Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

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.

Roles of calpain-calpastatin system (CCS) in human T cell activation

“…It was also suggested that m-calpain can actually be activated by active μ-calpain [4]. A corroborative evidence for direct relation between the activities of calpains and of ORAI1 channels had been demonstrated in murine model of Duchenne muscular dystrophy, where inhibition of ORAI1 by its specific inhibitor BTP-2 greatly reduced the cytosolic μ-calpain activity in the muscle fibres [39], and in skin keratinocytes, for which Ca 2+ - and calpain-dependent pathway is necessary for proliferation, differentiation and migration [40]. …”

Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy

“…Cytosolic calcium concentration is tightly regulated in muscle fibers and the plasma membrane plays an important role in this regulation through integral membrane proteins controlling both calcium entry and efflux. Defective calcium influx or efflux across the plasma membrane has already been shown to be involved in elevated calcium concentration occurring in Duchenne muscular dystrophy resulting from dystrophin deficiency (Deval et al, 2002; Vandebrouck et al, 2002; Zhao et al, 2012). Among the integral membrane proteins, voltage-activated, store-operated, and stretch-regulated channels represent the main ways of entry whereas Na + /Ca 2+ -exchanger or Ca 2+ -ATPase pump are involved in calcium extrusion (Vallejo-Illarramendi et al, 2014).…”

Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

“…It was already proposed that a pool of TRPV2 at the sarcolemma could be constitutively active and that Ca 2+ entry may promote the addressing of cytoplasmic TRPV2 in dystrophic cardiomyocytes [43]. Moreover, the constituents of SOCs, Orai and TRPC1 proteins, were shown to support elevated calcium influx after store depletion in dystrophic myotubes and fibres, when deficient in dystrophin [29,31,32]. Interestingly, TRPC1 was also suggested to support a constitutive calcium entry in skeletal muscle fibres when deficient in homer-1 [64], and we showed that the scaffolding protein α1-syntrophin prevents exacerbated TRPC1-dependent cation entry in skeletal myotubes [31].…”

“…In dystrophic fibres, TRPC1 have been involved in abnormal Ca 2+ influx [29] and Ward et al [30] have shown that expression level of TRPC1 was increased in aged mdx -DCM heart. TRPC1 was also shown to support elevated store-operated calcium entry in dystrophin deficient myotubes and fibres [29,31] as well as Orai1 in FDB fibres [32]. Studies in heterologous system demonstrated that TRPC1 expression induced a 10-fold increase of SAC ns currents and TRPC1 inhibition through RNA anti-sens reduced these currents [33].…”