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Different capacity for store-dependent Ca2+ influx and Ca2+ extrusion across the plasma membrane of wild-type and dystrophic mdx mouse muscle

T.R. Cully,1 J.N. Edwards,1 R.M. Murphy2 and B.S. Launikonis,1 1School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia and 2Department of Zoology, La Trobe University, Melbourne, VIC 3086, Australia.

Store-operated Ca2+ entry (SOCE) is a ubiquitously expressed signalling system that is highly specialized in skeletal muscle to respond rapidly to depletions of Ca2+ within the internal store. "Dysregulated" SOCE had been proposed as a pathway for Ca2+ entry into dystrophic muscle that leads to fibre degradation. We recently showed that the integral SOCE proteins, STIM1 and Orai1, are upregulated approximately three-fold in dystrophic mdx mouse muscle. However the muscle still responds with normal activation and deactivation of SOCE during Ca2+ depletion from skinned fibres (Edwards et al., 2010). We now test our findings in intact fibres where conditions may be different to those in skinned fibres. Furthermore, a recent paper has described a previously unidentified STIM1 isoform that is specific to excitable cells, STIM1L, that could be conferring the fast kinetics of SOCE observed in muscle (Darbellay et al., 2011). We have also probed for STIM1L in mdx and wild-type (WT) muscle.

All experiments were approved by The Animal Ethics Committee at The University of Queensland. C57BL/6 and C57BL/10-mdx mice were killed by asphyxiation following inhalation of CO2. Western blotting for STIM1L was conducted on homogenates of EDL muscle from WT and mdx. For Ca2+ imaging experiments, interossei muscle from these mice were removed and individual fibres were enzymatically isolated. Isolated fibres were loaded with 10 μM fluo-4AM and imaged on a confocal microscope. Fibres were depleted of Ca2+ in a K+-based physiological solution with 20 μM cyclopiazonic acid (CPA) and no added Ca2+, conditions favourable to Ca2+ extrusion by the plasma membrane Ca2+ pump (PMCA). After 20 min exposure to CPA, 10 mM caffeine was applied to the fibres to ensure thorough depletion of sarcoplasmic reticulum (SR). Store-dependent Ca2+ influx and subsequent deactivation were then observed upon reintroduction of external Ca2+ to the fibres in a 0 Ca2+, Na+-based physiological solution. The reintroduction of Ca2+ evoked fluo-4 transients in both mdx and WT fibres. 10 mM caffeine was subsequently applied to assay SR Ca2+ content. In other experiments cytoplasmic fura-2 was imaged by exciting at 340 and 380 nm during application of 50 μM CPA on a wide-field fluorescence microscope. We also imaged fluo-5N in the tubular system of skinned fibres during SR Ca2+ release in a low Mg2+ solution containing rhod-2 to estimate the store-dependent influx using confocal microscopy (Edwards et al., 2010). Statistical differences between data sets were determined by two-tailed t-test and n values are represented in parentheses.

We found that STIM1L was upregulated 1.8-fold in mdx muscle (P<0.05). Consistent with the higher levels of STIM in mdx fibres, the store-dependent Ca2+ influx recorded in skinned fibres was 2-fold greater in mdx compared with WT for the same amplitude of SR Ca2+ release (P<0.05). In contrast experiments in intact fibres showed a greater amplitude (1.16±0.07 (7) vs 1.60±0.13 (6); P<0.01) and d[fluo-4 fluorescence]/dt (1.02±0.5 (7) vs 5.1±1.4 (6) s-1; P<0.05) during SOCE in WT compared to mdx fibres. However both fibre types displayed a similar time integral of the caffeine-induced fluo-4 transient (210±20 (7) vs 165±25 (6); P>0.1) following SOCE suggesting a similar level of SR Ca2+ reloading. Imaging of cytoplasmic fura-2 in the presence of 50 μM CPA and no added Ca2+ showed that more Ca2+ remained in the cytoplasm of mdx compared to WT fibres (fura-2 ratio after CPA application in WT and mdx: 0.50±0.05 (7) vs 0.56±0.02 (8); P<0.05) suggesting that Ca2+ extrusion by PMCA is restricted in mdx. This also explains the apparent lower store-dependent Ca2+ influx in mdx fibres, as the washout of caffeine and CPA resulted in more cytoplasmic Ca2+ re-entering SR to partially deactivate SOCE before externally applied Ca2+ could enter the fibre via SOCE. These results suggest that store-dependent Ca2+ influx is greater and PMCA is restricted in its capacity to extrude Ca2+ in mdx compared to WT fibres.

Edwards JN, Friedrich O, Cully TR, von Wegner F, Murphy RM, Launikonis BS (2010). American Journal of Physiology - Cell Physiology 299: C42-C50.

Darbellay B, Arnaudeau S, Bader CR, Konig S, Bernheim L (2011). Journal of Cell Biology 194: 335-346.

Supported by Muscular Dystrophy Queensland.