DNA methylation changes during a sprint interval exercise performed under normobaric hypoxia or with blood flow restriction: A pilot study in men.

This crossover study evaluated DNA methylation changes in human salivary samples following single sprint interval training sessions performed in hypoxia, with blood flow restriction (BFR), or with gravity-induced BFR. Global DNA methylation levels were evaluated with an enzyme-linked immunosorbent assay. Methylation-sensitive restriction enzymes were used to determine the percentage methylation in a part of the promoter of the gene-inducible nitric oxide synthase (p-iNOS), as well as an enhancer (e-iNOS). Global methylation increased after exercise (p < 0.001; dz = 0.50). A tendency was observed for exercise × condition interaction (p = 0.070). Post hoc analyses revealed a significant increase in global methylation between pre- (7.2 ± 2.6%) and postexercise (10.7 ± 2.1%) with BFR (p = 0.025; dz = 0.69). Methylation of p-iNOS was unchanged (p > 0.05). Conversely, the methylation of e-iNOS increased from 0.6 ± 0.4% to 0.9 ± 0.8% after exercise (p = 0.025; dz = 0.41), independently of the condition (p > 0.05). Global methylation correlated with muscle oxygenation during exercise (r = 0.37, p = 0.042), while e-iNOS methylation showed an opposite association (r = -0.60, p = 0.025). Furthermore, p-iNOS methylation was linked to heart rate (r = 0.49, p = 0.028). Hence, a single sprint interval training increases global methylation in saliva, and adding BFR tends to increase it further. Lower muscle oxygenation is associated with augmented e-iNOS methylation. Finally, increased cardiovascular strain results in increased p-iNOS methylation.

Feasibility and early effects of bed-cycling eccentric training: Potential clinical applications.

PURPOSE: This study aimed to evaluate feasibility and early effects of moderate intensity bed-cycling eccentric training on healthy individuals, and establish whether this training modality could be implemented into bedridden patients' routine care. METHODS: Longitudinal study with prepost exercise intervention measurements. The development of a bed-adapted eccentric ergometer allowed to conduct five training sessions during 3 weeks at increasing intensity on 11 healthy individuals. Force-speed relationship, maximal voluntary knee extension force and neural activation of subjects were evaluated before and after the programme. RESULTS: Five training sessions were sufficient to decrease the rate of perceived exertion whereas eccentric power output increased (+40%). After training, maximal voluntary isometric contraction force measured during knee extension had significantly improved in all subjects, with a mean increase of 17%. Maximal cycling power was also significantly higher (+7%) after the training programme. CONCLUSION: Taken together, these results show that moderate load eccentric bed cycling (i) was feasible and efficient, (ii) did not generate excessive individual perception of effort during exercise nor develop major muscular or joint pain after training and (iii) allowed early force and power gains in healthy subjects.

Does the interference phenomenon affect strength development during same-session combined rehabilitation program in hemodialysis patients?

INTRODUCTION: This study aimed to assess if an interference effect could blunt the neuromuscular gains induced by a same-session combined rehabilitation in hemodialysis (HD) patients. METHODS: Patients exercised twice a week, for 16 weeks, over their HD sessions. They were either always trained with resistance and endurance exercises (continuous training, "CONT") or alternatively with 1 week of resistance alternated with 1 week of endurance (discontinuous training, "DISC"). Adherence and workload were continuously recorded. Short Physical Performance Battery (SPPB) score, one-leg balance test, and handgrip and quadriceps strength were evaluated before and after training intervention. RESULTS: Adherence to both programs was high (>90%). SPPB score had significantly improved (CONT: +1.5 point, DISC: +1.2 pt, p < 0.001), like one-leg balance test (CONT: +3.7 s, DISC: +5.5 s, p < 0.05), handgrip strength of exercised (CONT: +5.5 kg, DISC: +5.6 kg, p < 0.001) and of nonexercised arm (CONT: +4.4 kg, DISC: +2.8 kg, p < 0.01) as well as maximal quadriceps strength (+22 N·m for dominant and +29 N·m for nondominant leg in both groups, p < 0.001) bearing no difference between the trainings. CONCLUSION: Same-session combined training does not induce an interference effect in HD patients and temporal separation of exercises does not optimize strength gains. These practical data may be relevant for clinicians and practitioners to alternate endurance and resistance exercises.

Molecular Regulation of Skeletal Muscle Growth and Organelle Biosynthesis: Practical Recommendations for Exercise Training.

The regulation of skeletal muscle mass and organelle homeostasis is dependent on the capacity of cells to produce proteins and to recycle cytosolic portions. In this investigation, the mechanisms involved in skeletal muscle mass regulation-especially those associated with proteosynthesis and with the production of new organelles-are presented. Thus, the critical roles of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway and its regulators are reviewed. In addition, the importance of ribosome biogenesis, satellite cells involvement, myonuclear accretion, and some major epigenetic modifications related to protein synthesis are discussed. Furthermore, several studies conducted on the topic of exercise training have recognized the central role of both endurance and resistance exercise to reorganize sarcomeric proteins and to improve the capacity of cells to build efficient organelles. The molecular mechanisms underlying these adaptations to exercise training are presented throughout this review and practical recommendations for exercise prescription are provided. A better understanding of the aforementioned cellular pathways is essential for both healthy and sick people to avoid inefficient prescriptions and to improve muscle function with emergent strategies (e.g., hypoxic training). Finally, current limitations in the literature and further perspectives, notably on epigenetic mechanisms, are provided to encourage additional investigations on this topic.

Recent Data on Cellular Component Turnover: Focus on Adaptations to Physical Exercise.

Significant progress has expanded our knowledge of the signaling pathways coordinating muscle protein turnover during various conditions including exercise. In this manuscript, the multiple mechanisms that govern the turnover of cellular components are reviewed, and their overall roles in adaptations to exercise training are discussed. Recent studies have highlighted the central role of the energy sensor (AMP)-activated protein kinase (AMPK), forkhead box class O subfamily protein (FOXO) transcription factors and the kinase mechanistic (or mammalian) target of rapamycin complex (MTOR) in the regulation of autophagy for organelle maintenance during exercise. A new cellular trafficking involving the lysosome was also revealed for full activation of MTOR and protein synthesis during recovery. Other emerging candidates have been found to be relevant in organelle turnover, especially Parkin and the mitochondrial E3 ubiquitin protein ligase (Mul1) pathways for mitochondrial turnover, and the glycerolipids diacylglycerol (DAG) for protein translation and FOXO regulation. Recent experiments with autophagy and mitophagy flux assessment have also provided important insights concerning mitochondrial turnover during ageing and chronic exercise. However, data in humans are often controversial and further investigations are needed to clarify the involvement of autophagy in exercise performed with additional stresses, such as hypoxia, and to understand the influence of exercise modality. Improving our knowledge of these pathways should help develop therapeutic ways to counteract muscle disorders in pathological conditions.

eIF3f depletion impedes mouse embryonic development, reduces adult skeletal muscle mass and amplifies muscle loss during disuse.

KEY POINTS: In muscular cells, eukaryotic initiation factor subunit f (eIF3f) activates protein synthesis by allowing physical interaction between mechanistic target of rapamycin complex 1 (MTORC1) and ribosomal protein S6 kinase 1 (S6K1), although its physiological role in animals is unknown. A knockout approach suggests that homozygous mice carrying a null mutation of the eIF3f gene fail to develop and consequently die at early embryonic stage, whereas heterozygous mice associated with a partial depletion of eIF3f gene grow normally and are phenotypically indistinguishable from wild-type mice. Heterozygous mice express reduced eIF3f mRNA and protein levels in skeletal muscles and show diminished muscle mass associated with a decrease in the protein synthesis rate and an inhibition of the MTORC1 pathway. During hindlimb immobilization, heterozygous eIF3f mice display an exacerbated immobilization-induced muscle atrophy associated with reduced protein synthesis. These results highlight the essential role of eIF3f during embryonic development and its involvement in muscular homeostasis via protein synthesis regulation. ABSTRACT: Eukaryotic translation initiation factor 3, subunit F (eIF3f), a component of eIF3 complex, plays an important role in protein synthesis regulation, although its physiological functions are unknown. We generated and analysed mice carrying a null mutation in the eIF3f gene. We showed that homozygous eIF3f knockout fail to develop and that eIF3f-/- embryos die at an early stage of development but after the pre-implantation stage. However, disrupting one eIF3f allele does not affect growth, viability and fertility of heterozygous mice but, instead, reduces eIF3f mRNA and protein levels in all tissues examined. Although heterozygous mice are phenotypically indistinguishable from wild-type mice, they present a diminished body weight and a lean mass reduction associated with normal body size. Interestingly, skeletal muscles are mainly affected and display an altered cell size without modification of fibre number. Skeletal muscles of heterozygous mice show a deficiency in polysome content, a decrease in protein synthesis rate and an inhibition of the mechanistic target of rapamycin (MTOR) pathway. We then studied the effects of hindlimb immobilization that mimic muscle disuse on heterozygous mice aiming to further explore the involvement of eIF3f in protein synthesis. We found that eIF3f partial depletion amplifies muscle atrophy compared to wild-type mice. Mass and cross-sectional area decreases were associated with reduced MTOR pathway activation and protein synthesis rate. Taken together, our data indicate that eIF3f is essential for mice embryonic development and controls adult skeletal muscle mass via protein synthesis regulation in a MTOR-dependent manner.

AMP-activated protein kinase stabilizes FOXO3 in primary myotubes.

AMP-activated protein kinase (AMPK) is a critical enzyme in conditions of cellular energy deficit such as exercise, hypoxia or nutritional stress. AMPK is well known to regulate protein degradation pathways notably through FOXO-related axis. In this study, we investigated the implication of AMPK activation in FOXO3 expression and stability in skeletal muscle primary myotubes. First, time course and dose response studies revealed optimal AICAR treatment duration and dose in skeletal muscle cells. Then, experiments with cycloheximide treatment of primary myotubes highlighted that AICAR infusion extends FOXO3 protein half-life. Our results also showed that AICAR treatment or nutrient depletion increases FOXO3 expression in primary myotubes and the expression of the mitochondrial E3 ligase Mul1 involved in mitochondrial turnover (mitophagy). In AMPK KO cells, nutrient depletion failed to alter the level of some FOXO3-dependent atrophic genes, including LC3B, BNIP3, and the mitochondrial E3 ligase Mul1, but not the expression of other genes (i.e. FOXO1, Gabarapl1, MAFbx, MuRF1). In summary, our data highlight that AMPK stabilizes FOXO3 and suggest a role in the first initiation step of mitochondrial segregation in muscle cells.

Autophagy is essential to support skeletal muscle plasticity in response to endurance exercise.

Physical exercise is a stress that can substantially modulate cellular signaling mechanisms to promote morphological and metabolic adaptations. Skeletal muscle protein and organelle turnover is dependent on two major cellular pathways: Forkhead box class O proteins (FOXO) transcription factors that regulate two main proteolytic systems, the ubiquitin-proteasome, and the autophagy-lysosome systems, including mitochondrial autophagy, and the MTORC1 signaling associated with protein translation and autophagy inhibition. In recent years, it has been well documented that both acute and chronic endurance exercise can affect the autophagy pathway. Importantly, substantial efforts have been made to better understand discrepancies in the literature on its modulation during exercise. A single bout of endurance exercise increases autophagic flux when the duration is long enough, and this response is dependent on nutritional status, since autophagic flux markers and mRNA coding for actors involved in mitophagy are more abundant in the fasted state. In contrast, strength and resistance exercises preferentially raise ubiquitin-proteasome system activity and involve several protein synthesis factors, such as the recently characterized DAGK for mechanistic target of rapamycin activation. In this review, we discuss recent progress on the impact of acute and chronic exercise on cell component turnover systems, with particular focus on autophagy, which until now has been relatively overlooked in skeletal muscle. We especially highlight the most recent studies on the factors that can impact its modulation, including the mode of exercise and the nutritional status, and also discuss the current limitations in the literature to encourage further works on this topic.

Autophagy and protein turnover signaling in slow-twitch muscle during exercise.

PURPOSE: The aim of this study was to characterize skeletal muscle protein breakdown and mitochondrial dynamics markers at different points of endurance exercise. METHODS: Mice run at 10 m·min(-1) during 1 h, and running speed was increased by 0.5 m·min(-1) every minute during 40 min and then by 1 m·min(-1) until exhaustion. Animals were killed by cervical dislocation at 30, 60, 90, and 120 min; at time to exhaustion (Te); and at 3 and 24 h during recovery. The soleus and the deep red regions of the quadriceps muscles were pooled. RESULTS: AMPK phosphorylation (Thr172) increased from 30 min to Te, and FoxO3a phosphorylation (Thr32 and Ser253) decreased from 120 min to 3 h after exercise. FoxO3a-dependent E3 ligases Mul1 and MuRF1 proteins increased from 30 min to Te and at Te and 3 h after exercise, respectively, whereas MAFbx/atrogin-1 protein expression did not change significantly. The autophagic markers LC3B-II increased at 120 min and Te, and p62 significantly decreased at Te. The AMPK-dependent phosphorylation of Ulk1 at Ser317 and Ser555 increased from 60 min to Te and at 30 and 60 min, respectively. Akt (Ser473), MTOR (Ser2448), and 4E-BP1 (Thr37/46) phosphorylation decreased from 90 min to Te, and the MTOR-dependent phosphorylation of Ulk1 (Ser757) decreased from 120 min to Te. Ser616 phosphorylation of the mitochondrial fission marker DRP1 increased from 60 min to Te, but protein expression of the fusion markers mitofusin-2, a substrate of Mul1, and OPA1 did not significantly change. CONCLUSIONS: These results fit with a regulation of protein breakdown triggered by FoxO3a and Ulk1 pathways after AMPK activation and Akt/MTOR inhibition. Furthermore, our data suggest that mitochondrial fission is quickly increased, and mitochondrial fusion is unchanged during exercise.

FoxO transcription factors: their roles in the maintenance of skeletal muscle homeostasis.

Forkhead box class O family member proteins (FoxOs) are highly conserved transcription factors with important roles in cellular homeostasis. The four FoxO members in humans, FoxO1, FoxO3, FoxO4, and FoxO6, are all expressed in skeletal muscle, but the first three members are the most studied in muscle. In this review, we detail the multiple modes of FoxO regulation and discuss the central role of these proteins in the control of skeletal muscle plasticity. FoxO1 and FoxO3 are key factors of muscle energy homeostasis through the control of glycolytic and lipolytic flux, and mitochondrial metabolism. They are also key regulators of protein breakdown, as they modulate the activity of several actors in the ubiquitin­proteasome and autophagy­lysosomal proteolytic pathways, including mitochondrial autophagy, also called mitophagy. FoxO proteins have also been implicated in the regulation of the cell cycle, apoptosis, and muscle regeneration. Depending of their activation level, FoxO proteins can exhibit ambivalent functions. For example, a basal level of FoxO factors is necessary for cellular homeostasis and these proteins are required for adaptation to exercise. However, exacerbated activation may occur in the course of several diseases, resulting in metabolic disorders and atrophy. A better understanding of the precise functions of these transcriptions factors should thus lead to the development of new therapeutic approaches to prevent or limit the muscle wasting that prevails in numerous pathological states, such as immobilization, denervated conditions, neuromuscular disease, aging, AIDS, cancer, and diabetes.

eIF3f: a central regulator of the antagonism atrophy/hypertrophy in skeletal muscle.

The eukaryotic initiation factor 3 subunit f (eIF3f) is one of the 13 subunits of the translation initiation factor complex eIF3 required for several steps in the initiation of mRNA translation. In skeletal muscle, recent studies have demonstrated that eIF3f plays a central role in skeletal muscle size maintenance. Accordingly, eIF3f overexpression results in hypertrophy through modulation of protein synthesis via the mTORC1 pathway. Importantly, eIF3f was described as a target of the E3 ubiquitin ligase MAFbx/atrogin-1 for proteasome-mediated breakdown under atrophic conditions. The biological importance of the MAFbx/atrogin-1-dependent targeting of eFI3f is highlighted by the finding that expression of an eIF3f mutant insensitive to MAFbx/atrogin-1 polyubiquitination is associated with enhanced protection against starvation-induced muscle atrophy. A better understanding of the precise role of this subunit should lead to the development of new therapeutic approaches to prevent or limit muscle wasting that prevails in numerous physiological and pathological states such as immobilization, aging, denervated conditions, neuromuscular diseases, AIDS, cancer, diabetes. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.

The role of AMP-activated protein kinase in the coordination of skeletal muscle turnover and energy homeostasis.

The AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that acts as a sensor of cellular energy status switch regulating several systems including glucose and lipid metabolism. Recently, AMPK has been implicated in the control of skeletal muscle mass by decreasing mTORC1 activity and increasing protein degradation through regulation of ubiquitin-proteasome and autophagy pathways. In this review, we give an overview of the central role of AMPK in the control of skeletal muscle plasticity. We detail particularly its implication in the control of the hypertrophic and atrophic signaling pathways. In the light of these cumulative and attractive results, AMPK appears as a key player in regulating muscle homeostasis and the modulation of its activity may constitute a therapeutic potential in treating muscle wasting syndromes in humans.

AMPK promotes skeletal muscle autophagy through activation of forkhead FoxO3a and interaction with Ulk1.

In skeletal muscle, protein levels are determined by relative rates of protein synthesis and breakdown. The balance between synthesis and degradation of intracellular components determines the overall muscle fiber size. AMP-activated protein kinase (AMPK), a sensor of cellular energy status, was recently shown to increase myofibrillar protein degradation through the expression of MAFbx and MuRF1. In the present study, the effect of AMPK activation by AICAR on autophagy was investigated in muscle cells. Our results show that FoxO3a transcription factor activation by AMPK induces the expression of the autophagy-related proteins LC3B-II, Gabarapl1, and Beclin1 in primary mouse skeletal muscle myotubes and in the Tibialis anterior (TA) muscle. Time course studies reveal that AMPK activation by AICAR leads to a transient nuclear relocalization of FoxO3a followed by an increase of its cytosolic level. Moreover, AMPK activation leads to the inhibition of mTORC1 and its subsequent dissociation of Ulk1, Atg13, and FIP200 complex. Interestingly, we identify Ulk1 as a new interacting partner of AMPK in muscle cells and we show that Ulk1 is associated with AMPK under normal conditions and dissociates from AMPK during autophagy process. Moreover, we find that AMPK phosphorylates FoxO3a and Ulk1. In conclusion, our data show that AMPK activation stimulates autophagy in skeletal muscle cells through its effects on the transcriptional function of FoxO3a and takes part in the initiation of autophagosome formation by interacting with Ulk1. Here, we present new evidences that AMPK plays a crucial role in the fine tuning of protein expression programs that control skeletal muscle mass.

Wnt4 activates the canonical ß-catenin pathway and regulates negatively myostatin: functional implication in myogenesis.

Expression of Wnt proteins is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Previous studies have shown that Wn4 was involved in the myogenic fate of somites, in the myogenic proliferation, and differentiation of skeletal muscle. However, the function of this factor in adult muscle homeostasis remains not well understood. Here, we focus on the roles of Wnt4 during C2C12 myoblasts and satellite cells differentiation. We analyzed its myogenic activity, its mechanism of action, and its interaction with the anti-myogenic factor myostatin during differentiation. Established expression profiles indicate clearly that both types of cells express a few Wnts, and among these, only Wnt4 was not or barely detected during proliferation and was strongly induced during differentiation. As attested by myogenic factors expression pattern analysis and fusion index determination, overexpression of Wnt4 protein caused a strong increase in satellite cells and C2C12 myoblast differentiation leading to hypertrophic myotubes. By contrast, exposure of satellite and C2C12 cells to small interfering RNA against Wnt4 strongly diminished this process, confirming the myogenic activity of Wnt4. Moreover, we reported that Wnt4, which is usually described as a noncanonical Wnt, activates the canonical ß-catenin pathway during myogenic differentiation in both cell types and that this factor regulates negatively the expression of myostatin and the regulating pathways associated with myostatin. Interestingly, we found that recombinant myostatin was sufficient to antagonize the differentiation-promoting activities of Wnt4. Reciprocally, we also found that the genetic deletion of myostatin renders the satellite cells refractory to the hypertrophic effect of Wnt4. These results suggest that the Wnt4-induced decrease of myostatin plays a functional role during hypertrophy. We propose that Wnt4 protein may be a key factor that regulates the extent of differentiation in satellite and C2C12 cells.

The atypical alpha2beta2 IGF receptor expressed in inducible c2.7 myoblasts is derived from post-translational modifications of the mouse IGF-I receptor.

OBJECTIVE: Unlike parental permissive C2.7 myoblasts, inducible C2.7 myoblasts require IGF-I or IGF-II to differentiate and expression of MyoD is not constitutive. Our previous studies indicated that inducible myoblasts express an atypical alpha2beta2 IGF receptor that differs from the classical IGF-I receptor by its higher affinity for IGF-II compared with IGF-I and the higher molecular weight of its alpha and beta subunits. Expression of this atypical IGF-I receptor is developmentally regulated; hence this receptor is lost upon terminal differentiation. Muscle cell differentiation is a system in which IGF-II plays an essential role and developmentally regulated atypical IGF-I receptor may represent a candidate for mediating differentiation signals provided by IGF-II. To further understand the structure and the role of the atypical IGF-I receptor, (i) we investigated for a putative IGF-I receptor transcript polymorphism by extensive sequencing of RT-PCR products; (ii) we overexpressed cloned mouse IGF-I receptor in permissive and inducible C2.7 myoblasts and characterized the binding and structural properties of overexpressed IGF-I receptor and (iii) we analysed the effects of this overexpression on myoblasts differentiation. DESIGN: Cultured mouse myoblasts C2.7 and subclone variant inducible C2.7 cell lines were used. Mouse IGF-I receptor cDNA was cloned by cDNA library screening. Gene expression was measured by semi-quantitative RT-PCR analysis and receptor affinity by ligand binding. Receptor protein autophosphorylation of IGF-IR was analysed by immunoprecipitation and Western blot. Myoblastic differentiation was accessed by myogenic factors expression and immunofluorescence study. RESULTS: Atypical IGF-I receptor may correspond to a new receptor belonging to the insulin/IGF-I receptor family, or it may also derive from alternate splicing of the gene of the insulin/IGF-I receptors and/or post-translational modifications of the insulin/IGF-I receptors. Our results exclude the existence of a polymorphism of the IGF-I receptor transcripts in inducible and permissive myoblasts. In embryo and cancer cells IGF-II binds to insulin receptor (IR) isoform A, RT-PCR experiments show that IR is expressed in permissive but not in inducible myoblasts. We demonstrated here that post-translational processing of the mouse IGF-I receptor is responsible for the existence of the mouse atypical IGF-I receptor in inducible myoblasts. Overexpressed mouse IGF-I receptor in permissive myoblasts has the same biochemical and binding characteristics as the classical IGF-I receptor whereas in inducible myoblasts, overexpressed mouse IGF-I receptor has the biochemical, binding and functional characteristics of the atypical IGF-I receptor. CONCLUSIONS: Our results provide experimental evidence that the atypical IGF-I receptor variant expressed in subclone inducible C2.7 is issued from a post-translational processing of mouse IGF-I receptor. We show that this post-translational modification is closely associated with the cell lines indeed permissive C2.7 myoblasts process mouse cDNA IGF-I receptor as a classical IGF-I receptor whereas inducible C2.7 myoblasts process mouse cDNA IGF-I receptor as an atypical IGF-I receptor. On other hand, we show that overexpression of mouse IGF-I receptor in inducible myoblasts does not abrogate IGF-I or IGF-II requirement to differentiate.

Inhibition of myoblast differentiation by Sfrp1 and Sfrp2.

Secreted Frizzled-related proteins (Sfrps) are extracellular regulators of Wnt signalling and play important roles in developmental and oncogenic processes. They are known to be upregulated in regenerating muscle and in myoblast cultures but their function is unknown. Here, we show that the addition of recombinant Sfrp1 or Sfrp2 to C2C12 cell line cultures or to primary cultures of satellite cells results in the inhibition of myotube formation with no significant effect on the cell cycle or apoptosis. Even though at confluence, treated and untreated cultures are identical in appearance, analyses have shown that, for maximum effect, the cells have to be treated while they are proliferating. Furthermore, removal of Sfrp from the culture medium during differentiation restores normal myotube formation. We conclude that Sfrp1 and Sfrp2 act to prevent myoblasts from entering the terminal differentiation process.

Inhibition of autocrine secretion of myostatin enhances terminal differentiation in human rhabdomyosarcoma cells.

Rhabdomyosarcomas (RMSs) are one of the most common solid tumor of childhood. Rhabdomyosarcoma (RMS) cells fail to both complete the skeletal muscle differentiation program and irreversibly exit the cell cycle as a consequence of an active repression exerted on the muscle-promoting factor MyoD. Myostatin is a negative regulator of normal muscle growth, we have thus studied its possible role in RMS cells. Here, we present evidence that overexpression of myostatin is a common feature of RMS since both subtypes of RMS (embryonal RD and alveolar Rh30 cells) express high levels of myostatin when compared to nontumoral skeletal muscle cells. Interestingly, we found that inactivation of myostatin through overexpression of antisense myostatin or of follistatin (a myostatin antagonist) constructs enhanced differentiation of RD cells. In addition, RD and Rh30 cells treated with blocking antimyostatin antibodies progress into the myogenic terminal differentiation program. Finally, our results suggest that high levels of myostatin could impair MyoD function in RMS cells. These results show that an autocrine myostatin loop contributes to maintain RMS cells in an undifferentiating stage and suggest that new therapeutic approaches could be exploited for the treatment of RMS based on inactivation of myostatin protein.

Mechanisms involved in the inhibition of myoblast proliferation and differentiation by myostatin.

Muscle growth results from a set of complex processes including myogenic transcription factor's expression and activity, cell cycle withdrawal, myoblast fusion in myotubes, and acquisition of an apoptosis-resistant phenotype. Myostatin, a member of the TGFbeta family, described as a strong regulator of myogenesis in vivo Nature 387 (1997), 83; FEBS Lett. 474 (2000), 71 is upregulated during in vitro differentiation Biochem. Biophys. Res. Commun. 280 (2001), 561. To improve characterization of myostatin's myogenic influence, we stably transfected vectors expressing myostatin and myostatin antisense in C2C12 myoblasts. Here, we found that myostatin inhibits cell proliferation and differentiation. Our results also indicate that myogenin is an important target of myostatin. In addition, overexpressed but not endogenous myostatin decreases MyoD protein levels and induces changes in its phosphorylation pattern. We also established that myostatin overexpression reduces the frequency of G0/G1-arrested cells during differentiation. Conversely, inhibition of myostatin synthesis leads to enhanced cell cycle withdrawal and consequently stimulates myoblast differentiation. We examined the expression patterns of the pRb, E2F1, p53, and p21 proteins involved in cell cycle withdrawal. We found that myostatin overexpression increases p21 and p53 expression, as it does accumulation of hypophosphorylated Rb. Interestingly, myostatin overexpression strongly reduced low-mitogen-induced apoptosis, whereas antisense expression induced contrary changes. In conclusion, these data show the influence of overexpressed myostatin on myoblast proliferation, differentiation, and apoptosis is extended to endogenous myostatin. Though some differences in overexpression or inhibition of endogenous myostatin were observed, it appears that myogenin and p21 are essential targets of this growth factor.