Calcium homeostasis in muscle is crucial for function. Ryanodine receptors, such as RyR1, regulate calcium release into skeletal muscle fibers. Alterations in ryanodine receptors, genetic or post-translational, impair muscle force generation and contractility. Sensitive physiological measures are critical for testing treatments of muscle disorders with dysregulated calcium. Our objective is to identify sensitive contractility measures in muscle of mice with genetically altered RyR1 proteins. We hypothesized that mice with one mutated RYR1 allele would have intermediate skeletal muscle force-generating and contractile properties compared to those with zero or two mutated RYR1 alleles, and that properties dependent upon calcium dynamics would be most affected. We used a knock-in mouse model carrying the malignant hyperthermia–associated RyR1 p.G2435R missense mutation in MH region 2. These mice display increasingly severe RyR1 calcium leak in skeletal muscle fibers in a gene-dosage dependent manner. In vitro physiological measurements of baseline force production and contractile dynamics were performed on live whole soleus muscle from homozygous (RYR1WT/WT; HOM), heterozygous (RYR1KI/WT; HET), and wild-type littermates (RYR1WT/WT; WT). Mice ranged in age from 14-18 wk, and each group consisted of n=5-9. All contractility metrics were performed at 25°C. Key outcomes include maximal and submaximal force generation as well as contraction and relaxation dynamics. All data were analyzed using one-way ANOVA with Holm-Sidak post hoc analysis. For maximal tetanic force generation, female HOM mice generated 78% less isometric, 42% less concentric, and 61% less eccentric forces than WT (p≤0.033). Female HET generated 31% more isometric, 54% more concentric, and 30% more eccentric forces than WT (p≤0.008). HOM mice reached 50% of their maximum force at 27.5 Hz, while WT and HET mice reached 50% of maximum force at ~10 Hz (p≤0.001). Rates of isometric tetanic contraction and relaxation were also different between genotypes. Compared to WT, the rate of contraction was 36% faster for HET and 80% slower for HOM mice (p≤0.001). The rate of relaxation was 28% faster for HET and 79% slower for HOM mice (p≤0.006). When normalized to peak twitch force, HOM mice had 400% greater time-to-peak twitch and 150% greater half relaxation time than WT (p≤0.001). In male mice, no significant differences were measured between WT and HET mice in baseline force production or contractile dynamics (p≥0.387). Similar to females, HOM males had up to 83% lower tetanic and twitch forces (p≤0.001), and 88% slower rates of contraction and relaxation (p≤ 0.001). Male HOM mice reached 50% of their maximum force at 33.3 Hz, while WT and HETs reached 50% of maximum force at ~18.5 and 16.8 Hz, respectively (p≤0.001). This data reveals how altered RyR1 calcium leak impacts force generation and muscle contractility in a genotype-specific manner. This research will provide a sensitive, physiologically relevant platform for identifying therapeutic drugs that target muscle disorders by correcting calcium leak. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

AIM:This study investigates the activation and regulation of phasic store-operated calcium entry (pSOCE) in fast- and slow-twitch skeletal muscle fibers. Specifically, we aimed to enhance the sensitivity of pSOCE detection in slow-twitch fibers by optimizing ionic conditions and to compare the physiological relevance of pSOCE between fiber types. METHODS:We employed mechanically skinned fast-twitch extensor digitorum longus (EDL) muscle fibers loaded with spectrally distinct Ca2+-sensitive dyes to simultaneously measure action potential-induced sarcoplasmic reticulum Ca2+ release and t-tubular system Ca2+ dynamics with millisecond resolution. Experimental conditions were optimized by reducing cytosolic Mg2+ and EGTA buffering to enhance Ca2+ release in slow-twitch soleus fibers. Confocal microscopy was used to track t-tubular system Ca2+ depletion and reuptake during electric field stimulation. RESULTS:Skinned soleus fibers exhibited ~8-fold lower Ca2+ release per action potential compared to EDL fibers, yet pSOCE amplitudes were comparable. Reducing Mg2+ and EGTA levels increased Ca2+ release and left pSOCE kinetics in EDL fibers unaltered, but enabled pSOCE measurements in soleus fibers. While pSOCE in EDL fibers followed a linear dependence on the ambient Ca2+ concentration in the t-tubular system, such a relationship was violated in soleus fibers. CONCLUSION:These findings reveal a novel, fiber-type-specific difference in pSOCE regulation. When compared to EDL fibers, soleus fibers exhibited a higher sensitivity to SOCE activation despite releasing less Ca2+ from the sarcoplasmic reticulum upon an action potential. These differences may allow soleus fibers to sustain Ca2+ homeostasis more effectively, be more resilient against disruptions in Ca2+ handling, and entail protection against disease states.

For physiological processes in the vital organs of eutherian mammals to function, it is important to maintain constant core body temperature at ∼37°C. Mammals generate heat internally by thermogenesis. The focus of this review is on heat generated in resting skeletal muscles, using the same cellular components that muscles use to regulate cytoplasmic calcium concentrations [Ca2+] and contraction. Key to this process, known as muscle-based nonshivering thermogenesis (MB-NST), are tiny Ca2+ movements and associated ATP turnover coordinated by the plasma membrane, sarcoplasmic reticulum (SR), and the mitochondria. MB-NST has made mammals with gain-of-function SR ryanodine receptor (RyR) variants vulnerable to excessive heat generation that can be potentially lethal, known as malignant hyperthermia. Studies of RyR variants using recently developed techniques have advanced our understanding of MB-NST.

Ryanodine receptor 1 Ca2+ leak is a signal in skeletal muscle, but chronic leak can underlie pathology. Here we show that in healthy male mouse, limb-girdle muscle presents higher sympathetic input, elevated ryanodine receptor 1 basal phosphorylation, Ca2+ leak and mitochondrial Ca2+ content compared to distal leg muscles. These regional differences are consistent with heat generation in resting muscle to maintain core temperature. The dysferlin-null mouse develops severe pathology in the limb-girdle but not leg muscles. Absence of dysferlin disrupts dihydropyridine receptors' inhibitory control over ryanodine receptor 1 leak, synergistically increasing leak through the already phosphorylated channel of limb-girdle muscle. This alters Ca2+ handling and distribution leading to reactive oxygen species production prior to disease onset. With age, oxidation of Ca2+ -handling proteins in dysferlin-null limb-girdle muscle alters basal Ca2+ movements. Our results show that muscle-specific pathology in dysferlin-null mice is linked to increased ryanodine receptor 1 Ca2+ leak.

AIM:A fraction of the Ca2+ released from the sarcoplasmic reticulum (SR) enters mitochondria to transiently increase its [Ca2+ ] ([Ca2+ ]mito ). This transient [Ca2+ ]mito increase may be important in the resynthesis of ATP and other processes. The resynthesis of ATP in the mitochondria generates heat that can lead to hypermetabolic reactions in muscle with ryanodine receptor 1 (RyR1) variants during the cyclic releasing of SR Ca2+ in the presence of a RyR1 agonist. We aimed to analyse whether the mitochondria of RYR1 variant muscle handles Ca2+ differently from healthy muscle. METHODS:We used confocal microscopy to track mitochondrial and cytoplasmic Ca2+ with fluorescent dyes simultaneously during caffeine-induced Ca2+ waves in extensor digitorum longus muscle fibres from healthy mice and mice heterozygous (HET) for a malignant hyperthermia-causative RYR1 variant. RESULTS:Mitochondrial Ca2+ -transient peaks trailed the peak of cytoplasmic Ca2+ transients by many seconds with [Ca2+ ]mito not increasing by more than 250 nM. A strong linear relationship between cytoplasmic Ca2+ and [Ca2+ ]mito amplitudes was observed in HET RYR1 KI fibres but not wild type (WT). CONCLUSION:Our results indicate that [Ca2+ ]mito change within the nM range during SR Ca2+ release. HET fibre mitochondria are more sensitive to SR Ca2+ release flux than WT. This may indicate post-translation modification differences of the mitochondrial Ca2+ uniporter between the genotypes.

Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis, a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca2+-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) iso-forms for Ca2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca2+ pump, which is amplified by increasing RyR1 Ca2+ leak in mammals, subsequently increasing cytoplasmic [Ca2+] ([Ca2+]cyto). For ther-mogenesis to be functional, rising [Ca2+]cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca2+ activated, regenerative Ca2+ release that is robust in lower vertebrates needs to be suppressed in mammals. However, it has not been clear whether: i) the RyR1 can be opened by local increases in [Ca2+]cyto; and ii) downstream effectors of the SNS increase RyR Ca2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca2+]cyto under identical conditions opti-mized for activating regenerative Ca2+ release as Ca2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca2+]cyto, resulting in increased SR Ca2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.SignificanceA major evolutionary event in the rise of mammals was developing muscle-based thermogenesis. Mammals use muscle for heat generation while the muscle remains relaxed. The process of generating heat requires raising cytoplasmic [Ca2+] via RyR leak to hydrolyze ATP. We hypothesized that the transition to using muscle for heat generation required resistance to Ca2+- activated regenerative Ca2+ release, a robust mechanism in lower vertebrates, and sensitivity to leak regulation by the sympathetic nervous system. We developed approaches that demonstrate resistance of RyR to opening by abrupt rises in [Ca2+], and heat generation via cAMP-activated RyR Ca2+ leak in mammals. We conclude the evolutionary loss of a Ca2+- sensitive RyR isoform was important in RyR Ca2+ leak-based thermogenesis in mammals.

Cycling of Ca2+ between the sarcoplasmic reticulum (SR) and myoplasm is an important component of skeletal muscle resting metabolism. As part of this cycle, Ca2+ leaks from the SR into the myoplasm and is pumped back into the SR using ATP, which leads to the consumption of O2 and generation of heat. Ca2+ may leak through release channels or ryanodine receptors (RYRs). RYR Ca2+ leak can be monitored in a skinned fiber preparation in which leaked Ca2+ is pumped into the t-system and measured with a fluorescent dye. However, accurate quantification faces a number of hurdles. To overcome them, we developed a mathematical model of Ca2+ movement in these preparations. The model incorporated Ca2+ pumps that move Ca2+ from the myoplasm to the SR and from the junctional space (JS) to the t-system, Ca2+ buffering by EGTA in the JS and myoplasm and by buffers in the SR, and Ca2+ leaks from the SR into the JS and myoplasm and from the t-system into the myoplasm. The model accurately simulated Ca2+ uptake into the t-system, the relationship between myoplasmic [Ca2+] and steady-state t-system [Ca2+], and the effect of blocking RYR Ca2+ leak on t-system Ca2+ uptake. The magnitude of the leak through the RYRs would contribute ∼5% of the resting heat production of human muscle. In normal resting fibers, RYR Ca2+ leak makes a small contribution to resting metabolism. RYR-focused pathologies have the potential to increase RYR Ca2+ leak and the RYR leak component of resting metabolism.

Ca2+ is an integral component of the functional and developmental regulation of the mitochondria. In skeletal muscle, Ca2+ is reported to modulate the rate of ATP resynthesis, regulate the expression of peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC1α) following exercise, and drive the generation of reactive oxygen species (ROS). Due to the latter, mitochondrial Ca2+ overload is recognized as a pathophysiological event but the former events represent important physiological functions in need of tight regulation. Recently, we described the relationship between [Ca2+]mito and resting [Ca2+]cyto and other mitochondrial Ca2+-handling properties of skeletal muscle. An important next step is to understand the triggers for Ca2+ redistribution between intracellular compartments, which determine the mitochondrial Ca2+ load. These triggers in both physiological and pathophysiological scenarios can be traced to the coupled activity of the ryanodine receptor 1 (RyR1) and store-operated Ca2+ entry (SOCE) in the resting muscle. In this piece, we will discuss some issues regarding Ca2+ measurements relevant to mitochondrial Ca2+-handling, the steady-state relationship between cytoplasmic and mitochondrial Ca2+, and the potential implications for Ca2+ handling by muscle mitochondria and cellular function.

Mammals rely on nonshivering thermogenesis (NST) from skeletal muscle so that cold temperatures can be tolerated. NST results from activity of the sarcoplasmic reticulum (SR) Ca2+ pump in skeletal muscle, but the mechanisms that regulate this activity are unknown. Here, we develop a single-fiber assay to investigate the role of Ca2+ leak through ryanodine receptor 1 (RyR1) to generate heat at the SR Ca2+ pump in resting muscle. By inhibiting a subpopulation of RyR1s in a single-fiber preparation via targeted delivery of ryanodine through transverse tubules, we achieve in-preparation isolation of RyR1 Ca2+ leak. This maneuver provided a critical increase in signal-to-noise of the SR-temperature-sensitive dye ER thermoyellow fluorescence signal from the fiber to allow detection of SR temperature changes as either RyR1 or SR Ca2+ pump activity was altered. We found that RyR1 Ca2+ leak raises cytosolic [Ca2+] in the local vicinity of the SR Ca2+ pump to amplify thermogenesis. Furthermore, gene-dose-dependent increases in RyR1 leak in RYR1 mutant mice result in progressive rises in leak-dependent heat, consistent with raised local [Ca2+] at the SR Ca2+ pump via RyR1 Ca2+ leak. We also show that basal RyR Ca2+ leak and the heat generated by the SR Ca2+ pump in the absence of RyR Ca2+ leak is greater in fibers from mice than from toads. The distinct function of RyRs and SR Ca2+ pump in endothermic mammals compared to ectothermic amphibians provides insights into the mechanisms by which mammalian skeletal muscle achieves thermogenesis at rest.

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.

Muscle contraction depends on tightly regulated Ca2+ release. Aberrant Ca2+ 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 Ca2+ 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 Ca2+ leak initiates a cascade of events that cause precise redistribution of Ca2+ among the SR, cytoplasm, and mitochondria through altering the Ca2+ permeability of the transverse tubular system membrane. This redistribution of Ca2+ 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.

The N-terminal region (NTR) of the ryanodine receptor (RyR) calcium channels is critical to the regulation of Ca 2+ release during excitation-contraction coupling. NTR hosts numerous mutations linked to skeletal and cardiac myopathies (RyR1 and RyR2, respectively), highlighting its potential as therapeutic target. Here, we labeled the NTR of mouse RyR2 at subdomains A, B, and C with donor and acceptor pairs for fluorescence resonance energy transfer (FRET), obtaining two biosensors. Using fluorescence lifetime (FLT)-detection of intramolecular FRET, we developed high-throughput screening (HTS) assays with the biosensors to identify small-molecule modulators of RyR. We screened a 1280-compound validation library and identified several hits. Hits with saturable FRET dose-response profiles, and previously unreported effects on RyR activity, were further tested using [ 3 H]ryanodine binding to isolated sarcoplasmic reticulum vesicles, to measure their effects on full-length RyR opening in its natural membrane environment. We identified three novel inhibitors of both RyR1 and RyR2, and two RyR1-selective inhibitors at nanomolar Ca 2+ . These compounds may function as inhibitors of leaky RyRs in muscle. Two of these hits activated RyR1 only at micromolar Ca 2+ , highlighting them as potential activators of excitation-contraction coupling. These results indicate that large-scale HTS using this platform can lead to compounds with potential for therapeutic development.

Skeletal muscle possesses a unique, rapidly activating store-operated Ca2+ entry (SOCE) mechanism. SOCE is activated when the activity of the ryanodine receptors increase, following action potential-induced Ca2+release or even increased RyR Ca2+ leak (Koenig et al 2018, Comm Biol; Cully et al 2018, PNAS). The role of SOCE in muscle remains poorly defined, but a role in calcium content regulation is likely, which means it must work in concert with fibre Ca2+ extrusion mechanisms for balance. Kinetics of Ca2+ movements in imaging experiments performed with high spatiotemporal resolution have shown that the t-system PMCA exists in the same microdomain as RyRs and the SOCE protein components; and all components are equally distributed throughout the muscle fibre (Cully et al 2012, AJP). We studied RyR mutant mice (RyR KI; Lopez et al 2018, BJA) and calsequestrin 1 null (CSQKO) mice (Paolini et al 2007, J.Physiol) to examine the regulatory relationship of RyR Ca2+ leak with SOCE and PMCA activity using mechanically skinned fibres. We determined that RyR Ca2+ leak increased in the order WT

Obesity and other metabolic conditions are known to have an effect on a host of physiological mechanisms, in particular skeletal muscle thermoregulation. Heat generation in skeletal muscle during rest and contraction is driven primarily by the reuptake of Ca2+ via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. ATP consumption during this process contributes to a large amount of energy expenditure. The upregulation of this Ca2+ cycling process has been described in obesity and is thought to be an adaptive mechanism in response to high energy stores, however it is not well understood. Therefore, we aimed to assess cellular adaptations that may favour resting thermogenesis by quantifying ryanodine receptor (RyR1) Ca2+ leak and the associated basal Ca2+ handling properties of rat muscle under the stress of obesity. Wistar rats were fed a high fat diet to induce a metabolically altered model. Plasma free fatty acid, plasma triglyceride, plasma insulin and liver triglycerides were quantified and confirmed the obesity phenotype we targeted. Mechanically skinned extensor digitorum longus (EDL) muscle fibres were used to determine RyR Ca2+ leak. Compared with control fed rats, muscles in obese rats exhibited significant RyR leak and increased [Ca2+]t-system when presented with increasing concentrations of cytoplasmic Ca2+. Excessively leaky RyRs were present in long term obese rats and displayed spontaneous store operated calcium entry (SOCE) activation, which was inhibited by tetracaine. The introduction of caffeine following tetracaine induced a slowly depleting [Ca2+]t-system indicative of a well-buffered SR for Ca2+, suggesting an upregulation of calsequestrin (CSQ). Our results identified altered skeletal muscle Ca2+ handling properties that may be adaptations to enhance resting thermogenesis and subsequent energy expenditure in obese rats.

Elevated cytoplasmic [Ca2+] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca2+ 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 [3H]ryanodine binding to RyR1 at nanomolar Ca2+. Both compounds also decrease RyR1 Ca2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca2+ 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.