Knockdown of hypoxia-inducible factor-1α by siRNA inhibits C2C12 myoblast differentiation

We analyzed the role of Hypoxia-inducible factor (HIF)-1 alpha in myoblast differentiation by examining the expression and regulation of HIF-1 alpha in proliferating and differentiating C2C12 myoblast, and by knocking down HIF-1 alpha of C2C12 myoblasts with small interfering RNA(siRNA), given that HIF-1 alpha has been shown to be involved in differentiative process in non-muscle tissues. Although HIF-1 alpha mRNA was constantly expressed in C2C12 myoblasts both undergrowth\ and differentiating phase, HIF-1 alpha. protein was hardly detectable in the growth phase but became detectable only during myogenic differentiation even under normoxia. During early stage of C2C12 myogenesis, HIF-1 alpha. accumulated in the nuclei of myogenin-positive myoblasts. The inhibition of proteasome in the growth phase led to HIF-1 alpha protein accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF-1 alpha., suppressed HIF-1 alpha accumulation. Therefore, we suggest that the level of HIF-1 alpha protein expression is regulated by a proteasome-and chaperon-dependent pathway in C2C12 myoblast. Knockdown of HIF-1 alpha effectively blocked myotube formation and myosin heavy chain WHO expression. Finally, HIF-1 alpha/expression in vivo was confirmed in the regenerative Muscle tissue of mice after eccentric exercise. We conclude that HIF-1 alpha is required for C2C12 myogenesis in vitro, and suggest that HIF-1 alpha may have all essential role in regenerative muscle tissue in vivo.