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

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