Sarcoplasmic reticulum Ca2+ permeation explored from the lumen side in mdx muscle fibers under voltage control

Abstract

Under resting conditions, external Ca2+ is known to enter skeletal muscle cells, whereas Ca2+ stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the sarcolemmal Ca2+ entry and in the SR Ca2+ leak is still a matter of debate, but several lines of evidence suggest that these Ca2+ fluxes are up-regulated in Duchenne muscular dystrophy. 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 mouse model of Duchenne muscular dystrophy. Using the cytosolic Ca2+ dye Fura2, we first demonstrated that the rate of Ca2+ increase in response to cyclopiazonic acid (CPA)–induced inhibition of SR Ca2+-ATPases at resting potential was significantly higher in mdx fibers, which suggests an elevated SR Ca2+ leak. However, removal of external Ca2+ reduced the rate of CPA-induced Ca2+ increase in mdx and increased it in control fibers, which indicates an up-regulation of sarcolemmal Ca2+ influx in mdx fibers. Fibers were then loaded with the low-affinity Ca2+ dye Fluo5N-AM to measure intraluminal SR Ca2+ changes. Trains of action potentials, chloro-m-cresol, and depolarization pulses evoked transient Fluo5N fluorescence decreases, and recovery of voltage-induced Fluo5N fluorescence changes were inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR Ca2+ changes. Voltage dependence and magnitude of depolarization-induced SR Ca2+ depletion were found to be unchanged in mdx fibers, but the rate of the recovery phase that followed depletion was found to be faster, indicating a higher SR Ca2+ reuptake activity in mdx fibers. Overall, CPA-induced SR Ca2+ leak at −80 mV was found to be significantly higher in mdx fibers and was potentiated by removal of external Ca2+ in control fibers. The elevated passive SR Ca2+ leak may contribute to alteration of Ca2+ homeostasis in mdx muscle.

Dihydropyridine receptors actively control gating of ryanodine receptors in resting mouse skeletal muscle fibres

“…Indeed, it sets the steady‐state Ca 2+ level within the SR lumen, thus conditioning the magnitude of depolarization‐induced SR Ca 2+ release upon muscle activation, but also influencing store‐operated sarcolemmal Ca 2+ entry and cytosolic Ca 2+ resting level. In a physiopathological perspective, it is proposed that an elevated SR Ca 2+ leak may contribute to the development of muscular dystrophies (Bellinger et al 2009; Robin et al 2012).…”

“…Two previous studies have shown that fluo‐5N can be used as a relevant Ca 2+ indicator to monitor Ca 2+ changes in the SR lumen of mouse skeletal muscle fibres (Ziman et al 2010; Robin et al 2012). It has also been shown that this indicator is well suited for measuring SR Ca 2+ efflux and the resulting depletion at controlled resting membrane potentials (Robin et al 2012).…”

Selective inhibition of ATPase activity during contraction alters the activation of p38 MAP kinase isoforms in skeletal muscle

“…The finding that combined treatment of BTS and CPA prevented p38γ phosphorylation suggests that activation of the p38γ isoform but not p38α/β isoforms of MAPK could be highly dependent on changes in energetic demand since both major ATPases active in skeletal muscle during contraction were being inhibited. By extension, p38γ cannot be activated by changes in cytosolic calcium because CPA treatment results in large increases in cytosolic calcium (as shown in frog (Même et al, 1998) and murine muscle fibers (Robin et al, 2012) as well as evidenced by the mechanics data in Figure 4), yet muscle treated with both CPA and BTS have no increase in the p38γ signal.…”

“…Stimulating muscle in the presence of BTS alone, which eliminates force production but allows normal calcium transients to proceed, had no effect on the phosphorylation of any of the p38 isoforms when compared to just stimulation alone, confirming a previous report that force production is not responsible for the activation of p38 signaling (Dentel et al, 2005). However, when CPA and BTS were used together, a combination that leads to an increase in intracellular calcium (Robin et al, 2012) but may eliminate up to 97% of the free energy used in exercised muscle (Rall, 1982; Rall, 1985), the increase in p38γ phosphorylation typically found with stimulation was blunted. This demonstrates that p38 MAPK γ is not activated by increased contractile force or intracellular calcium concentration, but instead is responsive to changes in the balance of ATP usage since blocking ATPases also blunts phosphorylation.…”

“…Thirty minutes later, CPA was added (in 20 μl DMSO). Muscles were treated in this order because inhibition of SERCA by CPA causes a rise in intracellular calcium levels (Baudet et al, 1993; Robin et al, 2012) that may be sufficient to increase basal tension by activation of actomyosin ATPase. For experiments where a single ATPase was inhibited, 20 μl DMSO was added to the bath during the first 30 minute period, followed by addition of either 20 μl BTS or CPA stock solutions for the remaining 30 minutes.…”