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Gain-of-function missense variants in the cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), whereas RyR2 loss-offunction missense variants cause Ca2+ release deficiency syndrome (CRDS). Recently, truncating variants in RyR2 have also been associated with ventricular arrhythmias (VAs) and sudden cardiac death. However, there are limited insights into the potential clinical relevance and in vitro functional impact of RyR2 truncating variants. We performed genetic screening of patients presenting with syncope, VAs, or unexplained sudden death and in vitro characterization of the expression and function of RyR2 truncating variants in HEK293 cells. We identified two previously unknown RyR2 truncating variants (Y4591Ter and R4663Ter) and one splice site variant predicted to result in a frameshift and premature termination (N4717 + 15Ter). These 3 new RyR2 truncating variants and a recently reported RyR2 truncating variant, R4790Ter, were generated and functionally characterized in vitro. Immunoprecipitation and immunoblotting analyses showed that all 4 RyR2 truncating variants formed heteromers with the RyR2-wildtype (WT) protein. Each of these C-terminal RyR2 truncations was non-functional and suppressed [3H]ryanodine binding to RyR2-WT and RyR2-WT mediated store overload induced spontaneous Ca2+ release activity in HEK293 cells. The expression of these RyR2 truncating variants in HEK293 cells was markedly reduced compared with that of the full-length RyR2 WT protein. Our data indicate that C-terminal RyR2 truncating variants are non-functional and can exert a dominant negative impact on the function of the RyR2 WT protein through formation of heteromeric WT/truncation complex. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Reduced protein expression of the cardiac ryanodine receptor type 2 (RyR2) is thought to affect the susceptibility to stress-induced ventricular tachyarrhythmia (VT) and cardiac alternans, but direct evidence for the role of RyR2 protein expression in VT and cardiac alternans is lacking. Here, we used a mouse model (crrm1) that expresses a reduced level of the RyR2 protein to determine the impact of reduced RyR2 protein expression on the susceptibility to VT, cardiac alternans, cardiac hypertrophy, and sudden death. Electrocardiographic analysis revealed that after the injection of relatively high doses of caffeine and epinephrine (agents commonly used for stress test), wild-type (WT) mice displayed long-lasting VTs, whereas the crrm1 mutant mice exhibited no VTs at all, indicating that the crrm1 mutant mice are resistant to stress-induced VTs. Intact heart Ca2+ imaging and action potential (AP) recordings showed that the crrm1 mutant mice are more susceptible to fast-pacing induced Ca2+ alternans and AP duration alternans compared with WT mice. The crrm1 mutant mice also showed an increased heart-to-body-weight ratio and incidence of sudden death at young ages. Furthermore, the crrm1 mutant hearts displayed altered Ca2+ transients with increased time-to-peak and decay time (T50), increased ventricular wall thickness and ventricular cell area compared with WT hearts. These results indicate that reduced RyR2 protein expression suppresses stress-induced VTs, but enhances the susceptibility to cardiac alternans, hypertrophy, and sudden death. [ABSTRACT FROM AUTHOR]

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-Larginine 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. [ABSTRACT FROM AUTHOR]

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, Rcarvedilol 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. [ABSTRACT FROM AUTHOR]

CASQ2 (cardiac calsequestrin) is commonly believed to serve as the SR (sarcoplasmic reticulum) luminal Ca2+ sensor. Ablation of CASQ2 promotes SCWs (spontaneous Ca2+ waves) and CPVT (catecholaminergic polymorphic ventricular tachycardia) 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. In the present study, we assessed the role of the newly identified RyR2 (ryanodine receptor 2)-resident luminal Ca2+ sensor in determining SCW propensity, CPVT susceptibility and cardiac hypertrophy in Casq2-KO (knockout) 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 VTs (ventricular tachyarrhythmias), 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, whereas 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. [ABSTRACT FROM AUTHOR]

RyR2 (cardiac ryanodine receptor)-mediated Ca2+ release in cardiomyocytes terminates when the sarcoplasmic reticulum Ca2+ content depletes to a threshold level, known as the termination threshold. Despite its importance, little is known about the mechanism that regulates the termination threshold. CaM (calmodulin), by inhibiting RyR2, has been implicated in Ca2+ -release termination, but whether CaM modulates the termination threshold is unknown. To this end, we monitored the endoplasmic reticulum Ca2+ dynamics in RyR2-expressing HEK (human embryonic kidney)-293 cells transfected with WT (wild-type) CaM or mutants. We found that WT CaM or CaM mutations which abolish Ca2+ binding to the N-lobe (N-terminal lobe) of CaM increased the termination threshold (i.e. facilitated termination), but had no effect on the activation threshold at which spontaneous Ca2+ release occurs. On the other hand, CaM mutations that diminish Ca2+ binding to both the N-lobe and C-lobe (C-terminal lobe), or the C-lobe only, decreased the termination threshold (i.e. delayed termination) with a similar activation threshold. Furthermore, deletion of residues 3583- 3603 or point mutations (W3587A/L3591D/F3603A, W3587A, or L3591D) in the CaM-binding domain of RyR2 that are known to abolish or retain CaM binding all reduced the termination thresholdwithout having a significant impact on the activation threshold. Interestingly, the RyR2-F3603A mutation affected both the activation and termination threshold. Collectively, these data indicate that CaM facilitates the termination of Ca2+ release by increasing the termination threshold, and that this action of CaM depends on Ca2+ binding to the C-lobe, but not to the N-lobe, of CaM. The results of the present study also suggest that the CaM-binding domain of RyR2 is an important determinant of Ca2+ -release termination and activation. [ABSTRACT FROM AUTHOR]