Karim Hnia
email: karim.hnia@inserm.fr

Tim Clausen

Christel Moog-Lutz
email: christel.lutz@ipbs.fr

Shaping striated muscles with ubiquitin proteasome system in health and disease

For long-lived contractile cells, such as striated muscle cells, maintaining proteome integrity is a challenging task. These cells require hundreds of components that must be properly synthesized, folded and incorporated into the basic contractile unit, the sarcomere.

Muscle protein-quality-control in cells is mainly guaranteed by the ubiquitin-proteasome system (UPS), the lysosome-autophagy and various molecular chaperones. Recent studies establish the concept of dedicated UPS in the regulation of sarcomere assembly during development and in adult life to maintain the intricate and interwoven organization of protein complexes in muscle. Failure of sarcomere protein-quality-control often represents the basis of severe myopathies and cardiomyopathies in human, further highlighting its importance in producing and maintaining the contractile machinery of muscle cells in shape.

Protein homeostasis contributes to sarcomere development in striated muscles

Proteostasis or protein homeostasis network (PN) is a multi-compartmental system with a central role in regulating protein synthesis, folding, trafficking, disaggregation, and ultimately degradation (for review see [START_REF] Powers | Diversity in the origins of proteostasis networks--a driver for protein function in evolution[END_REF]). The main function of PN is to ensure the maintenance of proteome fidelity and integrity in order to preserve homeostasis in the cell and protect it from alterations induced by the exposure to environmental stress. One of the most studied PN components is protein quality control (PQC), a process assured by molecular chaperones and the two proteolytic systems: the ubiquitin-proteasome (UPS) (see Glossary) and the lysosome-autophagy systems [START_REF] Powers | Diversity in the origins of proteostasis networks--a driver for protein function in evolution[END_REF][START_REF] Sala | Shaping proteostasis at the cellular, tissue, and organismal level[END_REF]. PQC is challenged in longlived cells, such as muscle fibers, which are permanently and for extended time periods confronted to proteotoxic stress situations. The muscle contractile machinery, the sarcomere undergoes constant turnover to maintain the homeostatic condition and adapt to physiological changes. Here we discuss the contribution of UPS components i.e. E3 ubiquitin ligases, Deubiquitinating enzymes (DUBs) and the proteasome shuttle factors in building and remodeling the sarcomere in striated muscles. The action of UPS becomes evident at all stages of tissues lifespan, from (i) embryonic/fetal stages to safeguard stemness, (ii) to maintain muscle function in adults and finally, (iii) in disease contexts (myopathies and cardiomyopathies). Finally, we highlight the crosstalk between UPS and other PQC systems (autophagy/lysosome and chaperones/co-chaperones) in the preservation of muscle function.

The UPS is involved in striated muscle formation

Early in cardiac and skeletal muscle development, many sarcomeric proteins are expressed and assembled into contractile apparatus (Box1). Increasing evidence supports that the UPS regulates sarcomere remodelling during muscle development. A hallmark of UPS is the marking of proteins by poly-ubiquitin chains for degradation by the proteasome.

Addition of the ubiquitin mark follows a cascade of events that involves three classes of enzymes: the E1 ubiquitin-activating enzyme UBA1, the E2 ubiquitin-conjugating enzymes and the E3 ubiquitin ligases (E3s) [START_REF] Komander | The ubiquitin code[END_REF]. E3 ligases are the most numerous and heterogeneous class of enzymes in the ubiquitination pathway and many of these enzymes are highly active in muscle cells. E3 ligases are classified into 3 main families based on structural similarities and on the mechanism of ubiquitin transfer to its substrate.

The RING (Really Interesting New Gene) E3 ligases form the largest family and encompass the RING E3 ubiquitin ligases, the Cullin-RING ubiquitin ligases (CRLs) and the U-box containing E3 ligases (also known as E4 ligases). The two other families are the E3 HECT (Homologous to the E6AP Carboxyl Terminus) ligases and the RBR (RINGin Between-RING) E3 ligases [START_REF] Zheng | Ubiquitin Ligases: Structure, Function, and Regulation[END_REF][START_REF] Scheffner | Mammalian HECT ubiquitin-protein ligases: biological and pathophysiological aspects[END_REF][START_REF] Dove | Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms[END_REF].

Cardiac muscle

Mammalian heart development begins with the specification of cardiac progenitor cells and ends with the maturation of specialized cells i.e. cardiomyocytes (CM) (Figure 1A). CMs undergo extensive remodeling before assuming their unique function as contractile cells.

UPS could drive CMs maturation by targeting key proteins involved in this process.

Proteasome inhibition in zebrafish embryos disrupts myofibril formation [START_REF] Fukuda | Proteolysis regulates cardiomyocyte maturation and tissue integration[END_REF] and further analyses identified the involvement Asb2b (Ankyrin repeat and Suppressor Of Cytokine Signalling/SOCS box protein-2 as a key regulator of sarcomere organization controlling CM maturation. Asb2b protein, as the substrate-binding component of a CRL E3 ubiquitin ligase complexe induces the proteasome-dependent degradation of the transcription factor TCF3 (Transcription factor 3). TCF3 dimerizes with MESP1 (Mesoderm Posterior BHLH Transcription Factor 1) to regulate downstream target genes required for the specification of the cardiac lineage to mature CM [START_REF] Chan | Mesp1 patterns mesoderm into cardiac, hematopoietic, or skeletal myogenic progenitors in a context-dependent manner[END_REF] (Figure 1B). Asb2b deletion or TCF3 overexpression in cardiomyocytes leads to the disassembly of sarcomeres, disruption of cell-cell junctions and upregulation of immature markers (phenotypes associated with cardiomyocyte dedifferentiation) [START_REF] Fukuda | Proteolysis regulates cardiomyocyte maturation and tissue integration[END_REF]. Furthermore, Asb2α, the Asb2b homologue in mice) controls the stability of the actin-binding protein Filamin A (FLNA) in vivo, which marks a crucial step in CM differentiation when the premyofibril building occurs (Figure 1B). Loss of Asb2α, impairs sarcomere formation and cell morphology, leading to heartbeat dysfunction and early embryonic lethality in mice [START_REF] Metais | Asb2alpha-Filamin A Axis Is Essential for Actin Cytoskeleton Remodeling During Heart Development[END_REF]. In contrast to Asb E3 ligase proteins, the Muscle RING-Finger ubiquitin ligases MuRF1 and MuRF2 play redundant roles during sarcomere maturation in mice [START_REF] Willis | Muscle ring finger 1 and muscle ring finger 2 are necessary but functionally redundant during developmental cardiac growth and regulate E2F1-mediated gene expression in vivo[END_REF]. Transient knockdown of MuRF2 impacts microtubule dynamics, intermediate filaments integrity and the assembly of the M-lines [START_REF] Mcelhinny | Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development[END_REF]. Additional observations in zebrafish embryos suggest that MuRF1/2 could regulate sequentially several steps of cytoskeleton organization in CMs [START_REF] Shimizu | The Calcineurin-FoxO-MuRF1 signaling pathway regulates myofibril integrity in cardiomyocytes[END_REF] but further studies will be needed to identify MuRF substrates during cardiac development.

Skeletal muscle

In mouse embryos, some cells of the somites become myocytes (Figure 2A), then elongate and start to express specialized sarcomeric proteins such as Actin, Myosin, and Desmin [START_REF] Chal | Making muscle: skeletal myogenesis in vivo and in vitro[END_REF]. Similar to CMs, several ubiquitin ligases have been described to regulate early stages of skeletal muscle differentiation by targeting directly sarcomeric proteins turnover or myogenic transcription program that controls myofibrillar protein expression.

Indeed, the protein level of PAX3 (Paired Box 3) accumulates in response to inhibition of proteasome activity in muscle progenitors. PAX3 is forwarded for proteasomal degradation by the RAD23B (Ras Associated with Diabetes 23 Homolog B) shuttle factor in adult muscle stem cells (satellite cells) [START_REF] Boutet | Regulation of Pax3 by proteasomal degradation of monoubiquitinated protein in skeletal muscle progenitors[END_REF] (Figure 2B). PAX3 monoubiquitination is mediated by the ubiquitin-activating/conjugating activity of TAF1 (TATA-Box Binding Protein Associated Factor 1), a component of the core transcriptional machinery. TAF1 binds directly to PAX3 and overexpression of TAF1 increases the level of ubiquitinated PAX3 leading to its degradation by the proteasome [START_REF] Boutet | Taf1 regulates Pax3 protein by monoubiquitination in skeletal muscle progenitors[END_REF]. Like PAX3, PAX7 (Paired Box 7) regulates myogenesis via its differential effects on the transcription factor MyoD (Myogenic Differentiation 1) [START_REF] Bustos | NEDD4 Regulates PAX7 Levels Promoting Activation of the Differentiation Program in Skeletal Muscle Precursors[END_REF]. Therefore, it is believed that the PAX7-to-MyoD ratio plays a key role in the regulation of myogenesis. NEDD4-1 (Neural Precursor Cell Expressed, Developmentally Down-Regulated 4-1) ubiquitinates and suppress PAX7 activation, shifting the balance in favor of MyoD and promoting myogenesis (Figure 2B). MyoD is also a direct target of Atrogin-1/MAFbx (also known as MAFbx/Muscle Atrophy F-Box Protein or FBXO32/F-Box Protein 32) and HUWE1 (HECT, UBA And WWE Domain Containing 1).

Both ubiquitinate and targets MyoD for degradation, thereby impairing myoblast fusion and differentiation [START_REF] Lagirand-Cantaloube | Inhibition of atrogin-1/MAFbx mediated MyoD proteolysis prevents skeletal muscle atrophy in vivo[END_REF][START_REF] Noy | HUWE1 ubiquitinates MyoD and targets it for proteasomal degradation[END_REF] (Figure 2B). In addition, HUWE1-dependent ubiquitination and USP10-dependent deubiquitination activities may finely balance terminal muscle cell differentiation in vitro by regulating TBP (TAT-binding protein) levels. HUWE1 knockdown in cells showed decreased levels of main myogenic transcription factors, which promote an arrest of cell differentiation due to aberrant myofibrillogenesis [START_REF] Li | A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation[END_REF]. Finally, the Neuralized E3 Ubiquitin Protein Ligase 2 Neurl2/Ozz, a substrate-binding component of a CRL E3 ubiquitin ligase complex, regulates early steps of myofibrillogenesis by targeting for degradation β-catenin [START_REF] Nastasi | Ozz-E3, a muscle-specific ubiquitin ligase, regulates beta-catenin degradation during myogenesis[END_REF] and the embryonic myosin heavy chain (MyH3) (Figure 2B).

Ozz did not bind the soluble pool of myosin but induces specifically the degradation of MyH3 allowing its replacement with neonatal or adult isoforms [START_REF] Campos | Ozz-E3 ubiquitin ligase targets sarcomeric embryonic myosin heavy chain during muscle development[END_REF]. These observations suggest that in striated muscle, the UPS could regulate protein isoform exchanges within complex cytoskeletal structures and participate in the adaptation of cell contractile properties.

The UPS controls sarcomeric protein turnover in striated muscle

In myofibrils, the sarcomere is organized in a highly ordered structure composed of actin (thin filaments) and myosin (thick filaments) polymers that associate into a complex, called acto-myosin. The sarcomere is delimited by two Z-discs, situated in the middle of the I-band, which is positioned on both sides of the A-band. The M-line is located in the center of the sarcomere between two Z-discs (Figure 3A). Each of those compartments contains specific structural and regulatory proteins required for sarcomere function.

Ubiquitin ligase targets and specificity in the sarcomere

In general, the cellular localization of E3 ligases determines its preferential substrates.

However, depending on the physiological context, a particular E3 ligase could target several proteins located in different compartments of the sarcomere (Figure 3, key Figure ).

For example, MuRF1 localizes mainly to the M-band where it interacts with Titin, but it can also be found at the Z-disc [START_REF] Centner | Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain[END_REF]. MuRF1 binds to and ubiquitinates Myosin-Binding Protein C and Myosin Light Chains 1 and 2 (MyBPC, MLC1/2, respectively) [START_REF] Cohen | During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation[END_REF], and Troponin I3/TNNI3 [START_REF] Kedar | Muscle-specific RING finger 1 is a bona fide ubiquitin ligase that degrades cardiac troponin I[END_REF] (Figure 3B). Fitting to its preferred targets, a MuRF1 dominant-negative mutant could not level down the amounts of thick filaments in response to muscle denervation, whereas thin filaments were still degraded [START_REF] Cohen | During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation[END_REF]. MuRF1 is able to bind several E2 ubiquitin-conjugating enzymes and some of these interactions are facilitated by Telethonin/TCAP, a Z-disc protein that regulates the late assembly of sarcomere [START_REF] Polge | A muscle-specific MuRF1-E2 network requires stabilization of MuRF1-E2 complexes by telethonin, a newly identified substrate[END_REF]. The binding of MuRF1 to the E2 enzyme UBE2E1 or to UBE2J2 targets TCAP for degradation in atrophying muscle [START_REF] Polge | A muscle-specific MuRF1-E2 network requires stabilization of MuRF1-E2 complexes by telethonin, a newly identified substrate[END_REF]. MuRF2, which is localized at the M-band and Z-disc, could interact with several MuRF1-binding partners [START_REF] Witt | MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination[END_REF]. However, loss of MuRF1 but not MuRF2 in mice increased cardiac muscle hypertrophy (due to lack of protein degradation) [START_REF] Willis | Cardiac muscle ring finger-1 increases susceptibility to heart failure in vivo[END_REF]. MuRF3 has been shown to associate with Z-discs as well as glutamylated microtubules [START_REF] Spencer | Regulation of microtubule dynamics and myogenic differentiation by MURF, a striated muscle RING-finger protein[END_REF], suggesting some specificity for MuRFs targets at the sarcomere.

MuRF3 has an important role in maintaining cardiac integrity and function after acute myocardial infarction by controlling the turnover of Z-disc proteins (the four and a half LIM domain-2/FHL2 and Filamin C /FLNC) [START_REF] Fielitz | Loss of muscle-specific RING-finger 3 predisposes the heart to cardiac rupture after myocardial infarction[END_REF] (Figure 3B).

As a hub for mechanosignaling, the Z-disc compartment appears to be a preferred target of several ubiquitin ligases. Indeed, the E3 ligase TRIM32 (Tripartite Motif Containing 32) was reported to localise around the Z-disc in skeletal muscle of guinea pig, where it seems to promote the degradation of Actin/ACTA1, Tropomyosin/TPM1, TroponinI3/TNNI3 but also Z-disc proteins such as Actinins (ACTN1 and 3) and the Z-disc associated protein Desmin (Figure 1B) [START_REF] Cohen | Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy[END_REF] (Figure 3B). In a disuse (denervation) model, TRIM32 trigger myofibril breakdown in a two-phase process involving the initial disassembly of Desmin filaments and later to myofibril breakdown [START_REF] Volodin | Myofibril breakdown during atrophy is a delayed response requiring the transcription factor PAX4 and desmin depolymerization[END_REF]. Desmin filaments are critical for muscle architecture and function because they connect the Z-lines of adjacent myofibrils laterally to the sarcolemma, mitochondria, and nuclear membrane [START_REF] Hol | Type III Intermediate Filaments Desmin, Glial Fibrillary Acidic Protein (GFAP), Vimentin, and Peripherin[END_REF]. Mutations in Desmin or its chaperone the αB-crystallin/CRYAB/HSPB5 (CRYAB-R120G mutation) leads to Desmin aggregates causing myopathies or cardiomyopathies with impaired proteolytic function of proteasomes in cardiomyocytes [START_REF] Chen | Intrasarcoplasmic amyloidosis impairs proteolytic function of proteasomes in cardiomyocytes by compromising substrate uptake[END_REF]. Other main regulators of the Z-disc proteins are the Cullin-RING ubiquitin ligases (CRLs). The prototypical CRL is the SKP1/S-Phase Kinase-Associated Protein 1-CUL1-F-box protein (SCF) complex [START_REF] Genschik | The emerging family of CULLIN3-RING ubiquitin ligases (CRL3s): cellular functions and disease implications[END_REF], a modular complex, where each F-box protein has a distinct substrate spectrum, allowing the core SCF scaffold, by its modular architecture, to target a large number of substrate proteins in a selective manner [START_REF] Skaar | SCF ubiquitin ligase-targeted therapies[END_REF]. A prominent example for a F-box protein active in muscle is the Atrogin-1/MAFbx, a key player in muscle cell size regulation [START_REF] Bodine | Identification of ubiquitin ligases required for skeletal muscle atrophy[END_REF]. MAFbx is a CUL-1 adaptor that localizes to the sarcomeric Z-disc, ubiquitinates and targets for degradation structural -Actinin-2/ACTN2, Desmin/DES and regulatory (calcineurin/CALNA) proteins (Figure 3B), thereby controlling hypertrophic signaling [START_REF] Li | Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex[END_REF][START_REF] Bodine | Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1[END_REF]. MAFbx is also important for the degradation of cardiac myosin-binding protein C (cMyBP-C/MyBPC3), but only in disease states where protein becomes mutated or truncated, a cause of familial hypertrophic cardiomyopathy (FHC) [START_REF] Mearini | Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms[END_REF] (Table 1). The normal protein MyBPC3 decorates part of the A-band but mislocalizes to the Z-disc when mutated allowing its interaction with MAFbx and its subsequent degradation [START_REF] Mearini | Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms[END_REF].

The other two CRLs complexes involved in sarcomeric proteins turnover are the CUL3 and CUL5 based complexes. Loss of function of CUL3 results in neonatal lethality and proteomic analysis of embryonic muscle tissues suggested the main involvement in sarcomere maturation and extracellular matrix protein (ECM) turnover [START_REF] Papizan | Cullin-3-RING ubiquitin ligase activity is required for striated muscle function in mice[END_REF]. CUL3 based E3 ligase complexes contain adaptors, which recognize specifically the substrates to target. Indeed, the adaptor protein KLHL41 (Kelch Like Family Member 41 also known as KRP1) interacts with the sarcomeric Z-disc and M-band protein N-RAP (LIM domain and Nebulin repeat protein), a pr -Actinin connection during premyofibril assembly [START_REF] Greenberg | Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes[END_REF]. KLHL41-deficient mice display lethal disruption of sarcomeres and aberrant expression of skeletal muscle structural and contractile proteins. KLHL41 is poly-ubiquitinated and acts, at least in part, by preventing aggregation and degradation of Nebulin thus stabilizing sarcomeres by acting as a molecular chaperone [START_REF] Ramirez-Martinez | KLHL41 stabilizes skeletal muscle sarcomeres by nonproteolytic ubiquitination[END_REF]. Another CUL3 E3 ligase complex adaptor is the KCTD6 (BTB/POZ domain-containing protein 6).

The sarcomeric protein Obscurin was described to be a scaffold for KCTD6 and its substrate sAnk1.5 (small ankyrin-1 isoform 5) at the M-band in CMs [START_REF] Lange | Obscurin and KCTD6 regulate cullin-dependent small ankyrin-1 (sAnk1.5) protein turnover[END_REF]. Loss of Obscurin leads to the mislocalization of KCTD6 and sAnk1.5 to the Z-disc, causing CUL3 activation and subsequent sAnk1.5 protein degradation [START_REF] Lange | Obscurin and KCTD6 regulate cullin-dependent small ankyrin-1 (sAnk1.5) protein turnover[END_REF]. For CUL5 complexes firstadaptor the Z-disc where it interacts and trigger for degradation Desmin and Filamin B/FLNB (Figure 3B) [START_REF] Thottakara | The E3 ubiquitin ligase Asb2beta is downregulated in a mouse model of hypertrophic cardiomyopathy and targets desmin for proteasomal degradation[END_REF]. In a knock-in mouse model carrying a patient mutation in MYBPC3, -specific E3 ligase in cardiac muscle [START_REF] Thottakara | The E3 ubiquitin ligase Asb2beta is downregulated in a mouse model of hypertrophic cardiomyopathy and targets desmin for proteasomal degradation[END_REF] Finally, the U-box containing E3 ligases UFD-2 is also able to use the unc-45 chaperone as an adaptor protein to target the motor domain of the C. elegans muscle myosin heavy chain-B (MHC-B) (Figure 1B), in a specific manner [START_REF] Hellerschmied | UFD-2 is an adaptor-assisted E3 ligase targeting unfolded proteins[END_REF]. The unc-45 protein should thus operate as a specific co-factor of the UFD-2 ubiquitin ligase. Although UFD-2 could not initiate the ubiquitination of target proteins itself, the ligase can extend short ubiquitin chains previously added by its partner, the E3 enzyme UFD418 [START_REF] Koegl | A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly[END_REF]. To describe this unique ubiquitination activity, the term "E4" ligase was introduced. However, a recent study challenged this "E4" concept, showing that UFD-2 is a bona fide E3 ligase capable of polyubiquitinating substrate proteins present in a partially unfolded state [START_REF] Hellerschmied | UFD-2 is an adaptor-assisted E3 ligase targeting unfolded proteins[END_REF]. Accordingly, Ubox and CRL E3 ligases may have evolved a conceptually similar mechanism utilizing diverse molecular adaptors to select specific substrates.

Although little is known about HECT E3 ligase targets in muscle, NEDD4 was proposed to target thin filament integrity by regulating the turnover of PDLIM7 (PDZ and LIM domain7) [START_REF] D'cruz | PDLIM7 is a novel target of the ubiquitin ligase Nedd4-1 in skeletal muscle[END_REF] (Figure 3B). PDLIM7 binds tropomyosin (I-bands) and acts as an adapter protein contributing to the dynamics and to polymerization of the actin filament [START_REF] Guy | The PDZ domain of the LIM protein enigma binds to beta-tropomyosin[END_REF].

Deubiquitinating enzymes (DUBs) and shuttle factors

To date, several DUBs of the USP (Ubiquitin Specific Peptidase) family such as USP2, USP14, USP19, USP25, and USP28 have been identified as UPS regulators in muscle [START_REF] Wing | Deubiquitinases in skeletal muscle atrophy[END_REF], with USP19 being the most intensely studied DUB. USP19 is induced during muscle wasting and may control the levels of certain myofibrillar proteins [START_REF] Wing | Deubiquitinating enzymes in skeletal muscle atrophy-An essential role for USP19[END_REF]. The USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis [START_REF] Valero | Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25[END_REF].

USP25m interacts with three sarcomeric proteins: Actin alpha-1 /ACTN1, Filamin C/FLNC, and Myosin Binding Protein C1/MyBPC1 but only MyBPC1 degradation is prevented by overexpression of USP25m (Figure 3B) [START_REF] Bosch-Comas | The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins[END_REF].

The proteasomal shuttle factors including UBQLN1/2 and RAD23B have been shown to participate in muscle PQC. Although, there are no reported sarcomeric protein targets for UBQLN1, cardiomyocyte-restricted knockout of UBQLN1 accumulated oxidized and ubiquitinated proteins resulting in late-onset cardiomyopathy and exacerbated myocardial ischemia-reperfusion injury [START_REF] Hu | Inadequate ubiquitination-proteasome coupling contributes to myocardial ischemiareperfusion injury[END_REF]. However, a role of UBQLN2 in the degradation of Desmin was recently proposed [START_REF] Gavriilidis | The MTM1-UBQLN2-HSP complex mediates degradation of misfolded intermediate filaments in skeletal muscle[END_REF]. When misfolded Desmin filaments accumulate in skeletal muscle cells, UBQLN2 binds to the Desmin-binding protein (myotubularin, MTM1). The UBQLN2-MTM1 interaction promotes Heat shock protein (HSP) recruitment; thereby binding to misfolded Desmin and activates UBQLN2-cargo to shuttle to the proteasome (Figure 3B) [START_REF] Gavriilidis | The MTM1-UBQLN2-HSP complex mediates degradation of misfolded intermediate filaments in skeletal muscle[END_REF]. The E3 ligases involved in this cascade remains to be identified.

Sarcomere PQC in human diseases

Most of sarcomeric proteins are involved in striated muscle disease such as myopathies, cardiomyopathies or both. Therefore identifying how PQC systems manage the truncated or aberrantly folded protein is a critical issue to depict the disease molecular mechanisms underlying pathophysiology. Table 1 summarize UPS components that target sarcomeric proteins, the related muscle disease and how gene mutations impact on sarcomere integrity (Table 1). Interestingly, several diseases causing mutations were also found in ubiquitin ligase or their adaptors. For example, TRIM32 mutations in patients lead to Limb-Girdle Muscular Dystrophy type 2H (LGMD2H) [START_REF] Schoser | Commonality of TRIM32 mutation in causing sarcotubular myopathy and LGMD2H[END_REF]. Moreover, mutations in the adaptors proteins (Kelch Like Family Members) KLHL9, KLHL40, KLHL41 or in the Kelch Repeat And BTB Domain Containing 13 (KBTBD13) have been involved in a family of skeletal muscle disorders called Nemaline myopathies (Table 1) [START_REF] Gupta | Kelch proteins: emerging roles in skeletal muscle development and diseases[END_REF]. Molecular evidences support that mutation in these adaptors affect CUL3 interaction, which may in turn affect the ubiquitination and turnover of protein substrates targeted by the CUL3 complex [START_REF] Gupta | Kelch proteins: emerging roles in skeletal muscle development and diseases[END_REF].

UPS, autophagy and chaperone crosstalk to safeguard sarcomere integrity

UPS and Autophagy

The adaptor protein p62/SQSTM1 (Sequestosome1) and the autophagy Cargo Receptor NBR1 facilitate the crosstalk between the UPS and selective autophagy. Both proteins can mediate direct interactions between polyubiquitinated proteins and LC3-II allowing autophagosome formation (Figure 4A) [START_REF] Johansen | Selective autophagy mediated by autophagic adapter proteins[END_REF]. Selective autophagy could be an adaptive response in order to remove proteins when UPS function is impaired, but it may also offers an alternative degradation pathway to allow processing of a larger substrate pool at any given time [START_REF] Rogov | Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy[END_REF]. Indeed, the treatment of CMs with the proteasome inhibitor, MG-132 led to a specific enhancement of selective autophagy markers (p62, LC3-II) [START_REF] Zheng | Proteasome malfunction activates macroautophagy in the heart[END_REF]. On the other hand the activation (using rapamycin) and inhibition (using the 3-Methyladenine, 3-MA)

of autophagy in CMs overexpressing the mutant Desmin appears to modulate the ubiquitination profile of this protein, suggesting that autophagy regulates the fate of the ubiquitinated proteins under conditions of protein aggregation [START_REF] Tannous | Autophagy is an adaptive response in desmin-related cardiomyopathy[END_REF]. Consequently, silencing p62 in cardiomyocytes overexpressing mutant desmin removes UPS-autophagy crosstalk, decreases autophagic flux and leads to increase cell injury and decreased cell viability [START_REF] Zheng | Autophagy and p62 in cardiac proteinopathy[END_REF]. In a similar way, the knockdown of the COP9 signalosome (CSN) subunits CSN8 (which mediates Cullin-RING ligase deneddylation) suppressed the ubiquitination and degradation of CRYAB-R120G resulting in accumulation of protein aggregates and decreased autophagy [START_REF] Su | COP9 signalosome controls the degradation of cytosolic misfolded proteins and protects against cardiac proteotoxicity[END_REF]. A role of the HECT E3 ligase HACE1 (HECT Domain and Ankyrin Repeat Containing E3 Ubiquitin Protein Ligase 1) in the coordination of autophagy and UPS was proposed. HACE1 mediates the p62-dependent selective autophagic turnover of ubiquitinated proteins. Hace1(-/-) mice display abnormal cardiac hypertrophy and accumulation of LC3, p62 and ubiquitinated proteins enriched for cytoskeletal species, indicating impaired autophagy and UPS programs [START_REF] Zhang | HACE1-dependent protein degradation provides cardiac protection in response to haemodynamic stress[END_REF]. Moreover, depleting the DUB enzyme USP19 results in downregulation of autophagy-promoting genes in muscle [START_REF] Bedard | Inactivation of the ubiquitin-specific protease 19 deubiquitinating enzyme protects against muscle wasting[END_REF].

USP19 can also deubiquitinate and stabilize BECN1(Beclin 1), thereby promoting autophagy [START_REF] Jin | USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin-1[END_REF]. Many ubiquitin ligases can target directly proteins required for autophagy (Figure 4A). BECN1 can be polyubiquitinated by NEDD4, which affects the steady-state concentration of BECN1. Additionally, when the molecular partner of BECN1, the phosphoinositide-3-Kinase, Class 3 (PIK3C3/Vps34) expression is reduced, the ubiquitination and degradation of BECN1 by NEDD4 is enhanced, suggesting that NEDD4 could be part of a cellular quality control mechanism that targets free BECN1 [START_REF] Platta | Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1[END_REF]. The charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal-sorting complex (ESCRT) required for autophagy, is also a target of MAFbx-mediated degradation.

Mice lacking MAFbx failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation and subsequent cardiomyocyte apoptosis [START_REF] Zaglia | Atrogin-1 deficiency promotes cardiomyopathy and premature death via impaired autophagy[END_REF].

Finally, the CRL complex Cul3-KLHL20 coordinates the degradation of a set of autophagic proteins (ATG13/Autophagy-Related Protein13 and ULK1/Unc-51 Like Autophagy

Activating Kinase 1) in prolonged starvation condition to control autophagy termination [START_REF] Liu | Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination[END_REF] (Figure 4). Consistently, skeletal muscle-specific deletion of KLHL20 induces higher autophagy and potentiates muscle atrophy in an experimental model of diabetes [START_REF] Liu | Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination[END_REF].

These data indicate that some ubiquitin ligases could mediate the interplay between the UPS and autophagy/lysosome system.

Including molecular chaperones into the UPS and autophagy

Chaperone-Mediated Autophagy (CMA) and Chaperone-Assisted Selective Autophagy (CASA) connect the chaperone system to protein quality control (Figure 4B). CMA involves the delivery of a chaperone substrate across a lysosomal membrane in ubiquitinindependent manner [START_REF] Kaushik | The coming of age of chaperone-mediated autophagy[END_REF]. CASA involves the ubiquitin-dependent degradation of a chaperone substrate by the lysosome [START_REF] Arndt | Chaperone-assisted selective autophagy is essential for muscle maintenance[END_REF]. Unlike CMA, there is evidence for CASA in skeletal muscle. CASA mediates the turnover of FLNs (Filamins) in a CHIP (C-terminal Hsp70 binding protein) ubiquitination-dependent manner via the co-chaperone BAG-3 (Bcl-2-associated athanogene-3) [START_REF] Arndt | Chaperone-assisted selective autophagy is essential for muscle maintenance[END_REF]. In human, BAG-3 deletion leads to severe muscular dystrophy and gradual deterioration of Z-disc components [START_REF] Selcen | Mutation in BAG3 causes severe dominant childhood muscular dystrophy[END_REF]. The other member of the BAG family, BAG-1 was also involved in Chaperone-Assisted Proteasomal degradation (CAP) (Figure 4B), a process by which a chaperone client is ubiquitinated and subsequently degraded by the proteasome [START_REF] Behl | Breaking BAG: The Co-Chaperone BAG3 in Health and Disease[END_REF]. BAG-1 has been proposed to physically link chaperone and proteasome systems as it also associates with the 26S proteasome [START_REF] Luders | The ubiquitin-related BAG-1 provides a link between the molecular chaperones Hsc70/Hsp70 and the proteasome[END_REF]. BAG-1 could interact with CHIP, which provides a mechanism by which chaperone substrates are ubiquitinated and targeted directly to the proteasome [START_REF] Demand | Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling[END_REF].

Supporting a model where chaperone activity is critical to trigger or facilitate the degradation process, many chaperones remain associated with their client proteins or are localized on nearby sarcomere structures. When sarcomere damage or mis-assembly occurs, chaperones respond to their client protein regardless of the damage. Therefore, the chaperone response could refold the protein of interest to avoid aggregation after damage or will promote somehow the recruitment of specialized E3 ligases (or other factors) to trigger the client protein for degradation [START_REF] Gavriilidis | The MTM1-UBQLN2-HSP complex mediates degradation of misfolded intermediate filaments in skeletal muscle[END_REF]. Interestingly, some chaperone could play a direct role in the ubiquitination and the degradation of muscle proteins [START_REF] Gazda | The myosin chaperone UNC-45 is organized in tandem modules to support myofilament formation in C. elegans[END_REF].

Indeed, upon teaming up with the UFD-2 ubiquitin ligase, the UNC-45 chaperone functions as a specific E3 ligase co-factor. Moreover, the interplay of UFD-2 with UNC-45 enables the E3 ligase to target a distinct conformational state of the substrate, as the chaperone preferentially interacts with unfolded myosin [START_REF] Hellerschmied | UFD-2 is an adaptor-assisted E3 ligase targeting unfolded proteins[END_REF]. Altogether, these data consolidate the idea that pairs of chaperones and E3 ligases seem to constitute central nodes in the quality-control network of muscle cells.

Concluding remarks

Keeping the sarcomere orderly while allowing for contractility is apparently a challenging task that needs highly specialized quality control systems. In fact, sarcomere function relies on a finely regulated UPS that control the assembly, rearrangement, and maintenance during physiological and stress situations. Defective PQC is a hallmark of genetic and acquired skeletal and cardiac muscle diseases. In addition to E3 ubiquitin ligases, DUBs and shuttle factors, the proteasome functional insufficiency (PFI) plays a major role in cardiac pathologies [START_REF] Wang | Proteasome functional insufficiency in cardiac pathogenesis[END_REF][START_REF] Li | Cardiac proteasome functional insufficiency plays a pathogenic role in diabetic cardiomyopathy[END_REF]. Evidence now supports that failure of PQC can result in a decline of regenerative capacity of sarcopenic muscles [START_REF] Garcia-Prat | Autophagy maintains stemness by preventing senescence[END_REF]. Within the proteostasis networks, UPS has gained attention due to the increasing number of druggable targets together with the emergence of new treatments that target the proteasome, ubiquitin ligases and DUBs [START_REF] Wertz | From Discovery to Bedside: Targeting the Ubiquitin System[END_REF] (see Clinician's Corner). Some of these small molecules showed clinical benefits in cancer patients, yet very few have been tested in the context of cardiac or skeletal muscle diseases. One reason is probably due to the lack of information about the precise action by which the UPS regulates a particular sarcomeric protein in a normal and pathological context. To this end, delineating the molecular mechanism of ubiquitin ligases, DUBs, and chaperones on normal or truncated proteins would be crucial. Among the many questions that remain to be addressed (See Outstanding Questions), the possible role of UPS in sarcomeric isoform transition is critical in the context of diseases. Particularly, this is the case for failing heart, where evidence of embryonic-like gene re-expression was observed [START_REF] Yin | Sarcomeric protein isoform transitions in cardiac muscle: a journey to heart failure[END_REF]. Therefore, exploring the developmental regulation of sarcomeric proteins isoforms by PQC systems will be important in order to understand and modulate the molecular actors required for their turnover. We foresee that diverse future drug-based tools targeting UPS, autophagy, and chaperones will be exponentially explored and will positively result in novel and effective strategies muscle diseases.

Table1: UPS sarcomere proteins targets and involvement in human disease. 365 -Abnormal sarcomere formation.

-Loss of sarcomere connection with sarcolemma.

-Presence of protein aggregates [START_REF] Cohen | Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy[END_REF] [30] -Altered myofilament sliding.

-Altered sarcomere force * Not yet referenced in OMIM database: For ACTN2 only tow de novo mutations, a missense and a deletion, in two unrelated patients.

# CRYAB is not ubiquitinated by FBXO4 but functions as a cofactor.

Glossary

A-band: is responsible both for the regular packing of thick filaments and for the uniform distribution of the tension over the myosin filament lattice.

Deubiquitinating enzymes (DUBs): Specific proteases/isopeptidases that hydrolyse polyubiquitin chains on the substrates. E3 HECT (Homologous to the E6AP Carboxyl Terminus) ligases: Catalyse ubiquitin transfer to protein substrates through a two-step reaction. Ubiquitin is first transferred from the E2 to the E3 and then from the E3 to the substrate.

Lysosome-autophagy systems: a proteolytic compartment that degrades cytoplasmic material and organelles.

Myofibrils: Distinct sarcomere subunits that extend in a repeating pattern along the length of the muscle fibres.

M-band:

The transverse structure in the center of the sarcomeric A-band. M-band proteins provide stability during sarcomeric contraction.

MG-132:

A synthetic peptide aldehyde proteasome inhibitor (reversible, and cellpermeable).

3-MA: 3-Methyladenine is a pan inhibitor of phosphatidylinositol 3-kinases (PI3Ks). Inhibits autophagy by blocking autophagosome formation.

Proteasomal shuttle factors: a protein family that capture ubiquitynated substrates and trigger them to the proteasome.

RING (Really Interesting New Gene) E3 ligases:

Characterised by RING and U-box domains responsible for binding the ubiquitin-charged E2 and acting as adaptors to stimulate the transfer of ubiquitin directly from the E2 to the substrates.

RBR (RING-in Between-RING) E3 ligases: Contain two RING domains but functions as

RING/HECT hybrid. The RING1 domain binds the ubiquitin-charged E2 and the RING2 domain functions as a HECT-type E3s inducing the ubiquitin transfer through a two-step reaction.

Rapamycin: An allosteric inhibitor of mTORC1 the mammalian target of rapamycin (mTOR) complex 1.

Thick and Thin filaments: proteins assemble into polymeric filaments, which interact and generate force and shortening in response to Ca 2+ concentration.

Ubiquitin-proteasome system (UPS):

A multicomponent system that identifies and degrades unwanted proteins. The E1, E2, E3 enzymes, DUBs and the proteasome are the main components.

Z-disc:

The lateral borders of the sarcomere. Z-disc proteins are required for mechanical stability of the sarcomere with the sarcolemma (muscle plasma membrane) and organelles.

Box1: Striated muscle myogenesis

During embryonic development of the heart, the cardiomyocytes (CMs) originate from mesoderm-derived progenitors under the control of a transcriptional regulatory network.

During CM differentiation, components of the contractile sarcomere apparatus are expressed and assemble in myofibrils allowing the first contractions at E8.5 in mice. The sarcomere assembly process starts when the heart transforms from a tube-like structure into a chambered heart with two atrium-ventricle pairs. Concomitantly, progenitors adopt an elongated shape of CM allowing myofibrils to become aligned. CMs further maturate throughout embryonic and postnatal development. During this process, CMs adapt a more elongated and cylindrical shape, with more organized sarcomeres.

Skeletal muscles mainly derive from myogenic progenitors located in transient structures of the vertebrate (somites), under the control of a transcriptional regulatory network [START_REF] Buckingham | Gene regulatory networks and cell lineages that underlie the formation of skeletal muscle[END_REF].

Myogenesis starts with the expression of specialized cytoskeletal proteins such as skeletal muscle actin, heavy chains of myosin, and desmin in elongating myogenic precursors or myoblasts [START_REF] Chal | Making muscle: skeletal myogenesis in vivo and in vitro[END_REF]. In mice, a primary phase of myogenesis (E10.5-E12.5) is characterized by the fusion of myoblasts to produce multinucleated myotubes, which further mature into myofibers. The process of fusion starts to be elucidated and involves actin remodeling [START_REF] Sampath | Myoblast fusion confusion: the resolution begins[END_REF].

During the second phase of myogenesis (E14.5-17.5 in mouse), myoblasts fuse among themselves or to the primary fibers, which are used as a scaffold, and form secondary or fetal fibers. Developing skeletal muscles express transiently myosin isoforms, including embryonic and neonatal myosin heavy chains (MyHCemb/MYH3 and MyHCneo/MYH8, respectively). These myosin isoforms disappear after birth and are replaced by adult fast and slow myosins [START_REF] Schiaffino | Developmental myosins: expression patterns and functional significance[END_REF].

The sarcomere assembly is a complicated multistep process and a matter of debate. The most supported model starts with the construction of premyofibrils [START_REF] Sanger | Assembly and dynamics of myofibrils[END_REF].

Clinician's Corner

Small molecules targeting the ubiquitin proteasome system have been successfully developed and showed clinical benefits primarily in cancer patients. Therapeutic applications of these drugs in the muscle context are presented below:

Proteasome inhibitors

Proteasome inhibitors approved by FDA and administered systemically (bortezomib, carfilzomib or orally (ixazomib citrate) have substantially improved survival of patients suffering from multiple myeloma. They provide an efficient treatment option mainly for relapsed and refractory multiple myeloma (MM) [START_REF] Hideshima | The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells[END_REF][91][START_REF] Vogl | Phase I/II study of the novel proteasome inhibitor delanzomib (CEP-18770) for relapsed and refractory multiple myeloma[END_REF]. However, significant numbers of cardiac related events were reported, indicating that general proteasome inhibition is detrimental to heart function. [START_REF] Reneau | Cardiotoxicity risk with bortezomib versus lenalidomide for treatment of multiple myeloma: A propensity matched study of 1,790 patients[END_REF][START_REF] Waxman | Carfilzomib-Associated Cardiovascular Adverse Events: A Systematic Review and Meta-analysis[END_REF]. In mice a chronic treatment with bortezomib induces LV hypertrophy to a similar degree as transverse aortic constriction (TAC) [START_REF] Tang | Proteasome functional insufficiency activates the calcineurin-NFAT pathway in cardiomyocytes and promotes maladaptive remodelling of stressed mouse hearts[END_REF].

Proteasome inhibition decreases muscle inflammation, a cause of muscle fiber necrosis and degeneration in atrophic or dystrophic mouse models and in patient derived cells.

decreases muscle inflammation, a cause of muscle fiber necrosis and degeneration [START_REF] Caron | The proteasome inhibitor MG132 reduces immobilization-induced skeletal muscle atrophy in mice[END_REF][START_REF] Assereto | Pharmacological rescue of the dystrophin-glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment[END_REF]. However, proteasome inhibition by bortezomib could not prevent muscle wasting in cancer cachexia, as initially designed [START_REF] Penna | Effect of the specific proteasome inhibitor bortezomib on cancer-related muscle wasting[END_REF].

E1, E2 and E3 inhibitors

A potent inhibitor (MLN7243/TAK-243) of the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade was developed [START_REF] Hyer | A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment[END_REF] and has entered a phase I clinical trials in patients with advanced solid tumors (NCT02045095). The effect of MLN7243/TAK-1on muscle physiology is still unknown.

For E2 enzyme inhibitors such as CC0651, NSC697923, despite their promising effects in vitro, these require further optimization for future clinical trials.

Targeting of E3 enzymes is a challenging task and is under deep investigation but did not CASA also requires the presence of the co-chaperones BAG1/3 proteins (Bcl-2-associated athanogene 1 and 3). This degradation process was involved in the turnover of FLN in a CHIP ubiquitination-dependent way. CAP (Chaperone-Assisted Proteasomal degradation)

is the process by which a misfolded protein and associated chaperones are subsequently degraded by the proteasome. The co-chaperone BAG1 is involved in CAP, by targeting CHIP-ubiquitinated proteins to the proteasome. Finally, misfolded proteins can be directly degraded by the proteasome, which requires the action of specific ubiquitin ligases, chaperones and probably proteases (probably to reduce the size of the substrates) prior proteasome degradation. The shuttle factors can sense and targets directly misfolded proteins to the proteasome. The grey column showed an example of sarcomere proteins targeted by these degradation pathways.

1 -

 1 Miss shaped Z-disc.

  activating enzyme UBA1: Catalyses in ATP driven reaction the first step in ubiquitin conjugation to mark substrate proteins for degradation. The E1 enzyme uses ATP to form a thioester bond between the cysteine of its active site and the C-terminal glycine of ubiquitin. E2 ubiquitin-conjugating enzyme: Catalyses the second step reaction of ubiquitination cascade. A transesterification reaction transfers ubiquitin from the E1 enzyme in the active site of the E2 enzyme. E3 ubiquitin ligases (E3s): Catalyse the final step of the ubiquitination cascade. E3 ubiquitin ligases select protein substrates and mediate their polyubiquitination in cooperation with E2.

  Premyofibrils are composed of actin-based thin filaments attached to Z-bodies (enriched in α-Actinin) and associated with non-muscle myosin to form mini A-bands. The incorporation of Titin and muscle myosin II filaments transforms premyofibrils to nascent myofibrils with beaded Zbands. The addition of Myosin-binding protein C and M-band proteins, the alignment of the thick filaments into A-bands and the formation of continuous Z-bands lead to the formation of mature myofibrils [89].

  reach clinical trials. For example, MuRF1 inhibitor (P013222) was active in inhibiting MuRF1 in a cellular atrophy model [100]. Another compound that interferes with the MuRF1-titin interaction was recently identified and could attenuate muscle atrophy and contractile dysfunction in mice during cardiac cachexia [101]. However, neither have reached clinical trials. MLN4924 (Pevonedistat) is a recently discovered small molecule that acts as an indirect inhibitor of Cullin-RING E3 ligases. It is currently in Phase II clinical cancer trials (NCT03745352, NCT03862157, NCT03330821…). However, MLN4924 disrupts the muscle cell differentiation program [102], which may alter the muscle regenerative capacity in cancer treated patients. Inhibitors of Deubiquitinases (DUBs) Several inhibitors of DUBs such as USP7 (P5091 and HBX 41108), USP20 (VDU2), USP33 (VDU1), UCH-L3 USP14 (IU1), have been developed (for review see [103]), demonstrating the feasibility of developing relatively specific inhibitors to these enzymes. Treating cells with IU1 enhanced the degradation of misfolded and aggregated proteins in disease such as the neurodegeneration protein Tau [104]. These first-generation inhibitors are now approaching clinical trials for cancer patients.

Figure 1 :

 1 Figure 1: Role of ubiquitin ligases in cardiac development. (A) In mouse, cardiac muscle development starts at E6.5-E7 when cells from the Pre-Cardiac Mesoderm (PCM) migrate towards the anterior side of the embryo, along with the Primitive Streak (PS) to form a Midline (ML) cellular cord. They form the major cardiac progenitor cells located first into the First Heart Field (FHF) and the Second Heart Field (SHF) and later in the

Figure 2 :

 2 Figure 2: UPS targets in skeletal muscle myogenesis. Skeletal muscles originate fromthe paraxial mesoderm, which forms in the primitive streak (PS) along the neuronal tube (NT). Myogenesis is initiated in the anterior presomitic mesoderm (aPSM) segments from the somites. The early myotome, which spans each somite, is composed of primary myocytes in contact with the sclerotome (the origin of the axial skeleton and all parts of the ribs). During primary myogenesis, progenitors delaminate from the dorsal side of the dermomyotome (Epaxial DM) and contribute to the formation of large primary myofibers. Some progenitors also migrate from the ventral lip of the DM (Hypaxial DM) to populate the body wall and limb buds. During the secondary myogenesis, myogenic progenitors contribute to secondary fibers formation, using the primary fibers as a scaffold. During this phase, satellite cell (SC) precursors associate with myofibers and will be maintained throughout the life. For each step, markers for the early, intermediates and late differentiation are indicated. The genes indicated in blue encode transcription factors. Sarcomeric and associated proteins are indicated in red. The adhesion molecules Ncam1 (Neural Cell Adhesion Molecule 1) and VCAM1 (Vascular Cell Adhesion Molecule 1) are indicated in grey. The curved arrow indicates the self-renewal potential of progenitors. The box summarizes the UPS actors that regulate myogenic markers. HUWE1, MAFbx, NEDD4-1, and Ozz are Ubiquitin ligases (red). TAF1 has ubiquitin-activating and E2

Figure 3 : 2 (

 32 Figure 3: UPS-mediated remodelling of sarcomeric proteins in striated muscles. (A) Thin (Actin) and thick (Myosin) filaments are the main sarcomeric proteins in striated muscles. To generate contraction, the thick-thin filaments are associated with many regulatory and scaffolding proteins. The Troponin and Tropomyosin proteins regulate actin-myosin bridging to promote contraction in a Ca 2+ dependent manner. The Myosinbinding protein (MyBPC) and the Tropomyosin-binding protein PDLIM7 regulate actinmyosin interaction and sarcomere length. The Titin and Nebulin giant proteins spread longitudinally the entire sarcomere while the Myomesin connect transversally myosin thick filaments. The α-actinin and Filamins, are the main cytoskeleton components of the Z-disc while the Desmin filaments surround the myofibril at the Z-disk level to laterally register adjacent myofibrils. Z-disc contains also regulatory proteins such as Myotilin (MYOT), the capping actin protein (CAP-Z), the chaperone αB-crystallin (CRYAB) and the Titin capping protein TCAP/Telethonin. (B) Sarcomeric and associated proteins targeted by Ubiquitin ligases, Deubiquitinating enzymes (DUBs) and the shuttle factors such as Ubiquilin-2 (UBQLN2). UBQLN2 recognizes ubiquitin-conjugated chain on target proteins via their UBA domain (Ubiquitin-associated domain) and triggers them to the proteasome via its proteasome-binding domain (UBL, ubiquitin-like domain). This degradation process involves the action of heat shock proteins (HSPs).

Figure 4 :

 4 Figure 4: The crosstalk between UPS, autophagy, and chaperones in striated muscle. (A) The adaptor proteins p62 and NBR1 facilitate the crosstalk between the UPS and autophagy. Both proteins can mediate direct interactions between polyubiquitinated proteins and the autophagosome protein LC3-II allowing autophagosome formation and the degradation of substrates by the autophagolysosomes. The UPS controls the turnover of key proteins such as BECN1, PIK3C3, ULK1 and ATG13 involved in autophagosome formation and maturation. NEDD4, the CRL complex CUL3-KLHL20, and HUWE1 are Ubiquitin ligases and USP19 is a deubiquitinase. (B) Reported degradation pathways that trigger misfolded and aggregated proteins for degradation. Aggregates resistant to proteasome degradation and chaperone refolding could be partially or totally cleared by autophagy. CASA (Chaperone-Assisted Selective Autophagy) involves the ubiquitindependent degradation of misfolded proteins and associated chaperones by the lysosome.

Acknowledgments

Because the field of protein quality control, in particular, in muscle and associated disease is exponentially growing, we sincerely apologize to authors whose work is not cited owing space limitation. We warmly thank Dr. Constandina Arvanitis for manuscript editing and Dr.

Pierre Lutz for his helpful comments. Funding for K.H. is provided by INSERM and Association Française contre la Myopathie (AFM-20879). Funding for C. M-L. is provided by the Centre National de la Recherche Scientifique (CNRS) and the University of Toulouse.


Gianpiero Tebano
email: gianpiero.tebano@gmail.com

Grace Li
email: grace.ws.li@gmail.com

Bojana Beovic
email: bojana.beovic@kclj.si

Julia Bielicki
email: jbielick@sgul.ac.uk

Adrian Brink
email: brinka@ampath.co.za

Mushira A Enani
email: menani@kfmc.med.sa

Brian Godman
email: briangodman@btinternet.com

Sylvia Lemos Hinrichsen
email: sylviahinrichsen@hotmail.com

Dan Kibuule
email: dkibuule@unam.na

Levy-Hara Gabriel

Gabriel Levy Hara

Oyinlola Oduyebo
email: oyinoduyebo@yahoo.com

Mike Sharland
email: mike.sharland@stgeorges.nhs.uk

Sanjeev Singh
email: drsanjeevsingh7@yahoo.com

Heiman F L Wertheim
email: heiman.wertheim@gmail.com

Dilip Nathwani
email: dilip.nathwani@nhs.net

Céline Pulcini
email: celine.pulcini@univ-lorraine.fr

Gabriel Levy

Essential and forgotten antibiotics: An inventory in lowand middle-income countries

Keywords: Access, Antibiotic access, low-and middle-income countries, antibiotic stewardship, survey

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

BACKGROUND

Balancing access and excess is one of the most urgent and complex challenges currently facing antibiotic use worldwide, particularly in low-and middle-income countries (LMICs) [START_REF] Mendelson | Maximising access to achieve appropriate human antimicrobial use in low-income and middleincome countries[END_REF].

Reliable access to efficacious medicines is a human right and a health priority; it is considered a cornerstone in the achievement of sustainable human development and has been included by the United Nations in Millennium Development Goal 8E [START_REF] Mendelson | Maximising access to achieve appropriate human antimicrobial use in low-income and middleincome countries[END_REF][START_REF]Transforming our world: the 2030 Agenda for Sustainable Development : Sustainable Development Knowledge Platform[END_REF][START_REF]United Nations Millennium Development Goals[END_REF][START_REF] Pulcini | Ensuring universal access to old antibiotics: a critical but neglected priority[END_REF]. Nonetheless, inequity of access to antimicrobials remains a global public health problem [START_REF] Mendelson | Maximising access to achieve appropriate human antimicrobial use in low-income and middleincome countries[END_REF]. It was recently estimated, for example, that adequate provision of antibiotics could avert 75% of deaths attributable to community-acquired pneumonia in children younger than 5 years, equivalent to 445,000 deaths [START_REF] Laxminarayan | Access to effective antimicrobials: a worldwide challenge[END_REF].

The World Health Organization (WHO) Essential Medicines List (EML) was developed in order to provide guidance to country health systems for selecting priority drugs to be provided to the population [START_REF]Essential medicines[END_REF][START_REF]The Selection and Use of Essential Medicines[END_REF]. The WHO-EML was updated in 2017 (20 th edition for adult, 6 th edition for children) [START_REF]Model Lists of Essential Medicines[END_REF][START_REF] Sharland | Classifying antibiotics in the WHO Essential Medicines List for optimal use-be AWaRe[END_REF], with re-categorisation of antibiotic agents into groups labelled 'Access', 'Watch' and 'Reserve', to highlight both the need for measures guaranteeing access to these essential antibiotics, and for stewardship programmes to promote responsible use. 'Access' antibiotics are those most frequently used to treat common bacterial infections, hence those which should be available everywhere.

At the same time, misuse and overuse of antibiotics is threatening their efficacy, in both high income countries (HICs) and LMICs [START_REF]Antimicrobial resistance: global report on surveillance[END_REF], calling for antibiotic stewardship (ABS) efforts. In 2011, the ESCMID Study Group for Antimicrobial stewardshiP (ESGAP) examined the availability of so called 'Forgotten antibiotics' in selected HICs [START_REF] Pulcini | Forgotten Antibiotics: An Inventory in Europe, the United States, Canada, and Australia[END_REF]. These are old and often off-patent antibiotics, selected for their unique value in targeting specific pathogens or in specific clinical indications (Appendix A).

In clinical practice, 'Forgotten' antibiotics offer the possibility of treating certain infections with narrower-spectrum agents (e.g. pivmecillinam for cystitis), and/or provide therapeutic options for difficult-to-treat infections due to multidrug resistant (MDR) pathogens (e.g. IV fosfomycin for carbapenem-resistant bacteria). A follow-up ESGAP study in 2015 found that the availability of these 'Forgotten' antibiotics (again in the HIC setting) was further decreased [START_REF] Pulcini | Forgotten antibiotics: a follow-up inventory study in Europe, the USA, Canada and Australia[END_REF].

Neither the availability of WHO-EML 'Access' antibiotics, nor that of 'Forgotten' antibiotics has been detailed in LMICs. Because the approval by an official drug regulatory agency and/or the Ministry of Health is the first step in enabling an official and regulated supply of an antimicrobial in a given country, our study investigated which antibiotics are officially approved among WHO-EML 'Access' antibiotics and 'Forgotten' antibiotics in a large number of LMICs.

METHODS

This was an international, Internet-based, cross-sectional survey. The final questionnaire was in English and was hosted on SurveyMonkey® (Palo Alto, CA, USA). It is available in Appendix B.

Definitions

An antibiotic was defined as approved if a country's official drug regulatory agency and/or the Ministry of Health approved its use, making it at least theoretically available on the market. The inclusion in the national Essential Medicines List was considered as a form of official approval. Only formulations for systemic use, i.e. oral, intramuscular (IM) and intravenous (IV) routes of administration were considered.

Paediatric formulations were defined as those specifically intended for paediatric use, i.e. dispersible tablets, syrups, and powders for reconstitution as syrups [START_REF] Bennett | Bennett's Principles and Practice of Infectious Diseases[END_REF]. Antibiotics usually administered to children using smaller doses of the same formulation intended for adults (e.g. IV formulations) were not considered as specifically intended for paediatric use.

Antibiotic selection

A consensus procedure via an Internet-based questionnaire was developed within the scientific committee, to come up with a short list of antibiotics to explore (see the list of authors for members of the scientific committee). The full list of WHO-EML 'Access' and ESGAP 'Forgotten' antibiotics was not used, since collecting data on these 54 antibiotics was considered to be too much work for the respondents. The antibiotics we included were considered by the scientific committee to be those most interesting for LMICs, with a high level of agreement (≥ 80%) among the experts.

A total of 41 antibiotics were selected (Table 1), while 13 antibiotics were rejected (Appendix C).

Selected antibiotics included: a) 24 WHO-EML 'Access' antibiotics; b) 15 'Forgotten' antibiotics; c) imipenem and erythromycin (which are listed as alternatives to meropenem and clarithromycin respectively in the EML). Eight of the WHO-EML 'Access' antibiotics (Table 1) were also originally included in the 'Forgotten' antibiotics list [START_REF] Pulcini | Forgotten Antibiotics: An Inventory in Europe, the United States, Canada, and Australia[END_REF][START_REF] Pulcini | Forgotten antibiotics: a follow-up inventory study in Europe, the USA, Canada and Australia[END_REF]. These antibiotics, as well as erythromycin and imipenem, are included among WHO-EML 'Access' antibiotics in the further paragraphs and figures.

Among the antibiotics included in the study, 14 had formulations specifically intended for paediatric use [START_REF] Bennett | Bennett's Principles and Practice of Infectious Diseases[END_REF].

A pilot version of the survey was circulated to the Scientific Committee to assess it for clarity and time requirement.

Survey deployment and double checking of results

A convenience sample of 40 national LMICs' contacts with recognised expertise in the field of the study was selected and received the invitation to participate. These national contacts were part of the scientific committee for 6 countries (Argentina, Brazil, India, Namibia, Nigeria, South Africa), or were chosen among the professional networks of the members of the scientific committee. One person per country was contacted. The questionnaire was circulated from October to December 2017 and one reminder was sent to non-responders.

In order to ensure the reliability of collected data, respondents were asked to provide one or more official source document and/or website to support their answers, mainly national formularies, drug regulatory agency websites or the national Essential Medicines List. These documents/websites were double checked by one of the researchers (GT or GL) to ensure accuracy. Country representatives were contacted in case of discrepancy.

Data analysis

Data were reported as numbers and percentages or medians and interquartile range [IQR], as appropriate.

The Mann-Whitney U test was used for comparisons between different groups (different continents 200

and different income groups), using the software Prism 5.0 (GraphPad Software, La Jolla, CA, USA). 201

RESULTS

Study participants

The invitation to participate was sent to 40 national contacts and 28 (70%) accepted to participate: 11 (39%) from Africa, 11 (39%) from Asia, 6 (22%) from America (Table 2). Five (18%) countries were classified as low income countries (LIC, gross national income -GNI -per capita: $1,005 or less), 10 (36%) as low middle-income countries (LoMIC, GNI per capita: $1,006 to $3,955), and 13 (46%) as upper middle-income countries (UpMIC, GNI per capita: $3,956 to $12,235) [START_REF]World Bank Country and Lending Groups[END_REF]. The respondents are listed in the Acknowledgement section and their complete affiliations are detailed in the Appendix D.

Approval of antibiotics

Figure 1 shows the number of countries having approved each of the WHO-EML 'Access' antibiotics and 'Forgotten' antibiotics.

Among the WHO-EML 'Access' antibiotics, 9/26 (35%) were approved in all countries (amoxicillin, ampicillin, benzylpenicillin, ceftriaxone, clarithromycin, ciprofloxacin, doxycycline, gentamicin, metronidazole), and all (100%) were approved in more than two thirds of countries. Among the 'Forgotten' antibiotics not included in the 'Access' list, only 1/15 (cefepime) was approved in more than two thirds of countries.

Figure 2 shows the number of antibiotics approved per country. The median number was: 30

[interquartile range [START_REF]Model List of Essential Medicines. 19th List[END_REF][START_REF] Tängdén | Unavailability of old antibiotics threatens effective treatment for common bacterial infections[END_REF][START_REF] Mendelson | The One Health stewardship of colistin as an antibiotic of last resort for human health in South Africa[END_REF][START_REF] Manenzhe | The spread of carbapenemase-producing bacteria in Africa: a systematic review[END_REF][START_REF] Zellweger | A current perspective on antimicrobial resistance in Southeast Asia[END_REF][START_REF]Access to medicine foundation[END_REF][START_REF] Årdal | International cooperation to improve access to and sustain effectiveness of antimicrobials[END_REF][START_REF]Human Development Index (HDI)[END_REF][START_REF]Human Development Reports[END_REF][32][33][34][35] overall; 24 [START_REF]Model List of Essential Medicines. 19th List[END_REF][START_REF] Tängdén | Unavailability of old antibiotics threatens effective treatment for common bacterial infections[END_REF][START_REF] Mendelson | The One Health stewardship of colistin as an antibiotic of last resort for human health in South Africa[END_REF][START_REF] Manenzhe | The spread of carbapenemase-producing bacteria in Africa: a systematic review[END_REF][START_REF] Zellweger | A current perspective on antimicrobial resistance in Southeast Asia[END_REF] in Africa; 31 [START_REF]Access to medicine foundation[END_REF][START_REF] Årdal | International cooperation to improve access to and sustain effectiveness of antimicrobials[END_REF][START_REF]Human Development Index (HDI)[END_REF][START_REF]Human Development Reports[END_REF][32][33][34][35][36] in America; 34 [START_REF]Model List of Essential Medicines. 19th List[END_REF][START_REF] Tängdén | Unavailability of old antibiotics threatens effective treatment for common bacterial infections[END_REF][START_REF] Mendelson | The One Health stewardship of colistin as an antibiotic of last resort for human health in South Africa[END_REF][START_REF] Manenzhe | The spread of carbapenemase-producing bacteria in Africa: a systematic review[END_REF][START_REF] Zellweger | A current perspective on antimicrobial resistance in Southeast Asia[END_REF][START_REF]Access to medicine foundation[END_REF][START_REF] Årdal | International cooperation to improve access to and sustain effectiveness of antimicrobials[END_REF][START_REF]Human Development Index (HDI)[END_REF][START_REF]Human Development Reports[END_REF][32][33][34][35][36][37] in Asia; 24 [START_REF]Model List of Essential Medicines. 19th List[END_REF][START_REF] Tängdén | Unavailability of old antibiotics threatens effective treatment for common bacterial infections[END_REF][START_REF] Mendelson | The One Health stewardship of colistin as an antibiotic of last resort for human health in South Africa[END_REF][START_REF] Manenzhe | The spread of carbapenemase-producing bacteria in Africa: a systematic review[END_REF][START_REF] Zellweger | A current perspective on antimicrobial resistance in Southeast Asia[END_REF][START_REF]Access to medicine foundation[END_REF][START_REF] Årdal | International cooperation to improve access to and sustain effectiveness of antimicrobials[END_REF][START_REF]Human Development Index (HDI)[END_REF] in LIC; 25 [START_REF]Médecins sans frontières[END_REF][START_REF]Medicines shortages. Global approaches to addressing shortages of essential medicines in health systems[END_REF][START_REF]Model List of Essential Medicines. 19th List[END_REF][START_REF] Tängdén | Unavailability of old antibiotics threatens effective treatment for common bacterial infections[END_REF][START_REF] Mendelson | The One Health stewardship of colistin as an antibiotic of last resort for human health in South Africa[END_REF][START_REF] Manenzhe | The spread of carbapenemase-producing bacteria in Africa: a systematic review[END_REF][START_REF] Zellweger | A current perspective on antimicrobial resistance in Southeast Asia[END_REF][START_REF]Access to medicine foundation[END_REF][START_REF] Årdal | International cooperation to improve access to and sustain effectiveness of antimicrobials[END_REF][START_REF]Human Development Index (HDI)[END_REF][START_REF]Human Development Reports[END_REF][32][33][34][35][36] in LoMIC; 31 [START_REF]Human Development Index (HDI)[END_REF][START_REF]Human Development Reports[END_REF][32][33][34][35][36] in UpMIC. Medians were not significantly different among continents and different income groups (data not shown).

Approval of antibiotics specifically intended for paediatric use

Figure 3 shows the number of countries having approved a formulation specifically intended for paediatric use for each of the WHO-EML 'Access' antibiotics and 'Forgotten' antibiotics.

Six out of fourteen antibiotics (43%: amoxicillin, amoxicillin-clavulanic acid, cotrimoxazole, erythromycin, metronidazole, oral antistaphylococcal penicillin) were approved in more than two thirds of countries.

Figure 4 shows the number of formulations specifically intended for paediatric use approved per country.

The median number was: 8 [interquartile range [START_REF]The Selection and Use of Essential Medicines[END_REF][START_REF]Model Lists of Essential Medicines[END_REF][START_REF] Sharland | Classifying antibiotics in the WHO Essential Medicines List for optimal use-be AWaRe[END_REF] overall; 8 [START_REF]The Selection and Use of Essential Medicines[END_REF][START_REF]Model Lists of Essential Medicines[END_REF][START_REF] Sharland | Classifying antibiotics in the WHO Essential Medicines List for optimal use-be AWaRe[END_REF][START_REF]Antimicrobial resistance: global report on surveillance[END_REF] in Africa; 7 [START_REF] Laxminarayan | Access to effective antimicrobials: a worldwide challenge[END_REF][START_REF]Essential medicines[END_REF][START_REF]The Selection and Use of Essential Medicines[END_REF][START_REF]Model Lists of Essential Medicines[END_REF] in America; 8 [START_REF]The Selection and Use of Essential Medicines[END_REF][START_REF]Model Lists of Essential Medicines[END_REF][START_REF] Sharland | Classifying antibiotics in the WHO Essential Medicines List for optimal use-be AWaRe[END_REF][START_REF]Antimicrobial resistance: global report on surveillance[END_REF][START_REF] Pulcini | Forgotten Antibiotics: An Inventory in Europe, the United States, Canada, and Australia[END_REF] in Asia; 7 [START_REF]The Selection and Use of Essential Medicines[END_REF][START_REF]Model Lists of Essential Medicines[END_REF][START_REF] Sharland | Classifying antibiotics in the WHO Essential Medicines List for optimal use-be AWaRe[END_REF][START_REF]Antimicrobial resistance: global report on surveillance[END_REF][START_REF] Pulcini | Forgotten Antibiotics: An Inventory in Europe, the United States, Canada, and Australia[END_REF] in LIC; 8 [START_REF]Model Lists of Essential Medicines[END_REF][START_REF] Sharland | Classifying antibiotics in the WHO Essential Medicines List for optimal use-be AWaRe[END_REF][START_REF]Antimicrobial resistance: global report on surveillance[END_REF] in LoMIC; 7 [START_REF] Laxminarayan | Access to effective antimicrobials: a worldwide challenge[END_REF][START_REF]Essential medicines[END_REF][START_REF]The Selection and Use of Essential Medicines[END_REF][START_REF]Model Lists of Essential Medicines[END_REF] in UpMIC. Information about paediatric formulations was not available for Vietnam.

Double checking of results

The results were double checked by investigators in 21/28 cases (75%), consulting official source documents and websites provided by respondents. Double checking was not possible in 3 cases because of linguistic barriers (China, Iran, Vietnam) and in 4 cases because of a lack of electronic versions of source documents, or websites (Azerbaijan, Nepal, Panama, Thailand). The list of documents and websites is provided in Appendix E.

DISCUSSION

This large international survey gives an overview on the official approval by local drug regulatory agencies and/or the Ministries of Health of 41 useful antibiotics, including paediatric formulations, in a sample of 28 LMICs from 3 continents. The explored antibiotics were selected among those included as 'Access' antibiotics in the EML by WHO in 2017 [START_REF]Model Lists of Essential Medicines[END_REF][START_REF] Sharland | Classifying antibiotics in the WHO Essential Medicines List for optimal use-be AWaRe[END_REF], and those included in the list of 'Forgotten' antibiotics in the ESGAP studies by Pulcini et al. in 2011 [START_REF] Pulcini | Forgotten Antibiotics: An Inventory in Europe, the United States, Canada, and Australia[END_REF] and 2015 [START_REF] Pulcini | Forgotten antibiotics: a follow-up inventory study in Europe, the USA, Canada and Australia[END_REF]. We found that most of the WHO-EML 'Access' antibiotics are approved in the 28 surveyed LMICs, including the most clinically relevant paediatric formulations; on the contrary many 'Forgotten' antibiotics are not approved in these countries.

This study assessed if a given systemic antibiotic was approved for human use in a given country. We did not aim to collect information about effective availability at different levels of each country's health care system. The appraisal of drug availability is challenging for several reasons: health care systems in LMICs are known to have complex organisations, with different levels of health care provision (e.g. reference hospitals, district hospitals, peripheral primary health care facilities), having different aims and therefore providing different assortments of medications [START_REF]Health services[END_REF][START_REF] Das | The Quality of Medical Care in Low-Income Countries: From Providers to Markets[END_REF]; private for-profit health care providers are variably present and do not necessarily follow the policies implemented in the public system [START_REF] Forsberg | Moving towards in-depth knowledge on the private health sector in low-and middle-income countries[END_REF]; the circulation of drugs in the informal sector may be significant and difficult to assess [START_REF] Ahmed | Informal sector providers in Bangladesh: how equipped are they to provide rational health care?[END_REF]; other relevant actors in the non-profit sector may adhere to independent drug policies and provide their own supply channels [START_REF]social welfare of Tanzania. Mapping of the medicines procurement and supply management system in Tanzania[END_REF][START_REF]Guide to Global Fund Policies on Procurement and Supply Management of Health Products[END_REF][START_REF]Médecins sans frontières[END_REF]; drug shortages and troubles in supply chain may affect effective access, particularly in remote and poorest areas [START_REF]Medicines shortages. Global approaches to addressing shortages of essential medicines in health systems[END_REF]. Availability therefore needs to be assessed on a country-by-country basis. For example, a report by the Ministry of Health and Social Welfare of Tanzania showed that there are more than 25 stakeholders involved in the procurement of medicines in that country [START_REF]social welfare of Tanzania. Mapping of the medicines procurement and supply management system in Tanzania[END_REF]. This complex scenario revealed that investigations of drug approval is an important first step, since the approval by the local drug authority is needed to further enable a reliable and regulated access to a given antibiotic in a particular country.

Our study demonstrated that overall LMICs comply with WHO-EML, since the vast majority of 'Access' antibiotics were approved by an official drug regulatory agency and/or the Ministry of Health (Figure 1). Actually the list of 'Access' antibiotics was published in March 2017 and amended in August 2017, hence we can rather argue that surveyed countries complied with the previous WHO-EML (2015 version) [START_REF]Model List of Essential Medicines. 19th List[END_REF]. However, as illustrated in Table 1, the vast majority of this 'Access' antibiotics were already listed as 'Core antibiotics' in the previous WHO-EML, with the exception of few antibiotics (cefotaxime, clindamycin, carbapenems and vancomycin) which were anyway in the 'Complementary list' [START_REF]Model List of Essential Medicines. 19th List[END_REF].

The situation is significantly different for 'Forgotten' antibiotics. Our study explored the approval of 23 'Forgotten' antibiotics; 8 were included also among the WHO-EML 'Access' antibiotics and were approved in the large majority of countries (Figure 1); on the other hand, the remaining 15 'Forgotten' antibiotics were approved only in a minority of countries (Figure 1). 'Forgotten' antibiotics can have a role in daily practice in different clinical situations: enabling reduction in the use of other broader spectrum and sometimes less effective antimicrobials (e.g. antistaphylococcal penicillins, oral fosfomycin, pivmecillinam); offering the opportunity to treat MDR pathogens (e.g. aztreonam, IV fosfomycin, polymyxins, temocillin and many others); acting as niche agent for specific pathogens (e.g. ampicillin-sulbactam for Acinetobacter baumannii, spectinomycin for Neisseria gonorrhoeae, tobramycin for Pseudomonas aeruginosa) (Appendix A) [START_REF] Pulcini | Ensuring universal access to old antibiotics: a critical but neglected priority[END_REF][START_REF] Pulcini | Forgotten Antibiotics: An Inventory in Europe, the United States, Canada, and Australia[END_REF][START_REF] Tängdén | Unavailability of old antibiotics threatens effective treatment for common bacterial infections[END_REF]. This can be of added value in LMICs, where prevalence of MDR bacteria is often very high, calling for both effective treatments and antibiotic stewardship efforts [START_REF] Mendelson | The One Health stewardship of colistin as an antibiotic of last resort for human health in South Africa[END_REF][START_REF] Manenzhe | The spread of carbapenemase-producing bacteria in Africa: a systematic review[END_REF][START_REF] Zellweger | A current perspective on antimicrobial resistance in Southeast Asia[END_REF]. The availability of these mainly out-of-patent products is threatened by a vicious circle including low economic incentives; limited market size; low level of scientific evidence (particularly on pharmacokinetics/pharmacodynamics and clinical efficacy); low use by physicians; and absence of inclusion in guidelines [START_REF] Pulcini | Ensuring universal access to old antibiotics: a critical but neglected priority[END_REF][START_REF] Pulcini | Forgotten Antibiotics: An Inventory in Europe, the United States, Canada, and Australia[END_REF][START_REF] Tängdén | Unavailability of old antibiotics threatens effective treatment for common bacterial infections[END_REF].

The last version of the WHO-EML marked a step forward since 3 of these 'Forgotten' antibiotics (ertapenem, teicoplanin and ticarcillin-clavulanic acid) were included in the newly designed 'Watch' category and 5 (aztreonam, cefepime, colistin and polymyxin B, IV fosfomycin) in the 'Reserve' group [START_REF]Essential medicines[END_REF]. This is a significant change compared to the previous WHO-EML list [START_REF]Model List of Essential Medicines. 19th List[END_REF] and could help improve the availability of these molecules for selected conditions.

Overall American and Asian countries, as well as countries classified as upper middle-income countries approved more antibiotics than African countries and lower income countries. These data suggest that countries having higher incomes could guarantee access to more antibiotics, even if these differences were not statistically significant, probably due to the relatively small number of explored countries.

Concerning formulations specifically intended for paediatric use (Figures 3 and4), the antibiotics more frequently used for the most common paediatric bacterial infections (amoxicillin, amoxicillinclavulanic acid, antistaphylococcal penicillins, macrolides, cotrimoxazole and metronidazole) were approved in the majority of countries. However some countries approved the use of few paediatric formulations, suggesting that the availability of appropriate paediatric drugs could be threatened in some settings. Due to the enormous impact on children's health of proper access to medicines [START_REF] Laxminarayan | Access to effective antimicrobials: a worldwide challenge[END_REF] and the paucity of data about the availability of paediatric drugs, this aspect urgently deserves further efforts in research, as well as targeted policies.

There is still a long way to go. Coordinated efforts with multi-stakeholder approaches will be needed to improve antimicrobial availability in LMICs. Areas include: continuous monitoring of the availability of all antibiotics included in the WHO-EML; consideration of the possible role of 'Forgotten' antibiotics in these settings; access gap analysis; strengthening of health systems; promotion of universal health coverage; promotion of alternative economic models to delink payments for antimicrobials from the volumes sold; and the evaluation of the potential role and feasibility of a Global Antibiotic Access and Conservation Fund [START_REF] Mendelson | Maximising access to achieve appropriate human antimicrobial use in low-income and middleincome countries[END_REF][START_REF] Pulcini | Ensuring universal access to old antibiotics: a critical but neglected priority[END_REF][START_REF]Access to medicine foundation[END_REF][START_REF] Årdal | International cooperation to improve access to and sustain effectiveness of antimicrobials[END_REF].

This study has some limitations. Only one respondent was contacted for each country, thus the reliability of some of the collected data could be questioned. However, respondents provided one or more official document and/or website as source to corroborate their answers and these texts were double checked by the investigators in 75% of cases; all discrepancies were discussed and solved. The study investigated the approval of paediatric formulations overall, without detailing if different types of formulations (intended for children of different age groups) were approved. We did not distinguish between the approval of an antibiotic by a drug regulatory agency and/or the Ministry of Health and the inclusion in the national EML, even if this difference could influence the effective drug availability. We did not collect data on how many brands and/or generics are approved for each antibiotic, to avoid excessive workload for the respondents. It is however possible that if only a limited number had been approved, this would indicate greater vulnerability, when compared to many approvals per medicine. Finally we screened a sample of 28 countries, and our results cannot be generalized to other countries. Our sample nevertheless included countries from 3 continents, with a good geographic distribution and a huge variability in terms of health care, demographic and econometric characteristics (Table 2).

In conclusion, most of the WHO-EML 'Access' antibiotics are approved in the 28 surveyed LMICs, including the most relevant paediatric formulations. On the contrary, many 'Forgotten' antibiotics are not approved in these countries, despite their important role in some specific clinical conditions, particularly in areas with high prevalence of MDR bacteria. The WHO-EML therefore seems to have a very positive impact regarding approval of antibiotics at national level. Further studies are needed to verify in each LMIC if approved antibiotics are effectively available in daily clinical practice, with reliable supply chain, affordable prices, and acceptable formulations, at different levels of the health care system.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. WHO-EML 'Access' antibiotics and 'Forgotten' antibiotics approved in the 28 surveyed countries

Acknowledgements

Partial results were presented as a poster (n° 2935) at the 2018 ECCMID in Madrid.

The authors would like to thank all national contacts who participated in the survey: Fidan Huseynova (Azerbaijan), Abu Hena Mostafa Kamal (Bangladesh), Cossi Angelo Attinsounon (Benin), Joyce Kgatlwane (Botswana), Paul Turner (Cambodia), Mei Zeng (China), Carlos Robledo (Colombia), Daniel Kwame Afriyie and Emmanuel Sarkodie (Ghana), Mojdeh Hakemi-Vala (Iran), Vilada Chansamouth (Lao), Buddha Basnyat (Nepal), Silvio Vega (Panama), Elena Candia Florentín (Paraguay), Luis Cuellar (Peru), Awa Ndir (Senegal), Amu Adefolarin Ayodeji (Swaziland), Stanley Mwita (Tanzania), Visanu Thamlikitku (Thailand) Eric Vreede and Flavio Brandao De Araujo (Timor-Leste), Vu Dinh Phu (Vietnam), Aubrey

Chichonyi Kalungia (Zambia), Blessmore Vimbai Chaibva (Zimbabwe).

Declarations

Funding: No funding Competing Interests: The authors have no conflict of interest to disclose related with the topic.

Ethical Approval: Not required Table 1. List of the 41 included antibiotics Antibiotic (in alphabetical order) WHO-EML (2017) [START_REF]Essential medicines[END_REF] Former WHO-EML (2015) [START_REF]Model List of Essential Medicines. 19th List[END_REF] Forgotten antibiotics list Access antibiotics list

Watch antibiotics list

Reserve antibiotics list

Core list Compleme ntary list

Meropenem (and imipenem as an alternative) • UpMIC: upper middle-income countries (GNI per capita: $3,956 to $12,235)

• LoMIC: low middle-income countries (GNI per capita: $1,006 to $3,955)

• LIC: low income countries (GNI per capita: $1,005 or less)

2. HDI: Human development index. This is a composite indicator including a health dimension (life expectancy at birth), an education dimension (mean of years of schooling for adults aged 25 years and more and expected years of schooling for children of school entering age) and a standard of living dimension (gross national income per capita). The rank in the table refers to 188 screened countries (the higher the better). The HDI is endorsed by the United Nations Development Programme [START_REF]Human Development Index (HDI)[END_REF][START_REF]Human Development Reports[END_REF].