Muscle contraction depends on tightly regulated Ca 2+ release. Aberrant Ca 2+ leak through ryanodine receptor 1 (RyR1) on the sarcoplasmic reticulum (SR) membrane can lead to heatstroke and malignant hyperthermia (MH) susceptibility, as well as severe myopathy. However, the mechanism by which Ca 2+ leak drives these pathologies is unknown. Here, we investigate the effects of four mouse genotypes with increasingly severe RyR1 leak in skeletal muscle fibers. We find that RyR1 Ca 2+ leak initiates a cascade of events that cause precise redistribution of Ca 2+ among the SR, cytoplasm, and mitochondria through altering the Ca 2+ permeability of the transverse tubular system membrane. This redistribution of Ca 2+ allows mice with moderate RyR1 leak to maintain normal function; however, severe RyR1 leak with RYR1 mutations reduces the capacity to generate force. Our results reveal the mechanism underlying force preservation, increased ATP metabolism, and susceptibility to MH in individuals with gain-of-function RYR1 mutations.

Elevated cytoplasmic [Ca 2+ ] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca 2+ leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [ 3 H]ryanodine binding to RyR1 at nanomolar Ca 2+. Both compounds also decrease RyR1 Ca 2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca 2+ release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance. In striated muscle, contraction requires an intracellular Ca 2+-release event mediated by ryanodine receptors (RyR) that are embedded in the sarcoplasmic reticulum (SR) membrane. Dysregulation of skeletal (RyR1) and cardiac (RyR2) isoforms, via mutations or excess posttranslational modification, has been linked to severe muscle pathologies, including malignant hyperthermia (MH), central core disease, muscular dystrophy (MD), sarcopenia, catecholaminergic polymorphic ventricular tachycardia, heart failure, and more recently RyR2 has been recognized as a potentially significant contributor to diabetes and Alzheimer's disease 1-9. In most of these clinical indications, pathogenesis can be fueled by excess SR Ca 2+ "leak" via RyR under resting cellular conditions, which leads to toxic intracellular basal [Ca 2+ ] and insufficient SR Ca 2+ load. As a result, RyR is intensely studied as a therapeutic target. Indeed, the therapeutic potential of pharmaceutically targeting RyR1-mediated SR Ca 2+ leak in skeletal muscle has been shown in animal models of Duchenne MD, limb-girdle MD, and sarcopenia 5,10,11. The therapeutic potential of targeting RyR2-mediated SR Ca 2+ leak for treating heart failure and arrhythmia is also very well documented 12-16. Additionally, targeting RyR2 (which is abundant in the brain 17,18) may have therapeutic potential for treating neurodegenerative diseases. To introduce a systematic and efficient approach for identifying novel small-molecule chemical scaffolds with potential to mitigate RyR1 dysfunction, we developed and implemented a high-throughput screening (HTS) assay that uses fluorescence lifetime (FLT) detection of FRET 19. This assay was designed to identify compounds that bind to the RyR1 channel complex to allosterically correct its pathologically leaky state (without affecting normal channel function) 19. This FRET-based method is based on monitoring RyR binding of fluorescent...

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Contusion injuries in skeletal muscle commonly occur in contact sport and vehicular and industrial workplace accidents. Icing has traditionally been used to treat such injuries under the premise that it alleviates pain, reduces tissue metabolism, and modifies vascular responses to decrease swelling. Previous research has examined the effects of icing on inflammation and microcirculatory dynamics following muscle injury. However, whether icing influences angiogenesis, collateral vessel growth, or myofiber regeneration remains unknown. We compared the effects of icing vs. a sham treatment on the presence of neutrophils and macrophages; expression of CD34, von Willebrands factor (vWF), vascular endothelial growth factor (VEGF), and nestin; vessel volume; capillary density; and myofiber regeneration in skeletal after muscle contusion injury in rats. Muscle tissue was collected 1, 3, 7, and 28 d after injury. Compared with uninjured rats, muscles in rats that sustained the contusion injury exhibited major necrosis, inflammation, and increased expression of CD34, vWF, VEGF, and nestin. Compared with the sham treatment, icing attenuated and/or delayed neutrophil and macrophage infiltration; the expression of vWF, VEGF, and nestin; and the change in vessel volume within muscle in the first 7 d after injury (P < 0.05). By contrast, icing did not influence capillary density in muscle 28 d after injury (P = 0.59). The percentage of immature myofibers relative to the total number of fibers was greater in the icing group than in the sham group 28 d after injury (P = 0.026), but myofiber cross-sectional area did not differ between groups after 7 d (P = 0.35) and 28 d (P = 0.30). In conclusion, although icing disrupted inflammation and some aspects of angiogenesis/revascularization, these effects did not result in substantial differences in capillary density or muscle growth.

Store‐operated Ca2+ entry (SOCE) is critical to cell function. In skeletal muscle, SOCE has evolved alongside excitation–contraction coupling (EC coupling); as a result, it displays unique properties compared to SOCE in other cells. The plasma membrane of skeletal muscle is mostly internalized as the tubular system, with the tubules meeting the sarcoplasmic reticulum (SR) terminal cisternae, forming junctions where the proteins that regulate EC coupling and SOCE are positioned. In this review, we describe the properties and roles of SOCE based on direct measurements of Ca2+ influx during SR Ca2+ release and leak. SOCE is activated immediately and locally as the [Ca2+] of the junctional SR terminal cisternae ([Ca2+]jSR) depletes. [Ca2+]jSR changes rapidly and steeply with increasing activity of the SR ryanodine receptor isoform 1 (RyR1). The high fidelity of [Ca2+]jSR with RyR1 activity probably depends on the SR Ca2+‐buffer calsequestrin that is located immediately behind RyR1 inside the SR. This arrangement provides in‐phase activation and deactivation of SOCE with a large dynamic range, allowing precise grading of SOCE flux. The in‐phase activation of SOCE as the SR partially depletes traps Ca2+ in the cytoplasm, preventing net Ca2+ loss. Mild presentation of RyR1 leak can occur under physiological conditions, providing fibre Ca2+ redistribution without changing fibre Ca2+ content. This condition preserves normal contractile function at the same time as increasing basal metabolic rate. However, higher RyR1 leak drives excess cytoplasmic and mitochondrial Ca2+ load, setting a deleterious intracellular environment that compromises the function of the skeletal muscle. image