Single RyR2 Research Articles

Expression level of cardiac ryanodine receptors dictates properties of Ca2+-induced Ca2+ release

The type 2 ryanodine receptor (RyR2) is the major Ca2+ release channel required for Ca2+-induced Ca2+ release (CICR) and cardiac excitation-contraction coupling. The cluster organization of RyR2 at the dyad is critical for efficient CICR. Despite its central role in cardiac Ca2+ signaling, the mechanisms that control CICR are not fully understood. As a single RyR2 Ca2+ flux dictates local CICR that underlies Ca2+ sparks, RyR2 density in a cluster, and therefore the distance between RyR2s, should have a profound impact on local CICR. Here, we studied the effect of the RyR2 expression level ([RyR2]) on CICR activation, termination, and amplitude. The endoplasmic reticulum (ER)-targeted Ca2+ sensor RCEPIA-1er was used to directly measure the ER [Ca2+] (Ca2+]ER) in the T-Rex-293 the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) stable cell line expressing human RyR2. Cells coexpressing RyR2 and SERCA2a produced periodic [Ca2+]ER depletions in the form of spontaneous Ca2+ waves due to propagating CICR. For each studied cell, the [Ca2+]ER at which Ca2+ waves are activated and terminated was analyzed as a function of [RyR2]. CICR parameters, such as [Ca2+]ER activation, termination, and amplitude, were inversely proportional to [RyR2] at low-intermediate levels. Increasing the sensitivity of RyR2 to cytosolic Ca2+ lowered the [Ca2+]ER at which CICR is activated and terminated. Decreasing the sensitivity of RyR2 to cytosolic Ca2+ had the opposite effect on CICR. These results suggest that RyR2 density in the release cluster should have a significant impact on local CICR activation and termination. Since SR Ca2+ load is evenly distributed throughout the SR network, clusters with higher RyR2 density would have a higher probability of initiating spontaneous CICR.

Isolated Cardiac Ryanodine Receptor Function Varies Between Mammals.

Concerted robust opening of cardiac ryanodine receptors' (RyR2) Ca2+ release 1oplasmic reticulum (SR) is fundamental for normal systolic cardiac function. During diastole, infrequent spontaneous RyR2 openings mediate the SR Ca2+ leak that normally constrains SR Ca2+ load. Abnormal large diastolic RyR2-mediated Ca2+ leak events can cause delayed after depolarizations (DADs) and arrhythmias. The RyR2-associated mechanisms underlying these processes are being extensively studied at multiple levels utilizing various model animals. Since there are well-described species-specific differences in cardiac intracellular Ca2+ handing in situ, we tested whether or not single RyR2 function in vitro retains this species specificity. We isolated RyR2-rich heavy SR microsomes from mouse, rat, rabbit, and human ventricular muscle and quantified RyR2 function using identical solutions and methods. The single RyR2 cytosolic Ca2+ sensitivity was similar across these species. However, there were significant species differences in single RyR2 mean open times in both systole and diastole-like solutions. In diastole-like solutions, single rat/mouse RyR2 open probability and frequency of long openings (> 6ms) were similar, but these values were significantly greater than those of either single rabbit or human RyR2s. We propose these in vitro single RyR2 functional differences across species stem from the species-specific RyR2 regulatory environment present in the source tissue. Our results show the single rabbit RyR2 functional attributes, particularly in diastole-like conditions, replicate those of single human RyR2 best among the species tested.

Ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic in Casq2 -/- Mice.

Intracellular Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in RyR2 single channel and [3H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single channels and RyR2-mediated spontaneous Ca2+ release in Casq2 -/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 minutes and half-life of 45 minutes after intraperitoneal administration of 3 mg/kg in mice. In vivo, ent-B1 significantly reduced catecholamine-induced ventricular arrhythmias in Casq2 -/- mice in a dose-dependent manner. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. SIGNIFICANCE STATEMENT: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

Diverse Phenotypic Manifestations in a Family with a Novel RYR2 E4107A Variant.

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.

Generation of two induced pluripotent stem cell lines from catecholaminergic polymorphic ventricular tachycardia patients carrying RYR2 mutations

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a congenital arrhythmic syndrome caused by the RYR2 gene encoded ryanodine receptor. Mutations on RYR2 are commonly associated with ventricular tachycardia after adrenergic stimulation, leading to lethal arrhythmias and sudden cardiac death. We generated two human induced pluripotent stem cell (iPSC) lines from CPVT affected patients carrying single missense heterozygote RYR2 mutations, c.1082 G > A and c.100 A > C. Pluripotency and differentiation capability into derivatives of three germ layers were evaluated along with karyotype stability in the report. The generated patient-specific iPSC lines provide a reliable tool to investigate the CPVT phenotype and understand underlaying mechanisms.

Modeling Calcium Cycling in the Heart: Progress, Pitfalls, and Challenges.

Intracellular calcium (Ca) cycling in the heart plays key roles in excitation-contraction coupling and arrhythmogenesis. In cardiac myocytes, the Ca release channels, i.e., the ryanodine receptors (RyRs), are clustered in the sarcoplasmic reticulum membrane, forming Ca release units (CRUs). The RyRs in a CRU act collectively to give rise to discrete Ca release events, called Ca sparks. A cell contains hundreds to thousands of CRUs, diffusively coupled via Ca to form a CRU network. A rich spectrum of spatiotemporal Ca dynamics is observed in cardiac myocytes, including Ca sparks, spark clusters, mini-waves, persistent whole-cell waves, and oscillations. Models of different temporal and spatial scales have been developed to investigate these dynamics. Due to the complexities of the CRU network and the spatiotemporal Ca dynamics, it is challenging to model the Ca cycling dynamics in the cardiac system, particularly at the tissue sales. In this article, we review the progress of modeling of Ca cycling in cardiac systems from single RyRs to the tissue scale, the pros and cons of the current models and different modeling approaches, and the challenges to be tackled in the future.

β2-adrenergic stimulation potentiates spontaneous calcium release by increasing signal mass and co-activation of ryanodine receptor clusters.

It is unknown how β-adrenergic stimulation affects calcium dynamics in individual RyR2 clusters and leads to the induction of spontaneous calcium waves. To address this, we analysed spontaneous calcium release events in green fluorescent protein (GFP)-tagged RyR2 clusters. Cardiomyocytes from mice with GFP-tagged RyR2 or human right atrial tissue were subjected to immunofluorescent labelling or confocal calcium imaging. Spontaneous calcium release from single RyR2 clusters induced 91.4%±2.0% of all calcium sparks while 8.0%±1.6% were caused by release from two neighbouring clusters. Sparks with two RyR2 clusters had 40% bigger amplitude, were 26% wider, and lasted 35% longer at half maximum. Consequently, the spark mass was larger in two- than one-cluster sparks with a median and interquartile range for the cumulative distribution of 15.7±20.1 vs 7.6±5.7 a.u. (P<.01). β2-adrenergic stimulation increased RyR2 phosphorylation at s2809 and s2815, tripled the fraction of two- and three-cluster sparks, and significantly increased the spark mass. Interestingly, the amplitude and mass of the calcium released from a RyR2 cluster were proportional to the SR calcium load, but the firing rate was not. The spark mass was also higher in 33 patients with atrial fibrillation than in 36 without (22.9±23.4 a.u. vs 10.7±10.9; P=.015). Most sparks are caused by activation of a single RyR2 cluster at baseline while β-adrenergic stimulation doubles the mass and the number of clusters per spark. This mimics the shift in the cumulative spark mass distribution observed in myocytes from patients with atrial fibrillation.

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.

RYR2 Channel Inhibition Is the Principal Mechanism of Flecainide Action in CPVT.

The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT. To determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. We synthesized N-methylated flecainide analogues (QX-flecainide and N-methyl flecainide) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes-lacking intact sarcolemma and devoid of sodium channel contribution-flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N-methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas N-methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

Abstract 15420: Resolving a Controversy: Inhibition of Cardiac Ryanodine Receptors is the Principal Mechanism of Antiarrhythmic Action of Flecainide in Catecholaminergic Polymorphic Ventricular Tachycardia

Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive cardiac ryanodine receptor (RyR2) calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro , reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide’s efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide’s efficacy for suppressing spontaneous sarcoplasmic reticulum (SR) Ca release and for preventing ventricular tachycardia in vivo . Methods and Results: We synthesized N -methyl flecainide analogues (QX-FL and NM-FL) and showed that N -methylation reduces flecainide’s inhibitory potency on RyR2 channels but not on cardiac sodium channels. Antiarrhythmic efficacy was tested utilizing a calsequestrin knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes — lacking intact sarcolemma and devoid of sodium channel contribution — flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin (TTX) to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous SR Ca release, while QX-FL and NM-FL did not. In vivo , flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas NM-FL did not, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone was not enough to prevent arrhythmias. Hence, RyR2 inhibition by flecainide is critical for its mechanism of antiarrhythmic action.

Pathological phosphorylation of the ryanodine receptor at s2808 increases the number of individual clusters activated per calcium spark and the calcium released per cluster

Abstract Background Atrial fibrillation (AF) has been associated with an increase in ryanodine receptor (RyR2) phosphorylation and local calcium release (sparks), but it is not known how calcium dynamics of individual RyR2 clusters affect spark dimensions and properties. Purpose This study aimed to test the hypothesis that pathological alterations in the phosphorylation of individual RyR2 clusters at s2808 facilitate the fusion of spontaneous calcium release events from neighboring RyR2 clusters. Methods Cardiomyocytes from mice with GFP-tagged RyR2 or human right atrial tissue were subjected to confocal calcium imaging or immunofluorescent labelling of total and s2808 phosphorylated RyR2. Calcium signals were measured at a frame rate of 240 Hz in a 0.5 x 0.5 μm region of interest (ROI) for each GFP-tagged RyR2 cluster and spontaneous calcium release events were detected using a custom-made algorithm. Results Calcium sparks recorded in 41 myocytes with GFP-tagged RyR2s was due to the spontaneous opening of a single RyR2 cluster in 91.2±2.2% of the cells and two neighbouring clusters in (6.2±1.6%) of the cells. Events with two clusters had bigger amplitude (0.14±0.01 vs. 0.10±0.01, p&amp;lt;0.05), were wider (1.43±0.03 vs. 1.13±0.04 μm, p&amp;lt;0.05), and lasted longer at half maximum (59.8±5.2 vs. 44.4±2.4 ms, p&amp;lt;0.01). Consequently, the calcium spark mass, measured as the time integral of the spark in each ROI increased from 9.2±1.6 for 1 cluster to 17.8±3.5 a.u. for 2 clusters (p&amp;lt;0.01). Interestingly, sparks lasted longer (79±5 vs. 61±4 ms, p&amp;lt;0.001) were wider (3.0±0.2 vs. 2.2±0.1 μm, p&amp;gt;0.001) and had bigger mass (31.5±3.3 vs. 21.9±3.3 a.u, p&amp;lt;0.01) in atrial myocytes from 21 patients with AF than in 27 without. Because phosphorylation of RyR2 clusters at s2808 (s2808/total RyR2) was higher in patients with than without AF (0.80±0.19 vs. 0.44±0.03, p&amp;lt;0.05), we tested how stimulation of RyR2 phosphorylation at s2808 with the β2-adrenergic agonist fenoterol (3μM) affected calcium release in individual RyR2 clusters. Fenoterol increased s2808 phosphoryaltion from 0.39±0.05 to 0.79±0.16 (p&amp;lt;0.05, n=9). It also increased the mass of sparks with 1 RyR2 cluster (from 9.2±1.1 to 16.0±2.3 a.u., p&amp;lt;0.01) and sparks with 2 clusters from 17.8±3.5 to 23.6±2.7 a.u. Moreover, it increased the fraction of sparks with 2 clusters from 6.2±1.6% to 19.3±3.3% (p&amp;lt;0.01) and sparks with 3 clusters reached 6.3±1.9% in the presence of fenoterol. Conclusions The calcium spark mass recorded in patients without AF is comparable to that recorded during activation of calcium release from one or two GFP-tagged RyR2 clusters. The larger mass and slower kinetics of sparks recorded in patients with AF is compatible with an increase in the calcium released from each RyR2 cluster and a 3-fold increase in sparks with 2 or 3 RyR2 clusters observed in GFP-tagged RyR2s when phosphorylation at s2808 is increased to levels observed 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; Generatlitat de Catalunya

Molecular basis of high glucose-mediated cardiac calcium mishandling

Epidemiological and preclinical studies have pointed out a correlation between hyperglycemia and increasing risk of heart failure and cardiac death. In cardiomyocytes, hyperglycemia has been shown to alter Ca2 + signalling via CaMKII. Yet, the underlying mechanisms are still unclear. To determine the molecular basis of high glucose-mediated Ca2 + mishandling. Ventricular cardiomyocytes were isolated from control and Epac2-KO mice. Parallel experiments were repeated in human cardiomyocytes differentiated from induced pluripotent stem cells (h-iPSC-CM). Ca2 + signaling was studied in cells loaded with a fluorescent Ca2 + dye and treated with high glucose (HG) ± ESI-05 (Epac2 inhibitor). We assessed ryanodine receptor (RyR2) phosphorylation state by western blot and measured single RyR2 channel activity incorporated into bilayers. HG-mediated SR Ca2 + leak and pro-arrhythmic events were prevented with Epac2 blocker (ESI-05) in normal mice cells and with Epac2 deletion (Epac2-KO) (Ca2 + sparks frequency N /100 μm/s: 0.23 ± 0.15 for HG + ESI-05, 0.09 ± 0.04 for Epac2-KO + HG vs. 0.16 ± 0.06 for normal glucose, P = N.S.). HG treatment increased RyR2 phosphorylation at the CaMKII site (S2814, ∼ 29% increase, P < 0.05) leading to a dramatic increase in RyR2 open probability (P0 = 0.76 ± 0.07 in HG vs. 0.22 ± 0.07 in NG, P < 0.01) fully prevented with ESI-05 (P0 = 0.15 ± 0.03, P = N.S.). Similarly, in h-iPSC-CM, chronic HG (1 week) increased the percentage of cells presenting pro-arrhythmic events (85% of cells). Electrically evoked [Ca2 + ]i transients were decreased in h-IPSC-CM (F/F0 = 2.10 ± 0.07 vs. 2.94 ± 0.17, P < 0.01) and associated to higher RyR-S2814 phosphorylation. Those effects were blunted by ESI-05. Our data suggests that HG alters Ca2 + signalling via Epac2–mediated pro-arrhythmic events due a CaMKII-dependent increase of RyR2 activity. Overtime, this newly described mechanism lowers systolic Ca2 + release as seen in diabetic-associated cardiomyopathy.

N-Acetylcysteine Prevents the Spatial Memory Deficits and the Redox-Dependent RyR2 Decrease Displayed by an Alzheimer's Disease Rat Model.

We have previously reported that primary hippocampal neurons exposed to synaptotoxic amyloid beta oligomers (AβOs), which are likely causative agents of Alzheimer’s disease (AD), exhibit abnormal Ca2+ signals, mitochondrial dysfunction and defective structural plasticity. Additionally, AβOs-exposed neurons exhibit a decrease in the protein content of type-2 ryanodine receptor (RyR2) Ca2+ channels, which exert critical roles in hippocampal synaptic plasticity and spatial memory processes. The antioxidant N-acetylcysteine (NAC) prevents these deleterious effects of AβOs in vitro. The main contribution of the present work is to show that AβOs injections directly into the hippocampus, by engaging oxidation-mediated reversible pathways significantly decreased RyR2 protein content but increased single RyR2 channel activation by Ca2+ and caused considerable spatial memory deficits. AβOs injections into the CA3 hippocampal region impaired rat performance in the Oasis maze spatial memory task, decreased hippocampal glutathione levels and overall content of plasticity-related proteins (c-Fos, Arc, and RyR2) and increased ERK1/2 phosphorylation. In contrast, in hippocampus-derived mitochondria-associated membranes (MAM) AβOs injections increased RyR2 levels. Rats fed with NAC for 3-weeks prior to AβOs injections displayed comparable redox potential, RyR2 and Arc protein contents, similar ERK1/2 phosphorylation and RyR2 single channel activation by Ca2+ as saline-injected (control) rats. NAC-fed rats subsequently injected with AβOs displayed the same behavior in the spatial memory task as control rats. Based on the present in vivo results, we propose that redox-sensitive neuronal RyR2 channels partake in the mechanism underlying AβOs-induced memory disruption in rodents.

High-Fat-Diet-Induced Obesity Produces Spontaneous Ventricular Arrhythmias and Increases the Activity of Ryanodine Receptors in Mice.

Ventricular arrhythmias are a common cause of sudden cardiac death, and their occurrence is higher in obese subjects. Abnormal gating of ryanodine receptors (RyR2), the calcium release channels of the sarcoplasmic reticulum, can produce ventricular arrhythmias. Since obesity promotes oxidative stress and RyR2 are redox-sensitive channels, we investigated whether the RyR2 activity was altered in obese mice. Mice fed a high fat diet (HFD) became obese after eight weeks and exhibited a significant increase in the occurrence of ventricular arrhythmias. Single RyR2 channels isolated from the hearts of obese mice were more active in planar bilayers than those isolated from the hearts of the control mice. At the molecular level, RyR2 channels from HFD-fed mice had substantially fewer free thiol residues, suggesting that redox modifications were responsible for the higher activity. Apocynin, provided in the drinking water, completely prevented the appearance of ventricular arrhythmias in HFD-fed mice, and normalized the activity and content of the free thiol residues of the protein. HFD increased the expression of NOX4, an isoform of NADPH oxidase, in the heart. Our results suggest that HFD increases the activity of RyR2 channels via a redox-dependent mechanism, favoring the appearance of ventricular arrhythmias.

True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors

SummarySignaling nanodomains rely on spatial organization of proteins to allow controlled intracellular signaling. Examples include calcium release sites of cardiomyocytes where ryanodine receptors (RyRs) are clustered with their molecular partners. Localization microscopy has been crucial to visualizing these nanodomains but has been limited by brightness of markers, restricting the resolution and quantification of individual proteins clustered within. Harnessing the remarkable localization precision of DNA-PAINT (<10 nm), we visualized punctate labeling within these nanodomains, confirmed as single RyRs. RyR positions within sub-plasmalemmal nanodomains revealed how they are organized randomly into irregular clustering patterns leaving significant gaps occupied by accessory or regulatory proteins. RyR-inhibiting protein junctophilin-2 appeared highly concentrated adjacent to RyR channels. Analyzing these molecular maps showed significant variations in the co-clustering stoichiometry between junctophilin-2 and RyR, even between nearby nanodomains. This constitutes an additional level of complexity in RyR arrangement and regulation of calcium signaling, intrinsically built into the nanodomains.

Omecamtiv mecarbil activates ryanodine receptors from canine cardiac but not skeletal muscle

Due to the limited results achieved in the clinical treatment of heart failure, a new inotropic strategy of myosin motor activation has been developed. The lead molecule of myosin activator agents is omecamtiv mecarbil, which binds directly to the heavy chain of the cardiac β-myosin and enhances cardiac contractility by lengthening the lifetime of the acto-myosin complex and increasing the number of the active force-generating cross-bridges. In the absence of relevant data, the effect of omecamtiv mecarbil on canine cardiac ryanodine receptors (RyR 2) has been investigated in the present study by measuring the electrical activity of single RyR 2 channels incorporated into planar lipid bilayer. When applying 100nM Ca2+ concentration on the cis side ([Ca2+]cis) omecamtiv mecarbil (1–10µM) significantly increased the open probability and opening frequency of RyR 2, while the mean closed time was reduced. Mean open time was increased moderately by 10µM omecamtiv mecarbil. When [Ca2+]cis was elevated to 322 and 735nM, the effect of omecamtiv mecarbil on open probability was evident only at higher (3–10µM) concentrations. All effects of omecamtiv mecarbil were fully reversible upon washout. Omecamtiv mecarbil (up to 10µM) had no effect on the open probability of RyR 1, isolated from either canine or rabbit skeletal muscles. It is concluded that the direct stimulatory action of omecamtiv mecarbil on RyR 2 has to be taken into account when discussing the mechanism of action or the potential side effects of the compound.

Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor

Ca2+ regulates ryanodine receptor’s (RyR) activity through an activating and an inhibiting Ca2+-binding site located on the cytoplasmic side of the RyR channel. Their altered sensitivity plays an important role in the pathology of malignant hyperthermia and heart failure. We used lanthanide ions (Ln3+) as probes to investigate the Ca2+ sensors of RyR, because they specifically bind to Ca2+-binding proteins and they are impermeable to the channel. Eu3+’s and Sm3+’s action was tested on single RyR1 channels reconstituted into planar lipid bilayers. When the activating binding site was saturated by 50 μM Ca2+, Ln3+ potently inhibited RyR’s open probability (Kd Eu3+ = 167 ± 5 nM and Kd Sm3+ = 63 ± 3 nM), but in nominally 0 [Ca2+], low [Eu3+] activated the channel. These results suggest that Ln3+ acts as an agonist of both Ca2+-binding sites. More importantly, the voltage-dependent characteristics of Ln3+’s action led to the conclusion that the activating Ca2+ binding site is located within the electrical field of the channel (in the vestibule). This idea was tested by applying the pore blocker toxin maurocalcine on the cytoplasmic side of RyR. These experiments showed that RyR lost reactivity to changing cytosolic [Ca2+] from 50 μM to 100 nM when the toxin occupied the vestibule. These results suggest that maurocalcine mechanically prevented Ca2+ from dissociating from its binding site and support our vestibular Ca2+ sensor-model further.

The Cytoplasmic Region of Inner Helix S6 Is an Important Determinant of Cardiac Ryanodine Receptor Channel Gating

The ryanodine receptor (RyR) channel pore is formed by four S6 inner helices, with its intracellular gate located at the S6 helix bundle crossing region. The cytoplasmic region of the extended S6 helix is held by the U motif of the central domain and is thought to control the opening and closing of the S6 helix bundle. However, the functional significance of the S6 cytoplasmic region in channel gating is unknown. Here we assessed the role of the S6 cytoplasmic region in the function of cardiac RyR (RyR2) via structure-guided site-directed mutagenesis. We mutated each residue in the S6 cytoplasmic region of the mouse RyR2 (4876QQEQVKEDM4884) and characterized their functional impact. We found that mutations Q4876A, V4880A, K4881A, and M4884A, located mainly on one side of the S6 helix that faces the U motif, enhanced basal channel activity and the sensitivity to Ca2+ or caffeine activation, whereas mutations Q4877A, E4878A, Q4879A, and D4883A, located largely on the opposite side of S6, suppressed channel activity. Furthermore, V4880A, a cardiac arrhythmia-associated mutation, markedly enhanced the frequency of spontaneous openings and the sensitivity to cytosolic and luminal Ca2+ activation of single RyR2 channels. V4880A also increased the propensity and reduced the threshold for arrhythmogenic spontaneous Ca2+ release in HEK293 cells. Collectively, our data suggest that interactions between the cytoplasmic region of S6 and the U motif of RyR2 are important for stabilizing the closed state of the channel. Mutations in the S6/U motif domain interface likely destabilize the closed state of RyR2, resulting in enhanced basal channel activity and sensitivity to activation and increased propensity for spontaneous Ca2+ release and cardiac arrhythmias.

Nebivolol suppresses cardiac ryanodine receptor-mediated spontaneous Ca2+ release and catecholaminergic polymorphic ventricular tachycardia.

β-Blockers are a standard treatment for heart failure and cardiac arrhythmias. There are ∼30 commonly used β-blockers, representing a diverse class of drugs with different receptor affinities and pleiotropic properties. We reported that among 14 β-blockers tested previously, only carvedilol effectively suppressed cardiac ryanodine receptor (RyR2)-mediated spontaneous Ca2+ waves during store Ca2+ overload, also known as store overload-induced Ca2+ release (SOICR). Given the critical role of SOICR in arrhythmogenesis, it is of importance to determine whether there are other β-blockers that suppress SOICR. Here, we assessed the effect of other commonly used β-blockers on RyR2-mediated SOICR in HEK293 cells, using single-cell Ca2+ imaging. Of the 13 β-blockers tested, only nebivolol, a β-1-selective β-blocker with nitric oxide synthase (NOS)-stimulating action, effectively suppressed SOICR. The NOS inhibitor (N-nitro-l-arginine methyl ester) had no effect on nebivolol's SOICR inhibition, and the NOS activator (histamine or prostaglandin E2) alone did not inhibit SOICR. Hence, nebivolol's SOICR inhibition was independent of NOS stimulation. Like carvedilol, nebivolol reduced the opening of single RyR2 channels and suppressed spontaneous Ca2+ waves in intact hearts and catecholaminergic polymorphic ventricular tachycardia (CPVT) in the mice harboring a RyR2 mutation (R4496C). Interestingly, a non-β-blocking nebivolol enantiomer, (l)-nebivolol, also suppressed SOICR and CPVT without lowering heart rate. These data indicate that nebivolol, like carvedilol, possesses a RyR2-targeted action that suppresses SOICR and SOICR-evoked VTs. Thus, nebivolol represents a promising agent for Ca2+-triggered arrhythmias.

Simvastatin Activates Single Skeletal RyR1 Channels but Exerts More Complex Regulation of the Cardiac Isoform, RyR2

Statins are widely prescribed for the treatment of hypercholesterolemia. Although effective in lowering cholesterol levels, statin use has been associated with muscle weakness, myopathies and, in rare cases, rhabdomyolysis. The mechanisms underlying toxicity have not been elucidated, however, cerivastatin, a compound removed from the market due to reports of rhabdomyolysis, has been shown to trigger Ca2+ release from isolated skeletal sarcoplasmic reticulum (SR) vesicles (Inoue et al., 2003). To investigate whether statins can directly activate RyR channels, we incorporated single sheep cardiac RyR2 or mouse skeletal RyR1 channels into planar phospholipid bilayers under voltage-clamp conditions and added simvastatin, a commonly prescribed statin, to the cytosolic channel side. In the presence of 10 μM cytosolic [Ca2+], 10 μM simvastatin (carboxylate form), significantly increased RyR1 open probability (Po) from 0.020±0.013 to 0.269±0.068 (SEM, n=13, p<0.001). This effect was fully reversible after washout of simvastatin (0.033±0.029, SEM, n=6). The ability of simvastatin to activate RyR1 was further highlighted by our experiments showing that simvastatin is more effective than caffeine at stimulating [3H]ryanodine binding to skeletal SR (n=3, p<0.01). Unexpectedly, we found that 10 µM simvastatin did not activate RyR2 but that higher concentrations were required to increase Po. Low concentrations of simvastatin (1 µM) tended to reduce Po. These results may explain why the most frequently reported adverse effects of statins are of skeletal rather than cardiac origin. Confirmation that the direct modulation of RyR1 and RyR2 observed in bilayer experiments is relevant in cells is shown in permeabilised cardiac and skeletal myocytes where simvastatin increased the frequency of Ca2+ sparks in skeletal cells but decreased frequency in cardiac cells (see poster by Lotteau et al.).Funded by the British Heart Foundation.