Pauline Henrot
email: pauline.henrot@u-bordeaux.fr

Léo Blervaque

Isabelle Dupin

Maéva Zysman

Pauline Esteves

Fares Gouzi

Maurice Hayot

Pascal Pomiès

Patrick Berger

Leo Blervaque

Pascal Pomies

Patrick Berger1

Cellular interplay in skeletal muscle regeneration and wasting: insights from animal models

Keywords: Sarcopenja, Cachexia, Myofibres, Neutrophils, Fibrocytes

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Skeletal muscle wasting is a prevalent condition, associated linked to an imbalance between anabolism and catabolism with increased hospitalizations and disability, and is thus a in myocytes 1 . ln terms of lexical field, it encompasses both sarcopenia, which is currently defined by a loss of both muscle mass (without body weight loss) and force, 2 and cachexia, which reflects an active state of body weight loss predominantly linked to skeletal muscle loss and associated with an underlying condit.Ion. 3 There is indeed a vast panel of conditions associated with muscle wasting, ranging from physiological ageing or muscle disuse to chronic diseases such as cancer (the most well-known cause of cachexia), but also metabolic and cardiovascular diseases (diabetes, chronic kidney disease, heart failure), chronic inflammatory diseases such as rheumatoid arthritis, infectious diseases (acquired immunodeficiency syndrome, sepsis) and respiratory diseases such as chronic obstructive pulmonary disease (COPD). 4 In COPD in particular, a chronic respiratory disease mainly linked to cigarette smoke exposure, muscle wasting affects lip to 35% of the patients5 and is associated with a dramatic increase in mortality even after matching for respiratory function. 6,7 Given the high prevalence of COPD (around 10%) as well as the fact that it currently represents the third cause of death in the world, 8 studying the cellular basis behind COPD muscle alterations is of particular interest.

In addition to being associated with several very different diseases, muscle wasting appears to be heterogeneous in terms of phenotypes or histolo8lcal and molecular alterations. This might be due to the multifactorial origin of muscle loss. For example, in COPD, muscle wasting can be attributed to both extrinsic factors (deconditioning, repeated corticosteroid use, cigarette smoke exposure, malnutrition) and intrinsic COPD-bound factors (inflammation, hypoxia, oxidative stress). 9 Overall, these factors lead to an imbalance between protein synthesis and degradation in myocytes, in favour of increased catabolism. Affected pathways are well characterized: Catabolism includes the myostatin pathway (signalling through activin type 11 receptor), nuclear factor-kappa a (NF-kB) pathway and angiotensin and glucocorticoid receptors, all leading to increased ubiquitin-proteasome system activity (via the transcription factor forkhead box 0 (FOXO)), autophagy and apoptosis. 10 .Aabolism mainly includes the insulin-like growth factor 1 (lGF1) receptor, signalling via AKT and mammalian target of rapamycin (mTOR}.

However, the initial trigger behind this increased catabolism n myocytes remains unclear. In this context, taking into account the role of tissular resident skeletal muscle cells other than myocytes appears essential. Recent molecular biology techniques such as single-cell RNA sequencing have allowed a complete characterization of the resident cell populations in skeletal muscle, as well as unravelling new populations 11,12 . Indeed, , although satellite cells are at the core of myonuclear turnover and muscle regeneration, they interact with many other cells in a finely tuned process. However, muscle wasting is also a highly dynamic process which investigation is hampered by the differential cellular and protein regulation according to the stage Of the disease, as illustrated by the shift in macrophages type in muscle repair after injury. 13 As skeletal muscle is not so easily accessible, and repeated muscle biopsies are not always possible in patients already suffering from physical limitations, animal models appear essential to study the dynamics of muscle wasting, which allow access to every muscle as well as time-course studies. Several models are available for muscle wasting, whether primarily devoted to muscle loss/repair or related to the underlying condition. 14 Here, we will discuss the potential role of tissular resident cells interacting with myocytes within skeletal muscle using data from animal models of muscle wasting. We will discuss the role of and cellular cross-talk between progenitor, Immune and vascular cells as well as other constitutional cells such as neural cells and report data concerning their role in homoeostasis and regeneration as well as several sarcopenia models. We also focus our attention on fibrocytes, whose role has recently been highlighted in COPD lung by our team, 15,16 and sum up the current knowledge about the cellular interplay leading to muscle wasting in COPD. Finally, we present future promising leads for research, including the use of muscle organoids.

A summary of the respective role of each cell, as well as the preferential animal models used to study this role, can be found in Table 1.

Progenitor cells

Satellite Cells (SCS| are Skeletal muscle resident stem cells residing beneath the basal lamina of myofibres, quiescent at the steady state but upon activation, able to proliferate, differentiate and fuse with myofibres. They are thus responsible for both muscle maintenance via rnyonuclear turnover and functional muscle repair following damage. Their differentiation comes along with successive up-regulation and down-regulation of key transcription factors: The paired box transcription factor Pox7and the myogenic regulatory factors (MRFs) MyoD and Myr5 are required for myogenic determination (early stage), whereas the MRF myogenine is required for differentiation and MFF4 for myotube maturation (late stages). 17 This delicate balance is mediated by a cross-talk between the MRFs and the cell cycle regulators. Interestingly, it has recently been shown that a subset of SCs also express Pox3 and are resistant to environmental stress such as pollution. 18 SCs also participate to physiological muscle regeneration after endurance training, with a metabolic reprogramming favouring lower mitochondrial respiration and oxygen consumption. 19 Indeed, regular exercise increases SC pool and contribution to myofibres. 20 Concerning muscle regeneration Specifically, the crucial and no-redundant role Of SCs has been demonstrated by many studies, both in humans and animal models. 21 During regeneration, SCs also benefit from a reciprocal support of resident muscle cells such as fibroblasts 22 or endothelial cells (see below and Christov et al.,23 ). Recent data tend to precise the role of SC activation upon injury in the muscle tissue, as well as their plasticity. For example, I.t has been shown in a mouse model of cardiotoxin-induced injury that the muscle damage enabled in vivo reprogramming Of SCs to pluripotency. 24 Very interestingly, muscle repair was dependent on the prior local accumulation of tissular senescent cells and their Senescent-associated secretory phenotype, among which interleukin-6 {IL-6) plays a prominent role. This interesting observation counteracts the general idea that senescent cells are deleterious for tissue repair (see below).

In disease, SCs can be altered both in number and activity. Ageing mice rnodel5 indeed clearly point out a decrease in the SC pool2S Whether this is primarily linked to an intrinsic defect in SCs, such as alterations in genomic integrity and metabolic regulation, or alterations in the muscle micro-environment (stem cell niche) remains debated. On the one hand, engraftment of human SCs from both young and old donors in mice led to impaired renewal of the quiescent stem cell population of elderly donors, which was attributed to an increase in global DNA methylation, contributing to stem cell exhaustion. 26 Studies in mice models Of ageing also showed intrinsic defects in SCs, Such as pl6{lNK4a)/Rb-driven Stem cell senescence. S1 One the other hand, engraftment of post-mortem human muscle tissue from both young and old deceased donors into immunodeficient mice showed that SCs retained their regenerative capacity, 27 However, contribution of non-myogenic stem cells such as fibro-adipogenic progenitors (see below) cannot be excluded.

Overall, muscle micro-environment seems to play a prominent role in the muscle wasting process. Indeed, SCs isolated from cachectic muscles differentiate faster in vitro than controls. 28 Factors associated with the NFB pathway, such as pro-inflammatory cytokines {tumor necrosis alpha (TNF-), angiotensin Ill or myostatin are classically incriminated. 29 One study showed that enhancement of growth differentiation factor 11 (GDF11) circulating content, either by administrating recombinant protein or by parabiosis, could restore genomic integrity of SCs by reducing DNA damage, as well as their myogenic potential assessed by the number of myogenic colonies in vitro.3° Interestingly, no change in SC proliferation was observed. This study further showed that GDF11 could act through enhancement of mitochondrial biogenesis. The functional improvement opens therapeutic possibilities for GDFll. Oxvtoc.in was also pointed out as a promising candidate improving aged muscle regeneration. S2 Data from several other atrophy models such as denervation or cancer cachexia models suggest that impaired regulation of SCs and their ensuing myogenic program ls an important contributing factor to the muscle wasting process (for review, see Biressi and Gopinath 31 ). The early phase of myogenesis can be affected, with a decreased proliferation observed (n a chronic kidney disease, linked to a reduced insulin growth factor-1 (lGF-1) signalling. 32 0f note, differentiation was also impaired in this study. In addition, observational and experimental data from cancer models support the fact that differentiation is mainly impaired in atrophying muscle, 29 as an initial increase in Pax7+ cells 33 suggests that proliferation is not the limiting factor here. Blockade of the activin type 11 receptor pathway leads to active proliferation of SCs in vivo in mouse model of cancer-induced cachexia, 34 emphasizing both a preserved proliferative capacity and the mutual cross-talk between myocytes and SCs (Figure 1). The increase in Pax7 expression also reflects an increase of assisting non-SC populations, such as fibro-adipogenic progenitors (FAPs) and pericytes, which seem to undergo a lineage switch and adopt myogenic fate 35 (Figure 1). Of note, the number of SCs is reduced in latestage atrophy in both human and rodent models, 36 emphasizing the dynamic process of muscle wasting and the fact that timing is crucial, both to determine underlying mechanisms and as a therapeutic prospect. Finally, another potential cause for muscle atrophy is a defect of SC fusion with myoblasts, as observed in a denervation model. 36,S3 In another similar model, the decreased fusion was attributed to a lack of up-regulation of M-cadherin transcription. S4 A decreased fusion was also observed in vitro in senescent myob|asts. 37 Immune cells take part in muscle repair, initially by phagocyting debris (M1-like macrophages and neutrophils, coordinated by CD8+ T cells); at a later stage, they activate muscle regeneration through satellite cells proliferation, migration and differentiation |promoted by M2-like macrophages as well as T regulator lymphocytes (TRegs)). The vascular compartment also plays a n important role by regulating muscle nutrient uptake. Moreover, endothelial cells and pericytes carl promote both an8iogenesis and muscle regeneration, as well as recruit inflammatory cells. In addition, pericytes have been reported to undergo myogenic differentiation` Telocytes, which have been identified close to satellite cells, could activate their differentiation into myoblasts. Fibro-adipogenic progenitor cells (FAPs) Interact with immune cells and satellite cells to remote skeletal muscle regeneration. Finally, satellite cells are essential to muscle regeneration, by proliferating and differentiating into myotubes, which later fuse and give rise to functional myofibres.

Fibro-Adipogenic progenitor cells (FAPs) are mesenchymal extra-laminal stromal cells that are usually quiescent unless induced by injury or intense exercise. They can be identified thanks to their expression of stem cell antigen 1 (Sea-1) and platelet-derived growth factor receptor alpha |PDGFR), and their phenotypical plasticity allows them to differentiate into adipocvtes or myofibroblasts according to the environment. 38 Although they are quiescent at the steady state, FAPs are required for homoeostatic muscle maintenance, 39,40 notably by secreting extracellular matrix (ECM) components. In muscle injury repair, they play a physiological role by promoting first phagocytosis and later regenerative fibrogenesis by producing ECM, in a delicate interplay with macrophages and lymphocytes. 41 Failure of T regulator lymphocytes (Tregs) recruitment has notably been attributed to absence of lL-33 signalling by senescent FAPs. 42 They also sustain SC regenerative potential in a paracrine manner by secreting lL-6 and WNT1-inducible signalling pathway protein 1 (WISP1/CCN4) to promote their proliferation and follistatin and lL-10 to promote their differentiation. 38 ln turn, they are controlled by several surrounding cells, including macrophages via transforming growth factor-a (TGF-a) production (promoting their differentiation towards fibroblasts), myocytes via lL-15 secretion (inhibiting their adipogenic differentiation) and eosinophils producing lL-4 and lL-13 (also inhibiting adipogenic differentiation). 38 IL4 even directly promotes fibrogenic differentiation, Supporting myogenesis by clearing out necrotic debris. 43 In acute muscle injury, FAPs are thought to be activated at least partly by lL-4 produced by recruited eosinophils. IL-4/lL-13 signalling further promotes proliferation of FAPs to support myogenesis while inhibiting their differentiation into adipocytes. 43 This physiological response is disrupted in the case of chronic trauma or disease such as Duchenne muscular dy5trophy, where FAPs are responsible for prolonged connective tissue production and muscle fibrosis. 44 Moreover, CD34-/-' mice suffer from more severe muscle atrophy induced by hypoxia than wild-type mice, and this seems to be at least partly mediated by a lower number of FAps. 45 Overall, the role Of FAPs in skeletal muscle tissue has mainly been characterized in regeneration, where they take part in both phases by secreting growth factors and cytokines.

In other diseases, they are mainly associated wi.th muscle fibrosis, for example, in models of chronic kidney disease, upon stimulation by myostatin. S5 ln this study, myostatin was responsible for the transition of FAPs to a fibroblast-like phenotype. Importantly, specific deletion of FAPs leads to a premature ageing phenotype with sarcopenia features, 40 They have also been associated with skeletal muscle wasting in the context of obese mice models by promoting fibre-adipogenic tissue replacement in the diaphragm. S6 Quite interestingly, transplanting intra-peritoneally senescent FAPs into mice is sufficient to induce a sarcopenialike functional phenotype using functional outcomes such as decreased grip strength and walking speed. At the molecular level, the authors observed an increase in quadriceps muscle TNF- and lL-6. 46 0f note, senolytics `cocktail' using dasatinib and quercetin improved significantly the functional parameters and the mice lifespan.

Vascular cells

Endothelial cells

Endothelial cells (ECs) in healthy muscle act as sensors for metabolic demand to induce upstream arterial dilation, providing a blood flow perfectly matched to oxygen and nutrient requirements. This active hyperaemia accounts for a large part of the oxygen convective supply capacities and is as such a major determinant of muscle oxygen uptake.47 Moreover, muscle glucose uptake has also been strongly related to the endothelial insulin sensitivity. 48 EC5 are thus critical in muscle adaptation to exercise training, with a pivotal role in muscle angiogenesis. However, besides their role in substrate delivery, few studies investigate ECs in skeletal muscle homoeostasis. In contrast, the role of skeletal muscle microvasculature has been well described, notably physiological angiogenesis such as exercise and altitude induced. 49 Indeed, there is a complex interplay between the muscle hypertrophic process and angiogenesis (Figure 1), also referred to as the myogenic-endothelial cross-talk. The acute and transient hypoxia generated by exercise seems to be responsible for vascular endothelial growth factor (VEGF) secretion, notably within myofibre-derived extracellular vesicles, 50 which stimulates angiogenesis in a HIF-1a-dependent mamer. S7 Moreover, exercise induces PGC-1a expression, which induces indirectly MCP-1 secretion by macrophages, which in turn activates ECs, pericytes and smooth muscle cells. 51 Other pro-angiogenic stimuli induced by exercise include shear stress and oxidative stress. 52 During the regenerative process, SCs can produce pro-angiogenic stimuli that induce EC proliferation and migration. S3.S8 Conversely, an abnormal endothelial Cell response could compromise the muscle repair. Endothelial progenitor cells, mobilized at the site of muscle injury, may promote both angiogenesis and muscle regeneration. S4,59 ECs could also play a role in muscle mass regulation. S5 Indeed, they appear closely juxtaposed to SCs in the muscle compartment, 23 and an increase of the endothelial cell number during the myogenesis process appeared as a strong stimulant of myogenic progenitor cells migration and proliferation. 53 Another study demonstrated the close vicinity between endothelial cells and SCs using muscle tissue clearing. 56 They also showed that both type of cells interact through VEGF-A and Notch signalling. This cross-talk results on the one hand ln resulting in capillaries patterning by SCs and on the other hand in promoting SC self-renewal. M2-biased macrophages (or restorative) also take part in the myogenesis~angiogenesis coupling: Indeed, adding M2-like macrophages in a 3D model composed of SCs and endothelial cells stimulated both capillaries and myotube formation. S3 This effect could be inhibited by blocking oncostatin M secreted by macrophages.

Pericytes

Pericytes are perivascular cells wrapped around capillaries, responsible for various physiological functions in skeletal muscle, including regulation of vessel growth, stabilization and permeability. 57 Few data are available concerning their precise role in skeletal muscle homoeostasis. However, two points are to be considered: their myogenic potential and their role in exercise-induced angjogenesis. Indeed, a myogenic potential for pericytes has been demonstrated in skeletal muscle, both in animal (including murine and canine models of dystrophy) and human studies. 58,59,S10 A particular subtype of pericytes, the nerve/glial antigen-2 (NG2) + /Nestin + subtype, is responsible for this myogenic role. 60 Pericytes are responsive to skeletal muscle contraction and provide significant variation of gene expression, notably upregulation of genes involved in immunomodulation and ECM remodelling. 61 Moreover, pericyte transplantation in skeletal muscle associated with exercise training have been associated with better improvements in muscle rnaximal contraction force. 61 Regarding the role of skeletal muscle angiogenesis in the muscle perfusion and fatigability, pericytes appear as essential to obtain viable and functional vasculature with angiogenesis 62 (Figure 1).

Pericytes also participate to muscle regeneration independently of SCs, due to their myogenic capacity. 59 Moreover, pericytes are able to induce re-entry into quiescence on completion of regeneration through angiopoietin-1 (Ang1) secretion. 63 ln a context of muscle damage, consecutive to muscle dystrophy or after eccentric exercise, the activity of skeletal muscle pericytes is increased and participate to muscle repair. 63,S11 Moreover, they could contribute to muscular dystrophy, both by participating to fat accumulation due to the adipogenic potential of PDFGR + type 1 pericytes and contributing to fibrosis via a possible differentiation to myofibrob|asts. 64

Immune cells

Macrophages

Resident macrophages are a heterogeneous cell population, quiescent at the steady state, appearing to maintain tissue homoeostasis and promoting muscle growth and regeneration, although little is known about their precise role. 65 The latter has been more extensively characterized in acute muscle injury experimental models.

The regeneration process of acute muscle injury models is classically biphasic, with an initial inflammatory phase followed try a secondary resolutive phase (for review, see Tidball 13 ) During the initial phase, resident macrophages differentiate into pro-inflammatory M1-biased phenotype (CD163|, aiming to phagocyte debris. This is coordinated by Interferon- (IFN) a major component Of muscle inflammatory response, acting through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Indeed, lFN-levels are dramatically increased immediately after the injury, and blocking lFN-reduces M1-type macrophage activation. 13 TNF- is the second major mediator of the inflammatory phase by activating the NF-B pathway in macrophages. In addition to resident macrophages, circulating monocytes are also recruited during this initial inflammatory phase. 66 A study performed ln a cardiotoxin-induced acute injury model in mice showed that secretion of the IGF-1 propeptide lGF-1Ea by M 1-biased macrophages promoted SC proliferation in the acute inflammatory phase .67 lGF-1 secretion was later on sustained mainly by a population of non-mveloid, non-myogenic cells, which were probably fibroblasts.68 Interestingly, lGF-1 also had effects on macrophages themselves, via an autocrine feedback loop limiting inflammation and promoting muscle reconstruction. The inflammatory phase appears essential for muscle homoeostasis, as doxorubicin treatment in exercise-challenged mice provokes muscle loss and accelerated death specifically by depleting the M1-macrophage population. 69 The transition towards the regenerative phase is accompanied by a progressive shift from M1 to M2-biased phenotype (CD163+) within the muscle tissue, after 4-7 days, in order to support the production of connective tissue and promote regenerative fibrosis. This polarization shift is likely to occur in the same cells as in the inflammatory phase 66,70 and is possibly functionally coupled to the transition in stages of myogenesis (from proliferation to differentiation). This transition is paralleled by a shift in secreted factors, from lFNand TNF- to lL-10, amphiregulin, lGF-1 and TGF-P.71 Tress produce lL-10 and amphiregulin, whereas macrophages produce lL-10 and lGF-1. Apart from secreted factors, the phenotypic transmembrane marker CD163 also actively takes part in the regeneration process by binding to haemoglobin-haptoglobin complexes and enhancing lL-10 secretion.72 The polarization shift is also accompanied by 5'-adenosine monophosphate-activated protein kinase (AMPK) activation (at least partly via lL-10), which further enhances phagocytosis and promotes M2 polarization. Differentiated, M2-biased macrophages stimulate myogenesis and myotube fusion, 66,71 Thus, the sequence of events leading to muscle repair has been well identified. However, the specific reason for the polarization shift remains unclear, although phagocytosis of debris has been recognized as one direct mechanism, both by creating space for repair and by activating the secretion of myokines implicated in the later stages, such as TGF-P, through the activation of AMPK pathway. Perhaps most interesting is the hypothesis that phagocytosis of debris could generate a form of immunological memory in macrophages that would facilitate their ability to recognize and respond to tissue damage, as suggested by an elegant study performed in drosophi|a. 73 This physiological response to injury is disturbed in several sarcopenia models. For example, a recent study showed that in mice, ageing leads to reduced macrophage density in the muscle and impaired lGF-1 production. Furthermore, ageing mice do not display the classical polarization shift contrary to young mice .74 Another ageing mouse model recently allowed the identification of a novel subset of skeletal muscle macrophages, named interferon-responsive macrophages (lFNRMs), which promote the proliferation and differentiation of SCs via CXCL-10 secretion; such cells were less abundant in older mice muscle. 75 Of note, a particular subset of macrophages called fibrocytes, originating from the bone marrow and bearing features of both leukocytes and fibroblasts, 76 could also play a role in muscle homoeostasis. Fibrocytes are defined as monocytes-derived, double-positive for CD45 and collagen, 77 which allows to distinguish them from FAPs. Although they have initially been described as pro-fibrotic, matrix-producing cells, more recent data pointed towards an extended regulatory role of these cells, which are notably able to regulate ECM turnover by collagen endocytosis. 77 They also have an immune regulatory role, as they are able to secrete pro-inflammatory cytokines as well as interacting with immune cells. 78 Fibrocyte differentiation is associated with chronic inflammation, ECM remodelling, 79 and metabolic reprogramming towards increased oxidative phosphorylation capacity, 80 suggesting that intramuscular fibrocytes could play both physiological and pathological roles in skeletal muscle function. One study reports their presence and role in the early phase of muscle regeneration after acute injury, mainly through the secretion of pro-fibrotic and proinflammatory factors, in the mar murine model of Duchenne's myopathy. 79 Although the mdx model does not involve muscle wasting per se, our recent identification of fibrocytes in the quadriceps of a murine model of COPD (unpublished data) suggests that fibrocytes might play a role in Skeletal muscle regeneration and wasting.

Lymphocytes

Lymphocytes take part in skeletal muscle homoeostasis notably by removing necrotic cells and secreting growth factors necessary for SC proliferation and differentiation, although they are virtually absent from the muscle tissue at the steady state. S12 Interestingly, a recent study has shown a positive correlation between the number of CD8+ T cells in the muscle rectus abdominis (as well as the total T lymphocyte number) and muscle mass, as evaluated, respectively, by immunohistochemistry and fibres cross-sectional area/computed tomography. 81 ln microarray analysis, CD8+ T-cell genes were also negatively associated with genes from catabolic pathways in this study (notably ubiquitin-proteasome and autophagy pathways).

In muscle repair, lymphocytes mainly coordinate the immune response in order to restore muscle homoeostasis. T lymphocytes might also directly promote myogenesis, as they support migration and proliferation of muscle SCs in vitro notably via secretion of IL-1TFN-, IFN- and IL-3 82 . Specifically, CD8 + T-cell infiltration helps in repairing the damage by promoting monocyte recruitment through the enhancement of CTC motif chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1) production by resident macrophages. 82 CD4+ lymphocytes are notably represented by regulators (Tregs) which account for around 50% of CD4 + T cells. Trees accumulate in injured muscles and reach a peak at approximately 4 days post-injury, where they promote the polarization shift from Ml to M2 macrophages 83 by secreting lL-10 (cf supra). Upon lL-33 signalling, they are also able to activate SCs through the growth factor amphiregulin and play a critical role in skeletal muscle regeneration. Altogether, their depletion prevents muscle repair. 42,83 They also highly express C{ chemokine receptor type 2 (CCR2), the receptor for the chemokine CCL2/MCP-1, which attracts monocytes into damaged muscle, Thus, the same chemotactic axis could also be of importance for Tregs recruitment.

Few data are available regarding the role of lymphocytes in other muscular diseases. Historically, T lymphocytes are rather associated with a deleterious effect on skeletal muscle, such as dystrophic muscles as in the mdx mouse model 84 or idiopathic inflammatory myopathies. S13 However, given their important role in regeneration of healthy muscle, it could be that this negative effect would only occur in case of prolonged presence in the tissue 85 as in the dystrophic models. In an ageing model, a failure to recruit enough TRegs has been observed, which partly accounts for defective repair and was improved by lL-33 administration. 42 Other types of CD4+ T cells have been less investigated. However, conditioned media from the Th2 and Thl7 subsets was shown to enhance angiogenesis in vitro and in vivo in a mouse model of Skeletal muscle ischaemia. S14

A current theory ls that T cells are able to take part in a 'muscle damage memory' mechanism, by analogy with the antimicrobial immune response. Supporting this theory is that an identical T-cell receptor arrangement (indicating peptide specificity) was repeatedly found among different animals in separate experiments of repeated contraction-induced damages. 83 This phenomenon could at least partly be driven by lL-15, as later demonstrated in a mouse model of autoimmune myositis. S15 Altogether, T lymphocytes represent a growing research interest for the future, due to their recently highlighted therapeutic potential. 86 Other potential cells of interest, such as neutrophils, telacytes, interstitial tenocytes and neural cells (terminal Schwann cells, motor neurons, kranocytes), are discussed in Data S1.

Cellular interplay in COPD muscle wasting: An unmet need

COPD, a chronic respiratory disease mainly linked to cigarette smoke exposure, is a global burden and is currently the third cause of death worldwide. 8 Its pathophysiology is still partly unknown and combines genetic predisposition, epigenetic modifications and environmental factors. 87 Skeletal muscle dysfunction in COPD affects 4-35% of the patients 5 is responsible of a significant alteration of the quality of life and is associated with higher mortality. Notably, a mid-thigh muscle cross-sectional area of less than 70 cm2, a low fat-free mass index and a reduced quadriceps strength are all independent predictors of mortality, with odds ratio up to 13.fold. 6,7,88 Muscular wasting could appear only after a few days of hospitalization according to a recent study. 89 However, no specific and efficient pharmaceutical therapy is available to date, whereas pulmonary rehabilitation remains the only validated treatment. 5

As for many other diseases, the respective role of each cell in skeletal muscle wasting in COPD patients remains unclear. To our knowledge, no data have been reported concerning the role of immune cells, except for neutrophils, which have been found elevated in the quadriceps of patients compared with controls 90 and hypoxaemic patients versus nonhypoxaemic. 91 Vascular cells drew more interest in this field. In COPD patients, both convective and diffusive oxygen supply are .Impaired, contributing to the reduced V'02max. We and others have found reduced indices of muscle capillarization, which was correlated to the patients V'o2max. 92.S16.S17 in addition, the training-induced muscle angiogenesis was blunted in COPD patients, when compared with healthy age and sex-matched Sedentary subjects. 92 Thus, targeting capillary rarefaction and angiogenic impairment in COPD patients could be an interesting strategy to reduce muscle fatigability and improve maximal exercise capacity. We have also observed evidences of an impairment of the capillary maturation, with disturbed pericyte/endothelial interactions during exercise training. 93 ln contrast with healthy sedentary subjects, the pericyte coverage of muscle capillaries in COPD patients during exercise training has been found defective. 94 This capillary maturation impairment occurs early in the disease time course, as a consequence of deleterious muscle micro-environment. 94 Concerning progenitor cells, it has been shown that SCs exhibit alterations of their own in COPD muscle, notably a senescent phenotype and increased oxidative stress, although their density seems unchanged. 95,96 Furthermore, primary SCs isolated from COPD patients displayed a delayed activation in culture, a decreased expression of myosin heavy chain during myotube formation 97 and a reduced myotube diameter 95 compared with controls, pointing towards impaired differentiation, ln vitro proliferation capacity was unchanged in these reports. 95.97 Interestingly, a recent study Suggested that a deleterious micro-environment to SCs brought by serums from COPD patients could contribute to the atrophy of healthy human myotubes in culture. 98 A study also showed evidences of activated SCs as well as increased apoptotic rates in the quadriceps c)f sarcopenic patients compared with nonsarcopenic, as well as in COPD patients compared with control subjects. 99 These data pointed towards a lower regenerative potential. Last, recent data point towards impaired autophagy specifically ln satellite cells from a mouse model of emphysema exhibiting sarcopenic features .100 Of note, the muscle hypertrophic response to exercise training has been found impaired specifically in hypoxaemic patients compared with non-hypoxaemic ones, 101 pointing out altered muscle adaptive response in this specific subset. Finally, as some studies have found evidences of intra-muscular fat increase in COPD patients, notably at the tissular level evaluated by CT, S18 studying the role of FAPs could be of interest in COPD muscle, considering their adipogenic potential. Evidences for neuromuscular junction degeneration also exist in the quadriceps of COPD patients, and mice exposed to CS extract during 4 months exhibit early neuromuscular junction degeneration preceding fibre-type shift and muscle atrophy. 102 Interestingly, the transcript muscle specific kinase (MuSK), regulating neuromuscular transmission, has been found to be more than twofold increase in COPD quadriceps compared with healthy subjects. 103 Moreover, neuromuscular junction degeneration following ageing has been shown to be accompanied by increased cholinergic activity, and increased cholinergic environment is a well-known key feature of COPD. Reduction in acetylcholine level in mice showed larger neuromuscular junction but also reduced expression of the pro-atrophic FoxO1 transcription factor and increase in satellite cell number. S19 Altogether, increased cholinergic signalling should be investigated as a possible contributor for muscle wasting in COPD.

Modelling COPD to study its complex pathophysiology is a relevant challenge. Several animal models (mouse, rat, guinea pig, hamster) with various modalities (exposure to cigarette smoke extract, elastase, lipopolysaccharide or transgenic animals) are reported ln the literature. 104 Most of these models are primarily designed to investigate the pulmonary condition 105 ; however, some of these models are used to study muscle wasting in COPD and are able to recapitulate features of the human disease, whether phenotypic (decreased muscle mass and strength), histological (decrease in myofibres cross-sectional area, fibre-type switch from type I to 11, decreased capillarity) or molecular (increased catabolism, decreased mitochondrial metabolism). 14 To our knowledge, only one recent study investigated the role of resident skeletal muscle cells in muscle wasting and found a reduced proliferative capacity of SCs, linked to a deaccelerated autophagy. 100 Overall, no animal model perfectly recapitulates all features of the disease, as results are often heterogeneous between Studies, and within a same model, between the different muscles. Moreover, none of the models accurately reflect the complexity and the different phenotypes of the human disease. Table 2 recapitulates the pros and cons of animal versus human experimentation in addressing the question of peripheral skeletal muscle wasting ln COPD. Finally and importantly, neither animal models nor human studies currently address the specific question of the cellular interplay in muscle wasting.

Discussion and perspectives

Skeletal muscle wasting is a prevalent condition, whose diagnosis is hampered by the heterogeneity of clinical entities as well as under-diagnosis, and management suffers from lack of targeted therapeutics. In this review, we address the respective roles of resident tissular cells graviting around myocytes in skeletal muscle homoeostasis, regeneration and sarcopenia, thanks to data obtained from various animal models, such as COPD, cancer, acute muscle injury or ageing. Progenitor cells such as satellite cells and fibro-adipogenic progenitors are primarily implicated in maintaining the myocyte pool, but vascular, immune and nervous cells all play a role in progenitor cells proliferation and differentiation, in addition to having a potential myogenic fate for vascular cells. Overall, few mechanisms are common between homoeostasis and regeneration, as most resident cells are quiescent at the steady state but become activated upon disruption of homoeostatic condition, whether subtle (e.g. exercise) or substantial (muscle injury or disease). Such activation comes along with a metabolic switch from predominant oxidative phosphorylation to glycolysis, as seen in FAPs 41 or SCs. 19 This highlights the sensor role for nutrients and redox state for these cells, as well as the requirement of rapid energy production upon proliferation, as described in other cell types 106 . Interestingly, fibrocytes switch their metabolism towards predominant glycolysis upon clifferentiation. 80 Furthermore, inhibiting mitochondrial respiration in cultured SCs from control mice promoted the expression of stemness, self-renewal and returnto-quiescence markers. 19 ln contrast, stimulation of mitochondrial protein synthesis via the metabolic modulator trimetazidine promoted myogenesis markers expression in vivo in aged mice 107 This discrepancy might come from the fact that metabolic modulators act indifferently on every skeletal muscle resident cell ln vivo. Nevertheless, these data open new therapeutic avenues in the field of metabolic reprogramming in order to enhance skeletal muscle regeneration capacities. Such approach has already been successfully tested in a mouse model of amyotrophic lateral sclerosis. S20 Should this be translated to sarcopenia, studies should aim at identifying regulators of the different metabolic pathways involved in every resident muscle cell type, in order to develop a targeted therapy, which should be tested in vivo in order to examine the effect on the whole tissue. Finally, promoting regeneration should be handled with caution as it is possible that repeated events of micro-regeneration (e.g. due to hypoxia or toxicity of CS exposure, which could be incriminated in COPD) lead to premature SCs exhaustion. Current treatments for muscle wasting depend on the underlying condition. Efforts to safely increase both muscle mass and force tend to be unsuccessful. The anabolic hormone testosterone has shown to increase both muscle mass and function, 108 ; however, it is associated with important side effects such as increased cardiovascular events in older individuals. 109 Although blocking the activin type 11 receptor (ActRllB) pathway has been shown to be very promising in animal cancer cachexia models, in terms of not only muscle mass gain but also lifespan, 34 such treatment has not been implemented successfully yet jn clinical practice. Its use in patients with COPD in particular did not improve skelet3l muscle function .110 Moreover, blockade of ActRllB in wild-type mice led to severe fatigue, and even to decreased muscle force via decreased capillarization and oxidative metabolism in the mdx mouse model 111 Overall, there is currently no FDA-approved drug to treat muscle wasting. However, promising leads are still emerging, such as the use of 11-4 in a preclinical cancer model, allowing to rescue muscle function and prolong lifespan. 112 Overall, precising the role of tissular resident cells can be useful in order to develop specific therapies for diseases associated with muscle wasting, for example, by targeting cell recruitment or differentiation.

Pre-clinical models do not completely reflect the complexity of human disease and are often quite heterogeneous in their ability to reproduce every characteristic of disease induced muscle wasting, as illustrated in the field of COPD. Moreover, current research tends to replace animal models with models avoiding the use of living organisms, such as the use of organoids, whether animal or human derived (also referred to as 'BAM': bio-artificial muscles). Although the field of muscle organoids is not as advanced as in other organs such as lung organoids, 113 it is clearly expanding and a very promising lead to study the dynamics of cellular regeneration . Several models have been published in the last decade, ranging from mono-cellular to a complex functional multi-cellular organoid, Vascularized, innervated and able to contract. [114][115][116] such models have been used successfully to model exercise training,

showing an increase in lL-6 secretion and an AKT Signalling pathway activation. 117 0f note, secretime of tumour organoids can also exhibit characteristic features of cachexia, as shown by a recent study performed in pancreatic tumour organoids. 118 However, to our knowledge, there is currently no study investigating the molecular and cellular mechanisms of muscle wasting using such constructs. Nevertheless, there is no doubt that muscle organoids will be at the core of basic research in the future years.

To conclude, skeletal muscle wasting is a crucial issue for patients suffering from chronic diseases, but also ageing healthy individuals. Better characterizing the role of each cell population within skeletal muscle is of interest in order to develop specific therapies for muscle regeneration and growth. 119

Figure 1

 1 Figure 1 Cellular interplay in skeletal muscle homoeostasis. Tissular resident cells interact with myocytes and with each Other in a finely tuned process.

Acknowledgements

The authors of this manuscript certify that they comply with the ethical guidelines for authorship and publishing in the Journal of Cachexia. Sarcopenia and Muscle.

Conflict of interest

Pauline Henrot, Leo Blervaque, lsabelle Dupin, Ma6va Zysman, Pauline Esteves, Fares Gouzi, Maurice Hayot, Pascal Pomies and Patrick Berger declare that they have no conflict of interest regarding this work.


Hager Jaouadi

Lilia Kraoua2

Lilia Chaker3

Alexandre Atkinson

Valérie Delague4

Nicolas Levy4

Rym Benkhalifa5

Ridha Mrad2

Sonia Abdelhak1

Stéphane Zaffran
email: stephane.zaffran@univ-amu.fr

Rym Benkhalifa
email: rym.benkhalifa@pasteur.tn

Novel ALPK3 mutation in a Tunisian patient with pediatric cardiomyopathy and facio-thoraco-skeletal features

Pediatric cardiomyopathy is a complex disease with clinical and genetic heterogeneity. Recently, the ALPK3 gene was described as a new hereditary cardiomyopathy gene underlying pediatric cardiomyopathies. Only eight patients carrying mutations in ALPK3 have been reported to date. Here, we report a 3-year-old male patient with both hypertrophic and dilated cardiomyopathy. The patient presented dysmorphic features and skeletal deformities of hands and feet, pectus excavatum, and cleft palate. The genetic investigation was performed by whole-exome sequencing in the patient and his parents. We identified a novel homozygous mutation in ALPK3 (c.1531_1532delAA; p.Lys511Argfs*12). Our work extends the phenotypic spectrum of the ALPK3-associated cardiomyopathy by reporting additional clinical features. This is the first study of a Tunisian patient with mutation in the ALPK3 gene. In conclusion, ALPK3 should be included in the list of genes to be considered in genetic studies for patients affected with pediatric syndromic cardiomyopathy.

Introduction

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are the most common forms of primary cardiomyopathies and the major cause of heart failure and sudden cardiac death [START_REF] Kimura | Molecular genetics and pathogenesis of cardiomyopathy[END_REF]. HCM is characterized by increased ventricular wall thickness associated with diastolic dysfunction of ventricular chambers. DCM is defined by dilation and systolic dysfunction of the left ventricle (LV) but right-ventricular involvement was also observed [START_REF] Kimura | Molecular genetics and pathogenesis of cardiomyopathy[END_REF][START_REF] Mestroni | Familial dilated cardiomyopathy: evidence for genetic and phenotypic heterogeneity. Heart Muscle Disease Study Group[END_REF]. These two entities occur in children with an annual incidence of 1.13 cases per 100.000 children younger than higher incidence was found in boys and infants under 1 year of age for both HCM and DCM [START_REF] Wilkinson | The Pediatric Cardiomyopathy Registry: 1995-2007[END_REF]. HCM and DCM are the most frequent defects among pediatric probands (51% HCM and 30% DCM) [START_REF] Kindel | Pediatric cardiomyopathy: importance of genetic and metabolic evaluation[END_REF]. These findings were consistent with the results of the Pediatric Cardiomyopathy Registry (PCMR) [START_REF] Wilkinson | The Pediatric Cardiomyopathy Registry: 1995-2007[END_REF][START_REF] Wilkinson | Lessons learned from the Pediatric Cardiomyopathy Registry (PCMR) Study Group[END_REF]. HCM and DCM have genetic and phenotypic heterogeneity with incomplete penetrance and variable expressivity. Different cardiac phenotypes are caused by mutations in the same gene, and numerous genes can be responsible of the same cardiomyopathy [START_REF] Lee | Pediatric cardiomyopathies[END_REF]. Furthermore, pediatric cardiomyopathies have both genetic and nongenetic etiologies. The genetic make-up of pediatric cardiomyopathies is more diversified than in the adult population because additional syndromic and metabolic genetic causes can be implicated [START_REF] Mestroni | Pediatric cardiomyopathy[END_REF]. Hence, the identification of the mutation spectrum and causal associated-genes are challenging. The advent of next generation sequencing technologies, such as whole-exome sequencing (WES) allowed the identification of the genetic basis of pediatric cardiomyopathies. Using this technique and the homozygosity mapping, the alpha-kinase 3 (ALPK3) gene was recently discovered as a novel pediatric cardiomyopathy-associated gene [START_REF] Almomani | Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy[END_REF]. Indeed, homozygous mutations in the ALPK3 gene were identified in 5 patients with pediatric cardiomyopathy. This study hypothesized that ALPK3 might control initial induction of differentiation and maturation of cardiomyocytes by regulating the expression of cardiac transcription factors such as HEY2 and HAND [START_REF] Almomani | Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy[END_REF]. The ALPK3 gene encodes a 1907 amino acids protein, which contains three functional domains: one α-type protein kinase domain and two immunoglobin-like domains. ALPK3 appears to be involved in cardiomyocytes differentiation and heart development [START_REF] Hosoda | A novel myocyte-specific gene Midori promotes the differentiation of P19CL6 cells into cardiomyocytes[END_REF]. A recent study using human induced pluripotent stem cell showed that absence of ALPK3 results in abnormal intracellular calcium [START_REF] Phelan | ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy[END_REF]. Here, we report a novel homozygous frameshift mutation in the ALPK3 gene in a 3-year-old boy affected with mixed HCM/ DCM phenotype and dysmorphic features.

Materials and methods

This study was conducted according to the principles of the Declaration of Helsinki and to the ethical standards of the first author's institutional review board (Registration number: IRB00005445, FWA00010074). The parents provided their written informed consent to participate in this study. After informed consent was obtained, peripheral blood samples were collected from the affected child and his parents. Genomic DNA was extracted from the samples according to standard techniques. Whole-exome sequencing (WES) was performed for the trio (the affected child, the father and the mother), by the Genomics and Bioinformatics Platform (GBiM) from the UMR 1251/Marseille Medical Genetics facility, using the NimbleGen SeqCap EZ MedExome kit (total design size 47 Mb) according to the manufacturer's protocol (Roche Sequencing Solutions, Madison, USA). The SeqCap EZ MedExome kit targets the entire human exome with enhanced coverage of exons from medically relevant genes in Mendelian diseases. Enriched fragment libraries were sequenced on the Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA) using a 150 bp paired-end sequencing protocol. Raw data were mapped to the built of the human genome (hg19) by using BWA 0.7.5. Variant calling was subsequently performed using GATK and annotation was done with ANNOVAR. All subsequent steps were performed using our in-house software for variant annotation and segregation VarAFT (http:// varaft.eu). WES data from the patient and his parents were analyzed simultaneously and segregated using VarAFT. All modes of inheritance have been tested during the analysis of the data. In order to reduce the significant number of variants generated by WES sequencing and to identify relevant variants, a strategy of filtering was adopted. Variants were selected as following: [START_REF] Kimura | Molecular genetics and pathogenesis of cardiomyopathy[END_REF] variants with Minor Allele Frequency (MAF) ≥ 1% in the Exome Aggregation Consortium database (ExAC) (http://exac.broadinstitute.org/) were removed [START_REF] Mestroni | Familial dilated cardiomyopathy: evidence for genetic and phenotypic heterogeneity. Heart Muscle Disease Study Group[END_REF]. The remaining variants were filtered based on their type and genomic localization, thus, synonymous, intronic, variants in intergenic, 3′ and 5′ UTR regions were discarded [START_REF] Wilkinson | The Pediatric Cardiomyopathy Registry: 1995-2007[END_REF]. The obtained variants' list (including rare, nonsense, missense, splice site, and frameshift INDELs), was then subsequently filtered according to the in silico pathogenicity prediction. Thus, variants predicted as polymorphism according to UMDPredictor (http://umd-predictor.eu/), SIFT (http://sift.jcvi. org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), and Mutation Taster (http://www.mutationtaster.org/) were excluded.

Results

Clinical presentation

The patient, a 3-year-old Tunisian boy, is the only child of apparently healthy first cousins aged 21 (mother) and 28 (father) years. He was born at term by spontaneous vaginal delivery following an uncomplicated pregnancy. His birth weight was 3550 g. The patient was hospitalized at birth for 2 months and a week because of respiratory distress. At 7 days, he was diagnosed with mixed HCM/DCM cardiomyopathy, and began treatment by captopril (25 mg), furosemide (40 mg) and carvedilol (6.25 mg). Echocardiography performed at the age of 3 months revealed levocardia, situs solitus, LV dilation and concentric hypertrophy with altered systolic function (end-diastolic diameter (EDD) = 26 mm; end-systolic diameter (ESD) = 24 mm; inter-ventricular septum thickness at end-diastole (IVSd) = 9 mm; ejection fraction (EF) = 35%). The right-ventricular walls were enlarged. A patent foramen ovale with left to right shunt and mild tricuspid insufficiency (systolic pulmonary arterial pressure = 30 mmHg) were also reported. Neither aortic insufficiency nor valvular aortic stenosis were observed. There was no isthmic coarctation of the aorta. Cardiac examination at 6 months showed less dilation of the LV with improvement of the systolic function and concentric hypertrophy of both LV and RV (EDD =27 mm; ESD= 20 mm; IVSd = 12 mm, EF =50%). Minimal mitral valve insufficiency was noted. Echocardiographic indices at 8 months were as following: EDD= 26 mm; ESD=19 mm; IVSd = 12 mm, EF = 52% and the LV posterior wall showed a cardiomyopathy of predominantly hypertrophic LV without obstruction (Fig. 1b.). Abnormalities in left ventricular compliance were also observed and echocardiographic indices were as following: EDD = 25 mm; ESD = 16 mm; IVSd = 15 mm, EF = 65%, and the LV posterior wall thickness = 12 mm. Echocardiographic screening in the father revealed asymmetric septal hypertrophy (septal thickness = 16 mm) and minor congenital mitral anomaly without valvular dysfunction (Fig. 1c). The mother's echocardiography was without abnormalities. First examination by geneticists at the age of 2 months and a half revealed a weight at 3800 g (<3rd centile), a height at 54 cm (<3rd centile) and a head circumference at 38.5 cm (3rd-15th centile), dysmorphic features including broad forehead, protruding eyes, divergent strabismus, blue sclera, low-set ears, long philtrum, thin lips, cleft palate, microretrognathia, and bilateral camptodactyly of hands and axial hypotonia. On last examination, at the age of 2 years and 9 months, weight was on the 25th percentile; height and head circumference were on the 50th percentile. He had almost the same dysmorphic features characterized by flat occiput, triangular, and flat face, broad forehead, down-slanting palpebral fissures, protruding eyes, mild ptosis of the left eyelid, blue sclera, low set and backward-rotated small ears, broad nasal ridge and nasal tip, thick ala nasi, short columella, long philtrum, everted lips, cleft palate, microretrognathia, and full cheeks. He also had short neck, sloping shoulders, pectus excavatum, bilateral camptodactyly of hands, bilateral overlapping of the 2nd and 3rd toes, bilateral clinodactyly of the 4th and 5th toes, and knee stiffness. The psychomotor development of the patient was normal. R-banded chromosome analysis on cultured peripheral blood lymphocytes from the proband was normal 46, XY. Fluorescent In Situ Hybridization (FISH) analysis with MD DiGeorge T-box1 (22q11)/22q13 (SHANK3) kreatech probe was performed but no microdeletion was identified. Creatine phosphokinase, lactate dehydrogenase, serum creatinine, ammonia, and lactates levels and chromatographic analysis of amino and organic acids were normal.

Whole-exome analysis

Taking into account the large number of genes mutated in inherited cardiac diseases we have performed a WES for the family (trio). WES data were analyzed and segregated using the Variant Annotation and Filtering Tool VarAFT. On average, 92 and 87% of all targeted exome was covered to a depth of 20× and 30×, respectively. All variants identified in our patient using the filtering strategy described above (Material and methods) are listed in Table 1 (homozygous mutations) and Supplementary Table 1 (heterozygous mutations). Based on these lists of variant we prioritized the homozygous frame-shift deletion in exon 5 of the ALPK3 gene (NM_020778): c.1531_1532delAA; p.Lys511Argfs*12 as a likely causative variant. Indeed, both parents were found heterozygous for this variant (Fig. 2). Moreover, the ALPK3 c.1531_1532delAA; p.Lys511Argfs*12 is not reported in the following databases: ExAC, dbSNP (https://www.ncbi.nlm.nih.gov/ projects/SNP/), NHLBI Exome Sequencing Project (http:// evs.gs.washington.edu/EVS/), gnomAD (http://gnomad. broadinstitute.org), 1000 Genomes (http://www.interna tionalgenome.org), and GME Variome (http://igm.ucsd. edu/gme/). In order to exclude the possibility that the ALPK3 p.Lys511Argfs*12 deletion could be a frequent variant specific of the Tunisian population, our in-house database, gathering WES data from 70 Tunisian individuals (140 chr) was queried and the variant was not present.

Discussion

We report a 3-year-old boy born to consanguineous parents of Tunisian origin and affected with mixed HCM/DCM, dysmorphic features, pectus excavatum, and skeletal deformities of both hands and feet.

Through an autosomal recessive model of inheritance analysis, we identified a novel frameshift deletion in ALPK3 not previously described in any frequency datasets. The ALPK3 c.1531_1532delAA variant is predicted to result in a premature stop codon (p.Lys511Argfs*12) leading to nonsense-mediated mRNA decay. To our knowledge, this novel variant in ALPK3 c.1531_1532delAA; is the sixth mutation identified in patient with pediatric cardiomyopathy, as only five mutations in ALPK3 have been reported to date: c.4736-1 G > A, c.3781 C > T, c.5294 G > A, c.3792 G > A, and c.2018delC (Fig. 3) [START_REF] Almomani | Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy[END_REF][START_REF] Phelan | ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy[END_REF][START_REF] Çağlayan | ALPK3 gene mutation in a patient with congenital cardiomyopathy and dysmorphic features[END_REF]. The human ALPK3 gene (OMIM*617608) encodes the alpha-kinase 3 protein and is mainly expressed in heart and skeletal muscle. In 2001, Hosoda et al. identified the ALPK3 gene, also named MIDORI (murine Myocyte Induction Differentiation Originator) using the differential display technique in the mouse P19CL6 cell line system and demonstrated that its expression is enriched in the forming heart during early development. Different studies suggested that ALPK3 is involved in the organization of myofibrils and the assembly of intercalated discs [START_REF] Almomani | Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy[END_REF][START_REF] Phelan | ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy[END_REF][START_REF] Van Sligtenhorst | Cardiomyopathy in α-kinase 3 (ALPK3)-deficient mice[END_REF]. Indeed, a recent study using CRISPR/cas9 editing in a proband-derived iPSC line confirmed that the bi-allelic loss of function mutation in ALPK3 is pathogenic. Moreover, heterozygous correction of ALPK3 allowed the full restoration of the wild-type phenotype [START_REF] Phelan | ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy[END_REF]. The loss of function of ALPK3 disrupts also membrane repolarization by extending the extracellular field potential duration. Calcium mishandling was also noted, which could underlie the cardiac hypertrophy and arrhythmia [START_REF] Phelan | ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy[END_REF]. Mixed HCM/DCM phenotype was noted among children [START_REF] Lee | Pediatric cardiomyopathies[END_REF], in particular in patients mutated in the ALPK3 gene [START_REF] Almomani | Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy[END_REF][START_REF] Phelan | ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy[END_REF][START_REF] Çağlayan | ALPK3 gene mutation in a patient with congenital cardiomyopathy and dysmorphic features[END_REF]. Previous studies have reported cases with progression of HCM to DCM [START_REF] Spirito | Absence of progression of left ventricular hypertrophy in adult patients with hypertrophic cardiomyopathy[END_REF][START_REF] Hecht | Coexistence of sudden cardiac death and end-stage heart failure in familial hypertrophic cardiomyopathy[END_REF][START_REF] Freeman | Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene[END_REF]. Our patient displayed initially a mixed HCM/DCM phenotype that has evolved into a concentric hypertrophy of both left and right ventricles. Çag layan et al. reported a 2½-year-old male patient carrying a homozygous frameshift mutation in ALPK3 (c.2018delC; p.Gln675Serfs*30) with a progression of DCM to HCM [START_REF] Çağlayan | ALPK3 gene mutation in a patient with congenital cardiomyopathy and dysmorphic features[END_REF]. Moreover, three patients harboring mutations in ALPK3 displayed significant right-ventricular dysfunction [START_REF] Almomani | Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy[END_REF]. On the cardiac level, the phenotype of our patient is consistent with that of patients carrying mutations in ALPK3 previously described [START_REF] Almomani | Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy[END_REF][START_REF] Phelan | ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy[END_REF][START_REF] Çağlayan | ALPK3 gene mutation in a patient with congenital cardiomyopathy and dysmorphic features[END_REF]. In our study, both parents were found heterozygous for the ALPK3 c.1531_1532delAA; p.Lys511Argfs*12 mutation. Echocardiography of the mother did not show any cardiac dysfunction. However, the father presented hypertrophy of the inter-ventricular septum and minor congenital mitral anomaly. These observations are consistent with previous studies which found that heterozygous carriers of ALPK3 mutations displayed either no evidence of cardiomyopathy or later-onset cardiomyopathy or an atypical distribution of hypertrophy [START_REF] Almomani | Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy[END_REF][START_REF] Phelan | ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy[END_REF]. Hence, the c.1531_1532delAA; p.Lys511Argfs*12 in ALPK3 at a homozygous state is the likely pathogenic mutation underlying the cardiac phenotype of our patient. Furthermore, we hypothesized that ALPK3 gene could be responsible of the dysmorphic and skeletal features of our patient. Low-set ears and high arched palate were the unique dysmorphic features described in one patient harboring ALPK3 mutation [START_REF] Çağlayan | ALPK3 gene mutation in a patient with congenital cardiomyopathy and dysmorphic features[END_REF]. The clinical presentation of our patient is more diversified including cleft palate, pectus excavatum, bilateral camptodactyly, bilateral clinodactyly, and knee stiffness. Tissues expression of all identified variants (Table 1 and Supplementary Table 1) using GTEx database as well as related OMIM diseases did not reveal an association with skeletal deformities, pectus excavatum and cleft palate, except the ALPK3 variant. Indeed, previous studies in the mouse revealed that ALPK3 is expressed in the fetal and adult heart, as well as branchial arch and skeletal muscles [START_REF] Hosoda | A novel myocyte-specific gene Midori promotes the differentiation of P19CL6 cells into cardiomyocytes[END_REF][START_REF] Van Sligtenhorst | Cardiomyopathy in α-kinase 3 (ALPK3)-deficient mice[END_REF]. Similarly, a high level of ALPK3 transcript was detected in cardiac and skeletal muscles in humans [START_REF] Phelan | ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy[END_REF]. In conclusion, our study reinforces the implication of ALPK3 in pediatric cardiomyopathy and sheds light on the syndromic nature of the disease. Therefore, we have extended the mutational and phenotypic spectrum of ALPK3-related disease by reporting a novel homozygous mutation associated with additional clinical features.

Fig. 1

 1 Fig. 1 Echocardiographic images. a Echocardiography of the patient at the age of 11 months and b at 15 months. c Echocardiography of the father at the age of 30 years

Acknowledgements

We thank the patient's family for their collaboration. This work was supported by the Tunisian Ministry of Public Health, the Ministry of Higher Education and Scientific Research (LR16IPT05). The project leading to this publication has received funding from Excellence Initiative of Aix-Marseille University -A*MIDEX, a French "Investissements d'Avenir" programme (RAREMED project).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


Julien De Rosny
email: julien.derosny@espci.fr

Claire Debever

Stephane Conti

Philippe Roux

Diffusive Reverberant Acoustic Wave Spectroscopy with absorbing scatterers

Keywords: numbers: 43.35.+d, 43.20.+g, 43.90.+v, 43.80.+p cross sections, field fluctuations, diffusive wave spectroscopy, extinction paradox, acoustics

Strong interest has been shown on the propagation of transient ultrasonic waves inside a high reverberating cavity filled with moving scatterers. A diffusive model theoretically justifies that the elastic cross section and the dynamic of the scatterers can be deduced from field correlations. Recently, experiments have been performed to obtain also the inelastic cross section of the scatterers.

In this paper, we propose to justify this extension within the diffusive model. Experimental results obtained inside a 1.5 liter reverberant water tank at 900kHz central working frequency with spheres made of materials of different absorption are presented. In particular, an original illustration of the extinction paradox is highlighted.

I. INTRODUCTION

Recent interest has increased on the interaction of a pulse acoustic wave inside a strong reverberant cavity filled with moving scatterers. In such a configuration, the motion of scatterers induces fluctuations of the scattered and reverberated sound field. On a theoretical point of view, a statistical model based on a diffusive field assumption has been proposed [1].

It has been shown that the field autocorrelation is directly related to global parameters of the system such as the scatterers displacement, the cavity volume, the total elastic cross section of the scatterers. Different experimental works confirm this model [1,[START_REF] Demer | Absolute measurements of total target strength from reverberation in a cavity[END_REF]. This research leads to the development of the Diffusive Reverberant Acoustic Wave Spectroscopy (DRAWS).

DRAWS has been successfully applied to measure the elastic cross section as well as the displacement of fish swimming in a water tank [1,[START_REF] De Rosny | Multiple scattering in a reflecting cavity application to fish counting in a tank[END_REF]. Recently Conti et al. [START_REF] Conti | Measurement of the scattering and absorption cross sections of the human body[END_REF] have proposed an extension of DRAWS in order to also measure the inelastic total cross section (i.e., the scatterers absorption). The method has been applied to human body [START_REF] Conti | Measurement of the scattering and absorption cross sections of the human body[END_REF]. In this paper, we propose to confirm theoretically the Conti et al. method in the framework of diffusive model.

Experimental results seem to confirm this model. Especially, the elastic and inelastic total cross sections of a strong reflecting sphere and a strong absorbing sphere are measured. We show that the results are directly related to the so-called "Extinction Paradox".

II. THEORY

The time dependence of the pressure wave field, φ(t), can be decomposed into an infinite sum of φ n (t) wave fields. Here φ n (t) represents the wave field which is scattered n times and t is the propagation time of the wave after its emission from the source (at t = 0). Under the diffuse field assumption, the mean squared fields obey to the balance equation between the different scattered order field intensities:

d φ 2 n (t) dt = -φ 2 n (t) 1 τ E + 1 τ A + φ 2 n-1 (t) τ E (1)
where τ E is the elastic mean free time and τ A the inelastic one. Eq. 1 results from the following consideration: within time dt, the elastic scattering of the (n -1) th order scattered wave field feeds the n th order field. However at the same time, n th order field decreases due to its elastic scattering and its absorption. Obviously the "feeding" term in Eq. 1 is not present for the field that is not scattered (n = 0). The absorption is the sum of the inelastic scattering and the cavity absorption (boundaries+water absorption), i.e., 1/τ A = 1/τ A + 1/τ C . Moreover, it has been shown under the dilute approximation that

τ E = V /Nσ E c in a reverberant cavity of a volume V filled with N scatterers[1]. Straightforwardly, τ A = V /Nσ A c
where σ A is the scatterers' inelastic cross section. We assume that the different scattered field orders are decorrelated from one to one, therefore φ 2 (t) = n φ 2 n (t) . Two fields, φ(t) and φ (t) recorded while the scatterer positions have completely changed are only correlated through the part of the field that is not scattered, i.e., φ(t)φ (t) = φ 2 0 (t) . Using Eq. 1, it comes

φ(t)φ (t) / φ 2 (t) = e -t/τ E (2)
Summing the set of Eqs 1 for all n yields:

d φ 2 (t) /dt = -φ 2 (t) /τ A (3) d φ 2 C (t) /dt = -φ 2 C (t) /τ C (4)
φ C corresponds to the reverberated field obtained without scatterers. Finally from the two previous equations, it comes

φ 2 (t) / φ 2 C (t) = e -t/τ A (5)
Hence the experimental measure of φ 2 (t) , φ 2 C (t) and φ(t)φ (t) give an estimate of scatterers' elastic and inelastic cross sections σ E and σ A by the way of Eqs. ( 2) and [START_REF] Morse | Theoretical acoustics[END_REF].

III. EXPERIMENTS

The experimental setup is shown on Fig. 1. An acoustic source emits a few 900kHz periods. Synchronously, a digital scope records the scattered-reverberated pressure field on a hydrophone. This response is then recorded on the hard drive of a computer by the way of the parallel port (see Fig. 2). The acquisition is repeated 200 times, the ball position being changed between each acquisition. From the bank of responses, φ 2 (t) and φ(t)φ (t) are estimated. As for φ C is obtained from the record of the pressure field response with no ball in the tank. In Fig. 3 are plotted the ratios with respect to the propagation time given by Eqs. ( 2) and [START_REF] Morse | Theoretical acoustics[END_REF]. They are obtained from two spheres: a 18mm-diameter one made of Ureol and a 20mm-diammeter other one made of steel. From a linear fit, σ E and σ A are deduced. Experiments are also performed with plasticine and gelatin spheres whose diameters are equal to 19mm. The experimental cross-sections are presented in Table I.

They are compared to their theoretical values [START_REF] Morse | Theoretical acoustics[END_REF] when acoustic properties of materials are known. The Ureol ball was produced by injection molding. The sphere mold was not perfect. Moreover a few bubbles were trapped inside the Ureol resin. Consequently the theoretical and experimental total cross sections are slightly different.

IV. DISCUSSION

At 900kHz, the wavelength equals 1.67mm in water. Hence the 20mm ball diameter is large compare to the wavelength. In such a case, one may assume that the ray theory is valid. The impedance of steel is about 30 times higher than the water one. Hence a steel-ball can be seen as a nearly perfectly reflecting sphere. Under the ray theory assumption, the elastic cross section of such a scatterer would equal to its geometrical cross section (πR

2
where R is the sphere radius). For our 20mm diameter sphere, the geometric cross-section equals 314mm 2 . Nevertheless the experimental value for σ E (see Table I) is about twice larger than the geometrical one. This behavior is characteristic of the so-called "extinction paradox" [START_REF] Van De Hulst | Light Scattering by Small Particles[END_REF]. Contrary to the specular reflection on the scatterer, the ray theory cannot model the forward-diffraction on the edge of the scatterer. The diffraction forms an angular pattern that is identical with the diffraction through a hole of area πR 2 by Babinet's principle. Hence the reflection contribution and the diffraction contribution to the elastic cross section are equal [START_REF] Van De Hulst | Light Scattering by Small Particles[END_REF]. Here we give an experimental illustration. To this end, we exploit the results obtained with the 18mm-diameter ball made of Ureol (see Table I). The acoustic impedance of Ureol is almost equal to water one. Moreover the intrinsic attenuation of Ureol is extremely strong (240dB/m/MHz). Hence an Ureol ball acts as a nearly perfect absorbing scatterer due to its impedance matching and strong dissipation. In such a case, all the "rays" previously reflected penetrate in the ball and are absorbed. Hence rays now only contribute to the inelastic scattering whereas the diffraction process remains unchanged and still participate to the elastic cross-section. Therefore σ E and σ A should be almost equal to the geometric total cross-section, i.e. 254mm 2 . This is roughly experimentally observed, the difference originates the imperfect absorption, the slight mismatch of impedance between water and Ureol and finally the wavelength that is not infinitely small compare to the ball radius.

V. CONCLUSION

In conclusion, DRAWS is an original and simple technique in order to measure both the elastic and inelastic total cross sections. An experimental validation has been shown within the framework of the extinction paradox. The row Th corresponds to the theoretical values when material properties are known.

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Figures

  Figures

Figure 1 :

 1 Figure 1: Experimental set-up. A sphere is moved inside a metallic tank while acoustic pulsed signals are sequentially sent by a source. The reverberated signals are recorded on a hydrophone.

Figure 2 :

 2 Figure 2: One record of the reverberated pressure field in response to a 10µs pulse.

Table I :

 I Figure 3: Experimental mean intensity ratio, φ 2 (t) / φ2 C (t) (squares) and normalized correlation, φ(t)φ (t) / φ 2 (t) (triangles). Straight lines represents least-square linear fits. Plots (a) and (b) are obtained with 18mm diameter Ureol sphere and a 20mm stainless steel sphere, respectively. E σ A σ E σ A σ E σ A σ E σ A Table of tree measures (E1, E2 and E3) of σ E and σ A expressed in mm 2 for four materials.

	Tables
	Steel Ureol Plasticine Gelatin
	σ E1 498 11 251 214 302 206 350 38
	E2 543 20 256 214 296 203 311 23
	E3 551 17 252 207 315 204 313 24
	Th 510 0 288 256 x	x	x x

List of Figures