Store-operated Ca²⁺ entry (SOCE) in skeletal muscle involves signalling between stromal-interacting molecule 1 (STIM1) in the sarcoplasmic reticulum (SR) and Ca²⁺ selective Orai1 channels in the sarcolemma. Here we generate transgenic mice with muscle-specific expression of dominant-negative Orai1 (dnOrai1) and demonstrate that Orai1-dependent SOCE promotes growth and limits fatigue in adult skeletal muscle. dnOrai1 mice lack SOCE specifically in muscle but are fertile and thrive well into adulthood. Although muscle ultrastructure, excitation–contraction (EC) coupling, fibre type, and expression of other Ca²⁺ regulatory proteins are unaltered, dnOrai1 mice exhibit reduced body weight, muscle mass and fibre cross-sectional area. Importantly, during intense repetitive activity, dnOrai1 mice display increased susceptibility to fatigue at the single fibre, excised muscle and whole-animal levels. We further show that STIM1 and Orai1 proteins co-localize within the triad junction but do not exist in a preassembled context. These results show that Orai1-dependent SOCE has an important physiological role in muscles of adult mice.

Central core disease (CCD) is a human myopathy that involves a dysregulation in muscle Ca²+ homeostasis caused by mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the protein that comprises the calcium release channel of the SR. Although genetic studies have clearly demonstrated linkage between mutations in RyR1 and CCD, the impact of these mutations on release channel function and excitation-contraction coupling in skeletal muscle is unknown. Toward this goal, we have engineered the different CCD mutations found in the NH₂-terminal region of RyR1 into a rabbit RyR1 cDNA (R164C, I404M, Y523S, R2163H, and R2435H) and characterized the functional effects of these mutations after expression in myotubes derived from RyR1-knockout (dyspedic) mice. Resting Ca²+ levels were elevated in dyspedic myotubes expressing four of these mutants (Y523S > R2163H > R2435H R164C > I404M RyR1). A similar rank order was also found for the degree of SR Ca²+ depletion assessed using maximal concentrations of caffeine (10 mM) or cyclopiazonic acid (CPA, 30 μM). Although all of the CCD mutants fully restored L-current density, voltage-gated SR Ca²+ release was smaller and activated at more negative potentials for myotubes expressing the NH₂-terminal CCD mutations. The shift in the voltage dependence of SR Ca²+ release correlated strongly with changes in resting Ca²+, SR Ca²+ store depletion, and peak voltage–gated release, indicating that increased release channel activity at negative membrane potentials promotes SR Ca²+ leak. Coexpression of wild-type and Y523S RyR1 proteins in dyspedic myotubes resulted in release channels that exhibited an intermediate degree of SR Ca²+ leak. These results demonstrate that the NH₂-terminal CCD mutants enhance release channel sensitivity to activation by voltage in a manner that leads to increased SR Ca²+ leak, store depletion, and a reduction in voltage-gated Ca²+ release. Two fundamentally distinct cellular mechanisms (leaky channels and EC uncoupling) are proposed to explain how altered release channel function caused by different mutations in RyR1 could result in muscle weakness in CCD.

Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle‐specific expression of a novel mitochondrial‐targeted superoxide biosensor (mt‐cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and adenine nucleotide translocase functionality, but it was independent of cyclophilin‐D‐mediated mitochondrial permeability transition pore activity. The diverse spatial dimensions of individual mSOF events were found to reflect a complex underlying morphology of the mitochondrial network, as examined by electron microscopy. Muscle activity regulated mSOF activity in a biphasic manner. Specifically, mSOF frequency was significantly increased following brief tetanic stimulation (18.1 ± 1.6 to 22.3±2.0 flashes/1000 μm ² ·100 s before and after 5 tetani) and markedly decreased (to 7.7±1.6 flashes/1000 μm ² ·100 s) following prolonged tetanic stimulation (40 tetani). A significant temperature‐dependent increase in mSOF frequency (11.9±0.8 and 19.8±2.6 flashes/1000 μm ² ·100 s at 23°C and 37°C) was observed in fibers from RYR1 ^Y522S/WT mice, a mouse model of malignant hyperthermia and heat‐induced hypermetabolism. Together, these results demonstrate that mSOF activity is a highly sensitive biomarker of mitochondrial respiration and the cellular metabolic state of muscle during physiological activity and pathological oxidative stress.—Wei, L., Salahura, G., Boncompagni, S., Kasischke, K. A., Protasi, F., Sheu, S.‐S., Dirksen, R. T. Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease. FASEB J. 25, 3068–3078 (2011). www.fasebj.org