Short Communication: Flecainide Exerts an Antiarrhythmic Effect in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia by Increasing the Threshold for Triggered ActivityNovelty and Significance
Rationale: Flecainide prevents arrhythmias in catecholaminergic polymorphic ventricular tachycardia, but the antiarrhythmic mechanism remains unresolved. It is possible for flecainide to directly affect the cardiac ryanodine receptor (RyR2); however, an extracellular site of action is suggested because of the hydrophilic nature of flecainide.
Objective: To investigate the mechanism for the antiarrhythmic action of flecainide in a RyR2R4496C+/− knock-in mouse model of catecholaminergic polymorphic ventricular tachycardia.
Methods and Results: Flecainide prevented catecholamine-induced sustained ventricular tachycardia in RyR2R4496C+/− mice. Cellular studies were performed with isolated RyR2R4496C+/− myocytes. Isoproterenol caused the appearance of spontaneous Ca2+ transients, which were unaffected by flecainide (6 μmol/L). Flecainide did not affect Ca2+ transient amplitude, decay, or sarcoplasmic reticulum Ca2+ content. Moreover, it did not affect the frequency of spontaneous Ca2+ sparks in permeabilized myocytes. In contrast, flecainide effectively prevented triggered activity induced by isoproterenol. The threshold for action potential induction was increased significantly (P<0.01), which suggests a primary extracellular antiarrhythmic effect mediated by Na+ channel blockade.
Conclusions: Flecainide prevents catecholaminergic polymorphic ventricular tachycardia in RyR2R4496C+/− mice; however, at variance with previous reports, we observed minimal effects on intracellular Ca2+ homeostasis. Our data suggest that the antiarrhythmic activity of the drug is caused by reduction of Na+ channel availability and by an increase in the threshold for triggered activity.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by adrenergically mediated polymorphic ventricular tachycardia (VT) that leads to syncope and sudden cardiac death.1 Although β-adrenergic blockers are the mainstay therapy in CPVT patients, approximately 30% of patients receiving this treatment still experience cardiac arrhythmias and eventually require an implantable cardioverter-defibrillator.1 Abnormal diastolic calcium leak from the mutant cardiac ryanodine receptor (RyR2) is responsible for the induction of triggered activity (TA), which is the pivotal arrhythmogenic mechanism in CPVT.1 Potential antiarrhythmic strategies therefore include either targeting the abnormal calcium leak or targeting the ionic mechanisms responsible for the generation of TA. It has been proposed recently that flecainide prevents arrhythmias in calsequestrin knockout mice and in CPVT patients by blocking RyR2 and by decreasing its opening probability.2,3 In the present study, we systematically investigated the antiarrhythmic mechanism of flecainide in our RyR2R4496C+/− knock-in mouse model of CVPT4 and found that inhibition of TA by reducing the availability of sodium channels accounts for the unexpected antiarrhythmic efficacy of the drug in CPVT.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
ECG radiotelemetry monitors were implanted, and drug stress with coinjection of epinephrine and caffeine was performed as described previously in RyR2R4496C+/− (patent number US11/429167) mice.4 Ca2+ imaging and a patch clamp study were performed in isolated ventricular RyR2R4496C+/− myocytes. For flecainide diffusion across cell membrane, intact myocytes were incubated with flecainide for 30 minutes.
Flecainide Prevents Sustained VT in RyR2R4496C+/− Mice
Sustained VT occurred in 70% of RyR2R4496C+/− mice when challenged with epinephrine (2 mg/kg IP) and caffeine (120 mg/kg IP). In contrast, only 8% (1/12) of the animals had these arrhythmias when pretreated with flecainide (15 mg/kg; P<0.001; Figure 1A). Thus, flecainide exerts a powerful antiarrhythmic activity in RyR2R4496C+/− mice.
Effects of Flecainide on Intracellular Ca2+
The antiarrhythmic action of flecainide may be due to a direct action of the drug on intracellular Ca2+ homeostasis. We therefore evaluated this hypothesis in isolated ventricular RyR2R4496C+/− myocytes. Isoproterenol (1 μmol/L) increased the amplitude of Ca2+ transients, accelerated the Ca2+ transient decay, and increased the sarcoplasmic reticulum (SR) Ca2+ load. Despite the blockade of INa, flecainide (6 μmol/L, incubation for 30 minutes) had no effect on any of these parameters (Figures 1B and 1C), in agreement with Hilliard et al.3 In contrast, tetracaine (50 μmol/L) significantly increased the Ca2+ transient amplitude and SR Ca2+ load, which suggests that tetracaine but not flecainide is able to block RyR2 in intact myocytes.
We next investigated the effects of flecainide on SR function in permeabilized RyR2R4496C+/− myocytes. Tetracaine (50 μmol/L), but not flecainide (25 μmol/L), decreased the spontaneous Ca2+ wave frequency compared with untreated cells (Figure 2A). Flecainide had no effect on the frequency of the sparks, whereas tetracaine decreased the frequency of the sparks by ≈20%, which is consistent with the fact that tetracaine is a RyR2 blocker (Figure 2; Online Table I).5
Flecainide Does Not Prevent Spontaneous SR Ca2+ Release
Spontaneous Ca2+ transients (SCaTs) originating from abnormal SR Ca2+ release are a potential arrhythmogenic trigger. We next investigated the occurrence of SCaTs in intact RyR2R4496C+/− myocytes. Under basal conditions, only ≈4% of the myocytes (n=27 cells) developed SCaTs when paced at 1 Hz (Figure 3A). The incidence of SCaTs was increased to 87% (n=30 cells) by isoproterenol (1 μmol/L). Flecainide (6 μmol/L, incubation for 30 minutes) had no effect on the incidence of SCaTs, which occurred in ≈85% of myocytes (n=14 cells; Figure 3B). In contrast, tetracaine (50 μmol/L) reduced the incidence of SCaTs to ≈15% (P<0.001; n=23 cells).
SCaTs often led to the occurrence of triggered events (arrow in Figure 3A). In the presence of flecainide, 7% of myocytes developed triggered events in the presence of isoproterenol at 1 Hz pacing compared with 60% in the absence of flecainide (P<0.001; Figure 3C). These data demonstrate that the SCaTs elicited by isoproterenol were largely unaffected by flecainide, but the drug inhibited the development of triggered events.
Flecainide Abolishes Isoproterenol-Induced TA
We performed patch-clamp studies to investigate whether flecainide inhibits delayed afterdepolarization (DAD)–mediated TA. As reported previously,4 DADs and TA developed readily in RyR2R4496C+/− myocytes on exposure to isoproterenol (1 μmol/L), whereas exposure to flecainide (6 μmol/L) failed to abolish DADs but fully suppressed TA (n=6 cells; Figure 4A). In some cells, we performed simultaneous action potential (AP) recording and Ca2+ imaging to confirm that flecainide abolishes DAD-mediated TA with no effect on the underlying SCaTs (Figure 4B). Collectively, these data argue that at this concentration, flecainide preferentially alters AP properties rather than SR Ca2+ release in RyR2R4496C+/− myocytes. Flecainide directly affected AP properties by reducing the AP amplitude in a frequency-dependent manner (Figure 4C; Online Figure I). These data are consistent with the well-described use dependent block of the drug on the Na+ channel. We directly examined whether flecainide affects AP initiation by incrementally increasing the stimulus current to determine the threshold for AP induction. Flecainide (6 μmol/L) significantly augmented the minimal current required to elicit an AP (from 657±181 to 1171±269 pA, P<0.001; n=7 cells; Figure 4D).
Does Flecainide Have an Intracellular Effect on Excitability?
To determine whether flecainide exerts any effect on myocyte physiology once introduced into the intracellular compartment, we dialyzed flecainide (25 μmol/L) into RyR2R4496C+/− ventricular myocytes through the patch pipette (for >5 minutes). Intracellular flecainide had no effect on AP properties (Online Figure I and Table II), which suggests an extracellular mechanism of action. Moreover, under these conditions, isoproterenol (1 μmol/L) was still able to induce TA (Online Figure II); thus, only extracellular flecainide was able to abolish TA, which suggests the importance of its Na+ channel–blocking properties.
It has been agreed that RyR2 mutations cause instability of the RyR2 complex in response to adrenergic stimulation, which in turn facilitates abnormal diastolic Ca2+ release and development of DADs and TA. There is a pressing clinical need to identify new and effective antiarrhythmic approaches. We observed that flecainide has pronounced antiarrhythmic effects in vivo in our RyR2R4496C+/− mice, which supports the view that flecainide may be beneficial for the management of CPVT patients. The mechanisms of antiarrhythmic action of flecainide in patients with CPVT are not fully elucidated and may involve inhibition of the SR leak or an inhibitory effect of the electrophysiological consequences of abnormal diastolic calcium release.
Recently, Watanabe et al2 and Hilliard et al3 demonstrated that flecainide directly affects SR Ca2+ leak in myocytes derived from mice with a recessive form of CPVT. These authors showed that flecainide is able to inhibit SCaTs and Ca2+ sparks in intact myocytes derived from a calsequestrin knockout model of CPVT3 by blocking the release channel in its open state.4 However, we rationalized that given its high pKa (9.3), flecainide is charged primarily at physiological pH: thus, with only a mere 1% of neutral flecainide available for diffusion across the membrane, we considered it unlikely that an in vivo effect on RyR2 could account for the antiarrhythmic efficacy of the drug.6 We tested this hypothesis and found that when we applied flecainide to intact myocytes derived from our RyR2R4496C+/− knock-in mice,4 we failed to demonstrate a reduction of isoproterenol-induced SCaTs. Similarly, in permeabilized myocytes, we were unable to observe an effect on SR Ca2+ release events even at concentrations as high as 25 μmol/L. When we administered flecainide intracellularly through a patch pipette, we were still unable to observe any effect of flecainide on DADs and TA. However, although extracellular application of flecainide (6 μmol/L) failed to affect SCaTs or DADs, it rapidly inhibited TA, which demonstrates that it is the extracellular presence of flecainide that mediates its antiarrhythmic activity in our CPVT mouse model. It is theoretically possible that the discrepancy between the present findings and those of Knollmann et al are attributable to different experimental conditions or to the use of a different CPVT mouse model.
Our novel interpretation of the antiarrhythmic effect of flecainide is that it reduces the availability of sodium channels, thus preventing the development of triggered APs. It has been proposed that sodium channel blockers can prevent TA in cardiac myocytes by alleviation of elevated cytosol Na+ in some settings, such as ischemia and digitalis administration.7 Obviously, the efficacy of flecainide in preventing TA in the present study was independent of the cytosol Na+ level and therefore represents a novel antiarrhythmic mechanism of the compound. We found that the minimal current required to elicit an AP in RyR2R4496C+/− myocytes was increased significantly by flecainide, which suggests that an increased DAD amplitude leading to a more depolarized cell membrane would be necessary to elicit an AP in the presence of flecainide. Because the Na+ channel–blocking effect of flecainide is strongly frequency dependent, it is reasonable to propose that flecainide will have a dramatically enhanced effect to prevent TA at elevated heart rates. It makes the drug particularly effective during fast heart rates, ie, in the conditions in which CPVT patients develop cardiac arrhythmias. Whether the antiarrhythmic effect in CPVT observed with flecainide is shared by all class I antiarrhythmic agents deserves clinical evaluation. Some distinguishing properties of flecainide such as its high bioavailability and long half-life may give the drug a composite profile that makes it the class I antiarrhythmic agent of choice for CPVT patients.
The evidence that 30% of CPVT patients taking β-blocker therapy who also have an implantable cardioverter-defibrillator receive appropriate shocks to terminate life-threatening arrhythmias calls for the identification of drugs that act on a different target to potentiate the antiadrenergic effect of β-blockers and increase the “safety margin” to prevent VT. The sodium channel–blocking action of flecainide appears to be a good option to achieve control of arrhythmic episodes and reduce the need for an implantable cardioverter-defibrillator in CPVT patients.
Sources of Funding
This work was supported by Telethon grants Nos. GGP04066 and GGP06007 and Italian Ministero dell'Università e della Ricerca Scientifica e Tecnologica grants FIRB RBNE01XMP4 006, RBLA035A4X_002, PRIN 2006055828_002, RFMAU207641137D, and Fondation Leducq Award to the Alliance for Calmodulin Kinase Signaling in Heart Disease (08CVD01).
In May 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days.
This manuscript was sent to Gordon Tomaselli, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Non-standard Abbreviations and Acronyms
- action potential
- catecholaminergic polymorphic ventricular tachycardia
- delayed afterdepolarization
- ryanodine receptor type 2
- spontaneous Ca2+ transient
- sarcoplasmic reticulum
- triggered activity
- Received January 22, 2011.
- Received April 27, 2011.
- Revision received May 24, 2011.
- Accepted June 2, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal inherited arrhythmogenic disease. Present therapy is inadequate.
Abnormal diastolic calcium leak from the mutant cardiac ryanodine receptor (RyR2) is responsible for the induction of triggered activity, which is the pivotal arrhythmogenic mechanism in CPVT.
Flecainide, a sodium channel blocker, prevents ventricular arrhythmias in CPVT patients and in a CPVT transgenic mouse model. The leading hypothesis is that flecainide blocks RyR2 and therefore abolishes the abnormal diastolic calcium leak that generates cardiac arrhythmias.
What New Information Does This Article Contribute?
Flecainide prevents triggered activity by reducing Na+ channel availability and increasing the threshold for triggered activity.
Flecainide does not prevent abnormal diastolic calcium leak in RyR2R4496C+/− myocytes.
We investigated the mechanism by which flecainide prevents triggered activity induced by β-adrenergic stimulation in a knock-in animal model of CPVT that is heterozygous for the mutation R4496C in the RyR2 gene. Our data show that flecainide does not block abnormal diastolic calcium leak; however, it prevents the development of triggered beats by reducing the inward sodium current, thereby increasing the threshold for triggered activity. These observations bear important implications for the selection of therapeutic strategies in patients with CPVT.