AimTo identify microRNAs (miRs) involved in the regulation of skeletal muscle mass. For that purpose, we have initially utilized an in silico analysis, resulting in the identification of miR‐29c as a positive regulator of muscle mass.MethodsmiR‐29c was electrotransferred to the tibialis anterior to address its morphometric and functional properties and to determine the level of satellite cell proliferation and differentiation. qPCR was used to investigate the effect of miR‐29c overexpression on trophicity‐related genes. C2C12 cells were used to determine the impact of miR‐29c on myogenesis and a luciferase reporter assay was used to evaluate the ability of miR‐29c to bind to the MuRF1 3′UTR.ResultsThe overexpression of miR‐29c in the tibialis anterior increased muscle mass by 40%, with a corresponding increase in fibre cross‐sectional area and force and a 30% increase in length. In addition, satellite cell proliferation and differentiation were increased. In C2C12 cells, miR‐29c oligonucleotides caused increased levels of differentiation, as evidenced by an increase in eMHC immunostaining and the myotube fusion index. Accordingly, the mRNA levels of myogenic markers were also increased. Mechanistically, the overexpression of miR‐29c inhibited the expression of the muscle atrophic factors MuRF1, Atrogin‐1 and HDAC4. For the key atrogene MuRF1, we found that miR‐29c can bind to its 3′UTR to mediate repression.ConclusionsThe results herein suggest that miR‐29c can improve skeletal muscle size and function by stimulating satellite cell proliferation and repressing atrophy‐related genes. Taken together, our results indicate that miR‐29c might be useful as a future therapeutic device in diseases involving decreased skeletal muscle mass.

We characterized the metabolic profile of transgenic mice exhibiting enhanced muscle mass driven by increased mIGF-1 expression (MLC/mIGF-1). As expected, 6-month-old MLC/mIGF-1 mice were heavier than age-matched wild type (WT) mice (37.4 ± 0.3 versus 31.8 ± 0.6 g, resp.). MLC/mIGF-1 mice had higher respiratory quotient when compared to WT (0.9 ± 0.03 versus 0.74 ± 0.02, resp.) suggesting a preference for carbohydrate as the major fuel source. MLC/mIGF-1 mice had a higher rate of glucose disposal when compared to WT (3.25 ± 0.14 versus 2.39 ± 0.03%/min, resp.). The higher disposal rate correlated to ∼2-fold higher GLUT4 content in the extensor digitorum longus (EDL) muscle. Analysis of mRNA content for the glycolysis-related gene PFK-1 showed ∼3-fold upregulation in MLC/mIGF-1 animals. We also found a 50% downregulation of PGC1α mRNA levels in MLC/mIGF-1 mouse EDL muscle, suggesting less abundant mitochondria in this tissue. We found no difference in the expression of PPARα and PPARβ/δ, suggesting no modulation of key elements in oxidative metabolism. These data together suggest a shift in metabolism towards higher carbohydrate utilization, and that could explain the increased insulin sensitivity of hypertrophied skeletal muscle in MLC/mIGF-1 mice.

Lack of mechanical load leads to skeletal muscle atrophy, and one major underlying mechanism involves the myostatin pathway that negatively regulates protein synthesis and also activates Atrogin-1/MAFbx and MuRF1 genes. In hindlimb immobilization, leucine was observed to attenuate the upregulation of the referred atrogenes, thereby shortening the impact on fiber cross-sectional area, nonetheless, the possible connection with myostatin is still elusive. This study sought to verify the impact of leucine supplementation on myostatin expression. Male Wistar rats were supplemented with leucine and hindlimb immobilized for 3 and 7 days, after which soleus muscles were removed for morphometric measurements and analyzed for gene and protein expression by realtime PCR and Western blotting, respectively. Muscle wasting was prominent 7 days after immobilization, as expected, leucine feeding mitigated this effect. Atrogin-1/MAFbx gene expression was upregulated only after 3 days of immobilization, and this effect was attenuated by leucine supplementation. Atrogin-1/MAFbx protein levels were elevated after 7 days of immobilization, which leucine supplementation was not able to lessen. On the other hand, myostatin gene expression was upregulated in immobilization for 3 and 7 days, which returned to normal levels after leucine supplementation. Myostatin protein levels followed gene expression at a 3-day time point only. Follistatin gene expression was upregulated during immobilization and accentuated by leucine after 3 days of supplementation. Concerning protein expression, follistatin was not altered neither by immobilization nor in immobilized animals treated with leucine. In conclusion, leucine protects against skeletal muscle mass loss during disuse, and the underlying molecular mechanisms appear to involve myostatin inhibition and Atrogin-1 normalization independently of follistatin signaling.

Background Skeletal muscle regeneration is a powerful and highly synchronized process and it is well described that intracellular pathways related to anabolic responses are readily activated. On the other hand, the role of catabolic pathways in the skeletal muscle regenerative response is much less understood. In the present study, we hypothesized that MuRF1, a key gene involved in skeletal muscle mass loss, and its close counterpart MuRF2 play a role in skeletal muscle regeneration. Methods and results Wild type, single MuRF1 knock out, single MuRF2 knock out and double MuRF1&2 dKO mice had their tibialis anterior muscle injured by a single round of 4 sequential injections of cardiotoxin (CTX‐10ūM, 5ūl each injection) spread along the length of the muscle. The animals were killed after 1, 3, 10 and 28 days after injury and the muscles were used to address general histology, satellite cells, myogenic markers and apoptosis. In addition, we silenced MuRF1 and MuRF2 in a primary myogenic cell culture to evaluate myogenesis and BAF57, a SWI/SNF chromatin‐remodeling complex component. Finally, we employed Chromatin Immuno Precipitation assays to investigate whether MuRF1 associates with the myogenic promoters MyoD and myogenin. After cardiotoxin (CTX) injection, MuRF1 and MuRF2 expression was strongly increased and spread out inside the remaining fibers and also along satellite cells. In line with a critical role during regeneration, MuRF1&2 dKO deficient injured muscles were unable to properly regenerate. In addition, dKO injured muscle failed to express activators of the myogenic program, Myf‐5, FHL2 and MARP2, while myogenin levels were normally increased. Accordingly, siRNA reduced levels of MuRF1 and MuRF2 caused a severe myogenic deficit in primary myogenic cells, without any proliferation deficiency. Finally, in MuRF1&2 siRNA knockdown studies and Chromatin Immuno Precipitation analysis of primary myogenic cultures we have shown an impaired nuclear clearance of BAF57 and a shortage in chromatin remodeling as a likely mechanism underlying the deficient myogenic differentiation. Conclusion In summary, our results indicate a key cooperative role of the E3 ligases MuRF1 and MuRF2 in skeletal muscle regenerative response and myogenesis by inducing chromatin opening at myogenic promoters after BAF57 removal during muscle regeneration.