Orai1 Mediates Store-Operated Ca2+ Entry in Normal Skeletal Muscle and Exacerbated Ca2+ Entry in Dystrophic Muscle

Abstract

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy, in which loss of dystrophin expression results in compromised sarcolemmal integrity. Although evidence shows that defects in Ca2+ homeostasis is a causal factor for the progressive cell death observed in DMD, the mechanism of Ca2+ deregulation is still under debate. Several laboratories showed that enhanced Ca2+ entry might serve as a pathological factor in dystrophic muscles. In this study, we explored the role of store operated Ca2+ entry (SOCE) in Ca2+ deregulation of dystrophic muscles. We used real-time PCR and Western blotting to detect known isoforms of Orai and STIM1 and determined that Orai1 was the most abundant in skeletal muscle and was significantly upregulated in muscles from mdx mice, while STIM1 levels remained largely unchanged. Furthermore, Mn2+ quenching of fura-2 fluorescence was applied to measure SOCE activity in flexor digitorum brevis (FDB) fibers and a significant increase in SOCE activity was detected in mdx fibers. Similar levels of resting [Ca]i was identified in wt and mdx groups, while peak response to C/R was significantly higher in mdx fibers than wt. Furthermore, we electroporated shRNA probe against mouse Orai1 into FDB muscle of living mice to produce effective knockdown (KD) of Orai1 expression. Two weeks after Orai1 KD, SOCE activity was eliminated in both wt and mdx muscle fibers and peak response to caffeine and ryanodine in mdx fiber sreturned to a level comparable to wt muscle fiber. Therefore, our study established that Orai1 is an essential component of SOCE machinery in adult skeletal muscle and indicates that Orai1-mediated SOCE could be the major pathway for additional Ca2+ entry into mdx muscle fibers, which would eventually lead to progression of DMD.