Dysregulated intracellular Ca2+ signaling is implicated in a variety of cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia. Spontaneous diastolic Ca 2+ 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 (Ca 2+ 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 Ca 2+ 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.calcium-induced calcium release | luminal calcium | RyR2 deactivation | sarcoplasmic reticulum

Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α+/− myocytes resulted in reduced Ca2+ waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca2+ regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart.

These results suggest that the arrhythmogenic adverse effects of CGs on Ca(2+) handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.

Key pointsr Phosphorylation at Ser-2808 is suggested to result in RyR2 hyperactivity, i.e. 'leakiness' , thus contributing to the pathology of cardiac diseases.r We studied the effect of disabling phosphorylation at Ser-2808 of RyR2 in a genetic model of Ca 2+ -dependent cardiomyopathy, which was caused by leaky RyR2. r These results suggest a novel mode of RyR2 regulation via dephosphorylation at Ser-2808 in normal and diseased hearts.Abstract Phosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) at Ser-2808 is suggested to mediate the physiological 'fight or flight' response and contribute to heart failure by rendering the sarcoplasmic reticulum (SR) leaky for Ca 2+ . In the present study, we examined the potential role of RyR2 phosphorylation at Ser-2808 in the progression of Ca 2+ -dependent cardiomyopathy (CCM) by using mice genetically modified to feature elevated SR Ca 2+ leak while expressing RyR2s that cannot be phosphorylated at this site (S2808A). Surprisingly, rather than alleviating the disease phenotype, constitutive dephosphorylation of Ser-2808 aggravated CCM as manifested by shortened survival, deteriorated in vivo cardiac function, exacerbated SR Ca 2+ leak and mitochondrial injury. Notably, the deteriorations of cardiac function, myocyte Ca 2+ handling, and mitochondria integrity were consistently worse in mice with heterozygous ablation of Ser-2808 than in mice with complete ablation. Wild-type (WT) and CCM myocytes expressing unmutated RyR2s exhibited a high level of baseline phosphorylation at Ser-2808. Exposure of these CCM cells to protein phosphatase 1 caused a transitory increase in Ca 2+ leak attributable to partial dephosphorylation of RyR2 tetramers at Ser-2808 from more fully phosphorylated state. Thus, exacerbated Ca 2+ leak through partially dephosphorylated RyR2s accounts for the prevalence of the disease phenotype in the heterozygous S2808A CCM mice. These results do not support the importance of RyR2 hyperphosphorylation in Ca 2+ -dependent heart disease, and rather suggest roles for the opposite process, the RyR2 dephosphorylation at this residue in physiological and pathophysiological Ca 2+ signalling.

While the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction (EC) coupling that enhances the efficiency of cardiac SR Ca store utilization. This “efficient” mode of EC coupling involves reciprocal changes in the phosphorylation of RyR2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 (M2R) and activation of PKG, whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 (M3R) and decreased ROS-dependent activation of CaMKII. The overall effect of these changes in phosphorylation of RyR2 is an increase in systolic Ca release at the low SR Ca content, and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered RyR2 phosphorylation balance.