RyR2 Activity Research Articles

Efficacy of RyR2 inhibitor EL20 in induced pluripotent stem cell-derived cardiomyocytes from a patient with catecholaminergic polymorphic ventricular tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that often leads to sudden cardiac death. The most common form of CPVT is caused by autosomal‐dominant variants in the cardiac ryanodine receptor type‐2 (RYR2) gene. Mutations in RYR2 promote calcium (Ca2+) leak from the sarcoplasmic reticulum (SR), triggering lethal arrhythmias. Recently, it was demonstrated that tetracaine derivative EL20 specifically inhibits mutant RyR2, normalizes Ca2+ handling and suppresses arrhythmias in a CPVT mouse model. The objective of this study was to determine whether EL20 normalizes SR Ca2+ handling and arrhythmic events in induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) from a CPVT patient. Blood samples from a child carrying RyR2 variant RyR2 variant Arg‐176‐Glu (R176Q) and a mutation‐negative relative were reprogrammed into iPSCs using a Sendai virus system. iPSC‐CMs were derived using the StemdiffTM kit. Confocal Ca2+ imaging was used to quantify RyR2 activity in the absence and presence of EL20. iPSC‐CMs harbouring the R176Q variant demonstrated spontaneous SR Ca2+ release events, whereas administration of EL20 diminished these abnormal events at low nanomolar concentrations (IC50 = 82 nM). Importantly, treatment with EL20 did not have any adverse effects on systolic Ca2+ handling in control iPSC‐CMs. Our results show for the first time that tetracaine derivative EL20 normalized SR Ca2+ handling and suppresses arrhythmogenic activity in iPSC‐CMs derived from a CPVT patient. Hence, this study confirms that this RyR2‐inhibitor represents a promising therapeutic candidate for treatment of CPVT.

RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca2+− and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca2+-independent basal activity and Ca2+-dependent activation of [3H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.

Preliminary Studies Towards the Examination of Hypoxia‐related Transcriptional Regulation of Ryanodine Receptor Activity in Pulmonary Arteries of Fetal and Newborn Sheep

Before birth the pulmonary vasculature does not receive much blood supply and is relatively constricted. However, with birth the vasculature dilates and blood flow and oxygenation increase. Evidence indicates that pulmonary arterial dilation during birth is related to an increase in the activation of large conductance potassium channels that are activated by Ca2+ spark events after activation of ryanodine receptors (RyR) on the sarcoplasmic reticulum. This coupled mechanism is largely driven through activation of L-type Ca2+ channels (CaL), which stimulate RyRs. Depolarizing cells with 30 mM potassium activates CaL and drives RyR mediated Ca2+ events. Previous evidence from our group illustrates that long term maternal hypoxia disrupts Ca2+ sparks and causes pulmonary hypertension in newborn lambs. We intend to modulate key transcriptional processes to better understand the molecular mechanisms that disrupt Ca2+ spark, vasoreactivity, and pulmonary pressure following long term hypoxia. The current studies were therefore performed as a foundation for our upcoming studies that will examine transcriptional control of RyRs and potassium channels. We tested the hypothesis that we could replicate earlier work that showed membrane depolarization with 30 mM K increases Ca2+ spark activity through activation of RyRs. To address this hypothesis, pulmonary arterial myocytes of fetal and newborn sheep were examined in the presence and absence of 30K and 10 mM ryanodine, which is a RyR antagonist. The intracellular Ca2+ was recorded in myocytes of isolated pulmonary arteries that were loaded with Fluo-4 using line-scan techniques on a confocal microscope. Spatial and temporal characteristics of the events were analyzed using customized software. As predicted, 30K increased Ca2+ spark activity while ryanodine treatment decreased activity. This confirmatory evidence in fetus and newborns provides a platform for upcoming studies to evaluate the role of transcriptional regulation to cell excitability in response to maternal hypoxia.

TRPML Activation with MLSA1 Increases Ca 2+ Oscillations in Fetal Pulmonary Arterial Myocytes

Ca2+ signaling in vascular myocytes is a highly regulated process and includes whole-cell oscillations and focal sub-cellular Ca2+ events. Previous evidence shows that activation of ryanodine receptors on the sarcoplasmic reticulum contributes to Ca2+ oscillations in pulmonary arterial myocytes. The oscillatory activity is thought to contribute to arterial reactivity and the regulation of vascular blood flow. Evidence also shows that long-term fetal hypoxia results in restricted oscillatory activity in the pulmonary arterial myocytes. We hypothesize that MLSA-induced TRPML activation can increase oscillatory activity in normoxic fetal pulmonary arterial myocytes. Activation of TRPML channels on the lysosome may elicit a triggering-pulse of Ca2+ that would drive RyR activity, rescuing intracellular oscillations in the pulmonary arterial myocytes. To study oscillatory activity, pulmonary arteries were isolated from near-term fetal sheep exposed to low altitude (700 m) for 110+ days. Intracellular oscillatory activity in the presence and absence of 10 μm MLSA1, a TRPML activator, was then measured using laser scanning confocal microscopy in Fluo-4 loaded pulmonary arterial myocytes. In preliminary studies, MLSA1 statistically increased overall oscillatory activity in normoxic myocytes. Further statistical analysis also revealed modest decreases in duration and area under the curve of oscillations as well as a significant increase in amplitude. Whether TRPML activation can be used to rescue aberrant RyR mediated arterial reactivity along with aberrant Ca2+ signaling in fetal pulmonary myocytes following gestational hypoxia remains to be determined.

Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death.

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.

Oxidative stress in early metabolic syndrome impairs cardiac RyR2 and SERCA2a activity and modifies the interplay of these proteins during Ca2+ waves

We investigated how oxidative stress (OS) alters Ca2+ handling in ventricular myocytes in early metabolic syndrome (MetS) in sucrose-fed rats. The effects of N-acetyl cysteine (NAC) or dl-Dithiothreitol (DTT) on systolic Ca2+ transients (SCaTs), diastolic Ca2+ sparks (CaS) and Ca2+ waves (CaW), recorded by confocal techniques, and L-type Ca2+ current (ICa), assessed by whole-cell patch clamp, were evaluated in MetS and Control cells. MetS myocytes exhibited decreased SCaTs and CaS frequency but unaffected CaW propagation. In Control cells, NAC/DTT reduced RyR2/SERCA2a activity blunting SCaTs, CaS frequency and CaW propagation, suggesting that basal ROS optimised Ca2+ signalling by maintaining RyR2/SERCA2a function and that these proteins facilitate CaW propagation. Conversely, NAC/DTT in MetS recovered RyR2/SERCA2a function, improving SCaTs and CaS frequency, but unexpectedly decreasing CaW propagation. We hypothesised that OS decreases RyR2/SERCA2a activity at early MetS, and while decreased SERCA2a favours CaW propagation, diminished RyR2 restrains it.

Human Atrial Fibrillation Is Not Associated With Remodeling of Ryanodine Receptor Clusters

The release of Ca2+ by ryanodine receptor (RyR2) channels is critical for cardiac function. However, abnormal RyR2 activity has been linked to the development of arrhythmias, including increased spontaneous Ca2+ release in human atrial fibrillation (AF). Clustering properties of RyR2 have been suggested to alter the activity of the channel, with remodeling of RyR2 clusters identified in pre-clinical models of AF and heart failure. Whether such remodeling occurs in human cardiac disease remains unclear. This study aimed to investigate the nanoscale organization of RyR2 clusters in AF patients – the first known study to examine this potential remodeling in diseased human cardiomyocytes. Right atrial appendage from cardiac surgery patients with paroxysmal or persistent AF, or without AF (non-AF) were examined using super-resolution (dSTORM) imaging. Significant atrial dilation and cardiomyocyte hypertrophy was observed in persistent AF patients compared to non-AF, with these two parameters significantly correlated. Interestingly, the clustering properties of RyR2 were remarkably unaltered in the AF patients. No significant differences were identified in cluster size (mean ∼18 RyR2 channels), density or channel packing within clusters between patient groups. The spatial organization of clusters throughout the cardiomyocyte was also unchanged across the groups. RyR2 clustering properties did not significantly correlate with patient characteristics. In this first study to examine nanoscale RyR2 organization in human cardiac disease, these findings indicate that RyR2 cluster remodeling is not an underlying mechanism contributing to altered channel function and subsequent arrhythmogenesis in human AF.

Sarcoplasmic reticular Ca2+-ATPase inhibition paradoxically upregulates murine skeletal muscle Nav1.4 function

Skeletal muscle Na+ channels possess Ca2+- and calmodulin-binding sites implicated in Nav1.4 current (INa) downregulation following ryanodine receptor (RyR1) activation produced by exchange protein directly activated by cyclic AMP or caffeine challenge, effects abrogated by the RyR1-antagonist dantrolene which itself increased INa. These findings were attributed to actions of consequently altered cytosolic Ca2+, [Ca2+]i, on Nav1.4. We extend the latter hypothesis employing cyclopiazonic acid (CPA) challenge, which similarly increases [Ca2+]i, but through contrastingly inhibiting sarcoplasmic reticular (SR) Ca2+-ATPase. Loose patch clamping determined Na+ current (INa) families in intact native murine gastrocnemius skeletal myocytes, minimising artefactual [Ca2+]i perturbations. A bespoke flow system permitted continuous INa comparisons through graded depolarizing steps in identical stable membrane patches before and following solution change. In contrast to the previous studies modifying RyR1 activity, and imposing control solution changes, CPA (0.1 and 1 µM) produced persistent increases in INa within 1–4 min of introduction. CPA pre-treatment additionally abrogated previously reported reductions in INa produced by 0.5 mM caffeine. Plots of peak current against voltage excursion demonstrated that 1 µM CPA increased maximum INa by ~ 30%. It only slightly decreased half-maximal activating voltages (V0.5) and steepness factors (k), by 2 mV and 0.7, in contrast to the V0.5 and k shifts reported with direct RyR1 modification. These paradoxical findings complement previously reported downregulatory effects on Nav1.4 of RyR1-agonist mediated increases in bulk cytosolic [Ca2+]. They implicate possible local tubule-sarcoplasmic triadic domains containing reduced [Ca2+]TSR in the observed upregulation of Nav1.4 function following CPA-induced SR Ca2+ depletion.

Mitochondrial Calcium Uniporter Overexpression Exacerbates Arrhythmogenic Calcium Mishandling in Diseased Rat Ventricular Myocytes

Systolic dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with diminished Ca2+ transient amplitude and impaired mitochondrial function due to reduced mitochondrial [Ca2+]. Pharmacological or genetic facilitation of mito-Ca2+ uptake was shown to restore Ca2+ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that acute pharmacological enhancement of mito-Ca2+ uptake can exacerbate spontaneous Ca2+ release in myocytes from diseased animals, increasing propensity to stress-induced arrhythmias. To test whether chronic recovery of mito-[Ca2+] restores systolic Ca2+ release without adverse effects in diastole, we overexpressed mitochondrial Ca2+ uniporter (MCU) in ventricular myocytes (VMs) from rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca2+] using genetic probe mtRCamp1h revealed that mito-[Ca2+] in TAB VMs paced at 1 Hz under β-adrenergic stimulation is significantly lower than in Shams. Adenoviral MCU overexpression in TAB VMs restored mito-[Ca2+]. Confocal imaging of VMs loaded with fluorescent indicator Rhod-2 revealed that MCU overexpression in TAB VMs reversed the decrease in Ca2+ transient amplitude. Importantly, the decay of caffeine-induced Ca2+ transient in MCU-overexpressing TAB VMs was prolonged, indicative of decreased NCX activity. Moreover, MCU overexpression in TAB VMs was accompanied by a further increase in incidence of proarrhythmic spontaneous Ca2+ waves, suggesting exacerbation of RyR2 dysfunction. Experiments using genetic probe OMM-HyPer showed a 10-fold increase in mito-ROS emission in TAB VMs overexpressing MCU. Importantly, application of mito-ROS scavenger mitoTEMPO (20 µM, 10 min) restored Ca2+ transient amplitude, reduced spontaneous Ca2+ waves and restored mito-[Ca2+] in TAB VMs, rendering them normal. These findings suggest that restoration of mito-[Ca2+] in VMs from diseased hearts by MCU overexpression improves systolic Ca2+ release possibly via reduced NCX-mediated Ca2+ efflux, but exacerbates increased mito-ROS and RyR2 activity, increasing arrhythmic potential.

The Sarcoplasmic Reticulum Oxidoreductase System Modulates Luminal Ca2+ Regulation of the Ryanodine Receptor in Cardiac Disease

The sarcoplasmic reticulum (SR) Ca2+ release ryanodine receptor channel (RyR2) is susceptible to posttranslational modifications by reactive oxygen species (ROS), impairing luminal Ca2+ regulation and increasing channel activity. Despite significant research efforts, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment in the SR are involved in this process. In ventricular myocytes (VMs) from rats with hypertrophy induced by thoracic aortic banding (TAB), expression of SR oxidoreductase Ero1α was significantly increased compared to Sham VMs. Adenoviral expression of intra-SR ROS probe ERroGFP in cultured TAB rat VMs revealed enhanced oxidative stress in the vicinity of RyR2, in parallel with increased RyR2 oxidation, measured with DNP antibody. Ero1α overexpression in control VMs recapitulated a similar phenotype. Conversely, pharmacological inhibition of Ero1α with EN460 (20 μM) reduced arrhythmogenic potential in ex vivo optically mapped TAB rat hearts by reducing SR Ca2+ leak, and suppressed Ca2+ spark frequency in TAB VMs, indicating stabilization of RyR2 function. Utilizing a heterologous HEK293 cell system and intra-ER Ca2+ sensor R-CEPIAer to probe Ca2+ release dynamics of human RyR2, Ero1α overexpression significantly increased Ca2+ wave frequency, mimicking effects of strong oxidative agent DTDP. Knockdown of endogenous Ero1α had opposite effects. Furthermore, mutation of two intraluminal cysteines of RyR2 in the second intraluminal loop, potentially sensitive to modification by ROS, resulted in more frequent Ca2+ waves and reduced basal intra-ER [Ca2+]. These findings suggest upregulation of SR oxidoreductase Ero1α in cardiac disease is detrimental to luminal Ca2+ homeostasis by modulating RyR2 activity via ROS. Additionally, we have identified two potential intraluminal residues of the channel that may be sensitive to redox modification.

A Homology Modelling Study of Potential RyR2 Structural Changes in R2474S and V2475F CPVT1

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a disease caused by single-point mutations to cardiac ryanodine receptor Ca2+ release channels (RyR2). Life-threatening arrhythmias due to abnormal sarcoplasmic reticulum Ca2+ release can result. of the many CPVT1-causing mutations, two distinct CPVT1 gain-of-function mutations are found at neighbouring amino acid residues, R2474S and V2475F in the Helical Domain 1 (HD1), more than 43 Å from the transmembrane pore. To explore potential changes in the RyR2 HD1 structure caused by these mutations, we used MODELLER to construct HD1 homology models for the human wild-type (WT) as well as the two mutants, R2474S and V2475F. The models were based on resolved structures of the pig RyR2 HD1 (residues 2111 to 2679) from full-length channels in open and closed states (PDB ID: 5GOA, 5GO9). Subsequently, we generated a root mean square deviation (RMSD) matrix of modelled structures and used hierarchical clustering on the distance matrix to assess differences caused by mutants. Our results indicate that both mutations may alter the bending of the HD1 domain, particularly in the open state. From the full-length structures, it has previously been suggested that the motions of the HD1 are coupled to motions of the central domain (including the pore region). Thus, shifts in the HD1 may be translated into changes in the pore. As both mutations are reported to increase RyR2 activity, we suggest that these potential structural changes within the RyR2 HD1 domain are sufficient to subsequently alter the interactions between this domain and other regions of the channel so that channel opening is encouraged.

Interactive Roles of CaMKII/Ryanodine Receptor Signaling and Inflammation in Lung Diseases.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase and has been recently recognized to play a vital role in pathological events in the pulmonary system. CaMKII has diverse downstream targets that promote vascular disease, asthma, and cancer, so improved understanding of CaMKII signaling has the potential to lead to new therapies for lung diseases. Multiple studies have demonstrated that CaMKII is involved in redox modulation of ryanodine receptors (RyRs). CaMKII can be directly activated by reactive oxygen species (ROS) which then regulates RyR activity, which is essential for Ca2+-dependent processes in lung diseases. Furthermore, both CaMKII and RyRs participate in the inflammation process. However, their role in the pulmonary physiology in response to ROS is still an ambiguous one. Because CaMKII and RyRs are important in pulmonary biology, cell survival, cell cycle control, and inflammation, it is possible that the relationship between ROS and CaMKII/RyRs signal complex will be necessary for understanding and treating lung diseases. Here, we review roles of CaMKII/RyRs in lung diseases to understand with how CaMKII/RyRs may act as a transduction signal to connect prooxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of pulmonary disease.

Abstract 17220: Functional Roles and Molecular Mechanisms of Calcium Release Signaling in Hypoxic Cellular Responses in Pulmonary Artery Smooth Muscle Cells

Introduction: Hypoxia causes contractile and proliferative responses in pulmonary arterial smooth muscle cells (PASMCs), but not systemic artery SMCs, thereby leading to pulmonary hypertension (PH), but not systemic hypertension. However, the underlying mechanisms remain unclear. Hypothesis: Ca 2+ release signaling in PASMCs may mediate hypoxic pulmonary artery vasoconstriction, remodeling and hypertension. Methods: Single cell RT-PCR and Western blot analysis, FRET and immunoprecipitation, tissue contraction assay, immunohistochemistry staining and pressure-volume loop analysis, and genetic and pharmacological approaches, were, respectively, used to determine targeted molecular expression, interaction and function. Results: All ryanodine receptor (RyR1, RyR2 and RyR3) Ca 2+ release channels are expressed in mouse PASMCs. RyR1 or RyR3 knockout (KO) partially inhibits, while RyR2 KO completely blocks, hypoxic cellular responses. SMC-specific RyR2 KO restores abolishes hypoxic pulmonary artery vasoconstriction, remodeling and hypertension in mice. RyR2 KO also inhibits hypoxic nuclear factor (NF)-κB and downstream cyclin D1 activation. FK506 binding protein 12.6 (FKBP12.6) is an endogenous RyR2 inhibitor; its KO or FK506 treatment enhances, and treatment of S107 (a specific RyR2/FKBP12.6 stabilizer) inhibits hypoxic PH. Lentiviral shRNA-mediated SMC-specific Rieske iron-sulfur protein (RISP) knockdown diminishes mitochondrial reactive oxygen species (ROS) generation, RyR2/FKBP12.6 complex dissociation, and RyR2 activation, thereby blocking hypoxic PH. Conclusions: RyR1, RyR2 and RyR3 are all involved in hypoxic contractile and proliferative responses in PASMCs, but RyR2 is a most important player. RISP-dependent mitochondrial ROS - FKBP12.6/RyR2 complex dissociation - RyR2 hyperfunction signaling mediate PA vasoconstriction. RyR2 hyperfunction also activates Ca2+-dependent NF-κB/cyclinD1 pathway to provoke PA remodeling; PA vasoconstriction and remodeling together cause PH. Furthermore, RISP-mediated ROS inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be novel and effective options for the treatment of PH.

Treatment with beta-blockers normalizes RyR2 phosphorylation and calcium spark activity in atrial myocytes from patients with atrial fibrillation

Abstract Background Atrial fibrillation has been associated with an increase in ryanodine receptor (RyR2) phosphorylation and local calcium release (calcium sparks). Carvedilol, a nonselective beta-adrenergic receptor blocker also inhibits the cardiac ryanodine receptor (RyR2), but it has been suggested that the enantiomer R-carvedilol only inhibits RyR2 activity and hence has the potential to inhibit calcium sparks without affecting RyR2 phosphorylation. Purpose This study aimed to determine the ability of the enantiomers R- and S-carvedilol to reverse RyR2 phosphorylation at s2808 and calcium sparks induced by the β2-adrenergic agonist fenoterol, in order to determine the relationship between RyR2 phosphorylation at s2808 and calcium spark frequency, and to assess the efficacy of R- and S-carvedilol. Methods Human right atrial myocytes were isolated and subjected to immunofluorescent labelling of total and s2808 phosphorylated RyR2, or loaded with fluo-4 and subjected to confocal calcium imaging. Beta-adrenergic receptors were first activated with 3μM fenoterol and then inhibited by different concentrations of carvedilol R- or S-enantiomers. Results Incubation of myocytes with fenoterol increased the s2808/RyR2 ratio from 0.32±0.03 to 0.66±0.05 (n=18, p<0.001). Incubation with 0.1, 0.3, 1 or 3μM R-carvedilol in the presence of fenoterol changed the s2808/RyR2 ratio to 0.64±0.05, 0.44±0.04, 0.34±0.07 and 0.28±0.05 (p<0.01) respectively. For comparison 3μM S-carvedilol reduced the s2808/RyR2 ratio to 0.23±0.06 in myocytes from 5 patients (p<0.01). Confocal calcium imaging revealed that fenoterol increased the spark density from 0.28±0.04 to 1.24±0.25 events/s/1000μm2 (n=9, p<0.01) and addition of 0.1, 0.3, or 1μM R-carvedilol changed the frequency to 1.32±0.52, 0.38±0.05, and 0.15±0.05 events/s/1000μm2 (p<0.01) respectively. Analysis of atrial myocytes from patients without atrial fibrillation revealed that the s2808/RyR2 ratio was similar in 25 patients treated with beta-blockers (0.39±0.04) and 57 that did not receive beta-blockers (0.44±0.03, p=0.33) while the s2808/RyR2 ratio was significantly smaller in 16 patients with atrial fibrillation receiving beta-blockers (0.43±0.08) than in 5 patients that did not (0.80±0.19, p<0.05). Conclusions R-carvedilol reverses the effects of beta-adrenergic stimulation on s2808 phosphorylation and calcium sparks in human atrial myocytes, and treatment with beta-blockers reduces excessive RyR2 phosphorylation at s2808 in patients with atrial fibrillation to levels observed in those without the arrhythmia, pointing to beta-adrenergic receptors as a target for controlling RyR2 phophorylation and activity in atrial fibrillation. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Science and Innovation & Spanish Ministry of Health and Consume

The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability

Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.

Increased RyR2 activity is exacerbated by calcium leak-induced mitochondrial ROS

Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor (RyR2). The transfer of Ca2+ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca2+ leak, via altered SR-mitochondrial Ca2+ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under β-adrenergic stimulation. Expression of mitochondrial Ca2+ probe mtRCamp1h revealed diminished net mitochondrial [Ca2+] with enhanced SR Ca2+ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca2+ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca2+ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca2+ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca2+ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca2+ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.

Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes.

Increased protein phosphatase 1 (PP-1) activity has been found in end stage human heart failure. Although PP-1 has been extensively studied, a detailed understanding of its role in the excitation-contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP-1 activity on local Ca2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP-1 protein. We report that acute de-phosphorylation may increase the sensitivity of RyR2 channels to Ca2+ in situ, and that the RyR2-serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP-1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca2+ leak in the diseased heart. Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP-1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP-1 activity, we investigated how the PP-1 catalytic subunit (PP-1c) alters Ca2+ sparks in permeabilized cardiomyocytes and we also applied a PP-1-disrupting peptide (PDP3) to specifically activate endogenous PP-1, including the one anchored on the RyR2 macromolecular complex. We compared wild-type and transgenic mice in which the usually highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A+/+ ). In wild-type myocytes, PP-1 increased Ca2+ spark frequency by two-fold, followed by depletion of the sarcoplasmic reticulum Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A+/+ cardiomyocytes did not respond to both PP-1c and PDP3 treatment. Our results suggest an increased Ca2+ sensitivity of RyR2 upon de-phosphorylation by PP-1. Furthermore, we have confirmed the S2808 site as a target for PP-1 and as a potential link between RyR2s modulation and the cellular response.

Improvements of ER-Ca2+ Based High-Throughput Screening Method for Searching Novel RyR2 Inhibitors

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic reticulum (ER) and plays a central role in excitation-contraction coupling in the heart. When the RyR2 activity is abnormally enhanced by miss-sense mutations and/or excessive phosphorylation, spontaneous Ca2+ release occurs from the ER, which often results in fatal ventricular arrhythmias. Therefore, specific inhibitors of RyR2 are expected to have an antiarrhythmic effect on the RyR2-related arrhythmias, but currently such specific drugs have not been reported. We have recently established a screening method for isoform specific ryanodine receptor (RyR) inhibitors using ER Ca2+ indicator, R-CEPIA1er, in HEK293 cells (Murayama et al., Mol Pharmacol, 2018). Although this method is very useful, it requires 3 consecutive experimental days, i.e., culture of HEK293 cells expressing inducible RyR and R-CEPIA1er in 96-well plates on the first day, induction of protein expression by doxycycline on the next day, and assay using FlexStation3 on the third day. In order to screen a larger number of compounds more efficiently using a high-throughput screening device equipped with EM-CCD camera such as FDSS7000 (Hamamatsu), we made several improvements to this method. First, a novel bright green fluorescent indicator, G-CEPIA2er was used instead of R-CEPIA1er, which greatly improved signal-to-noise ratio. Second, measurements were done in 384 well plate with FDSS7000 just after seeding of cryopreserved cells which had been treated with doxycycline to induce RyR2 expression. These improvements resulted in a 48-fold increase in assay efficiency. This method, having allowed us to screen more than 20,000 compounds so far for both RyR1 and RyR2, proved to be a versatile and powerful tool for the development of specific RyR2-modifying reagents. This research was supported by JSPS Kakenhi grant (19K07105), AMED grant (19ek0109202s0203) and BINDS from AMED (0033).

Identifying Ryanodine Receptor Modulators: from High-Throughput Screening to Single Channel Recording

The ryanodine receptor Ca2+-release channels (RyRs) are central to cytoplasmic Ca2+ signalling in particular in the skeletal muscle of both mammals and insects. Regarding the latter, several insecticides target RyRs but recent studies have shown that specific RyR point mutations can confer insecticide resistance. Consequently, research for new RyR modulators remains crucial for crop protection. The full-length coding sequence of the Plutella xylostella RyR was synthesized and cloned into suitable mammalian expression vector. We engineered HEK-293 expressing the insect target and functionally selected stable clones by the use of Ca2+-sensitive fluorescence. In order to exclude any endogenous non-specific response, a related mock clone was also generated in parallel. Stimulation of the cells to activate the RyR elicited a Ca2+ mobilization, which was detected as a fluorescence increase using a FLIPR-TETRA instrument. This RyR cell-based assay was validated with RyR ligands and putative blockers and further optimized to make it suitable for high throughput screening. Drug discovery programs, which may involve the screening of >105 compounds, requires a high fidelity method of determining the effect of putative hits on the function of the target protein. Since RyR expression is targeted to internal membranes, patch-clamp of the nuclear membrane has been previously shown to be an effective method of isolating them for single-channel recording. We therefore set up this very challenging technique to measure the RyR activity single-channel properties (conductance, open probability -Po- and dwell time distribution) and to show the modulatory effect of Ca2+ on these above-mentioned parameters. From cell cloning to Mode of Action determination, we develop an assay funnel, optimized for hit sensitivity and specificity that is easily scalable to larger compound libraries.

Fifty-Hertz low frequency magnetic field modifies sarcoplasmic reticulum function

Among the putative mechanisms, by which extremely low frequency magnetic field (ELF-MF) modify calcium metabolism is that of affecting Ca2+ fluxes across cell membrane or internal Ca2+ stores. To test this hypothesis, whether ELF MF can modulate Ca2+ fluxes of isolated skeletal muscle sarcoplasmic reticulum vesicles (SR) was investigated in the present study. The Ca2+ transport and Ca2+-Mg2+-ATPase activity were observed by means of dynamic Ca2+ dye spectrum, the function of ryanodine receptor (RyR1) was monitored by [3H]-ryanodine binding assay. The membrane fluidity of SR was measured by fluorescence polarization. 50 Hz 0.4 mT MF exposure for 30 min led to a significant decrease in the Ca2+ uptake initial rate and Ca2+-Mg2+-ATPase activity compared to sham exposed SR. These results strongly suggest that prevention of SR Ca2+ uptake by ELF MF exposure was due to the inhibition of Ca2+-Mg2+- ATPase activity, and the increase of SR Ca2+ release was due to the activation of RyR1.