Mitochondrial calcium oscillations in C2C12 myotubes

Mitochondrial Ca2+ concentration ([Ca2+],) was monitored in C2C12 skeletal muscle cells stably expressing the Ca2+-sensitive photoprotein aequorin targeted to mitochondria. In myotubes, KCI-induced depolarization caused a peak of 3.03 +/- 0.14 muM [Ca2+], followed by an oscillatory second phase (5.1 +/- 0.1 per min). Chelation of extracellular Ca2+ or blockade of the voltage-operated Ca2+ channel attenuated both phases of the KCl response. The inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, cyclopiazonic acid, reduced the amplitude of the KCl-induced [Ca2+](m) peak and prevented the oscillations, suggesting that these were generated intracellularly, No such [Ca2+](m) oscillations occurred with the nicotinic agonist carbachol, cyclopiazonic acid alone, or the purinergic agonist ATP. In contrast, caffeine produced an oscillatory behavior, indicating a role of ryanodine receptors as mediators of the oscillations. The [Ca2+](m) response was desensitized when cells were exposed to two consecutive challenges with KCl separated by a 5-min wash, whereas a second pulse of carbachol potentiated [Ca2+](m) indicating differences in intracellular Ca2+ redistribution. Cross-desensitization between KCl and carbachol and cross-potentiation between carbachol and KCl were observed. These results suggest that close contacts between mitochondria and sarcoplasmic reticulum exist permitting Ca2+ ex changes during KCl depolarization. These newly demonstrated dynamic changes in [Ca2+](m) in stimulated skeletal muscle cells might contribute to the understanding of physiological and pathological processes in muscular disorders.