Andriy E. Belevych

Redox modification of ryanodine receptors contributes to sarcoplasmic reticulum Ca2+ leak in chronic heart failure.

Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca2+]c) and sarcoplasmic reticulum (SR) luminal Ca2+ ([Ca2+]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca2+ leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca2+]SR in HF compared to control cells. Both SR Ca2+ leak and [Ca2+]SR were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca2+ leak and decreased [Ca2+]SR in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca2+-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca2+ and was partially normalized by reducing agents through restoring luminal Ca2+ sensitivity oxidation of control RyR2s enhanced their luminal Ca2+ sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.

miR-1 Overexpression Enhances Ca2+ Release and Promotes Cardiac Arrhythmogenesis by Targeting PP2A Regulatory Subunit B56α and Causing CaMKII-Dependent Hyperphosphorylation of RyR2

MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation–contraction coupling and Ca2+ cycling in rat ventricular myocytes using methods of electrophysiology, Ca2+ imaging and quantitative immunoblotting. Adenoviral-mediated overexpression of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca2+ current, flattening of Ca2+ transients voltage dependence, and enhanced frequency of spontaneous Ca2+ sparks while reducing the sarcoplasmic reticulum Ca2+ content as compared with control. In the presence of isoproterenol, rhythmically paced, miR-1–overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca2+, events that occurred rarely in control myocytes under the same conditions. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Although phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor (RyR2) at S2814 (Ca2+/calmodulin-dependent protein kinase) but not at S2808 (protein kinase A). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56α involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 enhances cardiac excitation–contraction coupling by selectively increasing phosphorylation of the L-type and RyR2 channels via disrupting localization of PP2A activity to these channels.

Redox modification of ryanodine receptors underlies calcium alternans in a canine model of sudden cardiac death

AIMS Although cardiac alternans is a known predictor of lethal arrhythmias, its underlying causes remain largely undefined in disease settings. The potential role of, and mechanisms responsible for, beat-to-beat alternations in the amplitude of systolic Ca(2+) transients (Ca(2+) alternans) was investigated in a canine post-myocardial infarction (MI) model of sudden cardiac death (SCD). METHODS AND RESULTS Post-MI dogs had preserved left ventricular (LV) function and susceptibility to ventricular fibrillation (VF) during exercise. LV wedge preparations from VF dogs were more susceptible to action potential (AP) alternans and the frequency-dependence of Ca(2+) alternans was shifted towards slower rates in myocytes isolated from VF dogs relative to controls. In both groups of cells, cytosolic Ca(2+) transients ([Ca(2+)](c)) alternated in phase with changes in diastolic Ca(2+) in sarcoplasmic reticulum ([Ca(2+)](SR)), but the dependence of [Ca(2+)](c) amplitude on [Ca(2+)](SR) was steeper in VF cells. Abnormal ryanodine receptor (RyR) function in VF cells was indicated by increased fractional Ca(2+) release for a given amplitude of Ca(2+) current and elevated diastolic RyR-mediated SR Ca(2+) leak. SR Ca(2+) uptake activity did not differ between VF and control cells. VF myocytes had an increased rate of reactive oxygen species production and increased RyR oxidation. Treatment of VF myocytes with reducing agents normalized parameters of Ca(2+) handling and shifted the threshold of Ca(2+) alternans to higher frequencies. CONCLUSION Redox modulation of RyRs promotes generation of Ca(2+) alternans by enhancing the steepness of the Ca(2+) release-load relationship and thereby providing a substrate for post-MI arrhythmias.

MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex.

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca2+ release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca2+ cycling, and increased propensity to arrhythmogenesis.

The Relationship Between Arrhythmogenesis and Impaired Contractility in Heart Failure: Role of Altered Ryanodine Receptor Function

AIMS In heart failure (HF), abnormal myocyte Ca(2+) handling has been implicated in cardiac arrhythmias and contractile dysfunction. In the present study, we investigated the relationships between Ca(2+) handling, reduced myocyte contractility, and enhanced arrhythmogenesis during HF progression in a canine model of non-ischaemic HF. METHODS AND RESULTS Key Ca(2+) handling parameters were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) [Ca(2+)] in isolated ventricular myocytes at different stages of HF. The progression of HF was associated with an early and continuous increase in ryanodine receptor (RyR2)-mediated SR Ca(2+) leak. The increase in RyR2 activity was paralleled by an increase in the frequency of diastolic spontaneous Ca(2+) waves (SCWs) in HF myocytes under conditions of β-adrenergic stimulation. In addition to causing arrhythmogenic-delayed afterdepolarizations, SCWs decreased the amplitude of subsequent electrically evoked Ca(2+) transients by depleting SR Ca(2+). At late stages of HF, Ca(2+) release oscillated essentially independent of electrical pacing. The increased propensity for the generation of SCWs in HF myocytes was attributable to reduced ability of the RyR2 channels to become refractory following Ca(2+) release. The progressive alterations in RyR2 function and Ca(2+) cycling in HF myocytes were associated with sequential modifications of RyR2 by CaMKII-dependent phosphorylation and thiol oxidation. CONCLUSION These findings suggest that destabilized RyR2 activity due to excessive CaMKII phopshorylation and oxidation resulting in impaired post-release refractoriness is a common mechanism involved in arrhythmogenesis and contractile dysfunction in the failing heart.

Shortened Ca2+ Signaling Refractoriness Underlies Cellular Arrhythmogenesis in a Postinfarction Model of Sudden Cardiac Death

Rationale: Diastolic spontaneous Ca2+ waves (DCWs) are recognized as important contributors to triggered arrhythmias. DCWs are thought to arise when [Ca2+] in sarcoplasmic reticulum ([Ca2+]SR) reaches a certain threshold level, which might be reduced in cardiac disease as a consequence of sensitization of ryanodine receptors (RyR2s) to luminal Ca2+. Objective: We investigated the mechanisms of DCW generation in myocytes from normal and diseased hearts, using a canine model of post–myocardial infarction ventricular fibrillation (VF). Methods and Results: The frequency of DCWs, recorded during periodic pacing in the presence of a &bgr;-adrenergic receptor agonist isoproterenol, was significantly higher in VF myocytes than in normal controls. Rather than occurring immediately on reaching a final [Ca2+]SR, DCWs arose with a distinct time delay after attaining steady [Ca2+]SR in both experimental groups. Although the rate of [Ca2+]SR recovery after the SR Ca2+ release was similar between the groups, in VF myocytes the latency to DCWs was shorter, and the [Ca2+]SR at DCW initiation was lower. The restitution of depolarization-induced Ca2+ transients, assessed by a 2-pulse protocol, was significantly faster in VF myocytes than in controls. The VF-related alterations in myocyte Ca2+ cycling were mimicked by the RyR2 agonist, caffeine. The reducing agent, mercaptopropionylglycine, or the CaMKII inhibitor, KN93, decreased DCW frequency and normalized restitution of Ca2+ release in VF myocytes. Conclusions: The attainment of a certain threshold [Ca2+]SR is not sufficient for the generation of DCWs. Postrelease Ca2+ signaling refractoriness critically influences the occurrence of DCWs. Shortened Ca2+ signaling refractoriness due to RyR2 phosphorylation and oxidation is responsible for the increased rate of DCWs observed in VF myocytes and could provide a substrate for synchronization of arrhythmogenic events at the tissue level in hearts prone to VF.

Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex

AIMS Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. METHODS AND RESULTS In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca(2+) cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2(-/-)) mice. Casq2(-/-) mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca(2+) cycling, including abnormal Ca(2+) release, periods of significantly elevated diastolic Ca(2+) levels leading to pauses and unstable pacemaker rate. Importantly, Ca(2+) cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca(2+) transient upstrokes throughout the atrial pacemaker complex. CONCLUSIONS Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca(2+) release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients.

Effects of dietary omega–3 fatty acids on ventricular function in dogs with healed myocardial infarctions: in vivo and in vitro studies

Since omega-3 polyunsaturated fatty acids (n-3 PUFAs) can alter ventricular myocyte calcium handling, these fatty acids could adversely affect cardiac contractile function, particularly following myocardial infarction. Therefore, 4 wk after myocardial infarction, dogs were randomly assigned to either placebo (corn oil, 1 g/day, n = 16) or n-3 PUFAs supplement [docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) ethyl esters; 1, 2, or 4 g/day; n = 7, 8, and 12, respectively] groups. In vivo, ventricular function was evaluated by echocardiography before and after 3 mo of treatment. At the end of the 3-mo period, hearts were removed and in vitro function was evaluated using right ventricular trabeculae and isolated left ventricular myocytes. The treatment elicited significant (P < 0.0001) dose-dependent increases (16.4-fold increase with 4 g/day) in left ventricular tissue and red blood cell n-3 PUFA levels (EPA + DHA, placebo, 0.42 +/- 0.04; 1 g/day, 3.02 +/- 0.23; 2 g/day, 3.63 +/- 0.17; and 4 g/day, 6.97 +/- 0.33%). Regardless of the dose, n-3 PUFA treatment did not alter ventricular function in the intact animal (e.g., 4 g/day, fractional shortening: pre, 42.9 +/- 1.6 vs. post, 40.1 +/- 1.7%; placebo: pre, 39.2 +/- 1.3 vs. post, 38.4 +/- 1.6%). The developed force per cross-sectional area, changes in length- and frequency-dependent behavior in contractile force, and the inotropic response to beta-adrenoceptor activation were also similar for trabeculae obtained from placebo- or n-3 PUFA-treated dogs. Finally, calcium currents and calcium transients were the same in myocytes from n-3 PUFA- and placebo-treated dogs. Thus dietary n-3 PUFAs did not adversely alter either in vitro or in vivo ventricular contractile function in dogs with healed infarctions.

Decreased RyR2 refractoriness determines myocardial synchronization of aberrant Ca2+ release in a genetic model of arrhythmia

Dysregulated intracellular Ca2+ signaling is implicated in a variety of cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia. Spontaneous diastolic Ca2+ release (DCR) can induce arrhythmogenic plasma membrane depolarizations, although the mechanism responsible for DCR synchronization among adjacent myocytes required for ectopic activity remains unclear. We investigated the synchronization mechanism(s) of DCR underlying untimely action potentials and diastolic contractions (DCs) in a catecholaminergic polymorphic ventricular tachycardia mouse model with a mutation in cardiac calsequestrin. We used a combination of different approaches including single ryanodine receptor channel recording, optical imaging (Ca2+ and membrane potential), and contractile force measurements in ventricular myocytes and intact cardiac muscles. We demonstrate that DCR occurs in a temporally and spatially uniform manner in both myocytes and intact myocardial tissue isolated from cardiac calsequestrin mutation mice. Such synchronized DCR events give rise to triggered electrical activity that results in synchronous DCs in the myocardium. Importantly, we establish that synchronization of DCR is a result of a combination of abbreviated ryanodine receptor channel refractoriness and the preceding synchronous stimulated Ca2+ release/reuptake dynamics. Our study reveals how aberrant DCR events can become synchronized in the intact myocardium, leading to triggered activity and the resultant DCs in the settings of a cardiac rhythm disorder.

'Ryanopathy': causes and manifestations of RyR2 dysfunction in heart failure.

The cardiac ryanodine receptor (RyR2), a Ca(2+) release channel on the membrane of the sarcoplasmic reticulum (SR), plays a key role in determining the strength of the heartbeat by supplying Ca(2+) required for contractile activation. Abnormal RyR2 function is recognized as an important part of the pathophysiology of heart failure (HF). While in the normal heart, the balance between the cytosolic and intra-SR Ca(2+) regulation of RyR2 function maintains the contraction-relaxation cycle, in HF, this behaviour is compromised by excessive post-translational modifications of the RyR2. Such modification of the Ca(2+) release channel impairs the ability of the RyR2 to properly deactivate leading to a spectrum of Ca(2+)-dependent pathologies that include cardiac systolic and diastolic dysfunction, arrhythmias, and structural remodelling. In this article, we present an overview of recent advances in our understanding of the underlying causes and pathological consequences of abnormal RyR2 function in the failing heart. We also discuss the implications of these findings for HF therapy.