Carvedilol is one of the most effective beta-blockers for preventing ventricular tachyarrhythmias (VTs) in heart failure (HF), but the mechanisms underlying its favorable anti-arrhythmic benefits remain unclear. Spontaneous Ca2+ waves, also termed store-overload-induced Ca2+ release (SOICR), are known to evoke VTs in patients with HF. Here we show that carvedilol is the only beta-blocker that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol combined with its beta-blocking activity likely contributes to its favorable anti-arrhythmic effect. To allow individual and optimal titration of these beneficial activities, we developed a novel SOICR-inhibiting, minimally-beta-blocking carvedilol analogue VK-II-86. We found that VK-II-86 alone prevented stress-induced VTs in RyR2 mutant mice, and was more effective when combined with a selective beta-blocker metoprolol or bisoprolol. Thus, SOICR inhibition combined with optimal beta-blockade presents a new, promising and potentially patient-tailorable anti-arrhythmic approach.

Spontaneous Ca2+ release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca2+ release (SOICR) can result in Ca2+ waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here, we show that a point mutation E4872A in the helix bundle crossing (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, RyR2 Ca2+ activation. Introducing metal-binding histidines at this site converts RyR2 into a luminal Ni2+ gated channel. Mouse hearts harboring an RyR2 mutation at this site (E4872Q+/−) are resistant to store overload-induced Ca2+ waves and completely protected against Ca2+-triggered VTs. These data show that the RyR2 gate directly senses store Ca2+, explaining RyR2 store Ca2+ regulation, Ca2+ wave initiation, and Ca2+-triggered arrhythmias. This novel store-sensing gate structure is conserved in all RyRs and inositol 1,4,5-trisphosphate receptors.

Carvedilol is the current β-blocker of choice for suppressing ventricular tachyarrhythmia (VT). However, carvedilol’s benefits are dose-limited, attributable to its potent β-blocking activity that can lead to bradycardia and hypotension. The clinically used carvedilol is a racemic mixture of β-blocking S-carvedilol and non-β-blocking R-carvedilol. We recently reported that novel non-β-blocking carvedilol analogues are effective in suppressing arrhythmogenic Ca2+ waves and stress-induced VT without causing bradycardia. Thus, the non-β-blocking R-carvedilol enantiomer may also possess this favourable anti-arrhythmic property. To test this possibility, we synthesized R-carvedilol and assessed its effect on Ca2+ release and VT. Like racemic carvedilol, R-carvedilol directly reduces the open duration of the cardiac ryanodine receptor (RyR2), suppresses spontaneous Ca2+ oscillations in human embryonic kidney (HEK) 293 cells, Ca2+ waves in cardiomyocytes in intact hearts and stress-induced VT in mice harbouring a catecholaminergic polymorphic ventricular tachycardia (CPVT)-causing RyR2 mutation. Importantly, R-carvedilol did not significantly alter heart rate or blood pressure. Therefore, the non-β-blocking R-carvedilol enantiomer represents a very promising prophylactic treatment for Ca2+-triggered arrhythmia without the bradycardia and hypotension often associated with racemic carvedilol. Systematic clinical assessments of R-carvedilol as a new anti-arrhythmic agent may be warranted.

Carvedilol is a uniquely effective drug for the treatment of cardiac arrhythmias in patients with heart failure. This activity is in part due to its ability to inhibit store overload-induced calcium release (SOICR) through the RyR2 channel. We describe the synthesis, characterization and bioassay of ca. 100 compounds based on the carvedilol motif in order to identify features that correlate with and optimize SOICR inhibition. A single cell bioassay was employed based on the RyR2-R4496C mutant HEK-293 cell line, in which calcium release from the endoplasmic reticulum through the defective channel was measured. IC50 values for SOICR inhibition were thus obtained. The compounds investigated contained modifications to the three principal subunits of carvedilol, including the carbazole and catechol moieties, as well as the linker chain containing the β-amino alcohol functionality. The SAR results indicate that significant alterations are tolerated in each of the three subunits.

JNK2 activation up-regulates CaMKIIδ expression in the aged atrium. This JNK2 regulation in CaMKIIδ expression occurs at the transcription level through the JNK downstream transcription factor c-jun. The discovery of this novel molecular mechanism of JNK2-regulated CaMKII expression sheds new light on possible anti-arrhythmia drug development.

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 Ca waves during store Ca overload, also known as store overload-induced Ca 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 Ca 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 Ca 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 Ca-triggered arrhythmias.

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del+/−) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del+/− mice are not susceptible to CPVT. Ex3-del+/− cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca2+ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del+/− mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del+/− mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del+/− mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.

Cardiac calsequestrin (CASQ2) is commonly believed to serve as the sarcoplasmic reticulum (SR) luminal Ca2+ sensor. Ablation of CASQ2 promotes spontaneous Ca2+ waves (SCWs) and catecholaminergic polymorphic ventricular tachycardia (CPVT) upon stress but not at rest. How SCWs and CPVT are triggered by stress in the absence of the CASQ2-based luminal Ca2+ sensor is an important unresolved question. Here we assessed the role of the newly identified RyR2-resident luminal Ca2+ sensor in determining SCW propensity, CPVT susceptibility, and cardiac hypertrophy in Casq2 knockout (KO) mice. We crossbred Casq2 KO mice with RyR2 mutant (E4872Q+/−) mice, which lack RyR2-resident SR luminal Ca2+ sensing, to generate animals with both deficiencies. Casq2+/− and Casq2−/− mice showed stress-induced ventricular tachyarrhythmias (VTs), whereas Casq2+/−/E4872Q+/− and Casq2−/−/E4872Q+/− mice displayed little or no stress-induced VTs. Confocal Ca2+ imaging revealed that Casq2−/− hearts frequently exhibited SCWs after extracellular Ca2+ elevation or adrenergic stimulation, while Casq2−/−/E4872Q+/− hearts had few or no SCWs under the same conditions. Cardiac hypertrophy developed and CPVT susceptibility increased with age in Casq2−/− mice, but not in Casq2−/−/E4872Q+/− mice. However, the amplitudes and dynamics of voltage-induced Ca2+ transients in Casq2−/− and Casq2−/−/E4872Q+/− hearts were not significantly different. Our results indicate that SCWs, CPVT and hypertrophy in Casq2 null cardiac muscle are governed by the RyR2-resident luminal Ca2+ sensor. This implies that defects in CASQ2-based luminal Ca2+ sensing can be overridden by the RyR2-resident luminal Ca2+ sensor. This makes this RyR2-resident sensor a promising molecular target for the treatment of Ca2+-mediated arrhythmias.