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1.AbstractIn contrast to mice or zebrafish, trout exhibits post-larval muscle growth through hypertrophy and formation of new myofibers (hyperplasia). The muscle fibers are formed by the fusion of mononucleated cells (myoblasts) regulated by several muscle-specific proteins such as myomaker or myomixer. In this work, we identified a unique gene encoding a myomixer protein of 77 amino acids (aa) in the trout genome. Sequence analysis and phylogenetic tree, showed moderate conservation of the overall protein sequence across teleost fish (61% of aa identity between trout and zebrafish myomixer sequences). Nevertheless, the functionally essential motif, AxLyCxL is perfectly conserved in all studied sequences of vertebrates. Using in situ hybridization, we observed that myomixer was highly expressed in the embryonic myotome, particularly in the hyperplasic area. Moreover, myomixer remained readily expressed in white muscle of juvenile (1 and 20 g) although its expression decreased in mature fish. We also showed that myomixer is up-regulated during muscle regeneration and in vitro myoblasts fusion. Together, these data indicate that myomixer expression is consistently associated with the formation of new myofibers during somitogenesis, post-larval growth and muscle regeneration in trout.

The formation of new myofibers in vertebrates occurs by myoblast fusion and requires fusogenic activity of the muscle-specific membrane protein myomaker. Here, using in silico (BLAST) genome analyses, we show that the myomaker gene from trout includes 14 minisatellites, indicating that it has an unusual structure compared with those of other animal species. We found that the trout myomaker gene encodes a 434 –amino acid (aa) protein, in accordance with its apparent molecular mass (40 kDa) observed by immunoblotting. The first half of the trout myomaker protein (1–220 aa) is similar to the 221-aa mouse myomaker protein, whereas the second half (222–234 aa) does not correspond to any known motifs and arises from two protein extensions. The first extension (70 aa) apparently appeared with the radiation of the bony fish clade Euteleostei, whereas the second extension (up to 236 aa) is restricted to the superorder Protacanthopterygii (containing salmonids and pike) and corresponds to the insertion of minisatellites having a length of 30 nucleotides. According to gene expression analyses, trout myomaker expression is consistently associated with the formation of new myofibers during embryonic development, postlarval growth, and muscle regeneration. Using cell-mixing experiments, we observed that trout myomaker has retained the ability to drive the fusion of mouse fibroblasts with C2C12 myoblasts. Our work reveals that trout myomaker has fusogenic function despite containing two protein extensions., SCOPUS: ar.j, DecretOANoAutActif, info:eu-repo/semantics/published

Supplementary data to this article can be found online at : https://doi.org/10.1016/j.cbpb.2018.11.005; The understanding of muscle tissue formation and regeneration is essential for the development of therapeutic approaches to treat muscle diseases or loss of muscle mass and strength during ageing or cancer. One of the critical steps in muscle formation is the fusion of muscle cells to form or regenerate muscle fibres.To identify new genes controlling myoblast fusion, we performed a siRNA screen in c2c12 myoblasts. The genes identified during this screen were then studiedi n vivo by knock down in zebrafish using morpholino. We found that N-alpha-acetyl transferase 15 (Naa15) knockdown enhanced c2c12 myoblast fusion, suggesting that Naa15 negatively regulates myogenic cell fusion. We identified two Naa15 orthologous genes in the zebrafish genome: Naa15a and Naa15b. These two orthologs were expressed in the myogenic domain of the somite. Knockdown of zebrafish Naa15a and Naa15b genes induced a“U”-shaped segmentation of the myotome and alteration of myotome boundaries, resulting in the formation of abnormally long myofibres spanning adjacent somites. Taken together, these results show that Naa15 regulates myotome formation and myogenesis in fish.

International audience; Muscle inflammation as in idiopathic inflammatory myopathies (IIM) leads to muscle weakness, mononuclear cell infiltration, and myofiber dysfunction affecting calcium channels. The effects of interleukin-17A (IL-17) and tumor necrosis factor-alpha (TNF alpha) on inflammation and calcium changes were investigated in human myoblasts. Human myoblasts were exposed to IL-17 and/or TNF alpha with/without store-operated Ca2+ entry (SOCE) inhibitors (2-ABP or BTP2). For co-cultures, peripheral blood mononuclear cells (PBMC) from healthy donors activated or not with phytohemagglutinin (PHA) were added to myoblasts at a 5:1 ratio. IL-17 and TNF alpha induced in synergy CCL20 and IL-6 production by myoblasts (> 14-fold). PBMC-myoblast co-cultures enhanced CCL20 and IL-6 production in the presence or not of PHA compared to PBMC or myoblast monocultures. Anti-IL-17 and/or anti-TNF alpha decreased the production of IL-6 in co-cultures (p < 0.05). Transwell system that prevents direct cell-cell contact reduced CCL20 (p < 0.01) but not IL-6 secretion. IL-17 and/or TNF alpha increased the level of the ER stress marker Grp78, mitochondrial ROS and promoted SOCE activation by 2-fold (p < 0.01) in isolated myoblasts. SOCE inhibitors reduced the IL-6 production induced by IL-17/TNF alpha. Therefore, muscle inflammation induced by IL-17 and/or TNF alpha may increase muscle cell dysfunction, which, in turn, increased inflammation. Such close interplay between immune and non-immune mechanisms may drive and increase muscle inflammation and weakness.

BackgroundThe dramatic increase in myotomal muscle mass in post-hatching fish is related to their ability to lastingly produce new muscle fibres, a process termed hyperplasia. The molecular and cellular mechanisms underlying fish muscle hyperplasia largely remain unknown. In this study, we aimed to characterize intrinsic properties of myogenic cells originating from fish hyperplasic muscle. For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomic profile of myogenic precursors originating from hyperplasic muscle of juvenile trout (JT) and from non-hyperplasic muscle of fasted juvenile trout (FJT) and adult trout (AT).ResultsFor the first time, we showed that myogenic precursors proliferate in hyperplasic muscle from JT as shown by in vivo BrdU labeling. This proliferative rate was very low in AT and FJT muscle. Transcriptiomic analysis revealed that myogenic cells from FJT and AT displayed close expression profiles with only 64 differentially expressed genes (BH corrected p-val < 0.001). In contrast, 2623 differentially expressed genes were found between myogenic cells from JT and from both FJT and AT. Functional categories related to translation, mitochondrial activity, cell cycle, and myogenic differentiation were inferred from genes up regulated in JT compared to AT and FJT myogenic cells. Conversely, Notch signaling pathway, that signs cell quiescence, was inferred from genes down regulated in JT compared to FJT and AT. In line with our transcriptomic data, in vitro JT myogenic precursors displayed higher proliferation and differentiation capacities than FJT and AT myogenic precursors.ConclusionsThe transcriptomic analysis and examination of cell behavior converge to support the view that myogenic cells extracted from hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than myogenic cells extracted from non-hyperplasic muscle. The generation of gene expression profiles in myogenic cell extracted from muscle of juvenile trout may yield insights into the molecular and cellular mechanisms controlling hyperplasia and provides a useful list of potential molecular markers of hyperplasia.

Cultured meat is a technology with the potential—but not yet proven—ability to solve major upcoming problems with food security and environment when meat demand will increase according to FAO expectations. The technology is available as shown by a proof of concept. Turning cultured meat into a first commercial product available on the plate of consumers still requires different additional technical efforts to improve and complement the product, to scale-up production, to reproduce meat aging and to attain regulatory approval, and this will require time. These developments will be paralleled by replacing animal-derived materials by sustainable ones and by adding new components to the meat including fat tissue. Around the potential market introduction of the first product, consumer acceptance will be an important issue. If cultured meat enters the market, it will compete with conventional meat produced by more environmental friendly livestock practices, meat substitutes made from plant proteins, mycoproteins and insect proteins. Each type of meat substitute will have its own benefits and weaknesses. Apart from improved production of livestock meat, cultured meat is the only alternative that is based on muscle tissue from staple domesticated animals although muscle aging still needs to be investigated.

International audience; Multinucleated muscle fibres arise by fusion of precursor cells called myoblasts. We previously showed that CKIP-1 ectopic expression in C2C12 myoblasts increased cell fusion. In this work, we report that CKIP-1 depletion drastically impairs C2C12 myoblast fusion in vitro and in vivo during zebrafish muscle development. Within developing fast-twich myotome, Ckip-1 localises at the periphery of fast precursor cells, closed to the plasma membrane. Unlike wild-type myoblasts that form spatially arrayed multinucleated fast myofibres, Ckip-1-deficient myoblasts show a drastic reduction in fusion capacity. A search for CKIP-1 binding partners identified the ARPC1 subunit of Arp2/3 actin nucleation complex essential for myoblast fusion. We demonstrate that CKIP-1, through binding to plasma membrane phosphoinositides via its PH domain, regulates cell morphology and lamellipodia formation by recruiting the Arp2/3 complex at the plasma membrane. These results establish CKIP-1 as a regulator of cortical actin that recruits the Arp2/3 complex at the plasma membrane essential for muscle precursor elongation and fusion.

The muscle growth in mammals is regulated by several growth factors including myostatin (MSTN), a member of the transforming growth factor-beta (TGF-beta) superfamily. To date, it is unknown in fish whether MSTN could have any effect on proliferation or differentiation of myogenic cells. Using culture of trout satellite cells, we showed that mstn1a and mstn1b mRNA are expressed in myoblasts and that their expression decreased in differentiating myoblasts. We also demonstrated that a treatment with huMSTN decreased the proliferation of IGF1-stimulated myoblasts in a dose-dependent manner. By contrast, treatment of myoblasts with 100 nM of huMSTN for three days, did not affect the percentage of positive cells for myogenin neither the percentage of nuclei in myosin positive cells. Moreover, our results clearly indicated that huMSTN treatment had no effect on MyoD and myogenin protein levels, which suggests that huMSTN did not strongly affect MyoD activity. In conclusion, we showed that huMSTN inhibited proliferation but not differentiation of trout myoblasts, probably resulting from a lack of huMSTN effect on MyoD activity. Altogether, these results show high interspecies differences in the function of MSTN.

Many fish species experience long periods of fasting often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as proteins of this tissue act as a major energy source. However, the molecular components involved in muscle protein degradation as well as the regulatory networks that control their function are still incompletely defined in fish. The present work aimed to characterize the response of the autophagy-lysosomal degradative pathway to nutrient and serum availability in primary culture of rainbow trout myoblasts. In this aim, 4-day-old cells were incubated in a serum and amino acid-rich medium (complete medium), a serum and amino acid-deprived medium (minimal medium) or a minimal medium plus amino acids, and both the transcription-independent short-term response and the transcription-dependent long-term response of the autophagy-lysosomal degradative pathway were analyzed. We report that serum and amino acids withdrawal is accompanied by a rapid increase of autophagosome formation but also by a slower induction of the expression of several autophagy-related genes (LC3B, gabarapl1, atg4b). We also showed that this latter response is controlled by amino acid (AA) availability and that both TOR-dependent and TOR-independent pathways are involved in this effect. Together these results suggest an important role for AA released by muscle proteolysis during the fasting period in regulating the subtle balance between using proteins as disposable furniture to provide energy, and conserving muscle through protein sparing.

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International audience; Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated "reserve cells" are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

Soutenue le 27 avril 2007 Diplôme : Dr. d'Universite

International audience; Although physical interactions with other receptors have been reported, heterodimeric complexes of T3 nuclear receptors (TR) with retinoid X receptors (RXRs) are considered as major regulators of T3 target gene expression. However, despite the potent T3 influence in proliferating myoblasts, RXR isoforms are not expressed during proliferation, raising the question of the nature of the complex involved in TR alpha transcriptional activity. We have previously established that c-Jun induces TRalpha1 transcriptional activity in proliferating myoblasts not expressing RXR. This regulation is specific to the muscle lineage, suggesting the involvement of a musclespecific factor. In this study, we found that MyoD expression in HeLa cells stimulates TRalpha1 activity, an influence potentiated by c-Jun coexpression. Similarly, in the absence of RXR, MyoD or c-Jun overexpression in myoblasts induces TRalpha1 transcriptional activity through a direct repeat 4 or an inverted palindrome 6 thyroid hormone response element. The highest rate of activity was recorded when c-Jun and MyoD were coexpressed. Using c-Jun-negative dominants, we established thatMyoD influence on TRalpha1 activity needs c-Jun functionality. Furthermore, we demonstrated that TRalpha1 and MyoD physically interact in the hinge region of the receptor and the transactivation and basic helix loop helix domains of MyoD. RXR expression (spontaneously occurring at the onset of myoblast differentiation) in proliferating myoblasts abrogates these interactions. These data suggest that in the absence of RXR, TRalpha1 transcriptional activity in myoblasts is mediated through a complex including MyoD and c-Jun.

International audience; The regulation of gene expression by thyroid hormone (T-3) involves binding of the hormone to nuclear receptors [ thyroid hormone receptor (TR)] acting as T-3-dependent transcription factors encoded by TR alpha (NR1A1) and TR beta (NR1A2) genes. Several TR alpha variants have already been characterized, but only some of them display T-3 binding activity. In this study, we have identified another transcript, TR alpha-Delta E6, produced by alternative splicing with microexon 6b instead of exon 6. This splicing leads to the synthesis of a protein devoid of a hinge domain. The TR alpha-Delta E6 transcript is detected in all mouse tissues tested. Although TR alpha-Delta E6 did not bind DNA, its expression induced a TR alpha 1 sequestration in the cytoplasm. Functional studies demonstrated that TR alpha-Delta E6 inhibits the transcriptional activity of TR alpha 1 and retinoic X receptor-alpha, but not of retinoic acid receptor-alpha. We also found that TR alpha-Delta E6 efficiently decreased the ability of TR alpha to inhibit MyoD transcriptional activity during myoblast proliferation. Consequently, when overexpressed in myoblasts, it stimulated terminal differentiation. We suggest that this novel TR alpha variant may act as down regulator of overall T-3 receptor activity, including its ability to repress MyoD transcriptional activity during myoblast proliferation.

Parallèlement à son rôle dans le métabolisme énergétique, l’activité mitochondriale intervient également dans l’induction de l’apoptose, ainsi que dans la régulation de la prolifération et de la différenciation cellulaires. Il existe en particulier une véritable régulation de la différenciation des myoblastes par l’activité mitochondriale, indépendante de la production d’ATP. Elle implique notamment le contrôle de l’expression de myogénine et de l’activité des facteurs myogéniques. Dans cette étude, nous démontrons que l’expression du proto-oncogène c-Myc est respectivement stimulée ou diminuée par une inhibition ou une stimulation de l’activité mitochondriale. Cette régulation s’effectue en grande partie au niveau de la stabilité du messager, et au niveau de la localisation cellulaire de la protéine dans les myoblastes aviaires. De plus, la surexpression de c-Myc reproduit très exactement les effets d’une inhibition de l’activité mitochondriale : i) abrogation de la différenciation terminale ; ii) inhibition de l’expression de Myogénine, sans altération de celle de MyoD ; iii) blocage de l’aptitude des facteurs myogéniques à induire la différenciation ; iv) inhibition de la sortie des myoblastes du cycle cellulaire. Ces résultats démontrent que c-Myc est une cible importante de l’activité mitochondriale, impliquée dans l’influence de l’organite sur la différenciation des myoblastes. Nous avons également mis en évidence l’existence d’un autre gène cible de l’organite qui code la phosphatase calcium dépendante Calcineurine. Son expression est respectivement inhibée ou stimulée par l’inhibition ou la stimulation de l’activité mitochondriale. De plus, l’expression d’une forme constitutivement active de Calcineurine stimule la différenciation des myoblastes et l’expression de Myogénine, alors que ces deux événements sont bloqués par l’expression d’un ARN antisens Calcineurine. Enfin, la stimulation de l’activité mitochondriale, comme l’expression d’une forme constitutivement active de Calcineurine stimule spécifiquement l’expression de l’isoforme lente des chaînes lourdes de myosine. Ces données démontrent donc que, notamment via l’expression de Calcineurine, l’activité mitochondriale régule non seulement la différenciation des myoblastes, mais détermine également le type contractile des fibres musculaires, Mitochondrial activity is a major regulator of myoblast differentiation and of the expression of myosin isoforms. Besides their influence on fuel metabolism, mitochondrial activity is also involved in the induction of apoptosis and in the regulation of cell proliferation and differentiation. In particular, an actual regulation of myoblast differentiation by mitochondrial activity is well established, independently of ATP synthesis, through the control of myogenin expression and myogenic factor activity. In this study, we established that c-Myc expression is respectively up- or down-regulated by inhibition or stimulation of mitochondrial activity. This regulation essentially takes place at a post-transcriptional level and also concerns, in avian myoblast, the cellular localisation of the protein. In addition, c-Myc overexpression exactly mimics the influence of an inhibition of mitochondrial activity: i) inhibition of myoblast differentiation; ii) inhibition of myogenin expression; iii) inhibition of myogenic factor ability to induce differentiation; iv) inhibition of myoblast withdrawal from the cell cycle. These results demonstrate that c-Myc is an important target of mitochondrial activity involved in the myogenic influence of the organelle. We also identified another target gene of mitochondrial activity encoding the calcium-dependent phosphatase calcineurin. Its expression is respectively inhibited or stimulated by inhibition or stimulation of mitochondrial activity. In addition, expression of a "constitutively active form of calcineurin stimulates of myoblast differentiation and myosin isoform expression, whereas these two events are suppressed by expression of antisense calcineurin RNA. Lastly, stimulation of mitochondrial activity or overexpression of the constitutively active form of calcineurin specifically stimulates the expression of slow myosin heavy chain isoform. These data demonstrate that, partly via calcineurin expression, mitochondrial activity not only regulates myoblast differentiation, but also the contractile type of muscle fibres.

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From epigenetics to mecanotransduction, there is increasing evidence that mechanical properties play a central role in the development and the survival of organisms. To determine the mechanical properties of isolated living cells, we built a single cell rheometer. Individual cells, of various types, were strained under constant stress (creep experiment). Their creep function always scaled with a power law of time (J(t) = At^(α)). The exponent gave a normal distribution around 0.25, whereas the distribution of the prefactor was log-normal. As the mechanical response was linear, we could compare our creep parameters to those observed previously by dynamical rheology (magnetocytometry, AFM). Thus we confirmed, qualitatively as well as quantitatively, results obtained with local and oscillating perturbations. In order to identify the contribution of cytoskeletal elements to the mechanical properties, we compared wild-type and vimentin-deficient fibroblasts. We found that vimentin had no effect on the form of the creep function, but was necessary to maintain mechanical integrity at high strain. In addition, we measured the force generated by a cell upon spreading between two glass microplates. Preliminary results indicated that myoblasts adapted the force they applied to the stiffness of the microplates., De l'épigénétique (méthylation de l'ADN) à la mécanotransduction, il y a un nombre croissant de preuves que les propriétés mécaniques jouent un rôle essentiel dans le développement et la survie des organismes. Pour sonder les propriétés mécaniques d'une cellule vivante, nous avons mis au point un véritable rhéomètre à cellule unique. Ainsi avons-nous pu montrer, pour la première fois à l'échelle de la cellule entière, que la fonction de fluage suivait une loi de puissance fonction du temps (J(t) = At^(α)) pour tous les types cellulaires sollicités. L'exposant est distribué normalement autour de 0, 25. Le préfacteur est, pour sa part, distribué de manière log-normale. Ayant déterminé le régime linéaire, nous avons pu comparer nos résultats de fluage aux mesures de rhéologie dynamique (magnétocytométrie, AFM). Nous avons ainsi confirmé, qualitativement et quantitativement, les résultats obtenus par des techniques où la sollicitation est locale et oscillante. Par ailleurs, en vue d'identifier la contribution des éléments du cytosquelette à la réponse mécanique d'une cellule, nous avons réalisé des mesures sur des fibroblastes n'exprimant pas la vimentine et sur des fibroblastes sauvages. Il a été mis en évidence que la vimentine n'avait pas d'incidence sur la forme de la fonction de fluage mais qu'elle était nécessaire au maintient de l'intégrité mécanique à grande déformation. Enfin nous avons pu mesurer la force exercée par une cellule lorsqu'elle s'étale entre les deux lamelles de verres. Des résultats préliminaires semblent indiquer que les myoblastes sont capables d'adapter leur force à la raideur des lamelles.

To characterize the regulatory pathways involved in the inhibition of cell differentiation induced by the impairment of mitochondrial activity, we investigated the relationships occurring between organelle activity and myogenesis using an avian myoblast cell line (QM7). The inhibition of mitochondrial translation by chloramphenicol led to a potent block of myoblast differentiation. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone and oligomycin, which affect the organelle at different levels, exerted a similar influence. In addition, we provided evidence that this phenomenon was not the result of an alteration in cell viability. Conversely, overexpression of the mitochondrial T3 receptor (p43) stimulated organelle activity and strongly potentiated myoblast differentiation. The involvement of mitochondrial activity in an actual regulation of myogenesis is further supported by results demonstrating that the muscle regulatory gene myogenin, in contrast to CMD1 (chicken MyoD) and myf5, is a specific transcriptional target of mitochondrial activity. Whereas myogenin mRNA and protein levels were down-regulated by chloramphenicol treatment, they were up-regulated by p43 overexpression, in a positive relationship with the expression level of the transgene. We also found that myogenin or CMD1 overexpression in chloramphenicol-treated myoblasts did not restore differentiation, thus indicating that an alteration in mitochondrial activity interferes with the ability of myogenic factors to induce terminal differentiation.

Insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cell lines, and these actions are mostly mediated through the type I IGF receptor (type I IGF-R). To further investigate the role of this receptor in phenotypic characteristics of C2 murine myoblasts, we overexpressed the human type I IGF-R in the inducible clone of C2 cells, which requires IGFs in the differentiation medium to undergo terminal differentiation.Inducible myoblasts were transfected with either the eukaryotic expression vector pNTK or pNTK containing the human type I IGF-R complementary DNA, and we isolated two clones named Ind-Neo and Ind-R, respectively. Binding and autophosphorylation experiments indicate that Ind-R cells express about 10 times as much type I IGF-R compared with Ind-Neo control cells and that the transfected type I IGF-R is functional in Ind-R cells.We show that overexpression of the human type I IGF-R makes inducible myoblasts able to differentiate spontaneously, as assessed by expression of the myogenic transcription factors MyoD and myogenin, detection of the muscle-specific protein troponin T, and myotube formation. Moreover, when exposed to IGF-I, Ind-R cells lose contact inhibition, grow in the presence of a low level of growth factors and form colonies in soft agar, which is characteristic of a ligand-dependent transformed phenotype.It emerges from this study that 1) the type I IGF-R is strongly involved in the phenotypic differences between inducible and permissive cells with respect to the differentiation program; and 2) overexpression causes this receptor to act as a ligand-dependent transforming protein in muscle cells. We suggest that type I IGF-R abundance and level of activation may determine the efficiency of the autocrine mode of action of IGFs and discriminate their biological functions.