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 (light chain 3 beta) puncta, LC3B-II protein and LysoTracker fluorescence increased during C2C12 cell differentiation. Importantly, accumulation of LC3B-II protein occurred in CQ (chloroquine)-treated cells throughout differentiation. Furthermore, BECN1 (beclin 1), ATG7 (autophagy-related 7) and ATG12-5 protein increased, whereas SQSTM1/p62 (sequestosome 1) protein was rapidly reduced during differentiation. A transient decrease in BECN1–BCL2 association was observed from day 0.5 to 2 of differentiation. Chemical inhibition of JNK (c-Jun N-terminal kinase) during differentiation reduced LC3B-II protein and GFP–LC3B puncta and maintained BECN1–BCL2 association. Inhibition of autophagy by 3MA (3-methyladenine) 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 (caspase 3) activation, DNA fragmentation and the percentage of apoptotic nuclei. Similarly, shAtg7 cells had increased DNA fragmentation during differentiation compared with the controls. Collectively, these data demonstrate that autophagy increases and is required during myoblast differentiation. Moreover, autophagy protects differentiating myoblasts from apoptotic cell death.
- beclin 1 (BECN1)
- caspase 3 (CASP3)
- skeletal muscle
Abbreviations: AMPK, AMP-activated protein kinase; ATG, autophagy-related; AU, arbitrary unit; BECN1, beclin 1; BNIP3, BCL2/adenovirus E1B 19 kDa interacting protein 3; CASP3, caspase 3; CQ, chloroquine; D, day; DM, differentiation medium; DMEM, Dulbecco’s modified Eagle’s medium; FOXO3, forkhead box O3; GM, growth medium; HRP, horseradish peroxidase; IP, immunoprecipitation; JNK, c-Jun N-terminal kinase; LC3B, light chain 3 beta; 3MA, 3-methyladenine; MTOR, mammalian (or mechanistic) target of rapamycin; PI3K, phosphoinositide 3-kinase; PIK3C3/VPS34, class III phosphatidylinositol 3-kinase; SQSTM1/p62, sequestosome 1; ULK, unc-51 like kinase
- © The Authors Journal compilation © 2014 Biochemical Society