Sarcoplasmic Reticulum Calcium Leak in Normal and Dystrophic Skeletal Muscle

Abstract

Under resting conditions, external calcium is known to enter skeletal muscle cells while calcium stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the resting sarcolemmal calcium entry and in the SR calcium leak are still debated but several lines of evidence suggest that an up-regulation of these calcium fluxes occurs in Duchenne Muscular Dystrophy (DMD). We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the murine model of DMD. Using the cytosolic calcium dye Fura2, we first demonstrated that the rate of calcium increase induced by CPA (cyclopiazonic acid) inhibition of SR Ca2+-ATPases at resting potential is significantly higher in mdx fibers suggesting an elevated SR passive calcium leak. However, in these experiments, sarcolemmal calcium influx may contribute to the CPA-induced calcium increase and another series of experiments indicated that CPA-induced SR calcium leak was deeply modified in the absence of external calcium. Fibers were then loaded with the low affinity calcium dye Fluo5N-AM and dialyzed with 50 mM EGTA to measure intraluminal SR calcium changes. Depolarization pulses evoked voltage-dependent Fluo5N fluorescence decreases followed by a recovery phase which was inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR calcium changes. Voltage-dependence and magnitude of depolarization-induced SR calcium depletion were found to be unchanged in mdx fibers but the rate of the recovery phase that followed depletion was found to be faster, suggesting a higher SR calcium reuptake capacity in mdx fibers. Finally, CPA-induced SR calcium leak at −80 mV was found to be significantly higher in mdx fibers. The elevated SR passive calcium leak may participate to the muscle degenerative process in mdx muscle.