Mitochondrial pathway of apoptosis controls muscle differentiation

Abstract

Caspases are the core compartments of programmed cell death. Caspases also play non apoptotic roles in cell proliferation, migration, differentiation, and immunity. During myoblast differentiation, caspase-2, -9 and -3 are required for cell fusion, however, it was not clear whether caspases directly function during the myogenic program or indirectly by triggering signals from apoptotic cells at the site of myotube formation. Here caspase-3 activity was detected for the first time in differentiating myoblasts using two strategies. One used biosensors that are based on dimerization dependent protein exchange, that show altered fluorescence following cleavage by caspase-3. The second strategy used a cell permeant fluorogenic caspase-3 substrate to track caspase-3 activity in C2C12 cells using live cell microscopy. To investigate the pathway underlying caspase-3 activation, an RNAi-based silencing approach was used. Apaf-1 and cytochrome c were required for activation of caspase-3 as well as myoblast differentiation. Differentiation was associated with the formation of heptamer apoptosome as myotube formation was blocked using the apoptosome inhibitor, M50054. M50054 blocks caspase activation as well as apoptosis by binding to Apaf-1 in the apoptosome complex not the monomeric form. These data showed mitochondrial pathway of apoptosis aside from its apoptotic role, is responsible for activation of caspase-3 during C2C12 differentiation. Moreover, a transient increase in tBid, produced by caspase-2 mediated cleavage of BID, was detected during C2C12 differentiation. As caspase-2 inhibition supresses caspase-3 activity during differentiation this data linked caspase-2 to the mitochondrial pathway of apoptosis. DNA damage induced by caspase-3 is required for myoblast differentiation. Inhibition of caspase-3 or DNA-PK blocked Myomerger expression as well as myoblast fusion. These observations were in agreement with caspase-3-mediated DNA damage controlling gene regulation. In conclusion, my results showed for the first time the direct activation of caspase-3 inside differentiating myoblasts. In addition mitochondrial pathway of apoptosis was reported to directly promote myoblast differentiation and also regulates the expression of genes critical for differentiation.