Extinguishing intracellular calcium leak – a promising anti-arrhythmic approach

Ventricular arrhythmias remain a leading cause of sudden cardiac death (SCD) in the United States. Triggered activity (TA) is one of the major mechanisms causing tachyarrhythmias, the other two common causes being automaticity and reentry.¹ TA occurs when one or multiple spontaneously generated heart beats originate from an action potential that produces an afterdepolarization large enough to reach threshold membrane potential.² Historically, afterdepolarizations have been characterized as early (EAD) or delayed (DAD) depending on whether they originate before or after completion of repolarization, respectively.³ The cellular origin of DADs has long been attributed to abnormal Ca²⁺ release from the sarcoplasmic reticulum (SR), leading to activation of inward Na⁺/Ca²⁺-exchanger current (I_NCX). Several recent studies also have implicated abnormal SR Ca²⁺ release as a mechanisms underlying EADs, in addition to the ‘traditional’ mechanism involving reactivation of the L-type Ca²⁺ current by changes in membrane potential.^{4, 5}

The principal Ca²⁺ release channel on the SR is the type 2 ryanodine receptors (RyR2), which is activated by the relatively small amount of Ca²⁺ that enters the cytosol via voltage-dependent L-type Ca²⁺ channels during phase 2 of the action potential. This process of Ca²⁺-induced Ca²⁺ release (CICR) then results in a quantitatively much greater release of Ca²⁺ from the SR, which triggers myocyte contraction.⁶ Relaxation occurs during diastole when cytosolic Ca²⁺ is returned into the SR by the Ca²⁺-ATPase (SERCA2a) or extruded from the cell by NCX. An alternative theory for activation of RyR2 has been proposed by Jiang et al.⁷, who suggested that RyR2 can open independent of Ca²⁺ influx as a result of ‘store overload-induced Ca²⁺ release’ (SOICR). At this time, there is little or no solid experimental evidence for this SOICR phenomenon and its relevance for cardiac arrhythmias.⁸ However, it is clear that the level of SR Ca²⁺ loading sensitizes RyR2 channels to release, although the molecular mechanisms responsible for this feature remain unclear.

In patients with ‘catecholaminergic polymorphic ventricular tachycardia’ (CPVT), inherited mutations in RyR2 cause TA and lethal tachyarrhythmias.^{9, 10} Single channel recordings have revealed that these pathogenic RyR2 mutations increase the probabilistic opening of RyR2.¹⁰ Abnormal mutant RyR2 openings have also been shown to initiate spontaneous SR Ca²⁺ sparks and arrhythmogenic SR Ca²⁺ waves in ventricular myocytes from mice with RyR2 mutations.^{5, 11} Importantly, these abnormal SR Ca²⁺ release events occur in the absence of elevated SR Ca²⁺ contents.^{5, 12} It is believed that local elevations of [Ca²⁺] at one Ca²⁺ release unit can trigger Ca²⁺ release of neighboring RyR2 clusters, resulting in subcellular propagation and the formation of Ca²⁺ waves. Thus, abnormal Ca²⁺ waves underlying afterdepolarizations can occur as a result of ‘leaky’ RyR2 even in the absence of Ca²⁺ store overload. Under certain conditions such as elevated adrenergic tone, these Ca²⁺ waves occur in a sufficient number of myocytes leading to TA at the tissue level, which can lead to arrhythmia induction.^{11, 13}

In view of the important role of ‘leaky’ RyR2 in the development of afterdepolarizations and TA, a new class of antiarrhythmic drugs was developed that normalizes RyR2 channel functions without completely blocking the channel during CICR.⁶ The first example of a compound in this class is the benzothiazepine derivative JTV519 (also known as K201), which was shown to reduce RyR2 open probability and prevent ventricular tachycardia in a mouse model of FKBP12.6 deficiency.¹⁴ Lehnart et al.¹⁵ also demonstrated that JTV519 normalizes channel dysfunction caused by CPVT-linked missense mutations in RyR2. Subsequently, several JTV519 derivatives (referred to as ‘Rycals’) have been developed with improved RyR2-specificity, reduce off-target effects, and enhance oral availability.¹⁶ Compound S107 was shown to suppress premature ventricular contractions (PVCs) and spontaneous sustained VT in mdx mice, a mouse model of Duchenne’s muscular dystrophy.¹⁷

The class 1c antiarrhythmic drug flecainide was also shown to reduce the open probability of RyR2 in a calsquestrin-deficient (Casq2−/−) mouse model of CPVT and to reduce the occurrence of ventricular arrhythmias in CPVT patients.¹⁸ Hillard et al.¹⁹ subsequently demonstrated that flecainide reduces salutatory Ca²⁺ wave propagation between adjacent Ca²⁺ release units by inhibition of open state RyR2, without affecting SR Ca²⁺ content. Moreover, flecainide was shown to suppress spontaneous Ca²⁺ release events in RyR2 mutant mice heterozygous for CPVT-linked mutation R4496C (R4496/+).²⁰

Most recently, Dr. Chen’s group reported that the nonselective beta-blocker carvedilol and three related analogues (VK-II-86, CS-I-34 and CS-I-59) suppressed ventricular tachyarrhythmias in the R4496/+ mouse model of CPVT by reducing RyR2 open probability.²¹ Unlike other beta blockers, carvedilol suppressed the occurrence of spontaneous Ca²⁺ waves in ventricular myocytes isolated from R4496/+ mice. However, the mechanisms by which carvedilol analogous suppressed SR Ca²⁺ release and afterdepolarizations remained unclear.

In this issue of HeartRhythm, Maruyama et al.²² tested the hypothesis that a new carvedilol analogue VK-II-36 prevents TA through the suppression of EADs. Optical mapping studies of [Ca²⁺] and voltage in the epicardial surface of ventricles of R4496/+ mice revealed EADs and DADs coinciding with SCaEs at artificially slowed heart rates. Interestingly, the carvedilol analogue VK-II-36 prevented both types of afterdepolarizations, indicating that ‘leaky’ RyR2 were responsible for both EADs and DADs in this model. The findings are consistent with an important role for SR Ca²⁺ ‘leak’ in the pathogenesis of EADs, and they confirm that reactivation of L-type Ca²⁺ channels is not necessarily required for EAD genesis.

The paper also represents the first demonstration of antiarrhythmic effects of the carvedilol analogue VK-II-36 in a large animal model. In a rabbit model of acquired long QT syndrome induced by I_Kr blocker R-4031, VK-II-36 reduced action potential duration (APD) and dispersion. Those effects appeared to be caused by inhibition of abnormal SR Ca²⁺ release events, since the effects of VK-II-36 were primarily on the regions with long APDs. On the other hand, it remains to be established whether VK-II-36 has ‘off target’ effects on ion channels other than RyR2, which could modulate APD independent of VK-II-36’s effect on SR Ca²⁺ release. In addition, it would be interesting to determine in future studies how VK-II-86 compares to other carvedilol analogues in terms of its dose response, RyR2 selectivity, and oral availability.²¹

The findings of the paper by Maruyama et al.²² also are significant because they suggest that RyR2-modulating antiarrhythmic drugs could prevent triggered activity associated with EADs, not just DADs. It remains to be established whether this is true for carvedilol derivatives only, or for the entire class of RyR2 modulators that also includes JTV519, S107, and flecainide. The clinical indications for carvedilol derivatives and RyR2 modulators in general go beyond the rare inherited arrhythmia syndrome, as RyR2 gain-of-function activity has been causally linked to atrial fibrillation and lethal ventricular arrhythmias in failing hearts in various animal models. The lack of beta-blocking effects of the carvedilol analogue represents a favorable feature and suggests that these drugs could prevent arrhythmias without negative inotropic or chronotropic effects. Thus, inhibition of abnormal SR Ca²⁺ release via RyR2 represents a promising target for antiarrhythmic drug development.