RYR2 Channel Inhibition Is the Principal Mechanism 0f Flecainide Action in CPVT

Abstract

Rationale:

The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive cardiac ryanodine receptor (RyR2) 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.

Objective:

To determine whether RyR2 block independently contributes to flecainide’s efficacy for suppressing spontaneous sarcoplasmic reticulum (SR) Ca release and for preventing ventricular tachycardia in vivo.

Methods and Results:

We synthesized N-methylated flecainide analogues (QX-FL and NM-FL) 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 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 (TTX) to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous SR Ca release, while QX-FL and NM-FL did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2−/− mice, whereas NM-FL had no significant effect on arrhythmia burden, despite comparable sodium channel block.

Conclusions:

Flecainide 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.

Graphical Abstract

INTRODUCTION

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic arrhythmia syndrome caused by mutations in proteins that constitute the calcium (Ca) release unit of the cardiac sarcoplasmic reticulum (SR), such as ryanodine receptor (RyR2) SR Ca release channel,¹ calsequestrin (Casq2),^{2, 3} and calmodulin.^{4, 5} Mutations in these proteins cause spontaneous SR Ca release during adrenergic stress that can trigger potentially fatal ventricular arrhythmias.^{6, 7} The class Ic antiarrhythmic drug flecainide, a cardiac sodium channel blocker,⁸ exhibits striking efficacy for the prevention of ventricular arrhythmias in CPVT patients.^9–12 Despite its proven clinical efficacy in CPVT, the mechanism of flecainide action remains controversial. Based on our discovery that flecainide inhibits RyR2 Ca release channels, we have suggested that RyR2 block contributes independently to its mechanism of action in CPVT.⁹ This concept was supported by in vitro experiments showing that clinically-relevant concentrations of flecainide prevented spontaneous Ca release in CPVT cardiomyocytes even in the absence of extracellular sodium and Ca,⁹ or after cell membrane permeabilization which renders sodium channels inactive.^{13, 14} Subsequent studies have disputed the importance or action of RyR2 block by flecainide, and concluded that sodium channel block alone is responsible for flecainide’s efficacy in CPVT.^15–18 Those conclusions were based on observations that flecainide’s inhibitory potency in single RyR2 channel assays is too low to explain its clinical efficacy and that sodium channel blockers lacking single-channel RyR2-blocking properties, such as tetrodotoxin (TTX)¹⁸ or QX-flecainide (QX-FL) also suppressed spontaneous SR Ca release in intact cardiomyocytes.¹⁶

Flecainide’s mechanism of action is of major importance; hyperactive RyR2 channels are implicated in a variety of heart diseases associated with atrial and ventricular tachyarrhythmias,^19–21 but currently there is no clinically available RyR2-specific inhibitor. Uncovering whether RyR2 inhibition by flecainide plays a role for its antiarrhythmic activity would validate RyR2 as therapeutic target and facilitate the drug development of specific RyR2 inhibitors. Because of its proposed dual mode of action – sodium channel block and RyR2 block – it is difficult to resolve flecainide’s mechanism of action. It is well established that sodium channel blockers inhibit RyR2-mediated Ca release indirectly via the Na/Ca exchanger by altering intracellular sodium and hence cytosolic and intra-SR Ca concentrations.¹⁸ Remarkably, studies claiming that RyR2 block is not therapeutically relevant have not examined the effect of flecainide on RyR2-mediated SR Ca release in the complete absence of sodium channel activity.

To distinguish between these two mechanisms (sodium channel block vs RyR2 inhibition) and isolate flecainide’s direct effect on RyR2, we synthesized flecainide analogues that retain sodium channel block, but exhibit substantially lower potency of single channel RyR2 inhibition. We then tested the effects of flecainide and its analogues on spontaneous Ca release in cardiomyocytes from Casq2−/− mice, an established model of severe CPVT.²² To investigate the contribution of RyR2 block to its antiarrhythmic efficacy in vivo, we utilized our single N-methylated flecainide analogue that has pharmacological properties amenable to in vivo administration, inhibits sodium channel with similar potency as flecainide but does not block RyR2 Ca release in isolated cardiomyocytes.

METHODS

The materials and data from this study are available from the corresponding author upon reasonable request.

See supplement for expanded methods. The use of animals was approved by the Animal Care and Use Committee of Vanderbilt University Medical Center, USA (animal protocol #M1900057–00) and performed in accordance with NIH guidelines. Representative images/traces were selected from records that had values close to the mean/median measure. Statistical analyses were performed using Prism v6.01 (GraphPad Software, Inc.) or R (https://www.R-project.org/). Statistical tests were used as reported in the figure legends.

RESULTS

Generation of flecainide analogues with reduced RyR2 inhibitory activity.

In order to isolate the specific effects of flecainide on RyR2-mediated Ca release, we synthesized two flecainide analogues, a tetraalkylammonium salt derivative of flecainide, which is a quaternary ammonium cation also known as QX-flecainide (QX-FL)²³, and a single-methylated flecainide derivative, N-methyl flecainide (NM-FL), shown in Figure 1A. We hypothesized that the secondary amine on the piperidine ring in flecainide is necessary for its activity in RyR2 channels, but not sodium channels. This idea stems from work in which QX-FL has been shown to not block RyR2 in single channel studies performed at +40 mV membrane potential difference, where an effect with flecainide is observed.¹⁶ While both QX-FL and NM-FL have similar benzamide-derived structures compared to flecainide, there are key differences in the piperidine moiety. QX-FL, synthesized using a modified literature procedure, is doubly methylated at the piperidine nitrogen, resulting in a quaternary ammonium salt.²⁴ This modification renders QX-FL permanently charged and removes its ability to participate in hydrogen bonding at that nitrogen center. To test the hypothesis that the secondary amine is critical for flecainide RyR2 block as well as make a substrate suitable for in vivo administration, we synthesized NM-FL using an adapted Eschweiler-Clarke reaction to install a single methyl at the piperidine nitrogen.²⁴ This substrate maintains similar physiochemical properties to flecainide, yet also has the key N-H donor removed. Flecainide has a pKa of 9.3, resulting in 99% ionization at physiological pH (7.4). Although an exact pKa value for NM-FL has not been experimentally determined, the effect of N-methylation of amines on pKa has been extensively studied and documented. Hall shows that the pKa of 2-methylpiperidine and that of 1,2-dimethyl piperidine are less than one pKa unit apart.^{25, 26} This can be extended to the case of flecainide and NM-FL. Hence, like flecainide, NM-FL is predicted to be predominately ionized at physiological pH. Sodium channel or RyR2 block by NM-FL has not been characterized previously.

Figure 1.

N-Methylation of flecainide reduces its inhibitory activity on the single channel cardiac ryanodine receptor (RyR2). A) Chemical structures for flecainide and two N-methyl flecainide analogues: Double N-methyl flecainide, a quaternary ammonium cation (QX-FL) and single N-methyl flecainide (NM-FL). B) Representative single channel recordings from sheep RyR2 incorporated into lipid bilayers and treated with flecainide (top), QX-FL (middle), or NM-FL (bottom). Recordings were made in the presence of 0.1 μM free Ca and 2 mM ATP and the membrane potential was held at +40 mV. Open (o) and closed (c) states are labeled in the first recording. C) RyR2 open probability (Po) at various concentrations of flecainide or its analogues (normalized to maximum Po for each channel without drug). N_Flecainide = 13 (5 μM), 10 (10 μM), 7 (20 μM), 6 (30 μM), 8 (50 μM). N_NM-FL = 8 (10 μM), 10 (20 μM), 5 (50 μM), 6 (100 μM), 3 (150 μM), 4 (200 μM). N_QX-FL = 4 (10 μM), 4 (20 μM), 5 (50 μM), 5 (100 μM), 6 (150 μM), 12 (200 μM). Extra sum-of-squares F test was used to compare IC₅₀ values. P = 3.71E-09 for Flecainide vs NM-FL. P = 2.55E-05 for Flecainide vs QX-FL. P = 4.86E-02 for NM-FL vs QX-FL.

N-Methylation of flecainide reduces its inhibitory activity on RyR2 channels.

To determine the structure-activity relationship of the N-methylated flecainide analogues, we recorded single channel RyR2 openings and measured open probability (RyR2 P_o) from sheep RyR2 incorporated into lipid bilayers (representative traces shown in Figure 1B). At end-diastolic cytoplasmic Ca concentrations (0.1 μM), RyR2 open probability had a skewed distribution with a median of 0.042 and 25–75 percentiles at 0.009 and 0.18 (n = 60). Addition of flecainide to the cytoplasmic bath at a membrane potential of +40 mV inhibited RyR2 with an IC₅₀ of 15.9 μM (CI_95% = [10.9, 21.2]). Phosphorylation of single RyR2 channels had no significant effect on the potency of flecainide block (Supplemental Figure I). N-Methylation of flecainide substantially reduced its inhibitory potency on RyR2 channels. The IC₅₀ of the single-methylated NM-FL was 99 μM (CI_95% = [68.7, 144], P = 3.71E-09 vs flecainide). Double methylation further reduced the inhibitory potency, with an IC₅₀ of 182 μM for QX-FL (CI_95% = [128, 259], P = 2.55E-05 vs flecainide, P = 4.86E-02 vs NM-FL for QX-FL, Figure 1B & C).

We next tested flecainide and its analogues on RyR2 at a Ca concentration of 100 μM, which is the cytoplasmic Ca concentration in the dyadic cleft during systole.²⁷ Under those conditions, RyR2 was near maximally activated (median P_o = 0.68, 25–75 percentiles at 0.21 and 0.86, n = 32). As previously reported,⁹ high cytosolic Ca reduces the potency of RyR2 block by flecainide (IC₅₀ = 88 μM (CI_95% = [78.5, 99.5]), Supplemental Figure II), which may explain why flecainide does not inhibit systolic SR Ca release during normal excitation-contraction coupling.²⁸ N-Methylation significantly reduced the inhibitory potency of flecainide also at elevated Ca concentrations, with an IC₅₀ of 357 μM (CI_95% = [254, 502]) for the single-methylated NM-FL (P = 2.34E-14 vs flecainide), and 773 μM (CI_95% = [626, 952]) for the double-methylated QX-FL (P = 5.19E-24 vs flecainide, Supplemental Figure II).

To test whether reduced activity on single RyR2 channels translates into altered drug activity when the RyR2 channel is incorporated in its biological SR membrane, we recorded spontaneous Ca waves – which are generated by RyR2-mediated SR Ca release – in Casq2−/− cardiomyocytes subjected to saponin in order to selectively permeabilize the sarcolemma and enable equivalent access of flecainide or its analogues to RyR2. Importantly, treatment with saponin produces large pores in the sarcolemma, effectively clamping the sarcolemmal membrane potential at 0 mV and eliminating any effects of sodium channel block or sarcolemmal polarity on SR Ca release, while leaving the SR membrane intact.²⁹ Representative line scans of Ca waves are shown in Figure 2A. We observed a concentration-dependent decrease in Ca wave frequency (Figure 2B) following treatment with flecainide (IC₅₀ = 7.0 μM, CI_95% = [4.69, 10.3]). QX-FL and NM-FL did not inhibit Ca waves at the highest concentration tested, consistent with their reduced potency in the RyR2 single channel assay (Figure 1C). Taken together, these results indicate that N-methylation of flecainide reduces its ability to inhibit single RyR2 channels and RyR2 mediated Ca release.

Figure 2.

N-Methylation of flecainide abolishes its ability to inhibit sarcoplasmic reticulum (SR) calcium (Ca) waves in permeabilized Casq2−/− cardiomyocytes. A) Isolated Casq2−/− cardiomyocytes were subjected to saponin (40 μg/mL for 45 s) to selectively permeabilize the sarcolemma and enable equivalent access of flecainide or its analogues to cardiac ryanodine receptors (RyR2). Representative line scans show spontaneous Ca release events in the form of Ca waves after 5 minutes incubation in vehicle (Veh.) or 25 μM flecainide, QX-flecainide (QX-FL), or N-methyl flecainide (NM-FL). B) Ca wave frequency decreased in a dose-dependent manner following treatment with flecainide but not NM-FL or QX-FL. N = 28, 24, 31, 29, and 38 cells for flecainide; N = 23, 21, 40, and 63 cells for QX-FL; N = 21, 23, 26, and 34 cells for NM-FL. Two tailed t-tests were used to compare to vehicle. P = 1.96E-06 for a. P = 1.07E-10 for b. P = 8.59E-07 for c. Data are normalized to vehicle and shown as mean ± SEM. Normality was examined before statistical testing.

N-Methyl flecainide (NM-FL) inhibits cardiac sodium channels with properties comparable to flecainide.

Cardiac sodium channel block by QX-FL has been extensively characterized.²³ QX-FL is permanently charged at any pH, rendering it membrane impermeable. This requires it to be introduced intracellularly to exert any effect. Flecainide, while predominantly ionized at physiological pH, is in equilibrium with a small fraction that is neutral and can diffuse across the cell membrane. Once inside the cell, flecainide again reaches equilibrium with most of the compound being charged. Only the ionized form of flecainide is responsible for its characteristic sodium channel use-dependent block (i.e, the channel inhibition increases with higher channel activity).²³ Use-dependent block (UDB) of sodium channels is an important mechanism of action for class I antiarrhythmic drugs such as flecainide.⁸

Since NM-FL had not previously been characterized biologically, we compared its effect on the cardiac sodium channel (Na_V1.5) to that of flecainide. Experiments were carried out using whole cell patch clamp of human embryonic kidney (HEK293T) cells stably expressing human Na_V1.5.³⁰ Flecainide or NM-FL were applied in the extracellular perfusate and sodium currents were measured using standard protocols. Both flecainide and NM-FL exhibited strong use dependent block, with a similar concentration dependence (Figure 3A). With an IC₅₀ = 7.7 μM (CI_95% = [6.31, 9.49]) for UDB, NM-FL was modestly more potent than flecainide (IC₅₀ = 10.6 μM, CI_95% = [8.11, 13.9], P = 0.07), which is consistent with a slight difference in pKa. Rate of recovery from UDB (Supplemental Figure III) and voltage-dependence of UDB (Supplemental Figure IV) were not statistically different between flecainide and NM-FL. To assess the tonic block of sodium channels, we measured the effect of flecainide and NM-FL on the current-voltage relationship of I_Na activation (Figure 3B) and steady-state inactivation (Supplemental Figure V). At 6 μM, there were no statistically significant differences between flecainide and NM-FL (Figure 3B peak current t-test, P = 0.93). Taken together, these results demonstrate that N-methylation of flecainide’s piperidine moiety does not significantly alter its activity on cardiac sodium channels.

Figure 3.

Characterization of I_Na inhibition by flecainide and N-methyl flecainide (NM-FL) in HEK293T cells expressing human Na_V1.5. A) Representative current records (left) and concentration-response relationship (right) of use-dependent block (UDB) by flecainide and NM-FL. Currents were recorded before (control) and after (test, arrow) a train of conditioning pulse (300 pulses, 10 Hz frequency) in the presence of various concentrations (0.3, 1, 3, 10, and 30 μM) of flecainide or NM-FL. Data are normalized to the initial test pulse. IC₅₀ was 10.6 μM for flecainide (n = 3 cells for 0.3, 1, and 3 μM; n = 4 cells for 10 μM; n = 5 cells for 30 μM) and 7.7 μM for NM-FL (n = 4 cells/concentration). Extra sum-of-squares F test was used to compare IC₅₀ values. P = 0.067 for flecainide vs NM-FL. B) Current-voltage relationship of I_Na activation in the absence (vehicle, n = 10 cells) and presence of 6 μM flecainide (n = 5 cells) or NM-FL (n = 5 cells) applied to the bath solution (5 min. incubation). Left side of the panel shows voltage protocol for the experiment, as well as representative example traces. Peak current was compared using a two-tailed t-test. P = 0.93 for flecainide vs NM-FL.

Flecainide, but not QX-FL or NM-FL, inhibits spontaneous Ca release in intact cardiomyocytes under conditions of complete sodium channel block.

We next examined spontaneous SR Ca release inhibition in intact Casq2−/− cardiomyocytes using whole-cell patch clamp. Because QX-FL is permanently charged and does not cross cell membranes, flecainide, QX-FL, or NM-FL (6 μM) were applied in both the extracellular (perfused solution) and intracellular solutions (dialyzed via patch pipette) for 5 minutes. Cyclic AMP (200 μM) was included in the patch pipette to maximally activate protein kinase A (PKA) and mimic the catecholamine-mediated phosphorylation state that occurs during adrenergic response. Spontaneous Ca waves were recorded during a 48-second test period following termination of a short conditioning train of membrane-depolarizing steps (Figure 4A, Supplemental Figure VI). To abolish any contribution of sodium channel block by flecainide and its analogues and isolate potential effects from RyR2 inhibition, cells were pretreated with 30 μmol/L tetrodotoxin (TTX) for 15 minutes, which blocked I_Na completely (Supplemental Figure VII). TTX pretreatment (i.e., complete sodium channel block) significantly reduced Ca wave frequency by ~45% compared to vehicle treatment (Figure 4A). In the presence of TTX, flecainide further reduced Ca wave frequency (by ~90%, Figure 4A), which was significantly more than that of TTX alone (Figure 4A), indicating an additive effect. QX-FL and NM-FL, on the other hand, did not significantly reduce the frequency of spontaneous Ca waves beyond that observed with TTX alone (Figure 4A). Flecainide, QX-FL, and NM-FL had no effect on the SR Ca content, as estimated by a rapid 10 mM caffeine spritz at the end of every recording.

Figure 4.

Flecainide inhibits spontaneous Ca waves in intact Casq2−/− cardiomyocytes independently of sodium channel block. A) Top panels: Representative Ca fluorescent records from voltage-clamped ventricular myocytes after 5 minutes extracellular application and intracellular dialysis of either DMSO vehicle (Veh.), or 6 μM flecainide, QX-FL, or NM-FL. In 4 out of 5 experimental groups, cells were pre-incubated for 15 minutes with 30 μM tetrodotoxin (TTX) to completely block sodium channels. To elicit spontaneous calcium waves, a short conditioning train of 4 membrane depolarization steps from −70 mV to +10 mV with 1 Hz frequency was applied at the beginning of each recording (blue hash marks). Bottom panel: Spontaneous Ca wave frequency for each treatment. N = 11, 12, 13, 10, and 12 cells from 9 mice, respectively. B) Top panels: Representative traces showing the effect of flecainide and NM-FL in the absence of TTX. Bottom panel: Spontaneous Ca wave frequency for each treatment. Note the additive contributions from sodium channel and RyR2 block. N = 17, 11, and 12 cells from 9 mice. Statistical comparisons were made using a mixed model with Bonferroni correction to account for independent cardiomyocyte isolations.

We next compared flecainide and NM-FL in the absence of TTX (Figure 4B). The magnitude of Ca wave inhibition by Flecainide was significantly greater than that of NM-FL, further supporting the additive effect of RyR2 block on Ca wave inhibition (Figure 4B). Taken together, these results establish that flecainide inhibits arrhythmogenic RyR2-mediated SR Ca release in intact cardiomyocytes independent of its sodium channel blocking properties.

Flecainide, but not NM-FL, prevents ventricular tachycardia in Casq2−/− mice.

We next sought to determine if and to what extent the RyR2 block by flecainide influences its antiarrhythmic efficacy in vivo. Given its activity by extracellular application (Figure 3), equivalent block of cardiac sodium channels (Figure 3), and lack of direct effect on RyR2-mediated Ca waves (Figures 2 & 4) compared to flecainide, we used NM-FL as a tool to answer this question. Using the well-established Casq2−/− CPVT mouse model, Casq2−/− mice were randomized to a triple crossover study design and pretreated via intraperitoneal (i.p.) injection with vehicle, 15 mg/kg flecainide (Flec.), or 15 mg/kg NM-FL, followed by catecholamine challenge with isoproterenol (3 mg/kg i.p.) 90 min later. Informed by our previous studies of flecainide pharmacokinetics in mice,⁹ the dose was chosen to produce flecainide plasma concentrations in mice that are within the therapeutic range (0.4 – 1 mg/L) used clinically to prevent ventricular arrhythmias in CPVT patients.¹⁰ Figure 5A shows representative time courses of the heart rate in Casq2−/− mice before and after catecholamine challenge (time = 0). Injection of isoproterenol causes development of ventricular arrhythmia (inset), observed as a fluctuating heart rate due to variable R-R intervals with premature ventricular complexes and ventricular tachycardia. In vivo, use-dependent sodium channel block manifests itself as a widening of the QRS complex due to the reduction in depolarization rate and hence slowed conduction velocity.³¹ Figure 5B shows representative QRS waveforms from a Casq2−/− mouse. Both flecainide and NM-FL prolonged the QRS complex to a comparable degree (Figure 5C), indicating that the doses were bioequivalent and both drugs exhibit equivalent potency of use-dependent sodium channel block in vivo. Arrhythmia burden was classified on an ordinal scale from 0 – 4. Only flecainide, but not NM-FL, which lacks RyR2 blocking properties, significantly decreased arrhythmia burden (Figure 5D & E, Supplemental Figure VIII-A) and premature ventricular complexes (Supplemental Figure VIII-B), despite both drugs equivalently inhibiting the sodium channel. There were no differences in heart rates between treatment groups (Supplemental Figure VIII-C–E). Taken together, these results establish the importance of RyR2 inhibition by flecainide for its antiarrhythmic activity in vivo.

Figure 5.

Flecainide, but not N-methyl flecainide (NM-FL), prevents ventricular tachycardia in Casq2−/− mice. A) Heart rate in a Casq2−/− mouse pre-treated with DMSO (vehicle), 15 mg/kg flecainide, or 15 mg/kg NM-FL, before and after intraperitoneal (i.p.) injection with 3 mg/kg isoproterenol (time = 0). Arrythmias produce a variable beat-to-beat heart rate on electrocardiogram (ECG). B) QRS width for each drug treatment. Left panel: Representative ECG traces illustrating QRS morphology from the same mouse treated with DMSO (Veh.), flecainide (Flec.), or N-methyl flecainide (NM-FL). Waveforms are aligned to the initial moment of depolarization. Right panel: QRS width for each animal. Casq2−/− mice were randomized to a triple crossover design and injected i.p. with 15 mg/kg DMSO (equivalent volume), flecainide, or NM-FL. Repeated measures ANOVA with Tukey’s post-hoc test. Median values displayed as blue crossbars. C) Representative arrhythmias observed following catecholamine challenge. PVC = premature ventricular complex, bigeminy = alternating sinus beats with PVCs, couplet = 2 consecutive PVCs, non-sustained ventricular tachycardia (NSVT) = 3 or more consecutive PVCs. Scale bar = 250 ms. D) Classification of arrhythmia risk for each treatment group. E) Arrhythmia risk score. Normality was examined before statistical testing. Data in D and E compared with pairwise Wilcoxon rank sum test with Bonferroni correction. N = 10 mice in panels B, D & E.

DISCUSSION

Here, we report that flecainide remains a highly effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. Flecainide analogues that retain sodium channel block but have significantly reduced activity on RyR2 single channels fail to inhibit RyR2-mediated SR Ca release. These results indicate that RyR2 block by flecainide is important to suppress spontaneous Ca release in ventricular cardiomyocytes. In mice with CPVT, sodium channel block in the absence of RyR2 block did not prevent catecholamine-induced ventricular tachyarrhythmias. Hence, our results demonstrate that potent and direct inhibition of RyR2-mediated SR Ca release is required for flecainide’s antiarrhythmic action in preventing CPVT, and thus resolves an ongoing controversy in the field.

Previously, some investigators had concluded that RyR2 block is not a relevant mechanism of flecainide action,¹⁷ and that sodium channel block is solely responsible for its antiarrhythmic efficacy in CPVT.^{15, 17} This interpretation was based exclusively on in vitro experiments, mostly on single RyR2 channel data showing that flecainide does not inhibit RyR2 at a SR membrane potential of 0 mV but only at positive SR membrane potentials,¹⁷ which are considered non-physiological based on modeling studies.^{32, 33} However, the SR membrane potential during Ca release has never been measured experimentally and may not be 0 mV, at least not during initial phase of spontaneous SR Ca release. Thus, we carried out a careful biological assessment of flecainide inhibition of RyR2 in its native cellular environment as an extension of single channel studies, utilizing flecainide analogues that retain sodium channel inhibitory activity but compared to flecainide have reduced activity on RyR2 single channels at +40 mV. Treatment with saponin permeabilized cardiomyocytes and removed the contribution of sarcolemmal sodium channels – clamping intracellular [sodium] and rendering sodium channels inactivated – enabling us to resolve flecainide’s effect on RyR2-mediated Ca release directly (Figure 2). The direct action of flecainide on RyR2-mediated Ca release was confirmed in intact cardiomyocytes with complete inhibition of sodium channels by TTX (Figure 4). Importantly, our results in cardiomyocytes mirror the different inhibitory potency of flecainide and QX-FL or NM-FL on single RyR2 channels at positive membrane potentials (Figure 1 and reference¹⁶). Given that flecainide RyR2 block is steeply voltage-dependent,³⁴ our experimental results suggest that the junctional SR membrane polarizes to positive potentials during spontaneous Ca release, at least transiently. Future studies will have to confirm this hypothesis experimentally. The higher potency and efficacy of flecainide in intact (Figure 4) compared to permeabilized cardiomyocytes (Figure 3) can be explained by the additive effect of the sodium channel block in intact cardiomyocytes, as illustrated in Figure 4B.

Our discovery that methylation of a single amine residue abolishes flecainide’s activity against RyR2-mediated SR Ca release provides important information on flecainide’s structure-activity relationship of RyR2 inhibition. It establishes that the N-H in the piperidine ring of flecainide is necessary for its activity on RyR2. Additionally, the results of the methylation studies suggest that flecainide binding to the RyR2 macromolecular complex is selective and supports the existence of a discrete binding site in the RyR2 macromolecular complex. Future studies are warranted to identify the flecainide binding site on RyR2, which will enable further optimization of the RyR2-blocking properties of the flecainide molecule. This is important, because hyperactive RyR2 channels are implicated as an important arrhythmia mechanism in structural heart disease such as heart failure and ischemic heart disease^{19, 20} where sodium channel blockers are contraindicated.⁸

What is the relative need for sodium channel block versus RyR2 inhibition to achieve therapeutic benefit from flecainide therapy?

To answer this question, one should consider the mechanisms underlying the generation of catecholamine-induced ventricular arrhythmias in CPVT.³⁵ At the level of the cardiomyocyte, hyperactive RyR2 channels cause spontaneous RyR2 openings during diastole that coalesce into a propagated Ca wave, which in turn activates the electrogenic sodium calcium exchanger, resulting in a cell membrane depolarization. Membrane depolarizations of sufficient amplitude will activate voltage-gated sodium channels, which in turn will trigger a full cardiac action potential. The therapeutic benefit of RyR2 block by flecainide stems from its drastic reduction or complete prevention of arrhythmogenic calcium waves (Figure 4 and reference^{9, 13, 28}), which is the primary cellular event responsible for the generation of the spontaneous action potential in CPVT.³⁵ The therapeutic benefit of sodium channel block stems from two independent actions: (1) the reduction of cellular sodium and hence Ca loading, reducing the likelihood of spontaneous SR Ca release,^{16, 18} and (2), increasing the cell membrane potential threshold that can trigger a spontaneous action potential.¹⁵ Which of these mechanisms predominates depends largely on the experimental conditions, which has contributed to different investigators coming to various conclusions regarding the relative importance of each mechanism. For example, rapid pacing protocols favor sodium channel block as the primary mechanism because of the strong use-dependence of flecainide, leading to an overestimation of the contribution of sodium channel block.³⁶ To overcome this limitation, here we used a train of voltage-clamp pulses to stimulate cardiomyocytes and elicit arrhythmogenic Ca waves. We find that a complete block of sodium channels with TTX reduced the rate of spontaneous Ca waves by approximately 45% (Figure 4). A similar reduction was achieved by sodium channel block with NM-FL, which blocks sodium channels with similar potency as flecainide (Figure 4B) but does not inhibit RyR2 at the concentration tested. On the other hand, Flecainide reduced spontaneous Ca waves by over 90% (Figure 4), indicating that the additive effect of RyR2 block is substantial. In contrast, Bannister et. al. reported that sodium channel block by flecainide is solely responsible for its effect on Ca waves, based on a comparison of intracellular application of flecainide and QX-FL, which does not block RyR2 (Figure 1).¹⁶ However, QX-FL is a much more potent sodium channel blocker than flecainide when selectively applied to the intracellular side of the membrane, because flecainide rapidly diffuses out of the cell whereas quaternary ammonium QX-FL does not.²³ In our experiments, QX and flecainide were applied to both the intracellular and extracellular side of the sarcolemma at equal concentrations (Figure 4). Taken together, our results indicate that at least half of the therapeutic benefit at the level of the cardiomyocyte is derived from flecainide’s inhibition of RyR2-mediated Ca release, with a contribution by sodium channel inhibition that depends on the experimental conditions.

What is the relative contribution of sodium channel block versus RyR2 inhibition for therapeutic benefit from flecainide therapy in vivo in CPVT patients?

The collective evidence from experimental and clinical studies suggests that while RyR2 inhibition is beneficial, treatment with pure sodium channel blockers does not provide benefit and may even harm CPVT patients. In experimental studies, selective RyR2 block with ent-verticilide prevented catecholamine-induced ventricular arrhythmia in a murine CPVT model.³⁷ In clinical studies, based on case-control studies^{9, 11, 12} and a randomized clinical trial,⁹ the dual RyR2 and sodium channel blocker flecainide prevented ventricular arrythmias in patients with CPVT when used as a monotherapy or in combination with beta-blockers, and since 2013 is considered the standard of care in the treatment of CPVT patients.³⁸ Propafenone, another class Ic agent that inhibits RyR2 channels similar to flecainide, is also effective in CPVT patients.³⁹ Clinical experience with class Ia and Ib sodium channel blockers which lack activity on RyR2¹³ is limited, but in a case series of 29 CPVT patients, class Ia and Ib agents given alone or in combination with a β blocker were not protective, but rather significantly increased the risk of sudden death.⁴⁰ Two of the three cases given mexiletine and one case given disopyramide died suddenly. Our data testing the antiarrhythmic efficacy of class I agents in CPVT mouse models also indicate that sodium channel block alone is not effective in CPVT. Compared to flecainide or (R)-propafenone, sodium channel blockers that have less ((S)-propafenone) or no RyR2 blocking properties (lidocaine, procainamide) did not significantly reduce the incidence of exercise or catecholamine-induced ventricular tachycardia in CPVT mice.^{9, 39} The current study demonstrates that the N-methyl flecainide analogue, which lacks RyR2 block at clinically relevant concentrations (Figures 1, 2), did not reduce the severity of ventricular tachyarrhythmias in CPVT mice (Figure 5), even though the degree of sodium channel block as evidenced by the QRS widening on the ECG was comparable to that of flecainide (Figure 5). Why do sodium channel blockers reduce triggered activity in isolated CPVT cardiomyocytes^15–18 but do not protect against ventricular tachycardia in vivo? The answer is not clear but may relate to the well-established proarrhythmic liability of sodium channel blockers at the tissue level,⁶ which could explain why treatment with pure sodium channel blockers increased the risk of fatal arrhythmias in CPVT patients.⁴⁰ Collectively, these clinical and experimental data indicate that sodium channel block, by itself, likely confers little or no therapeutic benefit in CPVT. Rather, inhibition of RyR2 is the principal mechanism of antiarrhythmic action of flecainide in CPVT. Whether or not sodium channel block adds any therapeutic benefit when combined with RyR2 block – as is the case for flecainide – remains to be determined.

NOVELTY AND SIGNIFICANCE.

What Is Known?

• The class Ic antiarrhythmic drug flecainide is highly effective in preventing adrenergic stress–induced arrhythmias in mouse models of catecholaminergic polymorphic ventricular tachycardia (CPVT) and in CPVT patients refractory to standard drug treatment.

• Despite flecainide’s proven clinical efficacy in CPVT, the mechanism of its therapeutic action remains controversial: It is debated whether flecainide’s direct effect on ryanodine receptor (RyR2) is responsible for its antiarrhythmic efficacy or whether its efficacy can be solely attributed to flecainide’s inhibition of cardiac sodium channels.

What New Information Does This Article Contribute?

• In vitro, flecainide analogues that retain sodium channel block but have significantly reduced activity on RyR2 single channels fail to inhibit spontaneous RyR2-mediated SR Ca release when sodium channel activity is eliminated.

• In mice with CPVT, a flecainide analogue that lacks RyR2-inhibitory properties but is a potent Na channel blocker does not prevent ventricular tachycardia, whereas flecainide is highly effective in reducing arrhythmic events under the same experimental conditions.

• RyR2 channel inhibition likely is the principal mechanism of flecainide’s antiarrhythmic action.

Hyperactive RyR2 channels are implicated in a variety of heart diseases associated with atrial and ventricular tachyarrhythmias. The genetic syndrome of RyR2 hyperactivity, CPVT, is caused by mutations in RYR2 or in its binding partner calsequestrin (CASQ2). CPVT is characterized by spontaneous SR Ca release during adrenergic stress that can trigger atrial and ventricular tachyarrhythmia. Flecainide has been clinically proven to be effective in treating CPVT patients, but its mechanism of action remains debated. To distinguish between the two proposed mechanisms of flecainide action (sodium channel vs RyR2 inhibition), we synthesized flecainide analogues that retain sodium channel block, but exhibit substantially lower potency of RyR2 channel inhibition. In CPVT cardiomyocytes, flecainide 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 flecainide action in CPVT and may also be responsible for flecainide’s antiarrhythmic action in other atrial and ventricular arrhythmias.

Nonstandard Abbreviations and Acronyms:

Ca

calcium

Casq2

cardiac calsequestrin

CPVT

catecholaminergic polymorphic ventricular tachycardia

NM-FL

N-methyl flecainide analogue

QX-FL

quaternary charged flecainide analogue

RyR2

cardiac ryanodine receptor

SR

sarcoplasmic reticulum

TTX

tetrodotoxin