Post-Developmental Knockout of Orai1-Mediated Store-Operated Calcium Entry Improves Muscle Pathology in a Mouse Model of Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD), a genetic disorder caused by mutations in the dystrophin gene, is characterized by progressive muscle degeneration/weakness. Enhanced store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism coordinated by STIM1 and Orai1, is proposed to potentiate Ca2+-mediated damage in DMD. To determine the role of SOCE in promoting muscular dystrophy, we crossed mdx mice with tamoxifen-inducible, muscle-specific Orai1 knockout mice (mdx-Orai1 KO). Analysis of transcript levels revealed that Orai1 expression was ∼1.5x higher in muscle from mdx mice compared to WT mice, while nearly undetectable in muscle from mdx-Orai1 KO mice. Levels of Orai1 transcript correlated with SOCE activity measured using Mn2+ quench of fura-2 fluorescence in single flexor digitorum brevis muscle fibers. Specifically, SOCE was increased in mdx fibers and absent in fibers from mdx-Orai1 KO mice. Moreover, compared to WT fibers, mdx fibers exhibited: i) increased myoplasmic Ca2+ levels; ii) reduced Ca2+ store content; and iii) a significant prolongation in the rate of electrically-evoked Ca2+ transient decay. All of these effects were normalized in fibers from mdx-Orai1 KO mice. Ex vivo contractility experiments revealed that extensor digitorum longus muscles from mdx mice displayed a reduction in maximal tetanic specific force (150.3±3.5 mN/mm2) compared to WT muscles (199.6±11.1 mN/mm2), a parameter ameliorated in muscles from mdx-Orai1 KO mice (188.1±10.9 mN/mm2). When exposed to 10 consecutive eccentric contractions, mdx muscles exhibited a higher damage-induced force reduction than WT muscles (residual force after the 10th eccentric contraction was 35.5±3.8% and 61.8±1.9%, respectively). Interestingly, Orai1 ablation partially protected muscles from damage in mdx-Orai1 KO mice (residual force was 46.1±2.6% after 10 contractions). Our results indicate that enhanced Orai1-dependent SOCE exacerbates the dystrophic phenotype and Orai1 deficiency promotes sarcolemmal integrity/stability during eccentric contraction.