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 Ca2+-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) isoforms 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 thermogenesis 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 optimized 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.

Ryanodine receptor activity and store-operated Ca2+ entry: Critical regulators of Ca2+ content and function in skeletal muscle.

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.

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 Ca2+ 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. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose-response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation-contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation-contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.

Tiny changes in cytoplasmic [Ca2+] cause large changes in mitochondrial Ca2+: what are the triggers and functional implications?

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.

Comparative label-free mass spectrometric analysis of temporal changes in the skeletal muscle proteome after impact trauma in rats.

Proteomics offers the opportunity to identify and quantify many proteins and to explore how they correlate and interact with each other in biological networks. This study aimed to characterize changes in the muscle proteome during the destruction, repair, and early-remodeling phases after impact trauma in male Wistar rats. Muscle tissue was collected from uninjured control rats and rats that were euthanized between 6 h and 14 days after impact injury. Muscle tissue was analyzed using unbiased, data-independent acquisition LC-MS/MS. We identified 770 reviewed proteins in the muscle tissue, 296 of which were differentially abundant between the control and injury groups (P ≤ 0.05). Around 50 proteins showed large differences (≥10-fold) or a distinct pattern of abundance after injury. These included proteins that have not been identified previously in injured muscle, such as ferritin light chain 1, fibrinogen γ-chain, fibrinogen ß-chain, osteolectin, murinoglobulin-1, T-kininogen 2, calcium-regulated heat-stable protein 1, macrophage-capping protein, retinoid-inducible serine carboxypeptidase, ADP-ribosylation factor 4, Thy-1 membrane glycoprotein, and ADP-ribosylation factor-like protein 1. Some proteins increased between 6 h and 14 days, whereas other proteins increased in a more delayed pattern at 7 days after injury. Bioinformatic analysis revealed that various biological processes, including regulation of blood coagulation, fibrinolysis, regulation of wound healing, tissue regeneration, acute inflammatory response, and negative regulation of the immune effector process, were enriched in injured muscle tissue. This study advances the understanding of early muscle healing after muscle injury and lays a foundation for future mechanistic studies on interventions to treat muscle injury.

Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in 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.

RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays.

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.

Mechanistic insights into store-operated Ca2+ entry during excitation-contraction coupling in skeletal muscle.

Skeletal muscle fibres support store-operated Ca2+-entry (SOCE) across the t-tubular membrane upon exhaustive depletion of Ca2+ from the sarcoplasmic reticulum (SR). Recently we demonstrated the presence of a novel mode of SOCE activated under conditions of maintained [Ca2+]SR. This phasic SOCE manifested in a fast and transient manner in synchrony with excitation contraction (EC)-coupling mediated SR Ca2+-release (Communications Biology 1:31, doi: https://doi.org/10.1038/s42003-018-0033-7). Stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel 1 (ORAI1), positioned at the SR and t-system membranes, respectively, are the considered molecular correlate of SOCE. The evidence suggests that at the triads, where the terminal cisternae of the SR sandwich a t-tubule, STIM1 and ORAI1 proteins pre-position to allow for enhanced SOCE transduction. Here we show that phasic SOCE is not only shaped by global [Ca2+]SR but provide evidence for a local activation within nanodomains at the terminal cisternae of the SR. This feature may allow SOCE to modulate [Ca2+]SR during EC coupling. We define SOCE to occur on the same timescale as EC coupling and determine the temporal coherence of SOCE activation to SR Ca2+ release. We derive a delay of 0.3 ms reflecting diffusive Ca2+-equilibration at the luminal ryanodine receptor 1 (RyR1) channel mouth upon SR Ca2+-release. Numerical simulations of Ca2+-calsequestrin binding estimates a characteristic diffusion length and confines an upper limit for the spatial distance between STIM1 and RyR1. Experimental evidence for a 4- fold change in t-system Ca2+-permeability upon prolonged electrical stimulation in conjunction with numerical simulations of Ca2+-STIM1 binding suggests a Ca2+ dissociation constant of STIM1 below 0.35 mM. Our results show that phasic SOCE is intimately linked with RyR opening and closing, with only µs delays, because [Ca2+] in the terminal cisternae is just above the threshold for Ca2+ dissociation from STIM1 under physiological resting conditions. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Effects of Topical Icing on Inflammation, Angiogenesis, Revascularization, and Myofiber Regeneration in Skeletal Muscle Following Contusion Injury.

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.