Skeletal muscle specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1

Myogenin is required not for the initiation of myogenesis but instead for skeletal muscle formation through poorly understood mechanisms. We demonstrate in cultured cells and, for the first time, in embryonic tissue, that myogenic late genes that specify the skeletal muscle phenotype are bound by MyoD prior to the initiation of gene expression. At the onset of muscle specification, a transition from MyoD to myogenin occurred at late gene loci, concomitant with loss of HDAC2, the appearance of both the Mef2D regulator and the Brg1 chromatin-remodeling enzyme, and the opening of chromatin structure. We further demonstrated that ectopic expression of myogenin and Mef2D, in the absence of MyoD, was sufficient to induce muscle differentiation in a manner entirely dependent on Brg1. These results indicate that myogenin specifies the muscle phenotype by cooperating with Mef2D to recruit an ATP-dependent chromatin-remodeling enzyme that alters chromatin structure at regulatory sequences to promote terminal differentiation.     

Nitric oxide drives embryonic myogenesis in chicken through the upregulation of myogenic differentiation factors

The muscle-specific variant of neuronal nitric oxide (NO) synthase (NOS-I), is developmentally regulated in mouse suggesting a role of NO during myogenesis. In chick embryo, a good model of development, we found that the expression of NOS-I is up-regulated, but only in the early phase of development. Through a pharmacological intervention in ovo we found that NO signalling plays a relevant role during embryonic development. The inhibition of NOS-I decreased the growth of embryo, in particular of muscle tissue, while the restoring of physiological NO levels, via administration of a NO donor, reversed this effect. We found a selective action of NO, produced by NOS-I, on regulatory factors involved in myogenic differentiation in the early phase of chick embryo development: inhibition of NO generation leads to a decreased expression of the Myocyte enhancer factor 2a (Mef2a), Mef2c, Myogenin and Myosin, which was reversed by the administration of a NO donor. NO had no effects on Myf5 and MyoD, the myogenic regulatory factors necessary for myogenic determination. The action of NO on the myogenic regulatory factors was mediated via generation of cyclic GMP (cGMP) and activation of the cGMP-dependent protein kinase G (PKG). Finally we found in myoblasts in vitro that the activation of Mef2c was the key event mediating the NO-induced modulation of myogenesis.     

Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein

In this study we have investigated the effect and the mechanisms by which tumor necrosis factor-like weak inducer of apoptosis (TWEAK) modulates myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK inhibited their differentiation evident by a decrease in the expression of creatine kinase, myosin heavy chain-fast twitch, myogenin, and the formation of multinucleated myotubes. TWEAK also inhibited the differentiation of mouse primary myoblasts. Conversely, the proliferation of C2C12 myoblasts and the expression of a cell-cycle regulator cyclin D1 were increased in response to TWEAK treatment. Inhibition of cellular proliferation using hydroxyurea only partially reversed the inhibitory effect of TWEAK on myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK resulted in the activation of nuclear factor-kappaB (NF-kappaB), the (IkappaB) IkappaB kinase (IKK) complex, and the phosphorylation and degradation of IkappaBalpha protein. Inhibition of NF-kappaB activity by overexpression of a dominant negative mutant of IkappaBalpha (IkappaBalphaDeltaN) significantly increased the myogenic differentiation in TWEAK-treated C2C12 cultures. Furthermore, overexpression of a dominant negative mutant of IKKbeta (IKKbetaK44A) but not IKKalpha (IKKalphaK44M) reversed the inhibitory effect of TWEAK on myogenesis. TWEAK inhibited the expression of myogenic regulatory factors MyoD and myogenin and also induced the degradation of MyoD protein. Finally, inhibition of NF-kappaB activation through overexpression of IKKbetaK44A prevented the degradation of MyoD protein. Overall, our data suggest that TWEAK inhibits myogenesis through the activation of NF-kappaB signaling pathway and degradation of MyoD protein.     

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.