C2C12 myoblasts were cultured in DMEM (10 % FBS) and induced to differentiate into myotubes with DMEM (2 % horse serum) for 6 days. To disrupt autophagy, cells were (1) transfected with 50 nM Atg5 siRNA for 8 h twice over a 48 h period or (2) treated with 10 nM bafilomycin A1 or vehicle control for a 3 h/day for the first 3 days of differentiation. GFP-LC3 adenovirus was employed to visualize autophagy. Western blot and real-time qPCR were used to examine proteins and transcripts of interest. We observed increased LC3-II levels and GFP-LC3 puncta during the differentiation of C2C12 cells, suggesting the involvement of autophagy in this process. Transient inhibition of autophagy during the early stages of differentiation with either Atg5 siRNA or bafilomycin A1 interfered with myotube formation and attenuated the upregulation of myogenic transcription factors MyoD and myogenin. Differentiation was accompanied by an increase in PGC1α mRNA, mitochondrial mass, and oxygen consumption, all of which were blocked by disruption of autophagy. Autophagy coordinates transcription factor expression and mitochondrial turnover essential for cell differentiation (Fig. 15.1).

Fig. 15.1

Proposed mechanism for the role of autophagy in C2C12 cell differentiation. Upregulation of PGC1α, MyoD, and myogenin is a hallmark of cell differentiation. Transcription factor regulation by autophagy may affect mitochondrial turnover required (more...)