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

We hypothesize that both type-1 ryanodine receptor (RyR1) and IP -receptor (IP R) calcium channels are necessary for the mitochondrial Ca 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). Mitochondria matrix and cytoplasmic Ca levels were evaluated in fibers isolated from muscle using plasmids for the expression of a mitochondrial Ca sensor (CEPIA3 ) or a cytoplasmic Ca sensor (RCaMP). The role of intracellular Ca channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP R and Dantrolene for RyR1) and a genetic approach (shIP R1-RFP). O consumption was detected using Seahorse Extracellular Flux Analyzer. In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca levels. Mitochondrial Ca uptake required functional inositol IP R and RyR1 channels. Inhibition of either channel decreased basal O consumption rate but only RyR1 inhibition decreased ATP-linked O consumption. Cell membrane depolarization-induced Ca signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca -dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber. Ca -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.