Abstract:
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Myofilament organization into the highly structured sarcomeres requires the accurate assembly, folding and interaction of newly synthesized sarcomeric proteins. This complex process, termed myofibrillogenesis, occurs during striated muscle development and is necessary for contractility. Nascent polypeptide-associated complex (NAC) was initially purified as a heterodimeric complex (__NAC) binding to newly synthesized polypeptide chains as they emerge from the ribosome. skNAC, a prolinerich splice variant of the ubiquitous _NAC, is strongly expressed specifically in skeletal and cardiac muscle during myogenic differentiation and muscle repair. To date, little is known about a possible function of skNAC in mammalian tissues. In the current study, we investigated the regulation of the skNAC gene and the function of the skNAC protein in cultured skeletal myoblasts. We could demonstrate that in these cells, skNAC gene expression is regulated at the transcriptional level and that it is not a primary response gene in myogenic differentiation. By treatment of murine myoblasts with specific MAPK inhibitors we could further show that the skNAC gene is a target of p38 MAPK, which plays a crucial role in myogenesis. In addition, we observed that inhibition of skNAC gene expression in murine C2C12 cells via specific siRNAs caused a remarkably diffuse staining of myosin heavy chain (MyHC)-positive myotubes, whereas in control myoblasts, the typical punctate staining of newly formed sarcomeres was observed. However, surprisingly, we could not detect diminished expression of the MyHC gene at either the mRNA or the protein level in our si-RNA-treated cells. Consistent findings of disorganized sarcomeres after skNAC knockdown could be observed in a recent in vivo study in the zebrafish system (Li et al., FASEB J. 23,1988, 2009). These data suggest that skNAC might be a regulator of myofibrillogenesis. To test this hypothesis, we analyzed the effect of the skNAC knockdown on the expression of genes enconding molecular chaperones known to be involved in myofibril organization and genes encoding proteins involved in the degradation of misfolded sarcomeric proteins. Remarkably, we found that diminished skNAC expression has indeed pronounced and specific effects on the expression of these genes. Taken together, our data indicate that skNAC plays a critical role in sarcomere assembly during muscle cell differentiation.
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