CALPAIN AND CALPASTATIN IN MYOBLAST DIFFERENTIATION AND FUSION - EFFECTS OF INHIBITORS

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca2+-activated thi ol protease) induced proteolysis has been suggested to play a role in myoblast fusion, We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion, Using the red cell as a model, we found that red cell fusion required calpain a ctivation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly i n myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappe ars at a later stage (myotube formation). In the present study, the my oblast fusion inhibitors TGF-beta, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and f use in serum-poor medium containing insulin. TGF-beta and EGTA prevent ed the diminution of calpastatin. Calpeptin inhibited fusion without p reventing diminution of calpastatin, by inhibiting calpain activity di rectly. Protein levels of mu-calpain and m-calpain did not change sign ificantly in fusing myoblasts, nor in the inhibited, non-fusing myobla sts, The results indicate that calpastatin level is modulated by certa in growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion. (C) 1997 Elsevier Scien ce B.V.