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International Heart Journal 2024Cardiac ryanodine receptor (RyR2) gain-of-function mutations cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Conversely, RyR2 loss-of-function...
Cardiac ryanodine receptor (RyR2) gain-of-function mutations cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Conversely, RyR2 loss-of-function mutations cause a new disease entity, termed calcium release deficiency syndrome (CRDS), which may include RYR2-related long QT syndrome (LQTS). Importantly, unlike CPVT, patients with CRDS do not always exhibit exercise- or epinephrine-induced ventricular arrhythmias, which precludes a diagnosis of CRDS. Here we report a boy and his father, who both experienced exercise-induced cardiac events and harbor the same RYR2 E4107A variant. In the boy, an exercise stress test (EST) and epinephrine provocation test (EPT) did not induce any ventricular arrhythmias. QTc was slightly prolonged (QTc: 474 ms), and an EPT induced QTc prolongation (QTc-baseline: 466 ms, peak: 532 ms, steady-state: 527 ms). In contrast, in his father, QTc was not prolonged (QTc: 417 ms), and neither an EST nor EPT induced QTc prolongation. However, an EST induced multifocal premature ventricular contraction (PVC) bigeminy and bidirectional PVC couplets. Thus, they exhibited distinct clinical phenotypes: the boy exhibited LQTS (or CRDS) phenotype, whereas his father exhibited CPVT phenotype. These findings suggest that, in addition to the altered RyR2 function, other unidentified factors, such as other genetic, epigenetic, and environmental factors, and aging, may be involved in the diverse phenotypic manifestations. Considering that a single RYR2 variant can cause both CPVT and LQTS (or CRDS) phenotypes, in cascade screening of patients with CPVT and CRDS, an EST and EPT are not sufficient and genetic analysis is required to identify individuals who are at increased risk for life-threatening arrhythmias.
Topics: Humans; Ryanodine Receptor Calcium Release Channel; Male; Long QT Syndrome; Phenotype; Tachycardia, Ventricular; Electrocardiography; Pedigree; Adult; Exercise Test; Mutation
PubMed: 38825499
DOI: 10.1536/ihj.23-652 -
Biochemical and Biophysical Research... Sep 2024Excitation-contraction coupling in skeletal muscle myofibers depends upon Ca release from the sarcoplasmic reticulum through the ryanodine receptor/Ca-release channel...
Excitation-contraction coupling in skeletal muscle myofibers depends upon Ca release from the sarcoplasmic reticulum through the ryanodine receptor/Ca-release channel RyR1. The RyR1 contains ∼100 Cys thiols of which ∼30 comprise an allosteric network subject to posttranslational modification by S-nitrosylation, S-palmitoylation and S-oxidation. However, the role and function of these modifications is not understood. Although aberrant S-nitrosylation of multiple unidentified sites has been associated with dystrophic diseases, malignant hyperthermia and other myopathic syndromes, S-nitrosylation in physiological situations is reportedly specific to a single (1 of ∼100) Cys in RyR1, Cys in a manner gated by pO. Using mice expressing a form of RyR1 with a Cys→Ala point mutation to prevent S-nitrosylation at this site, we showed that Cys was the principal target of endogenous S-nitrosylation during normal muscle function. The absence of Cys S-nitrosylation suppressed stimulus-evoked Ca release at physiological pO (at least in part by altering the regulation of RyR1 by Ca/calmodulin), eliminated pO coupling, and diminished skeletal myocyte contractility in vitro and measures of muscle strength in vivo. Furthermore, we found that abrogation of Cys S-nitrosylation resulted in a developmental defect reflected in diminished myofiber diameter, altered fiber subtypes, and altered expression of genes implicated in muscle development and atrophy. Thus, our findings establish a physiological role for pO-coupled S-nitrosylation of RyR1 in skeletal muscle contractility and development and provide foundation for future studies of RyR1 modifications in physiology and disease.
Topics: Ryanodine Receptor Calcium Release Channel; Animals; Muscle, Skeletal; Mice; Calcium; Cysteine; Protein Processing, Post-Translational; Muscle Development; Mice, Transgenic; Calcium Signaling
PubMed: 38820626
DOI: 10.1016/j.bbrc.2024.150163 -
Frontiers in Cellular Neuroscience 2024Intracellular Ca-signaling in astrocytes is instrumental for their brain "housekeeping" role and astroglial control of synaptic plasticity. An important source for...
Intracellular Ca-signaling in astrocytes is instrumental for their brain "housekeeping" role and astroglial control of synaptic plasticity. An important source for elevating the cytosolic Ca level in astrocytes is a release from endoplasmic reticulum which can be triggered via two fundamental pathways: IP3 receptors and calcium-induced calcium release (CICR) mediated by Ca-sensitive ryanodine receptors (RyRs). While the physiological role for glial IP3 became a focus of intensive research and debate, ryanodine receptors received much less attention. We explored the role for ryanodine receptors in the modulation of cytosolic Ca-signaling in the cortical and hippocampal astrocytes, astrocyte-neuron communication and astroglia modulation of synaptic plasticity. Our data show that RyR-mediated Ca-induced Ca-release from ER brings substantial contribution into signaling in the functional microdomains hippocampal and neocortical astrocytes. Furthermore, RyR-mediated CICR activated the release of ATP and glutamate from hippocampal and neocortical astrocytes which, in turn, elicited transient purinergic and tonic glutamatergic currents in the neighboring pyramidal neurons. The CICR-facilitated release of ATP and glutamate was inhibited after intracellular perfusion of astrocytes with ryanodine and BAPTA and in the transgenic dnSNARE mice with impaired astroglial exocytosis. We also found out that RyR-mediated amplification of astrocytic Ca-signaling enhanced the long-term synaptic potentiation in the hippocampus and neocortex of aged mice. Combined, our data demonstrate that ryanodine receptors are essential for astrocytic Ca-signaling and efficient astrocyte-neuron communications. The RyR-mediated CICR contributes to astrocytic control of synaptic plasticity and can underlie, at least partially, neuroprotective and cognitive effects of caffein.
PubMed: 38812795
DOI: 10.3389/fncel.2024.1382010 -
Comparative Biochemistry and... Sep 2024Regional endothermy is the ability of an animal to elevate the temperature of specific regions of the body above that of the surrounding environment and has evolved...
Relative sarcolipin (SLN) and sarcoplasmic reticulum Ca ATPase (SERCA1) transcripts levels in closely related endothermic and ectothermic scombrid fishes: Implications for molecular basis of futile calcium cycle non-shivering thermogenesis (NST).
Regional endothermy is the ability of an animal to elevate the temperature of specific regions of the body above that of the surrounding environment and has evolved independently among several fish lineages. Sarcolipin (SLN) is a small transmembrane protein that uncouples the sarcoplasmic reticulum calcium ATPase pump (SERCA1b) resulting in futile Ca cycling and is thought to play a role in non-shivering thermogenesis (NST) in cold-challenged mammals and possibly some fishes. This study investigated the relative expression of sln and serca1 transcripts in three regionally-endothermic fishes (the skipjack, Katsuwonus pelamis, and yellowfin tuna, Thunnus albacares, both of which elevate the temperatures of their slow-twitch red skeletal muscle (RM) and extraocular muscles (EM), as well as the cranial endothermic swordfish, Xiphias gladius), and closely related ectothermic scombrids (the Eastern Pacific bonito, Sarda chiliensis, and Pacific chub mackerel, Scomber japonicus). Using Reverse Transcription quantitative PCR (RT-qPCR) and species-specific primers, relative sln expression trended higher in both the RM and EM for all four scombrid species compared to white muscle. In addition, relative serca1 expression was found to be higher in RM of skipjack and yellowfin tuna in comparison to white muscle. However, neither sln nor serca1 transcripts were higher in swordfish RM, EM or cranial heater tissue in comparison to white muscle. A key phosphorylation site in sarcolipin, threonine 5, is conserved in the swordfish, but is mutated to alanine or valine in tunas and the endothermic smalleye Pacific opah, Lampris incognitus, which should result in increased uncoupling of the SERCA pump. Our results support the role of potential SLN-NST in endothermic tunas and the lack thereof for swordfish.
Topics: Animals; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Proteolipids; Muscle Proteins; Thermogenesis; Calcium; Fish Proteins; Muscle, Skeletal; RNA, Messenger; Perciformes; Tuna
PubMed: 38782254
DOI: 10.1016/j.cbpa.2024.111667 -
Communications Biology May 2024Glycerophosphocholine (GPC) is an important precursor for intracellular choline supply in phosphatidylcholine (PC) metabolism. GDE5/Gpcpd1 hydrolyzes GPC into choline...
Glycerophosphocholine (GPC) is an important precursor for intracellular choline supply in phosphatidylcholine (PC) metabolism. GDE5/Gpcpd1 hydrolyzes GPC into choline and glycerol 3-phosphate; this study aimed to elucidate its physiological function in vivo. Heterozygous whole-body GDE5-deficient mice reveal a significant GPC accumulation across tissues, while homozygous whole-body knockout results in embryonic lethality. Skeletal muscle-specific GDE5 deletion (Gde5 skKO) exhibits reduced passive force and improved fatigue resistance in electrically stimulated gastrocnemius muscles in vivo. GDE5 deficiency also results in higher glycolytic metabolites and glycogen levels, and glycerophospholipids alteration, including reduced levels of phospholipids that bind polyunsaturated fatty acids (PUFAs), such as DHA. Interestingly, this PC fatty acid compositional change is similar to that observed in skeletal muscles of denervated and Duchenne muscular dystrophy mouse models. These are accompanied by decrease of GDE5 expression, suggesting a regulatory role of GDE5 activity for glycerophospholipid profiles. Furthermore, a DHA-rich diet enhances contractile force and lowers fatigue resistance, suggesting a functional relationship between PC fatty acid composition and muscle function. Finally, skinned fiber experiments show that GDE5 loss increases the probability of the ryanodine receptor opening and lowers the maximum Ca-activated force. Collectively, GDE5 activity plays roles in PC and glucose/glycogen metabolism in skeletal muscle.
Topics: Animals; Muscle, Skeletal; Mice; Phosphatidylcholines; Mice, Knockout; Muscle Contraction; Male; Mice, Inbred C57BL; Phosphoric Diester Hydrolases
PubMed: 38769369
DOI: 10.1038/s42003-024-06298-z -
Biochemical and Biophysical Research... Aug 2024Dexmedetomidine (DEX), a highly selective α2-adrenoceptor agonist, can decrease the incidence of arrhythmias, such as catecholaminergic polymorphic ventricular...
BACKGROUND
Dexmedetomidine (DEX), a highly selective α2-adrenoceptor agonist, can decrease the incidence of arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the underlying mechanisms by which DEX affects cardiac electrophysiological function remain unclear.
METHODS
Ryanodine receptor (RyR2) heterozygous R2474S mice were used as a model for CPVT. WT and RyR2 mice were treated with isoproterenol (ISO) and DEX, and electrocardiograms were continuously monitored during both in vivo and ex vivo experiments. Dual-dye optical mapping was used to explore the anti-arrhythmic mechanism of DEX.
RESULTS
DEX significantly reduced the occurrence and duration of ISO-induced of VT/VF in RyR2 mice in vivo and ex vivo. DEX remarkably prolonged action potential duration (APD) and calcium transient duration (CaTD) in both RyR2 and WT hearts, whereas it reduced APD heterogeneity and CaT alternans in RyR2 hearts. DEX inhibited ectopy and reentry formation, and stabilized voltage-calcium latency.
CONCLUSION
DEX exhibited an antiarrhythmic effect through stabilizing membrane voltage and intracellular Ca. DEX can be used as a beneficial perioperative anesthetic for patients with CPVT or other tachy-arrhythmias.
Topics: Animals; Dexmedetomidine; Ryanodine Receptor Calcium Release Channel; Calcium; Mice; Arrhythmias, Cardiac; Membrane Potentials; Isoproterenol; Tachycardia, Ventricular; Anti-Arrhythmia Agents; Male; Action Potentials; Mice, Inbred C57BL
PubMed: 38754163
DOI: 10.1016/j.bbrc.2024.150105 -
Nature Communications May 2024RyR1 is an intracellular Ca channel important in excitable cells such as neurons and muscle fibers. Ca activates it at low concentrations and inhibits it at high...
RyR1 is an intracellular Ca channel important in excitable cells such as neurons and muscle fibers. Ca activates it at low concentrations and inhibits it at high concentrations. Mg is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg-mediated inhibition, the molecular-level mechanisms remain unclear. In this work we determined two cryo-EM structures of RyR1 with Mg up to 2.8 Å resolution, identifying multiple Mg binding sites. Mg inhibits at the known Ca activating site and we propose that the EF hand domain is an inhibitory divalent cation sensor. Both divalent cations bind to ATP within a crevice, contributing to the precise transmission of allosteric changes within the enormous channel protein. Notably, Mg inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg inhibition is overcome during excitation.
Topics: Ryanodine Receptor Calcium Release Channel; Magnesium; Calcium; Cryoelectron Microscopy; Binding Sites; Animals; Molecular Dynamics Simulation; Adenosine Triphosphate; Humans; Rabbits
PubMed: 38750013
DOI: 10.1038/s41467-024-48292-3 -
Journal of Cachexia, Sarcopenia and... Jun 2024Autosomal-recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated...
Integrated multi-omics approach reveals the role of striated muscle preferentially expressed protein kinase in skeletal muscle including its relationship with myospryn complex.
BACKGROUND
Autosomal-recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). Loss of SPEG is associated with defective triad formation, abnormal excitation-contraction coupling, calcium mishandling and disruption of the focal adhesion complex in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi-omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions.
METHODS
Skeletal muscles from 2-month-old SPEG-deficient (Speg-CKO) and wild-type (WT) mice were used for RNA sequencing (n = 4 per genotype) to profile transcriptomics and mass spectrometry (n = 4 for WT; n = 3 for Speg-CKO mice) to profile proteomics and phosphoproteomics. In addition, interactomics was performed using the SPEG antibody on pooled muscle lysates (quadriceps, gastrocnemius and triceps) from WT and Speg-CKO mice. Based on the multi-omics results, we performed quantitative real-time PCR, co-immunoprecipitation and immunoblot to verify the findings.
RESULTS
We identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2 and RyR1, which are critical for triad formation, and that SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22 ± 3% for CMYA5 [P < 0.05] and 18 ± 3% for FSD2 [P < 0.01]). Furthermore, SPEG phosphorylates RyR1 at S2902 (phosphorylation level decreased to 55 ± 15% at S2902 in Speg-CKO mice; P < 0.05), and its loss affects JPH2 phosphorylation at multiple sites (increased phosphorylation at T161 [1.90 ± 0.24-fold], S162 [1.61 ± 0.37-fold] and S165 [1.66 ± 0.13-fold]; decreased phosphorylation at S228 and S231 [39 ± 6%], S234 [50 ± 12%], S593 [48 ± 3%] and S613 [66 ± 10%]; P < 0.05 for S162 and P < 0.01 for other sites). On analysing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix-receptor interaction (P < 1e) and peroxisome proliferator-activated receptor signalling (P < 9e).
CONCLUSIONS
We have elucidated the critical role of SPEG in the triad as it works closely with myospryn complex proteins (CMYA5, FSD2 and RyR1), it regulates phosphorylation levels of various residues in JPH2 and S2902 in RyR1, and its deficiency is associated with dysregulation of several pathways. The study identifies unique SPEG-interacting proteins and their phosphorylation functions and emphasizes the importance of using a multi-omics approach to comprehensively evaluate the molecular function of proteins involved in various genetic disorders.
Topics: Animals; Mice; Muscle, Skeletal; Mice, Knockout; Muscle Proteins; Ryanodine Receptor Calcium Release Channel; Proteomics; Phosphorylation; Multiomics; Myosin-Light-Chain Kinase
PubMed: 38725372
DOI: 10.1002/jcsm.13470 -
Frontiers in Physiology 2024The loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp...
Loose-patch clamp analysis applied to voltage-gated ionic currents following pharmacological ryanodine receptor modulation in murine hippocampal cornu ammonis-1 pyramidal neurons.
INTRODUCTION
The loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp micro-electrode, gap, or conventional patch voltage clamping. It demonstrated the feedback effects of pharmacological modification of ryanodine receptor (RyR)-mediated Ca release on the Na channel (Nav1.4) currents, initiating excitation-contraction coupling in native murine SkM. The effects of the further RyR and Ca-ATPase (SERCA) antagonists, dantrolene and cyclopiazonic acid (CPA), additionally implicated background tubular-sarcoplasmic Ca domains in these actions.
MATERIALS AND METHODS
We extend the loose-patch clamp approach to ion current measurements in murine hippocampal brain slice cornu ammonis-1 (CA1) pyramidal neurons. We explored the effects on Na currents of pharmacologically manipulating RyR and SERCA-mediated intracellular store Ca release and reuptake. We adopted protocols previously applied to native skeletal muscle. These demonstrated Ca-mediated feedback effects on the Na channel function.
RESULTS
Experiments applying depolarizing 15 ms duration loose-patch clamp steps to test voltages ranging from -40 to 120 mV positive to the resting membrane potential demonstrated that 0.5 mM caffeine decreased inward current amplitudes, agreeing with the previous SkM findings. It also decreased transient but not prolonged outward current amplitudes. However, 2 mM caffeine affected neither inward nor transient outward but increased prolonged outward currents, in contrast to its increasing inward currents in SkM. Furthermore, similarly and in contrast to previous SkM findings, both dantrolene (10 μM) and CPA (1 μM) pre-administration left both inward and outward currents unchanged. Nevertheless, dantrolene pretreatment still abrogated the effects of subsequent 0.5- and 2-mM caffeine challenges on both inward and outward currents. Finally, CPA abrogated the effects of 0.5 mM caffeine on both inward and outward currents, but with 2 mM caffeine, inward and transient outward currents were unchanged, but sustained outward currents increased.
CONCLUSION
We, thus, extend loose-patch clamping to establish pharmacological properties of murine CA1 pyramidal neurons and their similarities and contrasts with SkM. Here, evoked though not background Ca-store release influenced Nav and Kv excitation, consistent with smaller contributions of background store Ca release to resting [Ca]. This potential non-canonical mechanism could modulate neuronal membrane excitability or cellular firing rates.
PubMed: 38720787
DOI: 10.3389/fphys.2024.1359560 -
The Journal of Physical Chemistry. B May 2024Ryanodine receptor type 1 (RyR1) is a Ca-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger...
Ryanodine receptor type 1 (RyR1) is a Ca-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca, and caffeine binding, shedding light on the intricate relationship between Zn coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.
Topics: Ryanodine Receptor Calcium Release Channel; Molecular Dynamics Simulation; Zinc; Ligands; Calcium; Density Functional Theory; Binding Sites; Protein Binding; Zinc Fingers; Caffeine; Adenosine Triphosphate; Humans
PubMed: 38717304
DOI: 10.1021/acs.jpcb.4c01189