes impliqués dans le transport et la biosynthèse des AA, et l'autophagie pour fournir de nouveaux AA pour la synthèse de novo des protéines. De plus, ATF4 induit la transcription de PPP1R15A 59 (la protéine GADD34), la principale phosphatase d'eIF2α, agissant comme une importante boucle de rétrocontrôle négatif pour restaurer la synthèse protéique une fois le stress résolu [193] (Figure 54). Il a été proposé que la durée et l'intensité de la signalisation de l'ISR dicteraient la résultante biologique cellulaire. Par conséquent, ATF4 peut également faciliter l'exécution d'un programme transcriptionnel de mort cellulaire par apoptose lorsque l'homéostasie cellulaire ne peut être restaurée, en induisant la transcription de gènes apoptotiques [193] (Figure 54). 55 double-stranded RNA-dependent protein kinase 56 heme-regulated inhibitor 57 general control nonderepressible 2 58 activating transcription factor 4 59 protein phosphatase 1 regulatory subunit 15A de souris [99,207] (Figure 55). GADD45A est une protéine myonucléaire qui réprime des gènes impliqués dans l'anabolisme et induit des gènes impliqués dans le catabolisme (Figure 55). ATF4 est nécessaire et suffisante pour induire l'expression de Gadd45a en condition d'atrophie musculaire induite par le jeûne et l'immobilisation [207]. L'augmentation de Cdkn1a (protéine P21) dans les muscles de souris, est nécessaire et suffisante pour induire une atrophie par immobilisation médiée par ATF4 [207]. ATF4 induit également l'expression du gène Eif4ebp1, codant pour l'inhibiteur de la synthèse protéique 4E-BP1 [207] (Figure 55). Enfin, un autre gène cible d'ATF4, TRIB3 64 , a été associé à l'atrophie musculaire dans de nombreuses études. Par exemple, les souris déficientes pour Trib3 sont en partie résistante à l'induction de l'atrophie par le jeûne. À l'heure actuelle, les mécanismes par lesquels ATF4 est activé sur le plan transcriptionnel et traductionnel dans des conditions cataboliques ne sont pas clairs, mais peuvent impliquer différents mécanismes ou combinaisons de mécanismes.

Le modèle de résistance naturelle à l'atrophie musculaire de l'ours brun hibernant

Le biomimétisme est une approche qui cherche des solutions durables aux défis humains en imitant les modèles et les stratégies de la nature, ce qui a déjà permis des avancées et des progrès biomédicaux humains significatifs. L'hibernation est un parfait exemple de variabilité saisonnière pouvant receler des indices et solutions diverses pour les pathologies humaines.

Généralités de l'hibernation chez les ours. L'hibernation est une adaptation utilisée par certains

animaux pour faire face à un manque épisodique ou saisonnier d'énergie dû à des conditions environnementales défavorables (par exemple, faible disponibilité de nourriture/eau, forte pression de prédation) [273]. La torpeur est au coeur de l'hibernation, elle représente une période de suppression métabolique qui peut durer de quelques heures à plusieurs semaines. L'hibernation est un comportement plus élaboré, structuré en plusieurs longues périodes de torpeur souvent séparées par de brèves périodes d'éveil (IBA 65 ). Les IBA durent environ 24 heures et sont présents chez presque tous les petits hibernants (c'est-à-dire <10kg) mais pas chez les ours hibernants [273].

Les ours sont des mammifères de la famille des Ursidae. Les ours des climats chauds n'entrent pas en hibernation, pas plus que le panda géant ou l'ours polaire. Dans ce projet de thèse, nous nous sommes concentrés sur les ours hibernants, c'est-à-dire les ours bruns (Ursus arctos), les ours noirs américains 64

Abstract

Muscle wasting affects millions of people around the world, including the elderly, people with illnesses, and people who are immobilised for long periods of time. The loss of muscle mass leads to a decline in independence, promotes disease, increases resistance to treatment, and is associated with increased mortality. As a result, muscle wasting is a major public health problem. Many biomolecular mechanisms have been documented to explain the occurrence of muscle wasting, mainly through the use of laboratory rodent models. However, no treatment is really effective and/or adaptable for all today. The main objective of this thesis was to find new underlying mechanisms that could become therapeutic targets to combat muscle atrophy in humans. We chose an approach based on biomimicry.

Our strategy was (1) to perform a comparative physiology study between the brown bear model naturally resistant to atrophy during hibernation and the unloading mouse sensitive to muscle atrophy, (2) to study the role of the ATF4 and TGF-β/BMP signalling pathways in these two models, and finally (3) to initiate studies on human muscle cells to validate the hypotheses from the first two studies. In our first study, the strategy was to identify genes differentially regulated in brown bear muscle between the hibernation and active periods. Then we compared them to those differentially regulated in the muscles of the unloading mouse versus the control mouse. We showed that the concomitance of inhibition of TGF-β signalling and induction of BMP signalling appeared to be crucial for the maintenance of muscle mass under conditions of prolonged physical inactivity. In our second study, we showed that the induction of the ATF4 signalling pathway in muscle was uncoupled from muscle atrophy in healthy and physically inactive mice when previously treated with the halofuginone molecule, and also in hibernating bears. In all three situations, the maintenance of muscle mass was associated with both the induction of ATF4 and BMP signalling and the inhibition of TGF-β. Finally, preliminary results obtained by cultivating human muscle cells with hibernating brown bear serum suggest the presence of a circulating active compound that may mimic some of the characteristics observed in atrophy-resistant hibernating brown bear muscle. In conclusion, this work provides numerous perspectives in the modulation of the balance of TGF-β and BMP signalling pathways in situations of prolonged physical inactivity. In addition, it opens up new research on the identification of active compounds in bear serum that could be used in the human clinic to limit or prevent the onset of muscle atrophy during immobilisation or in other pathophysiological conditions.