Overexpression of calpastatin inhibits L8 myoblast fusion

The formation of skeletal muscle fibers involves cessation of myoblast division, myoblast alignment, and fusion to multinucleated myofibers. Calpain is one of the factors shown to be involved in myoblast fusion. Using L8 rat myoblasts, we found that calpain levels did not change significantly during myoblast differentiation, whereas calpastatin diminished prior to myoblast fusion and reappeared after fusion. The transient diminution in calpastatin allows the Ca²⁺-promoted activation of calpain and calpain-induced membrane proteolysis, which is required for myoblast fusion. Here we show that calpastatin overexpression in L8 myoblasts does not inhibit cell proliferation and alignment, but prevents myoblast fusion and fusion-associated protein degradation. In addition, calpastatin appears to modulate myogenic gene expression, as indicated by the lack of myogenin (a transcription factor expressed in differentiating myoblasts) in myoblasts overexpressing calpastatin. These results suggest that, in addition to the role in membrane disorganization in the fusing myoblasts, the calpain-calpastatin system may also modulate the levels of factors required for myoblast differentiation.