RYR2 Channel Inhibition Is the Principal Mechanism of Flecainide Action in CPVT
Graphical Abstract
Abstract
Rationale:
The 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.
Objective:
To 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 Results:
We 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.
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.
Introduction
Editorial, see p 332
In This Issue, see p 305
Meet the First Author, see p 306
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 RyR2 (cardiac ryanodine receptor) SR Ca release channel,1 Casq2 (cardiac calsequestrin),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 patients with CPVT.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 tetrodotoxin18 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 intraSR 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 2 mechanisms (sodium channel block versus 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 Data Supplement for expanded methods. The use of animals was approved by the Animal Care and Use Committee of Vanderbilt University Medical Center (animal protocol No. M1900057-00) and performed in accordance with National Institutes of Health 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
To isolate the specific effects of flecainide on RyR2-mediated Ca release, we synthesized 2 flecainide analogues, a tetraalkylammonium salt derivative of flecainide, which is a quaternary ammonium cation also known as 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 with 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 physicochemical 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 <1 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.
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 Po) from sheep RyR2 incorporated into lipid bilayers (representative traces shown in Figure 1B). At end-diastolic cytoplasmic Ca concentrations (0.1 µmol/L), RyR2 open probability had a skewed distribution with a median of 0.042 and 25 to 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 µmol/L (CI95%=[10.9–21.2]). Phosphorylation of single RyR2 channels had no significant effect on the potency of flecainide block (Figure I in the Data Supplement). N-Methylation of flecainide substantially reduced its inhibitory potency on RyR2 channels. The IC50 of the single-methylated NM-FL was 99 µmol/L (CI95%=[68.7–144], P=3.71×10−9 versus flecainide). Double methylation further reduced the inhibitory potency, with an IC50 of 182 µmol/L for QX-FL (CI95%=[128–259], P=2.55×10−5 versus flecainide, P=4.86×10−2 versus NM-FL for QX-FL, Figure 1B and 1C).
We next tested flecainide and its analogues on RyR2 at a Ca concentration of 100 µmol/L, which is the cytoplasmic Ca concentration in the dyadic cleft during systole.27 Under those conditions, RyR2 was near maximally activated (median Po=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 µmol/L (CI95%=[78.5–99.5]), Figure II in the Data Supplement), 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 µmol/L (CI95%=[254–502]) for the single-methylated NM-FL (P=2.34×10−14 versus flecainide), and 773 µmol/L (CI95%=[626–952]) for the double-methylated QX-FL (P=5.19×10−24 versus flecainide, Figure II in the Data Supplement).
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 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 µmol/L, CI95%=[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.
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 (UDB; ie, the channel inhibition increases with higher channel activity).23 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 (NaV1.5) to that of flecainide. Experiments were carried out using whole cell patch clamp of human embryonic kidney (HEK293T) cells stably expressing human NaV1.5.30 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 UDB, with a similar concentration dependence (Figure 3A). With an IC50=7.7 µmol/L (CI95%=[6.31–9.49]) for UDB, NM-FL was modestly more potent than flecainide (IC50=10.6 µmol/L, CI95%=[8.11–13.9], P=0.07), which is consistent with a slight difference in pKa. Rate of recovery from UDB (Figure III in the Data Supplement) and voltage dependence of UDB (Figure IV in the Data Supplement) 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 INa activation (Figure 3B) and steady-state inactivation (Figure V in the Data Supplement). At 6 µmol/L, 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.
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 µmol/L) were applied in both the extracellular (perfused solution) and intracellular solutions (dialyzed via patch pipette) for 5 minutes. Cyclic AMP (200 µmol/L) was included in the patch pipette to maximally activate PKA (protein kinase A) 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, Figure VI in the Data Supplement). 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 for 15 minutes, which blocked INa completely (Figure VII in the Data Supplement). Tetrodotoxin pretreatment (ie, complete sodium channel block) significantly reduced Ca wave frequency by ≈45% compared with vehicle treatment (Figure 4A). In the presence of tetrodotoxin, flecainide further reduced Ca wave frequency (by ≈90%, Figure 4A), which was significantly more than that of tetrodotoxin 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 tetrodotoxin alone (Figure 4A). Flecainide, QX-FL, and NM-FL had no effect on the SR Ca content, as estimated by a rapid 10 mmol/L caffeine spritz at the end of every recording.
We next compared flecainide and NM-FL in the absence of tetrodotoxin (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 and 4) compared with 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 injection with vehicle, 15 mg/kg flecainide (Flec.), or 15 mg/kg NM-FL, followed by catecholamine challenge with isoproterenol (3 mg/kg intraperitoneal) 90 minutes 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 patients with CPVT.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 because of 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 because of 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 to 4. Only flecainide, but not NM-FL, which lacks RyR2 blocking properties, significantly decreased arrhythmia burden (Figure 5D and 5E, Figure VIIIA in the Data Supplement) and premature ventricular complexes (Figure VIIIB in the Data Supplement), despite both drugs equivalently inhibiting the sodium channel. There were no differences in heart rates between treatment groups (Figure VIIIC through VIIIE in the Data Supplement). Taken together, these results establish the importance of RyR2 inhibition by flecainide for its antiarrhythmic activity in vivo.
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 nonphysiological 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 with 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 tetrodotoxin (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 study by Bannister et al16). 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 with 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 disease19,20 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 studies by Watanabe et al,9 Galimberti and Knollmann,13 and Hilliard et al28), 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 2 following independent actions: (1) the reduction of cellular sodium and hence Ca loading, reducing the likelihood of spontaneous SR Ca release16,18 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 about 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 tetrodotoxin reduced the rate of spontaneous Ca waves by ≈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 al16 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). 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 Patients With CPVT?
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 patients with CPVT. 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 studies9,11,12 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 β-blockers, and since 2013 is considered the standard of care in the treatment of patients with CPVT.38 Propafenone, another class Ic agent that inhibits RyR2 channels similar to flecainide, is also effective in patients with CPVT.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 patients with CPVT, 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 3 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 with 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 and 2), did not reduce the severity of ventricular tachyarrhythmias in CPVT mice (Figure 5), although 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 cardiomyocytes15–18 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 patients with CPVT.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.
Acknowledgments
The authors thank Paul Johnson for his assistance with the RyR2 single channel studies.
Novelty and Significance
•
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 patients with CPVT 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 RyR2 (ryanodine receptor) is responsible for its antiarrhythmic efficacy or whether its efficacy can be solely attributed to flecainide’s inhibition of cardiac sodium channels.
•
In vitro, flecainide analogues that retain sodium channel block but have significantly reduced activity on RyR2 single channels fail to inhibit spontaneous RyR2-mediated sarcoplasmic reticulum 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 Casq2 (calsequestrin). CPVT is characterized by spontaneous sarcoplasmic reticulum Ca release during adrenergic stress that can trigger atrial and ventricular tachyarrhythmia. Flecainide has been clinically proven to be effective in treating patients with CPVT, but its mechanism of action remains debated. To distinguish between the 2 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.
Footnote
Nonstandard Abbreviations and Acronyms
- Casq2
- cardiac calsequestrin
- CPVT
- catecholaminergic polymorphic ventricular tachycardia
- NM-FL
- N-methyl flecainide
- PKA
- protein kinase A
- QX-FL
- QX-flecainide
- RyR2
- cardiac ryanodine receptor
- SR
- sarcoplasmic reticulum
- UDB
- use-dependent block
Supplemental Material
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© 2020 American Heart Association, Inc.
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Received: 12 February 2020
Revision received: 4 December 2020
Accepted: 8 December 2020
Published online: 10 December 2020
Published in print: 5 February 2021
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This work was supported in part by National Institutes of Health (NIH) grants NHLBI R35 HL144980 (B.C. Knollmann), NHLBI R01 HL151223 (J.N. Johnston, B.C. Knollmann), NHLBI F32 HL140874 (D.J. Blackwell), T32—5T32GM007569-44 (B.C. Knollmann, C.L. Egly), NHLBI F31 HL151125 (A.N. Smith), the Leducq Foundation grant 18CVD05 (B.C. Knollmann), the American Heart Association grant 19SFRN34830019 (B.C. Knollmann), PhRMA Foundation Postdoctoral Award (D.J. Blackwell), and NSW Health infrastructure grant through the Hunter Medical Research Institute (D.R. Laver). Ca wave measurements were performed using the VUMC Cell Imaging Shared Resource (supported by NIH grants CA68485, DK20593, DK58404, DK59637, and Ey008126).
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