Muscle fibre mitochondrial [Ca ] dynamics during Ca waves in RYR1 gain-of-function mouse.

Aim: A fraction of the Ca released from the sarcoplasmic reticulum (SR) enters mitochondria to transiently increase its [Ca ] ([Ca ] ). This transient [Ca ] 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 Ca in the presence of a RyR1 agonist.

A bivalent remipede toxin promotes calcium release via ryanodine receptor activation.

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.

Evolutionary isolation of ryanodine receptor isoform 1 for muscle-based thermogenesis in mammals.

Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca pump, which is amplified by increasing RyR1 Ca leak in mammals, subsequently increasing cytoplasmic [Ca] ([Ca]). For thermogenesis to be functional, rising [Ca] must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca leak; nor should it compromise the muscle remaining relaxed.

Tiny changes in cytoplasmic [Ca] cause large changes in mitochondrial Ca: what are the triggers and functional implications?

Ca is an integral component of the functional and developmental regulation of the mitochondria. In skeletal muscle, Ca 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 Ca overload is recognized as a pathophysiological event but the former events represent important physiological functions in need of tight regulation.

A mathematical model to quantify RYR Ca2+ leak and associated heat production in resting human skeletal muscle fibers.

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).

Ryanodine receptor activity and store-operated Ca entry: Critical regulators of Ca content and function in skeletal muscle.

Store-operated Ca 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.

Ca leak through ryanodine receptor 1 regulates thermogenesis in resting skeletal muscle.

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) Ca 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 Ca leak through ryanodine receptor 1 (RyR1) to generate heat at the SR Ca pump in resting muscle.

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening.

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca release during excitation-contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs.

Ryanodine receptor leak triggers fiber Ca redistribution to preserve force and elevate basal metabolism in skeletal muscle.

Muscle contraction depends on tightly regulated Ca release. Aberrant Ca 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 leak drives these pathologies is unknown.

The Orai1 inhibitor BTP2 has multiple effects on Ca2+ handling in skeletal muscle.

BTP2 is an inhibitor of the Ca2+ channel Orai1, which mediates store-operated Ca2+ entry (SOCE). Despite having been extensively used in skeletal muscle, the effects of this inhibitor on Ca2+ handling in muscle cells have not been described. To address this question, we used intra- and extracellular application of BTP2 in mechanically skinned fibers and developed a localized modulator application approach, which provided in-preparation reference and test fiber sections to enhance detection of the effect of Ca2+ handling modulators.

Phasic Store-Operated Ca Entry During Excitation-Contraction Coupling in Skeletal Muscle Fibers From Exercised Mice.

Store-operated calcium entry (SOCE) plays a pivotal role in skeletal muscle physiology as, when impaired, the muscle is prone to early fatigue and the development of different myopathies. A chronic mode of slow SOCE activation is carried by stromal interaction molecule 1 (STIM1) and calcium-release activated channel 1 (ORAI1) proteins. A phasic mode of fast SOCE (pSOCE) occurs upon single muscle twitches in synchrony with excitation-contraction coupling, presumably activated by a local and transient depletion at the terminal cisternae of the sarcoplasmic reticulum Ca-stores.

A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction.

Key Points: Accumulation of inorganic phosphate (P ) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of P into SR are fully established.  We showed that P promoted Ca release at concentrations below 10 mm and decreased it at higher concentrations.

Multi-species transcriptomics reveals evolutionary diversity in the mechanisms regulating shrimp tail muscle excitation-contraction coupling.

The natural flight response in shrimp is powered by rapid contractions of the abdominal muscle fibres to propel themselves backwards away from perceived danger. This muscle contraction is dependent on repetitive depolarization of muscle plasma membrane, triggering tightly spaced cytoplasmic [Ca] transients and rapidly rising tetanic force responses. To achieve such high amplitude and high frequency of Ca transients requires a high abundance of sarcoplasmic/endoplasmic reticulum Ca ATPase (SERCA) to rapidly clear cytoplasmic Ca between each transient and an efficient Ca release system consisting of the Ryanodine Receptor (RyR), and voltage gated Ca channels (Cas).

A new selective pharmacological enhancer of the Orai1 Ca channel reveals roles for Orai1 in smooth and skeletal muscle functions.

Store operated calcium (Ca) entry is an important homeostatic mechanism in cells, whereby the release of Ca from intracellular endoplasmic reticulum stores triggers the activation of a Ca influx pathway. Mediated by Orai1, this Ca influx has specific and essential roles in biological processes as diverse as lactation to immunity. Although pharmacological inhibitors of this Ca influx mechanism have helped to define the role of store operated Ca entry in many cellular events, the lack of isoform specific modulators and activators of Orai1 has limited our full understanding of these processes.

RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca assays.

Elevated cytoplasmic [Ca] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca 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.

Mechanistic insights into store-operated Ca entry during excitation-contraction coupling in skeletal muscle.

Skeletal muscle fibres support store-operated Ca-entry (SOCE) across the t-tubular membrane upon exhaustive depletion of Ca from the sarcoplasmic reticulum (SR). Recently we demonstrated the presence of a novel mode of SOCE activated under conditions of maintained [Ca]. This phasic SOCE manifested in a fast and transient manner in synchrony with excitation contraction (EC)-coupling mediated SR Ca-release (Communications Biology 1:31, doi: https://doi.

Store-operated Ca entry is activated by every action potential in skeletal muscle.

Store-operated calcium (Ca) entry (SOCE) in skeletal muscle is rapidly activated across the tubular system during direct activation of Ca release. The tubular system is the invagination of the plasma membrane that forms junctions with the sarcoplasmic reticulum (SR) where STIM1, Orai1 and ryanodine receptors are found. The physiological activation of SOCE in muscle is not defined, thus clouding its physiological role.

Junctional membrane Ca dynamics in human muscle fibers are altered by malignant hyperthermia causative RyR mutation.

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca sensor of Ca dynamics in the junctional space. The sensor was used to assess the basal Ca-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant.

Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest.

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics.

Dantrolene requires Mg to arrest malignant hyperthermia.

Malignant hyperthermia (MH) is a clinical syndrome of skeletal muscle that presents as a hypermetabolic response to volatile anesthetic gases, where susceptible persons may develop lethally high body temperatures. Genetic predisposition mainly arises from mutations on the skeletal muscle ryanodine receptor (RyR). Dantrolene is administered to alleviate MH symptoms, but its mechanism of action and its influence on the Ca transients elicited by MH triggers are unknown.

Doublet stimulation increases Ca binding to troponin C to ensure rapid force development in skeletal muscle.

Fast-twitch skeletal muscle fibers are often exposed to motor neuron double discharges (≥200 Hz), which markedly increase both the rate of contraction and the magnitude of the resulting force responses. However, the mechanism responsible for these effects is poorly understood, likely because of technical limitations in previous studies. In this study, we measured cytosolic Ca during doublet activation using the low-affinity indicator Mag-Fluo-4 at high temporal resolution and modeled the effects of doublet stimulation on sarcoplasmic reticulum (SR) Ca release, binding of Ca to cytosolic buffers, and force enhancement in fast-twitch fibers.

Human skeletal muscle plasmalemma alters its structure to change its Ca-handling following heavy-load resistance exercise.

High-force eccentric exercise results in sustained increases in cytoplasmic Ca levels ([Ca]), which can cause damage to the muscle. Here we report that a heavy-load strength training bout greatly alters the structure of the membrane network inside the fibres, the tubular (t-) system, causing the loss of its predominantly transverse organization and an increase in vacuolation of its longitudinal tubules across adjacent sarcomeres. The transverse tubules and vacuoles displayed distinct Ca-handling properties.