Triple mode of action of flecainide in catecholaminergic polymorphic ventricular tachycardia

There has been a resurgence of interest in the class I_C antiarrhythmic agent flecainide fuelled by recent work identifying the drug as an effective treatment for catecholaminergic polymorphic ventricular tachycardia (CPVT).^1–3 CPVT is caused by mutations in the sarcoplasmic reticulum (SR) Ca²⁺ release channel (RyR2), or the associated proteins calsequestrin-2 or triadin, which lead to an increased propensity for spontaneous SR Ca²⁺ waves during exercise or stress. These in turn induce a transient inward current due to Ca²⁺ extrusion via the Na/Ca exchanger (NCX), delayed afterdepolarizations (DADs) and triggered action potentials. While it is increasingly accepted that flecainide is an effective treatment for CPVT, a degree of controversy has emerged regarding its mechanism of action.

Watanabe et al. provided the first evidence that, in addition to known inhibitory effects on the Na⁺ current (I_Na), flecainide also acts directly on RyR2 to prevent pro-arrhythmic SR release, thereby providing a dual mode of action.¹ Subsequent work demonstrated that flecainide only inhibits RyR2 gating when the channel is in the open state.⁴ The functional consequence of this effect on RyR2 is that the Ca²⁺ flux associated with each diastolic Ca²⁺ spark is reduced, while event frequency increases, such that there is no change in the SR Ca²⁺ leak, SR Ca²⁺ content, or systolic Ca²⁺ release. On the basis of these findings, we proposed a new, primary antiarrhythmic mechanism, whereby the flecainide-induced decrease in the spark mass reduces the probability that Ca²⁺ sparks will undergo salutatory propagation between junctional Ca²⁺ release sites, which is the basis of pro-arrhythmic Ca²⁺ waves.⁴

The importance of flecainide's action on RyR2 was recently questioned by Priori and colleagues, who reported that flecainide did not inhibit Ca²⁺ waves in a mouse model of CPVT.⁵ Instead, the antiarrhythmic effects of flecainide were attributed to inhibition of I_Na and a decrease in the probably of DADs triggering action potentials. However, as considered previously,⁶ diastolic spark frequency was sufficiently high in this study to suggest that the cells were very Ca²⁺ overloaded. Under Ca²⁺-overload conditions, we also find that flecainide is much less able prevent Ca²⁺ waves and effects mediated via I_Na inhibition would then dominate.⁷

In this volume, a study by Sikkel et al.⁸ also aims at addressing the mechanism by which flecainide influences Ca²⁺ sparks and waves. In contrast to the findings by the Priori group⁵ and consistent with our findings,⁴ the authors report a decrease in Ca²⁺ wave frequency in normal rat ventricular myocytes after exposure to flecainide. However, as other I_Na blockers were equally effective in reducing Ca²⁺ waves, the authors conclude that RyR2 inhibition is not relevant, and propose a mechanism whereby inhibition of I_Na is linked to indirect effects on Ca²⁺ sparks and waves via NCX. Unfortunately, contrary to the stated conclusions, the experimental conditions used by Sikkel et al. preclude any inferences regarding the role of RyR2 modulation by flecainide, for the following reasons:

Sikkel et al. use rapid pacing trains that will increase [Na⁺]_i and consequently [Ca]_i⁹ to elicit spontaneous Ca²⁺ sparks and Ca²⁺ waves in normal rat ventricular myocytes. While rapid pacing trains are a classic paradigm for triggering Ca²⁺ waves and DADs, studies in the 1980s have already shown that specific I_Na blockers, such as tetrodotoxin and lidocaine, can suppress pacing-train induced DADs in isolated Purkinje fibres, albeit without clarifying the underlying mechanism.¹⁰ The study by Sikkel et al. provides an explanation for the 30-year old results: I_Na blockers limit Na⁺ influx and hence cytosolic Ca²⁺ loading during rapid pacing trains, and thereby reduce Ca²⁺ sparks and Ca²⁺ waves. Since any form of I_Na inhibition is expected to reduce Ca²⁺ waves resulting from the rapid pacing protocol, the experiments were not designed to test the additional contribution of RyR2 inhibition by flecainide. This is also evidenced by the finding by Sikkel et al. that flecainide reduced Ca²⁺ spark frequency (which is the opposite of the direct effect of flecainide on RyR2 channels^4,11), indicating that any direct effect of flecainide on RyR2 was mitigated by the reported reduction in diastolic Ca²⁺ due to I_Na block. It is well established that reduced cytosolic [Ca²⁺] will reduce the rate of spontaneous RyR2 openings and Ca²⁺ sparks.⁹

Previous studies in intact myocytes from CPVT mice and isolated perfused rabbit hearts have shown that class I_C agents that also inhibit RyR2 (flecainide and R-propafenone) are significantly more effective at blocking Ca²⁺ waves and associated arrhythmias than drugs that only inhibit I_Na (e.g. lidocaine and procainamide).^12,13 Why then did Sikkel et al. not observe a more potent suppression of Ca²⁺ waves by flecainide compared with other I_Na inhibitors that lack RyR2 channel function (i.e. tetrodotoxin, lidocaine)? One important difference is that unlike Sikkel et al., Hwang et al.,⁷ and Lee et al. ¹² kept the pacing rate constant and used beta-adrenergic stimulation with isoproterenol to induce Ca²⁺ waves, experimental conditions that reduce the influence of Na⁺ channel block on cellular Ca²⁺ loading. Furthermore, Sikkel et al. applied 5 µM flecainide only for 5 min in the experiments comparing different Na⁺ channel blockers in the voltage clamp studies at −40 mV, the only experiment designed to test the contribution of RyR2 block on Ca²⁺ sparks and waves. However, as considered previously, flecainide enters myocytes slowly,^1,14 and it takes up to 30 min to reach maximal effects on Ca²⁺ waves in intact myocytes.¹⁵ Hence, in all previous studies evaluating the role of RyR2 modulation, intact cells were exposed to 6 µM flecainide between 15 and 30 min before the assessment of Ca²⁺ wave properties.^1,4,7 Sikkel et al. argue that 30 min exposure had no more effects on Ca²⁺ wave frequency than 5 min exposure. But those experiments were done in the same myocytes, and did not control for time dependent changes in wave frequency. In contrast to the results by Sikkel et al., we do not find any effect of flecainide on Ca²⁺ waves after 5 min flecainide exposure, ¹⁵ indicating that experimental conditions of rapid pacing induced Ca²⁺ waves are not comparable with our studies.

In permeabilized myocytes, the effect of flecainide on Ca²⁺ sparks is rapid, consistent with the removal of the sarcolemma as a barrier to diffusion.⁴ Sikkel et al. argue that effects of flecainide on RyR2 in permeabilized rat myocytes require cytosolic concentrations that are above those normally found within the plasma (i.e. 25 µM).⁸ However, in a relevant disease model of CPVT myocytes, we find a flecainide IC₅₀ for reducing Ca²⁺ wave frequency of 7 µM in permeabilized myocytes,¹¹ and of 2 µM in intact myocytes after 30 min incubation.¹³ Moreover, previous reports have shown a marked accumulation of flecainide in the ventricular myocardium.^1,16

Taken together, the experimental conditions (rapid pacing induced [Na]_i loading and short incubation times) chosen by Sikkel et al. strongly favour the detection of effects of I_Na inhibition on Ca²⁺ waves and hence the suppression of DADs, a property of Na⁺ channel blockers that had been observed several decades ago.¹⁰ On the other hand, the experiments by Sikkel et al. were not designed to detect the contribution of RyR2 block by class I_C agents to their antiarrhythmic effects on Ca²⁺ wave triggered arrhythmia, a contribution which has been confirmed independently by three groups.^7,12,17 We would caution that the absence of evidence does not constitute evidence for the absence of the therapeutic role of RyR2 modulation by flecainide. Rather, the mechanism of Ca²⁺ wave suppression by flecainide described by Sikkel et al. is in addition to Ca²⁺ wave suppression due to flecainide's direct action on RyR2 channels reported by us and others, and the reduced probability of DADs triggered action potentials described by the Priori group. This triple mode of action likely explains flecainide's striking clinical efficacy in CPVT patients.