Interplay between Autophagy and Apoptosis During Skeletal Muscle Differentiation

Abstract

Our previous studies showed that autophagy regulates apoptosis and is required for proper skeletal muscle differentiation. Previously, we inhibited autophagy in C2C12 cells using 3MA (3-methyladenine) treatment or shRNA against ATG7, both of which resulted in elevated apoptotic signaling and impaired skeletal muscle differentiation. In the present study, we treated C2C12 cells with ad-DN-ATG5 (adenovirus expressing the dominant negative form of the autophagy related protein 5) to interrupt autophagosome formation and inhibit autophagy. The ad-DN-ATG5 treated C2C12 cells displayed elevated apoptosis (increased CASP3 (caspase3) activation) as well as lower MYH (myosin heavy chain) expression and impaired myoblast fusion and differentiation. The increased CASP3 activation in ad-DN-ATG5 treated C2C12 cells was accompanied by significantly reduced BECN1 (beclin 1) levels. Studies in other cell types and contexts have indicated that CASP3 could cleave BECN1 and inactivate BECN1 autophagic function. Therefore, we investigated whether STS (staurosporine)-induced CASP3 activation is also accompanied by lower BECN1 levels. STS treatment also resulted in reduced BECN1 levels in differentiating C2C12 cells. Next, we investigated the importance of BECN1 regulator; AMBRA1 (activating molecule in BECN1 regulated autophagy protein 1), during C2C12 differentiation. Silencing AMBRA1 expression in C2C12 cells using an AMBRA1 siRNA, lead to reduced levels of anti-apoptotic protein BCL2 (BCL2 apoptosis regulator) as well as significantly lower MYH expression. Collectively, the result of this study i) confirm the previously observed protective role of autophagy against apoptosis during skeletal muscle differentiation, ii) introduce BECN1 as a potential factor contributing in the interplay between autophagy and apoptosis, and iii) indicate an important role for AMBRA1 in regulating apoptotic signaling and skeletal muscle differentiation.