MYOBLAST FUSION REQUIRES CYTOSOLIC CALCIUM ELEVATION BUT NOT ACTIVATION OF VOLTAGE-DEPENDENT CALCIUM CHANNELS

Many studies of in vitro skeletal myogenesis have demonstrated that fu sion of myoblasts into multinucleated myotubes is regulated by calcium -dependent processes. Calcium ions appear to be necessary at the outer face of the membrane, and an additional internal calcium increase see ms required to promote fusion of aligned myoblasts. It has been propos ed that a calcium influx could take place prior to fusion and that thi s may be mediated by voltage-dependent calcium channels. Previously, w e showed that two types of voltage-dependent calcium currents were exp ressed in multinucleated myotubes but not in rat myoblasts growing in primary culture before the withdrawal of the growth medium. We also sh owed that the previous formation of multinucleated synticia was not a prerequisite of developmental appearance of calcium currents, suggesti ng that the two events were time-correlated but not sequentially depen dent. These features led us to investigate changes in internal calcium activity and the possible appearance of voltage-dependent calcium inf lux pathways just after the promotion of fusion by the change of cultu re medium. The results confirm that a rise in cytosolic calcium activi ty occurs slightly before fusion in confluent myoblasts and remained i n newly formed myotubes. Reducing this elevation by internal calcium b uffering lowered myoblast fusion and, reciprocally, blocking cell fusi on prevented calcium increase. Treatment with the organic calcium chan nel blockers nifedipine (5 mu M) and PN 200-110 (1 mu M) did not alter cytosolic calcium changes nor cell fusion, and voltage-dependent calc ium currents were never observed by the perforated patch-clamp techniq ue in aligned fusion-competent myoblasts. Other voltage-operated mecha nisms of calcium rise were not detected since depolarization with hype rpotassium solutions failed to elicit increases in intracellular calci um. On the contrary, acetylcholine was able to promote extracellular c alcium-dependent calcium transients. Our results confirm the requireme nt of an increase in resting calcium during fusion, but do not support the hypothesis of an influx through voltage-dependent channels or oth er voltage-operated pathways. The elevation of internal calcium activi ty may result from other mechanisms, such as a cholinergic action for example.