Factors affecting SOCE activation in mammalian skeletal muscle fibers

Abstract

Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca2+ entry, SOCE. Sarcoplasmic reticulum (SR) Ca2+ stores were depleted by repetitive exposures to high K+ or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca2+. SR Ca2+ store replenishment was substantially reduced using 5 μM cyclopiazonic acid (CPA). Readmission of external Ca2+ (5 mM) increased basal [Ca 2+]i under two modalities. In mode 1 [Ca 2+]i initially increased at a rate of 0.8 ± 0.1 nM/s and later at a rate of 12.3 ± 2.6 nM/s, reaching a final value of 477.8 ± 36.8 nM in 215.7 ± 25.9 s. In mode 2, [Ca 2+]i increased at a rate of 0.8 ± 0.1 nM/s to a value of 204.9 ± 20.6 nM in 185.4 ± 21.1 s. FCCP, 2 μM, reduced this Ca2+ entry. In nine FCCP-poisoned fibers, the initial rate of Ca2+ increase was 0.34 ± 0.1 nM/s (mean ± SEM), reaching a plateau of 149.2 ± 14.1 nM in 217 ± 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca2+, we show that during depletion [Ca2+]mito initially increases and later diminishes. Finally, we show that the increase in basal [Ca2+]i, associated with SOCE activation, diminishes upon external Na+ withdrawal. Na+ entry through the SOCE pathway and activation of the reversal of Na+/Ca2+ exchanger could explain this SOCE modulation by Na+. © 2009 The Physiological Society of Japan and Springer.