Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

Xiaoli Zhao,Shinji Komazaki,Sai Zhang,Noah Weisleder,Joseph G Moloughney,Johnny Huard

doi:10.1371/journal.pone.0049862

Xiaoli Zhao, Shinji Komazaki + Show 4 more

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https://doi.org/10.1371/journal.pone.0049862

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Abstract

There is substantial evidence indicating that disruption of Ca2+ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca2+ deregulation and the Ca2+ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca2+ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca2+ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca2+ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca2+ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca2+ store contributes to the disrupted Ca2+ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.

Highlights

Muscular dystrophy is characterized by muscle degeneration and reduced contractile function due to the death of skeletal muscle fibers
Further studies show that the protein level of Orai1 is increased in flexor digitorum brevis (FDB) muscle (Fig. 1B) as well as gastrocnemius and extensor digitorum longus (EDL) muscle (Fig. S1), supporting the possibly that Orai1mediated Ca2+ entry may be elevated in mdx muscle fibers
10 different transient receptor potential channel (TRP) channels isoforms have been detected in skeletal muscle by RT-PCR, western blot or immunohistochemistry [59], and here we confirm that TRPC3 and TRPC6 were expressed in skeletal muscle (Fig. 6B)

Summary

Introduction

Muscular dystrophy is characterized by muscle degeneration and reduced contractile function due to the death of skeletal muscle fibers. The most common type, Duchenne muscular dystrophy (DMD), results from a loss of function of the dystrophin gene [1]. While the exact cause of muscle fiber death is not clearly established, there is an increasing body of evidence showing that a defect in Ca2+ homeostasis is a causal factor for the progressive damage observed in muscular dystrophy [5]. One of the early cellular defects observed in DMD muscle biopsies was an increase in the number of fibers positive for a histochemical Ca2+ staining [6], and later efforts established that DMD may be associated with increased influx of Ca2+ [7,8].

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