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Multiple modes of ryanodine receptor 2 inhibition by flecainide.

ABSTRACT: Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na(+) and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca(2+)], [Mg(2+)], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ?1 ms, and 2) a slow block consisting of closed events with a mean duration of ?1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca(2+)] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide.

SUBMITTER: Mehra D

PROVIDER: S-EPMC4244595 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Multiple modes of ryanodine receptor 2 inhibition by flecainide.

Mehra D D Imtiaz M S MS van Helden D F DF Knollmann B C BC Laver D R DR

Molecular pharmacology 20141001 6

Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na(+) and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating con ...[more]

PMID: 25274603

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Project description:Flecainide suppresses cardiac tachyarrhythmias including paroxysmal atrial fibrillation, supraventricular tachycardia and arrhythmic long QT syndromes (LQTS), as well as the Ca2+ -mediated, catecholaminergic polymorphic ventricular tachycardia (CPVT). However, flecainide can also exert pro-arrhythmic effects most notably following myocardial infarction and when used to diagnose Brugada syndrome (BrS). These divergent actions result from its physiological and pharmacological actions at multiple, interacting levels of cellular organization. These were studied in murine genetic models with modified Nav channel or intracellular ryanodine receptor (RyR2)-Ca2+ channel function. Flecainide accesses its transmembrane Nav 1.5 channel binding site during activated, open, states producing a use-dependent antagonism. Closing either activation or inactivation gates traps flecainide within the pore. An early peak INa related to activation of Nav channels followed by rapid de-activation, drives action potential (AP) upstrokes and their propagation. This is diminished in pro-arrhythmic conditions reflecting loss of function of Nav 1.5 channels, such as BrS, accordingly exacerbated by flecainide challenge. Contrastingly, pro-arrhythmic effects attributed to prolonged AP recovery by abnormal late INaL following gain-of-function modifications of Nav 1.5 channels in LQTS3 are reduced by flecainide. Anti-arrhythmic effects of flecainide that reduce triggering in CPVT models mediated by sarcoplasmic reticular Ca2+ release could arise from its primary actions on Nav channels indirectly decreasing [Ca2+ ]i through a reduced [Na+ ]i and/or direct open-state RyR2-Ca2+ channel antagonism. The consequent [Ca2+ ]i alterations could also modify AP propagation velocity and therefore arrhythmic substrate through its actions on Nav 1.5 channel function. This is consistent with the paradoxical differences between flecainide actions upon Na+ currents, AP conduction and arrhythmogenesis under circumstances of normal and increased RyR2 function.Linked articlesThis article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Project description:RationaleThe class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT.ObjectiveTo determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo.Methods and resultsWe synthesized N-methylated flecainide analogues (QX-flecainide and N-methyl flecainide) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes-lacking intact sarcolemma and devoid of sodium channel contribution-flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N-methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas N-methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block.ConclusionsFlecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

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Multiple modes of ryanodine receptor 2 inhibition by flecainide.

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Multiple modes of ryanodine receptor 2 inhibition by flecainide.

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