Ryanodine receptor leak triggers fiber Ca2+ redistribution to preserve force and elevate basal metabolism in skeletal muscle
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ABSTRACT:
SUBMITTER: Lamboley C
PROVIDER: S-EPMC8550231 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Various ryanodine receptor 2 (RyR2) point mutations cause catecholamine-induced polymorphic ventricular tachycardia (CPVT), a life-threatening arrhythmia evoked by diastolic intracellular Ca2+ release dysfunction. These mutations occur in essential regions of RyR2 that regulate Ca2+ release. The molecular dysfunction caused by CPVT-associated RyR2 mutations as well as the functional consequences remain unresolved. Here, we study the most severe CPVT-associated RyR2 mutation (K4750Q) known to date. We define the molecular and cellular dysfunction generated by this mutation and detail how it alters RyR2 function, using Ca2+ imaging, ryanodine binding, and single-channel recordings. HEK293 cells and cardiac HL-1 cells expressing RyR2-K4750Q show greatly enhanced spontaneous Ca2+ oscillations. An endoplasmic reticulum-targeted Ca2+ sensor, R-CEPIA1er, revealed that RyR2-K4750Q mediates excessive diastolic Ca2+ leak, which dramatically reduces luminal [Ca2+]. We further show that the K4750Q mutation causes three RyR2 defects: hypersensitization to activation by cytosolic Ca2+, loss of cytosolic Ca2+/Mg2+-mediated inactivation, and hypersensitization to luminal Ca2+ activation. These defects combine to kinetically stabilize RyR2-K4750Q openings, thus explaining the extensive diastolic Ca2+ leak from the sarcoplasmic reticulum, frequent Ca2+ waves, and severe CPVT phenotype. As the multiple concurrent defects are induced by a single point mutation, the K4750 residue likely resides at a critical structural point at which cytosolic and luminal RyR2 control input converge.
| S-EPMC5299618 | biostudies-literature
Project description: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.
| S-EPMC8794839 | biostudies-literature
Project description:Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca(2+)](c)) and sarcoplasmic reticulum (SR) luminal Ca(2+) ([Ca(2+)]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca(2+) leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca(2+)](SR) in HF compared to control cells. Both SR Ca(2+) leak and [Ca(2+)](SR) were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca(2+) leak and decreased [Ca(2+)](SR) in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca(2+)-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca(2+) and was partially normalized by reducing agents through restoring luminal Ca(2+) sensitivity oxidation of control RyR2s enhanced their luminal Ca(2+) sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.
| S-EPMC3274754 | biostudies-literature
Project description:High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.
| S-EPMC4687604 | biostudies-literature
Project description:Cognitive dysfunction (CD) in heart failure (HF) adversely affects treatment compliance and quality of life. Although, ryanodine receptor type 2 (RyR2) has been linked to cardiac muscle dysfunction, its role in CD remains unclear. Here, we show in hippocampal neurons from patients and mice with HF that the ryanodine receptor type 2 (RyR2)/intracellular Ca 2+ release channels were post-translationally modified (PTM) and leaky. RyR2 PTM was caused by hyper-adrenergic stress and activation of the transforming growth factor (TGF-β) pathway. HF mice treated with either a RyR2 stabilizer drug (S107), beta-blocker (propranolol) or TGF-β inhibitor (SD-208), or mice insensitive RyR2 Ca 2+ leak (RyR2-S2808A), were protected against HF-induced CD. Taken together, we propose that HF is a systemic illness that include cardiogenic dementia, and intracellular Ca 2+ leak is a common effector in multiple components of HF.
2023-08-30 | PXD042295 | Pride
Project description:Presynaptic Ca(2+) stores have been suggested to regulate Ca(2+) dynamics within the nerve terminals at certain types of the synapse. However, little is known about their mode of activation, molecular identity, and detailed subcellular localization. Here, we show that the ryanodine-sensitive stores exist in axons and amplify presynaptic Ca(2+) accumulation at the hippocampal mossy fiber synapses, which display robust presynaptic forms of plasticity. Caffeine, a potent drug inducing Ca(2+) release from ryanodine-sensitive stores, causes elevation of presynaptic Ca(2+) levels and enhancement of transmitter release from the mossy fiber terminals. The blockers of ryanodine receptors, TMB-8 or ryanodine, reduce presynaptic Ca(2+) transients elicited by repetitive stimuli of mossy fibers but do not affect those evoked by single shocks, suggesting that ryanodine receptors amplify presynaptic Ca(2+) dynamics in an activity dependent manner. Furthermore, we generated the specific antibody against the type 2 ryanodine receptor (RyR2; originally referred to as the cardiac type) and examined the cellular and subcellular localization using immunohistochemistry. RyR2 is highly expressed in the stratum lucidum of the CA3 region and mostly colocalizes with axonal marker NF160 but not with terminal marker VGLUT1. Immunoelectron microscopy revealed that RyR2 is distributed around smooth ER within the mossy fibers but is almost excluded from their terminal portions. These results suggest that axonal localization of RyR2 at sites distant from the active zones enables use dependent Ca(2+) release from intracellular stores within the mossy fibers and thereby facilitates robust presynaptic forms of plasticity at the mossy fiber-CA3 synapse.
| S-EPMC2575294 | biostudies-literature
Project description:The skeletal muscle dihydropyridine receptor (DHPR) and ryanodine receptor (RyR1) are known to engage a form of conformation coupling essential for muscle contraction in response to depolarization, referred to as excitation-contraction coupling. Here we use WT and Ca(V)1.1 null (dysgenic) myotubes to provide evidence for an unexplored RyR1-DHPR interaction that regulates the transition of the RyR1 between gating and leak states. Using double-barreled Ca(2+)-selective microelectrodes, we demonstrate that the lack of Ca(V)1.1 expression was associated with an increased myoplasmic resting [Ca(2+)] ([Ca(2+)](rest)), increased resting sarcolemmal Ca(2+) entry, and decreased sarcoplasmic reticulum (SR) Ca(2+) loading. Pharmacological control of the RyR1 leak state, using bastadin 5, reverted the three parameters to WT levels. The fact that Ca(2+) sparks are not more frequent in dysgenic than in WT myotubes adds support to the hypothesis that the leak state is a conformation distinct from gating RyR1s. We conclude from these data that this orthograde DHPR-to-RyR1 signal inhibits the transition of gated RyR1s into the leak state. Further, it suggests that the DHPR-uncoupled RyR1 population in WT muscle has a higher propensity to be in the leak conformation. RyR1 leak functions are to keep [Ca(2+)](rest) and the SR Ca(2+) content in the physiological range and thus maintain normal intracellular Ca(2+) homeostasis.
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Project description:The hepatitis C non-structural protein 5A (NS5A) is a Zn(2+)-binding phosphoprotein essential for viral replication. Expression of NS5A perturbs intracellular Ca(2+) levels by an undefined mechanism, activating transcription factors implicated in the chronic pathogenesis of hepatitis infections. Here, we demonstrate that regulated expression of NS5A enhanced the passive leak of Ca(2+) from a subset of the endoplasmic reticulum (ER) Ca(2+) stores. This action was not replicated by expression of the amphipathic NH(2)-membrane anchoring domain of NS5A alone, despite targeting to intracellular membranes. Depletion of the NS5A-targeted ER Ca(2+) store was prevented under conditions of ample ATP supply suggesting compensatory Ca(2+) ATPase activity, but observed under conditions of ATP insufficiency and in intact cells expressing NS5A.
| S-EPMC2292798 | biostudies-literature
Project description:Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.
| S-EPMC1137430 | biostudies-literature
Project description:Spontaneous Ca(2+) release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca(2+) release (SOICR) can result in Ca(2+) waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here we show that a point mutation, E4872A, in the helix bundle crossing region (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, Ca(2+) activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni(2+)-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca(2+)-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca(2+), explaining the regulation of RyR2 by luminal Ca(2+), the initiation of Ca(2+) waves and Ca(2+)-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.
| S-EPMC4269524 | biostudies-literature