Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism

Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP(3)-receptor (IP(3)R) calcium channels are necessary for the mitochondrial Ca(2+) 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 Ca(2+) levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca(2+) sensor (CEPIA3mt) or a cytoplasmic Ca(2+) sensor (RCaMP). The role of intracellular Ca(2+) channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP(3)R and Dantrolene for RyR1) and a genetic approach (shIP(3)R1-RFP). O(2) consumption was detected using Seahorse Extracellular Flux Analyzer. Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca(2+) levels. Mitochondrial Ca(2+) uptake required functional inositol IP(3)R and RyR1 channels. Inhibition of either channel decreased basal O(2) consumption rate but only RyR1 inhibition decreased ATP-linked O(2) consumption. Cell membrane depolarization-induced Ca(2+) signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca(2+) signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca(2+)-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber. Conclusion: Ca(2+)-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.