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.
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. image
Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca 2+ -handling proteins and membrane structures to conduct excitation–contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca 2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca 2+ pump, which is amplified by increasing RyR1 Ca 2+ leak in mammals, subsequently increasing cytoplasmic [Ca 2+ ] ([Ca 2+ ] cyto ). For thermogenesis to be functional, rising [Ca 2+ ] cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca 2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca 2+ activated, regenerative Ca 2+ 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 [Ca 2+ ] cyto ; and ii) downstream effectors of the SNS increase RyR Ca 2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca 2+ ] cyto under identical conditions optimized for activating regenerative Ca 2+ release as Ca 2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca 2+ ] cyto , resulting in increased SR Ca 2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca 2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca 2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.
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 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.
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