Alterations of Excitation-Contraction Coupling and Excitation Coupled Ca~(2+) Entry in Human Myotubes Carrying CAV3 Mutations Linked to Rippling Muscle Disease

摘要

Rippling muscle disease is caused by muta-tions in the gene encoding caveolin-3 (CAV3), the muscle-specific isoform of the scaffolding protein caveolin, a protein involved in the formation of caveolae. In healthy muscle, caveolin-3 is responsible for the formation of caveolae, which are highly organized sarcolemmal clusters influencing early muscle differentiation, signalling and Ca~(2+) homeostasis. In the present study we examined Ca~(2+) homeostasis and excitation-contraction (E-C) coupling in cultured myotubes derived from two patients with Rippling muscle disease with severe reduction in caveolin-3 expression; one patient har-boured the heterozygous c.84C >A mutation while the other patient harbored a homozygous splice-site mutation (c.102+ 2T>C) affecting the splice donor site of intron 1 of the CAV3 gene. Our results show that cells from control and rippling muscle disease patients had similar resting [Ca~(2+) ]_i and 4-chloro-m-cresol-induced Ca" release but reduced KCl-induced Ca~(2+) influx. Detailed analysis of the voltage-dependence of Ca~(2+) transients revealed a significant shift of Ca~(2+) release activation to higher depolarization levels in CAV3 mutated cells. High resolution immunofluorescence analysis by Total Internal Fluorescence microscopy supports the hypothesis that loss of caveolin-3 leads to microscopic disarrays in the colocalization of the voltage-sensing dihydropyridine receptor and the ryanodine receptor, thereby reducing the efficiency of excitation-contraction coupling.