Tightly Regulated Store-operated Ca2+ Entry In Healthy And Dystrophic Skeletal Muscle

Abstract

Store-operated Ca2+ entry (SOCE) is a mechanism that allows the entry of extracellular Ca2+ upon depletion of the internal stores. This mechanism has been described in skeletal muscle and the two main molecular players, Stim1 and Orai1, have been identified. Previous work, mainly on myotubes, suggested that SOCE may become deregulated in dystrophic skeletal muscle and result in cellular Ca2+ overload. The final result of such a process would be activation of proteolytic enzymes, cell necrosis and/or apoptosis. To examine the regulation of SOCE in healthy and dystrophic muscle we examined the biochemistry and physiology of skeletal muscle from wild-type (wt) and mdx mice (8-20 weeks old). Western blotting of single fibres showed that Stim1 and Orai1 were expressed at 2-3 times higher levels in mdx compared with wt muscle (normalized to total myosin). Consistent with this, enzymatically isolated interossei fibres loaded with fluo-4AM and depleted of Ca2+ in a solution containing 0 Ca2+, 20 μM cyclopiazonic acid and 10 mM caffeine showed a 3-fold higher rate of Ca2+ entry into the depleted mdx fibres compared to wt upon re-addition of 2 mM Ca2+. However, this does not imply Ca2+ overload will occur via SOCE in dystrophic cells if deactivation is unaffected. To study SOCE kinetics, skinned fibres with t-system trapped fluo-5N were bathed in an internal solution with rhod-2 and continuously imaged in xyt mode on a confocal microscope. Intracellular Ca2+ release was induced by lowering cytoplasmic [Mg2+]. Transient t-system Ca2+-depletion and reuptake was preceded by transient SR Ca2+-release and reuptake in fibres from both mdx and wt mice. This indicates robust activation and deactivation mechanisms of SOCE in both wt and dystrophic muscle which prevent not only depletion but also overloading of the internal Ca2+-stores.