Potential m-calpain substrates during myoblast fusion

Many studies have demonstrated that m-calpain was implicated in cell membra ne reorganization-related phenomena during fusion via a regulation by calpa statin, the specific Ca2+-dependent proteolytic inhibitor. However, the rea l biological role of this protease is unclear because many targeted protein s are still unknown. Using different digestion experiments we have demonstr ated that desmin, vimentin, talin, and fibronectin represent very good subs trates for this proteinase capable of cleaving them in fragments which are immediately degraded by other enzymatic systems. Concerning intermediate fi laments, we showed that during the phenomenon of fusion, the amount of desm in was significantly reduced while the concentration of vimentin presented a steady level. On the other hand, we have conducted biological assays on c ultured myoblasts supplemented by exogenous factors such as calpain inhibit ors or antisense oligonucleotides capable of stimulating or inhibiting m-ca lpain activity. The effect of such factors on fusion and concomitantly on t he targeted substrates was analyzed and quantified. When m-calpain activity and myoblast fusion were prevented by addition of calpain inhibitors enter ing the cells, the amounts of desmin, talin, and fibronectin were increased , whereas the amount of vimentin was unchanged. Using antisense strategy, s imilar results were obtained. In addition, when the phenomenon of fusion wa s enhanced by preventing calpastatin synthesis, the amounts of desmin, tali n, and fibronectin were significantly reduced. Taken together, these result s support the hypothesis that m-calpain is involved in myoblast fusion by c leaving certain proteins identified here. This cleavage could modify membra ne and cytoskeleton organization for the myoblasts to fuse, (C) 1999 Academ ic Press.