Increased Store-Operated Ca2+ Entry in Skeletal Muscle with Reduced Calsequestrin-1 Expression

Abstract

Store-operated Ca2+ entry (SOCE) contributes to Ca2+ handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn2+ fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca2+ store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39°C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca2+ permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca2+ as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca2+ entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca2+ signaling in MH.