Store-operated Ca2+ Entry In Skeletal Muscle Research Articles

Alteration of STIM1/Orai1-Mediated SOCE in Skeletal Muscle: Impact in Genetic Muscle Diseases and Beyond.

Intracellular Ca2+ ions represent a signaling mediator that plays a critical role in regulating different muscular cellular processes. Ca2+ homeostasis preservation is essential for maintaining skeletal muscle structure and function. Store-operated Ca2+ entry (SOCE), a Ca2+-entry process activated by depletion of intracellular stores contributing to the regulation of various function in many cell types, is pivotal to ensure a proper Ca2+ homeostasis in muscle fibers. It is coordinated by STIM1, the main Ca2+ sensor located in the sarcoplasmic reticulum, and ORAI1 protein, a Ca2+-permeable channel located on transverse tubules. It is commonly accepted that Ca2+ entry via SOCE has the crucial role in short- and long-term muscle function, regulating and adapting many cellular processes including muscle contractility, postnatal development, myofiber phenotype and plasticity. Lack or mutations of STIM1 and/or Orai1 and the consequent SOCE alteration have been associated with serious consequences for muscle function. Importantly, evidence suggests that SOCE alteration can trigger a change of intracellular Ca2+ signaling in skeletal muscle, participating in the pathogenesis of different progressive muscle diseases such as tubular aggregate myopathy, muscular dystrophy, cachexia, and sarcopenia. This review provides a brief overview of the molecular mechanisms underlying STIM1/Orai1-dependent SOCE in skeletal muscle, focusing on how SOCE alteration could contribute to skeletal muscle wasting disorders and on how SOCE components could represent pharmacological targets with high therapeutic potential.

Enhanced Ca2+ influx from STIM1–Orai1 induces muscle pathology in mouse models of muscular dystrophy

Muscular dystrophy is a progressive muscle wasting disease that is thought to be initiated by unregulated Ca(2+) influx into myofibers leading to their death. Store-operated Ca(2+) entry (SOCE) through sarcolemmal Ca(2+) selective Orai1 channels in complex with STIM1 in the sarcoplasmic reticulum is one such potential disease mechanism for pathologic Ca(2+) entry. Here, we generated a mouse model of STIM1 overexpression in skeletal muscle to determine whether this type of Ca(2+) entry could induce muscular dystrophy. Myofibers from muscle-specific STIM1 transgenic mice showed a significant increase in SOCE in skeletal muscle, modeling an observed increase in the same current in dystrophic myofibers. Histological and biochemical analysis of STIM1 transgenic mice showed fulminant muscle disease characterized by myofiber necrosis, swollen mitochondria, infiltration of inflammatory cells, enhanced interstitial fibrosis and elevated serum creatine kinase levels. This dystrophic-like disease in STIM1 transgenic mice was abrogated by crossing in a transgene expressing a dominant-negative Orai1 (dnOrai1) mutant. The dnOrai1 transgene also significantly reduced the severity of muscular dystrophy in both mdx (dystrophin mutant mice) and δ-sarcoglycan-deficient (Sgcd(-/-)) mouse models of disease. Hence, Ca(2+) influx across an unstable sarcolemma due to increased activity of a STIM1-Orai1 complex is a disease determinant in muscular dystrophy, and hence, SOCE represents a potential therapeutic target.

STIM1 negatively regulates Ca2+ release from the sarcoplasmic reticulum in skeletal myotubes

STIM1 (stromal interaction molecule 1) mediates SOCE (store-operated Ca²⁺ entry) in skeletal muscle. However, the direct role(s) of STIM1 in skeletal muscle, such as Ca²⁺ release from the SR (sarcoplasmic reticulum) for muscle contraction, have not been identified. The times required for the maximal expression of endogenous STIM1 or Orai1, or for the appearance of puncta during the differentiation of mouse primary skeletal myoblasts to myotubes, were all different, and the formation of puncta was detected with no stimulus during differentiation, suggesting that, in skeletal muscle, the formation of puncta is a part of the differentiation. Wild-type STIM1 and two STIM1 mutants (Triple mutant, missing Ca²⁺-sensing residues but possessing the intact C-terminus; and E136X, missing the C-terminus) were overexpressed in the myotubes. The wild-type STIM1 increased SOCE, whereas neither mutant had an effect on SOCE. It was interesting that increases in the formation of puncta were observed in the Triple mutant as well as in wild-type STIM1, suggesting that SOCE-irrelevant puncta could exist in skeletal muscle. On the other hand, overexpression of wild-type or Triple mutant, but not E136X, attenuated Ca²⁺ releases from the SR in response to KCl [evoking ECC (excitation-contraction coupling) via activating DHPR (dihydropyridine receptor)] in a dominant-negative manner. The attenuation was removed by STIM1 knockdown, and STIM1 was co-immunoprecipitated with DHRP in a Ca²⁺-independent manner. These results suggest that STIM1 negatively regulates Ca²⁺ release from the SR through the direct interaction of the STIM1 C-terminus with DHPR, and that STIM1 is involved in both ECC and SOCE in skeletal muscle.

Store‐operated Ca2+ entry is not required for store refilling in skeletal muscle

The present review describes store-operated Ca²⁺ entry (SOCE) in skeletal muscle. Fundamental discoveries in the field of skeletal muscle SOCE are described and the techniques that were used to make these. The advantages and limitations in these techniques are discussed to provide a means of questioning and determining the physiological role(s) of SOCE in skeletal muscle. It is concluded that SOCE has little or no role in the filling of the sarcoplasmic reticulum with Ca²⁺ at rest or during a single contracture. It is likely that SOCE is activated during fatigue, although direct measurements of SOCE are lacking and the physiological significance remains uncertain.

Toward the roles of store-operated Ca2+ entry in skeletal muscle

Store-operated Ca(2+) entry (SOCE) has been found to be a rapidly activated robust mechanism in skeletal muscle fibres. It is conducted across the junctional membranes by stromal interacting molecule 1 (STIM1) and Orai1, which are housed in the sarcoplasmic reticulum (SR) and tubular (t-) system, respectively. These molecules that conduct SOCE appear evenly distributed throughout the SR and t-system of skeletal muscle, allowing for rapid and local control in response to depletions of Ca(2+) from SR. The significant depletion of SR Ca(2+) required to reach the activation threshold for SOCE could only be achieved during prolonged bouts of excitation-contraction coupling (EC coupling) in a healthy skeletal muscle fibre, meaning that this mechanism is not responsible for refilling the SR with Ca(2+) during periods of fibre quiescence. While Ca(2+) in SR remains below the activation threshold for SOCE, a low-amplitude persistent Ca(2+) influx is provided to the junctional cleft. This article reviews the properties of SOCE in skeletal muscle and the proposed molecular mechanism, assesses its potential physiological roles during EC coupling, namely refilling the SR with Ca(2+) and simple balancing of Ca(2+) within the cell, and also proposes the possibility of SOCE as a potential regulator of t-system and SR membrane protein function.

Molecular Mechanism of Store Operated Ca2+ Entry in Adult Mammalian Skeletal Muscle

In skeletal muscle, store-operated Ca2+ entry (SOCE) is a trans-sarcolemmal Ca2+ influx pathway activated when sarcoplasmic reticulum (SR) Ca2+ stores are depleted. However, the mechanism of activation and physiological role of SOCE in adult skeletal muscle remains largely unknown. We recently reported that both STIM1 and Orai1 proteins are required for SOCE in skeletal myotubes. Here we investigated the mechanism of SOCE in adult skeletal muscles using mouse FDB fibers following electroporation of either cherry-tagged dnOrai1 cDNA or STIM1 siRNAs. Using a Mn2+ quench assay, we found that thapsigargin-induced SR Ca2+ store depletion activates a Ca2+ influx pathway in adult FDB fibers that is inhibited by: 1. multiple SOCE channel blockers (La3+, BTP-2 or SK&F96365) (3321±598 counts/sec, n=16 and 477±274 counts/sec, n=4 in the absence and presence of La3+, respectively), 2. expression of cherry-tagged dnOrai1 (571±130 counts/sec, n=12), or 3. STIM1 knockdown (7565±1590 counts/sec, n=3 and 217±247 counts/sec, n=5 in control and after STIM1 knockdown, respectively). To further assess the role of SOCE in muscle, we generated skeletal muscle-specific HA-tagged dominant negative Orai1 transgenic mice (HSAdnOrai) using a transgene driven by the human skeletal muscle actin (HSA) promoter (provided by Dr. J. Molkentin). HSAdnOrai mice survive beyond weaning and develop/breed normally. Western blot analysis using an HA antibody confirmed dnOrai1 transgene expression in skeletal muscle, but not in heart, lung, brain, spleen kidney, or liver. Primary myotubes derived from HSAdnOrai mice show significantly decreased SOCE following store depletion as assessed in Mn2+ quench (>92%) and Ca2+ influx (>95%) assays These results demonstrate that STIM1-Orai1 coupling mediates SOCE in adult skeletal muscle and that HSAdnOrai transgenic mice are a valuable tool for future studies of the physiological role of SOCE in skeletal muscle.

Increased Store-Operated Ca2+ Entry in Skeletal Muscle with Knockdown of Calsequestrin

Malignant hyperthermia (MH) is a life-threatening syndrome triggered by volatile anesthetics, in which uncontrolled elevation of myoplasmic Ca2+ leads to hypercontracture of skeletal muscle and elevation of body temperature. Our recent study showed that azumolene, an analog of dantrolene used to treat MH, inhibits a component of store-operated Ca2+ entry (SOCE) coupled to activation of the ryanodine receptor in skeletal muscle (JBC 281: 33477, 2006). Given our previous observation that overexpression of calsequestrin-1 (CSQ1) suppressed SOCE in skeletal muscle (JBC 278: 3286, 2003), here we tested the hypothesis that reduced CSQ1 expression would enhance an azumolene-sensitive SOCE in this tissue. A shRNA probe specific for CSQ1 (JBC 281: 15772, 2006) was introduced into flexor digitorum brevis (FDB) muscles of living mice using electroporation. Individual transfected FDB muscle fibers labeled with a fluorescent marker were isolated for SOCE measurements using Mn-quenching of Fura-2 fluorescence. At room temperature (20–22°C), SOCE induced by caffeine/ryanodine was significantly enhanced in CSQ1-knockdown muscle fibers (in 10−4 ΔF360/s, 9.36 ± 1.31) compared to those transfected with control (4.71 ± 1.29, p<0.05). Pre-incubation with azumolene (20 μM) completely inhibited the elevated SOCE detected in CSQ1-knockdown fibers (1.26 ± 0.38, p<0.01). To prevent muscle contraction, we used N-benzyl-p-toluene sulfonamide (BTS, 40 μM), a specific myosin II inhibitor. When temperature of the bathing solution was increased to 40°C, muscle fibers with knockdown of CSQ1 displayed a significant elevation in cytosolic Ca2+ over that seen in control fibers. Thus reduced CSQ1 expression is likely coupled to elevation of cytosolic Ca2+ due to increased SOCE function at higher temperatures. These results suggest that elevated SOCE activity in skeletal muscle may be linked to the pathophysiology of MH and the heat-sensitivity of MH-susceptible animals.

Differential dependence of store‐operated and excitation‐coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1

In non-excitable cells, agonist-induced depletion of intracellular Ca(2+) stores triggers Ca(2+) influx via a process termed store-operated Ca(2+) entry (SOCE). In T-lymphocytes, stromal interaction molecule 1 (STIM1) acts as the intra-store Ca(2+) sensor and Orai1 functions as the Ca(2+)-permeable SOCE channel activated by STIM1 following store depletion. Two functionally distinct Ca(2+) entry pathways exist in skeletal muscle; one activated by store depletion (SOCE) and a second by sustained/repetitive depolarization that does not require store depletion (excitation-coupled Ca(2+) entry, ECCE). However, the role of STIM1 and Orai1 in coordinating SOCE and ECCE activity in skeletal muscle and whether these two Ca(2+) entry pathways represent distinct molecular entities or two different activation mechanisms of the same channel complex is unknown. Here we address these issues using siRNA-mediated STIM1 knockdown, dominant-negative Orai1, and permeation-defective Orai1 to determine the role of STIM1 and Orai1 in store-operated and excitation-coupled Ca(2+) entry in skeletal myotubes. SOCE and ECCE activity were quantified from both intracellular Ca(2+) measurements and Mn(2+) quench assays. We found that STIM1 siRNA reduced STIM1 protein by more than 90% and abolished SOCE activity, while expression of siRNA-resistant hSTIM1 fully restored SOCE. SOCE was also abolished by dominant-negative Orai1 (E106Q) and markedly reduced by expression of a permeation-defective Orai1 (E190Q). In contrast, ECCE was unaffected by STIM1 knockdown, E106Q expression or E190Q expression. These results are the first to demonstrate that SOCE in skeletal muscle requires both STIM1 and Orai1 and that SOCE and ECCE represent two distinct molecular entities.