Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP₃-receptor (IP₃R) calcium channels are necessary for the mitochondrial Ca²⁺ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling). Methods: Mitochondria matrix and cytoplasmic Ca²⁺ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca²⁺ sensor (CEPIA3mt) or a cytoplasmic Ca²⁺ sensor (RCaMP). The role of intracellular Ca²⁺ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP₃R and Dantrolene for RyR1) and a genetic approach (shIP₃R1-RFP). O₂ consumption was detected using Seahorse Extracellular Flux Analyzer. Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca²⁺ levels. Mitochondrial Ca²⁺ uptake required functional inositol IP₃R and RyR1 channels. Inhibition of either channel decreased basal O₂ consumption rate but only RyR1 inhibition decreased ATP-linked O₂ consumption. Cell membrane depolarization-induced Ca²⁺ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca²⁺ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca²⁺-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber. Conclusion: Ca²⁺-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an "excitation-metabolism" coupling process in skeletal muscle fibers.

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