Anne Bonnieu

Myostatin and the skeletal muscle atrophy and hypertrophy signaling pathways

Myostatin, a member of the transforming growth factor-β superfamily, is a potent negative regulator of skeletal muscle growth and is conserved in many species, from rodents to humans. Myostatin inactivation can induce skeletal muscle hypertrophy, while its overexpression or systemic administration causes muscle atrophy. As it represents a potential target for stimulating muscle growth and/or preventing muscle wasting, myostatin regulation and functions in the control of muscle mass have been extensively studied. A wealth of data strongly suggests that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression. Moreover, myostatin plays a central role in integrating/mediating anabolic and catabolic responses. Myostatin negatively regulates the activity of the Akt pathway, which promotes protein synthesis, and increases the activity of the ubiquitin–proteasome system to induce atrophy. Several new studies have brought new information on how myostatin may affect both ribosomal biogenesis and translation efficiency of specific mRNA subclasses. In addition, although myostatin has been identified as a modulator of the major catabolic pathways, including the ubiquitin–proteasome and the autophagy–lysosome systems, the underlying mechanisms are only partially understood. The goal of this review is to highlight outstanding questions about myostatin-mediated regulation of the anabolic and catabolic signaling pathways in skeletal muscle. Particular emphasis has been placed on (1) the cross-regulation between myostatin, the growth-promoting pathways and the proteolytic systems; (2) how myostatin inhibition leads to muscle hypertrophy; and (3) the regulation of translation by myostatin.

Inhibition of Notch signaling induces myotube hypertrophy by recruiting a subpopulation of reserve cells

During muscle differentiation, a population of quiescent undifferentiated myoblasts (reserve cells) emerges among mature muscle cells. However, the molecular mechanisms underlying such cell segregation and the characterization of this subpopulation of myoblasts remain to be determined. Notch is known to control the behavior and fate of murine muscle stem cells. In this study, we examined the role of Notch in myoblast segregation. We showed that inhibition of Notch activity by either overexpressing Numb or by using a pharmacological γ‐secretase inhibitor (DAPT) enhanced differentiation of murine and human myoblasts. This effect was not restricted to in vitro culture systems since DAPT‐treated zebrafish embryos also showed increased differentiation. Using C2.7 myoblasts as a model, we showed that inhibition of Notch induced myotube hypertrophy by recruiting reserve cells that do not normally fuse. We further showed that endogenous Notch‐signaling components were differentially expressed and activated in reserve cells with respect to Notch 1 and CD34 expression. We identified CD34 negative reserve cells as the subpopulation of myoblasts recruited to fuse into myotubes during differentiation in response to Notch inhibition. Therefore, we showed here that the activation of Notch 1 is important to maintain a subpopulation of CD34 negative reserve cells in an undifferentiated state. J. Cell. Physiol. 208: 538–548, 2006.

Myostatin up-regulation is associated with the skeletal muscle response to hypoxic stimuli

Myostatin and hypoxia signalling pathways are able to induce skeletal muscle atrophy, but whether a relationship between these two pathways exists is currently unknown. Here, we tested the hypothesis that a potential mechanism for hypoxia effect on skeletal muscle may be through regulation of myostatin. We reported an induction of myostatin expression in muscles of rats exposed to chronic hypoxia. Interestingly, we also demonstrated increased skeletal muscle myostatin protein expression in skeletal muscle of hypoxemic patients with severe chronic obstructive pulmonary disease (COPD). Parallel studies in human skeletal muscle cell cultures showed that induction of myostatin expression in myotubes treated with hypoxia-mimicking agent such as cobalt chloride (CoCl(2)) is associated with myotube atrophy. Furthermore, we demonstrated that inhibition of myostatin by means of genetic deletion of myostatin or treatment with blocking antimyostatin antibodies inhibits the CoCl(2)-induced atrophy in muscle cells. Finally, addition of recombinant myostatin restored the CoCl(2)-induced atrophy in myostatin deficient myotubes. These results strongly suggest that myostatin can play an essential role in the adaptation of skeletal muscle to hypoxic environment.

Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells.

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant‐derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast‐CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56‐purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non‐human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)‐induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non‐toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage.

REDD1 deletion prevents dexamethasone-induced skeletal muscle atrophy

REDD1 (regulated in development and DNA damage response 1) has been proposed to inhibit the mechanistic target of rapamycin complex 1 (mTORC1) during in vitro hypoxia. REDD1 expression is low under basal conditions but is highly increased in response to several catabolic stresses, like hypoxia and glucocorticoids. However, REDD1 function seems to be tissue and stress dependent, and its role in skeletal muscle in vivo has been poorly characterized. Here, we investigated the effect of REDD1 deletion on skeletal muscle mass, protein synthesis, proteolysis, and mTORC1 signaling pathway under basal conditions and after glucocorticoid administration. Whereas skeletal muscle mass and typology were unchanged between wild-type (WT) and REDD1-null mice, oral gavage with dexamethasone (DEX) for 7 days reduced tibialis anterior and gastrocnemius muscle weights as well as tibialis anterior fiber size only in WT. Similarly, REDD1 deletion prevented the inhibition of protein synthesis and mTORC1 activity (assessed by S6, 4E-BP1, and ULK1 phosphorylation) observed in gastrocnemius muscle of WT mice following single DEX administration for 5 h. However, our results suggest that REDD1-mediated inhibition of mTORC1 in skeletal muscle is not related to the modulation of the binding between TSC2 and 14-3-3. In contrast, our data highlight a new mechanism involved in mTORC1 inhibition linking REDD1, Akt, and PRAS40. Altogether, these results demonstrated in vivo that REDD1 is required for glucocorticoid-induced inhibition of protein synthesis via mTORC1 downregulation. Inhibition of REDD1 may thus be a strategy to limit muscle loss in glucocorticoid-mediated atrophy.

Myostatin inactivation increases myotube size through regulation of translational initiation machinery

Myostatin deficiency leads in skeletal muscle overgrowth but the precise molecular mechanisms underlying this hypertrophy are not well understood. In this study, to gain insight into the role of endogenous myostatin in the translational regulation, we used an in vitro model of cultured satellite cells derived from myostatin knock‐out mice. Our results show that myostatin knock‐out myotubes are larger than control myotubes and that this phenotype is associated with an increased activation of the Akt/mTOR signaling pathway, a known regulator of muscle hypertrophy. These results demonstrate that hypertrophy due to myostatin deficiency is preserved in vitro and suggest that myostatin deletion results in an increased protein synthesis. Accordingly, the rates of global RNA content, polysome formation and protein synthesis are all increased in myostatin‐deficient myotubes while they are counteracted by the addition of recombinant myostatin. We furthermore demonstrated that genetic deletion of myostatin stimulates cap‐dependent translation by positively regulating assembly of the translation preinitiation complex. Together the data indicate that myostatin controls muscle hypertrophy in part by regulating protein synthesis initiation rates, that is, translational efficiency. J. Cell. Biochem. 112: 3531–3542, 2011.

Myostatin: Biology and Clinical Relevance

Myostatin is a negative regulator of muscle mass. Important advances in our understanding of the complex biology of this factor have revealed the therapeutic potential of antagonizing the myostatin pathway. Here we present the rationale for evaluating anti-myostatin therapies in human muscle-wasting disorders.

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.

Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis

The results reported here indicate that retinoic acid (RA) induces growth arrest and differentiation only in MyoD-expressing muscle cells. Transient transfection assays reveal a functional interaction between MyoD, a key myogenic regulator and RA-receptors, principal mediators of RA actions. Interestingly, we demonstrate that RXR-MyoD-containing complexes are recruited at specific MyoD DNA-binding sites in muscle cells. Furthermore, we also demonstrate that RA-receptors and the muscle basic helix–loop–helix (b-HLH) proteins interact physically. Mutational analysis suggests that this interaction occurs via the basic region of muscle b-HLH proteins and the DNA-binding domain of RA-receptors and is important for functional interactions between these two families of transcription factors. In conclusion, these results highlight novel interactions between two distinct groups of regulatory proteins that influence cell growth and differentiation.

Wnt4 activates the canonical β-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 β-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.