Mitochondria fine-tune the slow Ca²⁺ transients induced by electrical stimulation of skeletal myotubes

Mitochondria sense cytoplasmic Ca²⁺ signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca²⁺ signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca²⁺ signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca²⁺ signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca²⁺ transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca²⁺ signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca²⁺ signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45Hz) skeletal myotubes. Similar Ca²⁺ signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca²⁺ rise induced by stimulation was inhibited by pre-incubation with ryanodine, whereas the phospholipase C inhibitor U73122 blocked the slow mitochondrial Ca²⁺ signal, showing that mitochondria sense the two cytoplasmic Ca²⁺ signal components. The fast but not the slow Ca²⁺ transient enhanced mitochondrial ATP production. Inhibition of the mitochondrial Ca²⁺ uniporter prevented the emergence of mitochondrial Ca²⁺ transients and significantly increased the magnitude of slow cytoplasmic Ca²⁺ signals after stimulation. Precluding mitochondrial Ca²⁺ extrusion with the Na⁺/Ca²⁺ exchanger inhibitor CGP37157 decreased mitochondrial potential, increased the magnitude of the slow cytoplasmic Ca²⁺ signal and decreased the rate of Ca²⁺ signal propagation from one nucleus to the next. Over expression of the mitochondrial fission protein Drp-1 decreased mitochondrial size and the slow Ca²⁺ transient in mitochondria, but enhanced cytoplasmic and nuclear slow transients. The present results indicate that mitochondria play a central role in the regulation of Ca²⁺ signals involved in gene expression in myotubes.