Autophagy is required and protects against apoptosis during myoblast differentiation

Several degradative systems assist in formation of multinucleated, terminally differentiated myotubes. However, the role of autophagy in this process has not been examined. GFP-LC3B puncta, LC3B-II protein, and LysoTracker fluorescence increased during C2C12 differentiation. Importantly, accumulation of LC3B-II protein occurred in chloroquine (CQ) treated cells throughout differentiation. Furthermore, BECN1, ATG7, and ATG12-5 protein increased, while SQSTM1/p62 protein was rapidly reduced during differentiation. A transient decrease in BECN1:BCL2 association was observed from D0.5 to D2 of differentiation. Chemical inhibition of JNK during differentiation reduced LC3B-II protein and GFP-LC3B puncta, and maintained BECN1:BCL2 association. Inhibition of autophagy by 3MA or shRNA against Atg7 (shAtg7) resulted in lower myosin heavy chain expression, as well as impaired myoblast fusion and differentiation. Interestingly, 3MA treatment during differentiation increased transient CASP3 activation, DNA fragmentation, and the percentage of apoptotic nuclei. Similarly, shAtg7 cells had increased DNA fragmentation during differentiation compared to controls. Collectively, these data demonstrate that autophagy increases and is required during myoblast differentiation. Moreover, autophagy protects differentiating myoblast from apoptotic cell death.

Source: BJ Cell - May 28, 2014 Category: Biochemistry Authors: E M. McMillan, J Quadrilatero Tags: BJ Cell Source Type: research