MYOBLAST FUSION REQUIRES FIBRONECTIN DEGRADATION BY EXTERIORIZED M-CALPAIN

We recently reported that when myoblasts fuse, m-calpain could be exte riorized. Indeed, at present a number of works support this hypothesis because this enzyme was localized intercellularly and more particular ly associated to extracellular matrix components. Knowing that the cel l surface of the fusing myoblast is supposed to undergo many changes, we addressed the question whether m-calpain could be involved in the p henomenon of fusion via fibronectin cleavage or degradation. Using dif ferent digestion experiments, we demonstrated that soluble purified fi bronectin and highly insoluble fibronectin fibrils represent very good substrates for this proteinase; moreover, at the burst of fusion, fib ronectin proteolytic fragments could be identified. On the other hand, we have conducted biological assays on cultured myoblasts using a def ined medium supplemented by exogenous factors capable of stimulating o r inhibiting m-calpain activity. The effects of such factors on rat my oblast fusion and concomitantly on the targeted glycoprotein were anal yzed and quantified. When m-calpain activity and the phenomenon of fus ion were reduced (defined medium without insulin), the amount of the 2 20-kDa fibronectin band was increased by 43%. When m-calpain activity and myoblast fusion were prevented by addition of antibodies to m-calp ain or calpain inhibitor II, the fibronectin concentration was higher since it was increased by similar to 67 and similar to 71%, respective ly. In addition, when observed at the ultrastructural level, m-calpain seems to be localized at the potential fusion site of myoblasts and m ore particularly associated to the extracellular matrix when muscle ce lls were initially treated by anti-m-calpain IgG. Taken together, thes e results support the hypothesis that exteriorized m-calpain could be, in part, involved in myoblast fusion via fibronectin alteration or de gradation. (C) 1997 Academic Press.