CHANGES IN CYTOSOLIC RESTING IONIZED CALCIUM LEVEL AND IN CALCIUM TRANSIENTS DURING IN-VITRO DEVELOPMENT OF NORMAL AND DUCHENNE MUSCULAR-DYSTROPHY CULTURED SKELETAL-MUSCLE MEASURED BY LASER CYTOFLUOROMETRY USING INDO-1

Intracellular calcium activity was recorded during in vitro myogenesis of human normal and DMD muscle, using the calcium probe Indo-1 under laser illumination, at rest and during different kinds of stimulation (acetylcholine, high K+, caffeine). In myoblasts, the resting intracel lular calcium level was significantly larger in DMD cells (89 +/- 9 nM ; n = 40 vs 37 +/- 5 nM; n = 22) but there was no significant differen ce in myotubes, after fusion (44 +/- 4 nM; n = 34 vs 36 +/- 4 nM; n = 52). A similar evolution was observed in cells cultured from FSH biops ies. The amplitude of ACh- and high K+-induced calcium transients was significantly halved in DMD myotubes as compared to control ones and n on-significantly decreased for caffeine responses. Some alterations in the kinetics of responses were observed in DMD muscle: the rising pha ses of ACh- and high K+-elicited transients and the decaying phase of the ACh-responses were significantly slowed down. It is concluded that : (i) in aneurally cultured human muscle, an increase in the basal lev el of internal calcium can occur at early stages of myogenesis before the expression of the dystrophin gene; and (ii) the changes in calcium transients induced by depolarization or direct stimulation of sarcopl asmic reticulum are not susceptible of inducing a calcium overload in DMD cells.