Abstract
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.
Highlights
The skeletal muscle system plays a critical role in the energy balance of the organism
K+-induced depolarization of muscle fibers did not affect the maximal respiratory capacity, the proton leak (LH+ ) or the non-mitochondrial oxygen consumption rate (OCR) (Figures 1A–C). These results suggest that K+-induced membrane depolarization directly stimulates mitochondrial metabolism in skeletal muscle fibers
Dantrolene but not xestospongin B reduced the slope of the fluorescence increase from 0.55 ± 0.01 to 0.15 ± 0.12. These results are consistent with the kinetics of activation of ryanodine receptor type-1 (RyR1) and IP3R in muscle fibers. These findings suggest that Ca2+ release through both IP3R and RyR1 are involved in the mitochondrial Ca2+ increase produced by depolarizing stimuli
Summary
The skeletal muscle system plays a critical role in the energy balance of the organism. Its energy requirements can increase nearly instantaneously by more than 100-fold (Weibel and Hoppeler, 2005). This skeletal muscle characteristic suggests the need for a fast signaling mechanism responsible for the metabolic increase generated by muscle plasma membrane depolarization. We define as “excitationmetabolism coupling” this functional interaction between plasma membrane depolarization and the resulting increase in muscle metabolism. Mitochondrial function and other bioenergetics pathways such as glycolysis are likely to play critical roles in maintaining the energy balance between supply and demand. Mitochondrial dysfunction characterizes a wide spectrum of adult-onset degenerative diseases, including muscle atrophy, muscle dystrophy, insulin resistance, type 2 diabetes, age-related sarcopenia, among others (Arnould et al, 2015), which may affect the excitation-metabolism coupling process
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