Store-operated Ca²⁺ entry (SOCE), a ubiquitous mechanism that allows recovery of Ca²⁺ ions from the extracellular space, has been proposed to limit fatigue during repetitive skeletal muscle activity. However, the subcellular location for SOCE in muscle fibers has not been unequivocally identified. Here we show that exercise drives a significant remodeling of the sarcotubular system to form previously unidentified junctions between the sarcoplasmic reticulum (SR) and transverse-tubules (TTs). We also demonstrate that these new SR-TT junctions contain the molecular machinery that mediate SOCE: stromal interaction molecule-1 (STIM1), which functions as the SR Ca²⁺ sensor, and Orai1, the Ca²⁺-permeable channel in the TT. In addition, EDL muscles isolated from exercised mice exhibit an increased capability of maintaining contractile force during repetitive stimulation in the presence of 2.5 mM extracellular Ca²⁺, compared to muscles from control mice. This functional difference is significantly reduced by either replacement of extracellular Ca²⁺ with Mg²⁺ or the addition of SOCE inhibitors (BTP-2 and 2-APB). We propose that the new SR-TT junctions formed during exercise, and that contain STIM1 and Orai1, function as Ca²⁺Entry Units (CEUs), structures that provide a pathway to rapidly recover Ca²⁺ ions from the extracellular space during repetitive muscle activity.

Skeletal muscle contraction relies on both high-fidelity calcium (Ca²⁺) signals and robust capacity for adenosine triphosphate (ATP) generation. Ca²⁺ release units (CRUs) are highly organized junctions between the terminal cisternae of the sarcoplasmic reticulum (SR) and the transverse tubule (T-tubule). CRUs provide the structural framework for rapid elevations in myoplasmic Ca²⁺ during excitation–contraction (EC) coupling, the process whereby depolarization of the T-tubule membrane triggers SR Ca²⁺ release through ryanodine receptor-1 (RyR1) channels. Under conditions of local or global depletion of SR Ca²⁺ stores, store-operated Ca²⁺ entry (SOCE) provides an additional source of Ca²⁺ that originates from the extracellular space. In addition to Ca²⁺, skeletal muscle also requires ATP to both produce force and to replenish SR Ca²⁺ stores. Mitochondria are the principal intracellular organelles responsible for ATP production via aerobic respiration. This review provides a broad overview of the literature supporting a role for impaired Ca²⁺ handling, dysfunctional Ca²⁺-dependent production of reactive oxygen/nitrogen species (ROS/RNS), and structural/functional alterations in CRUs and mitochondria in the loss of muscle mass, reduction in muscle contractility, and increase in muscle damage in sarcopenia and a wide range of muscle disorders including muscular dystrophy, rhabdomyolysis, central core disease, and disuse atrophy. Understanding the impact of these processes on normal muscle function will provide important insights into potential therapeutic targets designed to prevent or reverse muscle dysfunction during aging and disease.

Store-operated Ca²⁺ entry (SOCE) is a ubiquitous Ca²⁺ influx mechanism triggered by depletion of Ca²⁺ stores from the endoplasmic/sarcoplasmic reticulum (ER/SR). We recently reported that acute exercise in WT mice drives the formation of Ca²⁺ entry units (CEUs), intracellular junctions that contain STIM1 and Orai1, the two key proteins mediating SOCE. The presence of CEUs correlates with increased constitutive- and store-operated Ca²⁺ entry, as well as sustained Ca²⁺ release and force generation during repetitive stimulation. Skeletal muscle from mice lacking calsequestrin-1 (CASQ1-null), the primary Ca²⁺-binding protein in the lumen of SR terminal cisternae, exhibits significantly reduced total Ca²⁺ store content and marked SR Ca²⁺ depletion during high-frequency stimulation. Here, we report that CEUs are constitutively assembled in extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of sedentary CASQ1-null mice. The higher density of CEUs in EDL (39.6 ± 2.1/100 µm² versus 2.0 ± 0.3/100 µm²) and FDB (16.7 ± 1.0/100 µm² versus 2.7 ± 0.5/100 µm²) muscles of CASQ1-null compared with WT mice correlated with enhanced constitutive- and store-operated Ca²⁺ entry and increased expression of STIM1, Orai1, and SERCA. The higher ability to recover Ca²⁺ ions via SOCE in CASQ1-null muscle served to promote enhanced maintenance of peak Ca²⁺ transient amplitude, increased dependence of luminal SR Ca²⁺ replenishment on BTP-2-sensitive SOCE, and increased maintenance of contractile force during repetitive, high-frequency stimulation. Together, these data suggest that muscles from CASQ1-null mice compensate for the lack of CASQ1 and reduction in total releasable SR Ca²⁺ content by assembling CEUs to promote constitutive and store-operated Ca²⁺ entry.

Central core disease (CCD) is a congenital myopathy linked to mutations in the ryanodine receptor type 1 (RYR1), the sarcoplasmic reticulum Ca²⁺ release channel of skeletal muscle. CCD is characterized by formation of amorphous cores within muscle fibers, lacking mitochondrial activity. In skeletal muscle of RYR1^Y522S/WT knock‐in mice, carrying a human mutation in RYR1 linked to malignant hyperthermia (MH) with cores, oxidative stress is elevated and fibers present severe mitochondrial damage and cores. We treated RYR1^Y522S/WT mice with N‐acetylcysteine (NAC), an antioxidant provided ad libitum in drinking water for either 2 or 6 months. Our results show that 2 months of NAC treatment starting at 2 months of age, when mitochondrial and fiber damage was still minimal, (i) reduce formation of unstructured and contracture cores, (ii) improve muscle function, and (iii) decrease mitochondrial damage. The beneficial effect of NAC treatment is also evident following 6 months of treatment starting at 4 months of age, when structural damage was at an advanced stage. NAC exerts its protective effect likely by lowering oxidative stress, as supported by the reduction of 3‐NT and SOD2 levels. This work suggests that NAC administration is beneficial to prevent mitochondrial damage and formation of cores and improve muscle function in RYR1^Y522S/WT mice.

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca ²⁺ release channel/RyR2 (type 2 ryanodine receptor) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2 ^R420Q . Objective: To determine the arrhythmogenic mechanisms of this mutation. Methods and Results: Ventricular tachycardias under stress conditions were observed in both patients with catecholaminergic polymorphic ventricular tachycardia and knock-in mice. During action potential recording (by patch-clamp in knock-in mouse cardiomyocytes and by microelectrodes in mutant human induced pluripotent stem cell-derived cardiomyocytes), we observed an increased occurrence of delayed afterdepolarizations under isoproterenol stimulation, associated with increased Ca ²⁺ waves during confocal Ca ²⁺ recording in both mouse and human RyR2 ^R420Q cardiomyocytes. In addition, Ca ²⁺ -induced Ca ²⁺ -release, as well as a rough indicator of fractional Ca ²⁺ release, were higher and Ca ²⁺ sparks longer in the RyR2 ^R420Q -expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum measured by gated stimulated emission depletion super-resolution and electron microscopy, respectively. The increase in junctional sarcoplasmic reticulum width might be due to the impairment of RyR2 ^R420Q binding to JPH2 (junctophilin-2), as there were less junctophilin-2 coimmunoprecipitated with RyR2 ^R420Q . At the single current level, the RyR2 ^R420Q channel dwells longer in the open state at low intracellular Ca ²⁺ ([Ca ²⁺ ] _i ), but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N terminus and the core solenoid, a RyR2 interdomain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death. Conclusions: The RyR2 ^R420Q catecholaminergic polymorphic ventricular tachycardia mutation modifies the interdomain interaction of the channel and weakens its association with JPH2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction.

In humans, hyperthermic episodes can be triggered by halogenated anesthetics [malignant hyperthermia (MH) susceptibility] and by high temperature [environmental heat stroke (HS)]. Correlation between MH susceptibility and HS is supported by extensive work in mouse models that carry a mutation in ryanodine receptor type‐1 (RYR1 ^Y522S/WT ) and calsequestrin‐1 knockout (CASQ1‐null), 2 proteins that control Ca ²⁺ release in skeletal muscle. As overheating episodes in humans have also been described during exertion, here we subjected RYR1 ^Y522S/WT and CASQ1‐null mice to an exertional‐stress protocol (incremental running on a treadmill at 34°C and 40% humidity). The mortality rate was 80 and 78.6% in RYR1 ^Y522S/WT and CASQ1‐null mice, respectively, vs. 0% in wild‐type mice. Lethal crises were characterized by hyperthermia and rhabdomyolysis, classic features of MH episodes. Of importance, pretreatment with azumolene, an analog of the drug used in humans to treat MH crises, reduced mortality to 0 and 12.5% in RYR1 ^Y522S/WT and CASQ1‐null mice, respectively, thanks to a striking reduction of hyperthermia and rhabdomyolysis. At the molecular level, azumolene strongly prevented Ca ²⁺ dependent activation of calpains and NF‐kB by lowering myoplasmic Ca ²⁺ concentration and nitro‐oxidative stress, parameters that were elevated in RYR1 ^Y522S/WT and CASQ1‐null mice. These results suggest that common molecular mechanisms underlie MH crises and exertional HS in mice.—Michelucci, A., Paolini, C., Boncompagni, S., Canato, M., Reggiani, C., Protasi, F. Strenuous exercise triggers a life‐threatening response in mice susceptible to malignant hyperthermia. FASEB J . 31, 3649–3662 (2017). www.fasebj.org