Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca ²⁺ ] _c ) and sarcoplasmic reticulum (SR) luminal Ca ²⁺ ([Ca ²⁺ ]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca ²⁺ leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca ²⁺ ] _SR in HF compared to control cells. Both SR Ca ²⁺ leak and [Ca ²⁺ ] _SR were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca ²⁺ leak and decreased [Ca ²⁺ ] _SR in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca ²⁺ -handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca ²⁺ and was partially normalized by reducing agents through restoring luminal Ca ²⁺ sensitivity oxidation of control RyR2s enhanced their luminal Ca ²⁺ sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.

The cardiac ryanodine receptor (RyR2) is the sarcoplasmic reticulum (SR) Ca²⁺ release channel which is responsible for generation of the cytosolic Ca²⁺ transient required for activation of cardiac contraction. RyR2 functional activity is governed by changes in [Ca²⁺] on both the cytosolic and luminal phase of the RyR2 channel. Activation of RyR2 by cytosolic Ca²⁺ results in Ca²⁺-induced Ca²⁺ release (CICR) from the SR. The decline in luminal [Ca²⁺] following release contributes to termination of CICR and Ca²⁺ signalling refractoriness through the process of luminal Ca²⁺-dependent deactivation of RyR2s. The control of RyR2s by luminal Ca²⁺ involves coordinated interaction of the channel with several SR proteins, including the Ca²⁺-binding protein calsequestrin (CASQ2), and the integral proteins triadin 1 (TRD) and junctin (JCN). CASQ2 in addition to serving as a Ca²⁺ storage site and a luminal Ca²⁺ buffer modulates RyR2 function more directly as a putative luminal Ca²⁺ sensor. TRD and JCN, stimulatory by themselves, mediate the interactions between CASQ2 and RyR2. Acquired and genetic defects in proteins of this junctional Ca²⁺ signalling complex lead to disease states such as cardiac arrhythmia and heart failure by impairing luminal Ca²⁺ regulation of RyR2.

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 Ca²⁺ transients (Ca²⁺ alternans) was investigated in a canine post-myocardial infarction (MI) model of sudden cardiac death (SCD). 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 Ca²⁺ alternans was shifted towards slower rates in myocytes isolated from VF dogs relative to controls. In both groups of cells, cytosolic Ca²⁺ transients ([Ca²⁺]_c) alternated in phase with changes in diastolic Ca²⁺ in sarcoplasmic reticulum ([Ca²⁺]_SR), but the dependence of [Ca²⁺]_c amplitude on [Ca²⁺]_SR was steeper in VF cells. Abnormal ryanodine receptor (RyR) function in VF cells was indicated by increased fractional Ca²⁺ release for a given amplitude of Ca²⁺ current and elevated diastolic RyR-mediated SR Ca²⁺ leak. SR Ca²⁺ 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 Ca²⁺ handling and shifted the threshold of Ca²⁺ alternans to higher frequencies. Redox modulation of RyRs promotes generation of Ca²⁺ alternans by enhancing the steepness of the Ca²⁺ release–load relationship and thereby providing a substrate for post-MI arrhythmias.

In heart failure (HF), abnormal myocyte Ca²⁺ handling has been implicated in cardiac arrhythmias and contractile dysfunction. In the present study, we investigated the relationships between Ca²⁺ handling, reduced myocyte contractility, and enhanced arrhythmogenesis during HF progression in a canine model of non-ischaemic HF. Key Ca²⁺ handling parameters were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) [Ca²⁺] 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 Ca²⁺ leak. The increase in RyR2 activity was paralleled by an increase in the frequency of diastolic spontaneous Ca²⁺ 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 Ca²⁺ transients by depleting SR Ca²⁺. At late stages of HF, Ca²⁺ 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 Ca²⁺ release. The progressive alterations in RyR2 function and Ca²⁺ cycling in HF myocytes were associated with sequential modifications of RyR2 by CaMKII-dependent phosphorylation and thiol oxidation. 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.

Rationale:: Diastolic spontaneous Ca ²⁺ waves (DCWs) are recognized as important contributors to triggered arrhythmias. DCWs are thought to arise when [Ca ²⁺ ] in sarcoplasmic reticulum ([Ca ²⁺ ] _SR ) reaches a certain threshold level, which might be reduced in cardiac disease as a consequence of sensitization of ryanodine receptors (RyR2s) to luminal Ca ²⁺ . 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 β-adrenergic receptor agonist isoproterenol, was significantly higher in VF myocytes than in normal controls. Rather than occurring immediately on reaching a final [Ca ²⁺ ] _SR , DCWs arose with a distinct time delay after attaining steady [Ca ²⁺ ] _SR in both experimental groups. Although the rate of [Ca ²⁺ ] _SR recovery after the SR Ca ²⁺ release was similar between the groups, in VF myocytes the latency to DCWs was shorter, and the [Ca ²⁺ ] _SR at DCW initiation was lower. The restitution of depolarization-induced Ca ²⁺ transients, assessed by a 2-pulse protocol, was significantly faster in VF myocytes than in controls. The VF-related alterations in myocyte Ca ²⁺ cycling were mimicked by the RyR2 agonist, caffeine. The reducing agent, mercaptopropionylglycine, or the CaMKII inhibitor, KN93, decreased DCW frequency and normalized restitution of Ca ²⁺ release in VF myocytes. Conclusions:: The attainment of a certain threshold [Ca ²⁺ ] _SR is not sufficient for the generation of DCWs. Postrelease Ca ²⁺ signaling refractoriness critically influences the occurrence of DCWs. Shortened Ca ²⁺ 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.