INVOLVEMENT OF THE DIHYDROPYRIDINE RECEPTOR AND INTERNAL CA2+ STORES IN MYOBLAST FUSION

The process of myoblast fusion during skeletal myogenesis is calcium r egulated. Both dihydropyridine receptor and ryanodine receptor are alr eady present on muscle precursors, at the prefusional stage, before th ey are required for excitation-contraction coupling. Previous pharmaco logical studies have shown the need for a special pool of Ca2+ associa ted with the membrane for the fusion process to occur. We hypothesized that this pool of Ca2+ is mobilized via a machinery similar to that i nvolved in excitation-contraction coupling. The process of fusion in r at L6 muscle precursors was either totally or partially abolished in t he presence of the L-type calcium channel inhibitors SR33557 and nifed ipine (half inhibition towards 2 mu M), respectively, The inhibition w as reversible and dose-dependent. Drugs able to deplete internal calci um stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusio n whereas ryanodine had no effect. This suggests that fusion may be co ntrolled by internal pools of Ca2+ but that its regulation may be inse nsitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms, Indeed, Western blot analysis of pre- and postfusional L 6 cells demonstrated the presence, at the prefusional stage, of a tran sient form of the ryanodine receptor protein with an apparent molecula r weight slightly different from those of the classical skeletal and c ardiac forms. Taken together, these results support the hypothesis tha t the fusion process is driven by a mechanism involving both the dihyd ropyridine receptor (alpha 1 subunit of the L-type Ca2+ channel) and t he internal stores of Ca2+, The machinery underlying this mechanism mi ght consist of slightly different forms of the classic molecules that in adult muscle ensure excitation-contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca2 + is triggered by the type of mechanism already described in skeletal muscle. (C) 1996 Academic Press, Inc.