Hypoxia inhibits myogenic differentiation through accelerated MyoD degradation

Cells undergo a variety of biological responses when placed in hypoxic conditions, including alterations in metabolic state and growth rate. Here we investigated the effect of hypoxia on the ability of myogenic cells to differentiate in culture. Exposure of myoblasts to hypoxia strongly inhibited multinucleated myotube formation and the expression of differentiation markers. We showed that hypoxia reversibly inhibited MyoD, Myf5, and myogenin expression. One key step in skeletal muscle differentiation involves the up-regulation of the cell cycle-dependent kinase inhibitors p21 and p27 as well as the product of the retinoblastoma gene (pRb). Myoblasts cultured under hypoxic conditions in differentiation medium failed to up-regulate both p21 and pRb despite the G1 cell cycle arrest, as evidenced by p27 accumulation and pRb hypophosphorylation. Hypoxia-dependent inhibition of differentiation was associated with MyoD degradation by the ubiquitin-proteasome pathway. MyoD overexpression in C2C12 myoblasts overrode the differentiation block imposed by hypoxic conditions. Thus, hypoxia by inducing MyoD degradation blocked accumulation of early myogenic differentiation markers such as myogenin and p21 and pRb, preventing both permanent cell cycle withdraw and terminal differentiation. Our study revealed a novel anti-differentiation effect exerted by hypoxia in myogenic cells and identified MyoD degradation as a relevant target of hypoxia.     

HDAC8 regulates canonical Wnt pathway to promote differentiation in skeletal muscles

Histone deacetylase 8 (HDAC8) is a class 1 histone deacetylase and a member of the cohesin complex. HDAC8 is expressed in smooth muscles, but its expression in skeletal muscle has not been described. We have shown for the first time that HDAC8 is expressed in human and zebrafish skeletal muscles. Using RD/12 and RD/18 rhabdomyosarcoma cells with low and high differentiation potency, respectively, we highlighted a specific correlation with HDAC8 expression and an advanced stage of muscle differentiation. We inhibited HDAC8 activity through a specific PCI-34051 inhibitor in murine C2C12 myoblasts and zebrafish embryos, and we observed skeletal muscles differentiation impairment. We also found a positive regulation of the canonical Wnt signaling by HDAC8 that might explain muscle differentiation defects. These findings suggest a novel mechanism through which HDAC8 expression, in a specific time window of skeletal muscle development, positively regulates canonical Wnt pathway that is necessary for muscle differentiation.     

pRb-dependent cyclin D3 protein stabilization is required for myogenic differentiation

The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb-/- myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3beta (GSK-3beta)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3beta to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances muscle-specific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.     

Ubiquitin-proteasome-mediated degradation, intracellular localization, and protein synthesis of MyoD and Id1 during muscle differentiation

Mammalian skeletal myogenesis results in the differentiation of myoblasts to mature syncytial myotubes, a process regulated by an intricate genetic network of at least three protein families: muscle regulatory factors, E proteins, and Id proteins. MyoD, a key muscle regulatory factor, and its negative regulator Id1 have both been shown to be degraded by the ubiquitin-proteasome system. Using C2C12 cells and confocal fluorescence microscopy, we showed that MyoD and Id1 co-localize within the nucleus in proliferating myoblasts. In mature myotubes, in contrast, they reside in distinctive subcellular compartments, with MyoD within the nucleus and Id1 exclusively in the cytoplasm. Cellular abundance of Id1 was markedly diminished from the very onset of muscle differentiation, whereas MyoD abundance was reduced to a much lesser extent and only at the later stages of differentiation. These reductions in MyoD and Id1 protein levels seem to result from a change in the rate of protein synthesis rather than the rate of degradation. In vivo protein stability studies revealed that the rates of ubiquitin-proteasome-mediated MyoD and Id1 degradation are independent of myogenic differentiation state. Id1 and MyoD were both rapidly degraded, each with a t 1/2 approximately = 1 h in myoblasts and in myotubes. Furthermore, relative protein synthesis rates for MyoD and Id1 were significantly diminished during myoblast to myotube differentiation. These results provide insight as to the interaction between MyoD and Id1 in the process of muscle differentiation and have implications for the involvement of the ubiquitin-proteasome-mediated protein degradation and protein synthesis in muscle differentiation and metabolism under abnormal and pathological conditions.     

FHL1 regulates myoblast differentiation and autophagy through its interaction with LC3

Four and a half LIM domain protein 1 (FHL1) belongs to the FHL protein family and is predominantly expressed in skeletal and cardiac muscle. FHL1 acts as a scaffold during sarcomere assembly and plays a vital role in muscle growth and development. Autophagy is key to skeletal muscle development and regeneration, with its dysfunction associated with a range of muscular pathologies and disorders. In this study, we constructed FHL1-silenced or FHL1-overexpressed myoblasts to investigate its role in autophagy during the differentiation of chicken myoblasts into myotubules. Our data showed that FHL1 contributes to myoblast differentiation as measured through MyoG, MyoD, Myh3, and Mb mRNA expression, MyoG and MyHC protein expression and the morphological characteristics of myoblasts. The results showed that FHL1 silencing inhibited the expression of ATG5 and ATG7, meanwhile, immunofluorescence and immunoprecipitation showed that FHL1 and LC3 interacted to regulate the correct formation of autophagosomes. FHL1 inhibition increased cleaved caspase-3 and PARP abundance and promoted myoblast apoptosis. Furthermore, FHL1 rescued skeletal muscle atrophy through regulating the expression of Atrogin-1 and MuRF1. Taken together, these data suggested that FHL1 regulates chicken myoblast differentiation through its interaction with LC3.