Flecainide exerts paradoxical effects on sodium currents and atrial arrhythmia in murine RyR2-P2328S hearts

Abstract

Aims: Cardiac ryanodine receptor mutations are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and some, including RyR2-P2328S, also predispose to atrial fibrillation. Recent work associates reduced atrial Nav 1.5 currents in homozygous RyR2-P2328S (RyR2(S/S) ) mice with slowed conduction and increased arrhythmogenicity. Yet clinically, and in murine models, the Nav 1.5 blocker flecainide reduces ventricular arrhythmogenicity in CPVT. We aimed to determine whether, and how, flecainide influences atrial arrhythmogenicity in RyR2(S/S) mice and their wild-type (WT) littermates.

Methods: We explored effects of 1 μm flecainide on WT and RyR2(S/S) atria. Arrhythmic incidence, action potential (AP) conduction velocity (CV), atrial effective refractory period (AERP) and AP wavelength (λ = CV × AERP) were measured using multi-electrode array recordings in Langendorff-perfused hearts; Na(+) currents (INa ) were recorded using loose patch clamping of superfused atria.

Results: RyR2(S/S) showed more frequent atrial arrhythmias, slower CV, reduced INa and unchanged AERP compared to WT. Flecainide was anti-arrhythmic in RyR2(S/S) but pro-arrhythmic in WT. It increased INa in RyR2(S/S) atria, whereas it reduced INa as expected in WT. It increased AERP while sparing CV in RyR2(S/S) , but reduced CV while sparing AERP in WT. Thus, RyR2(S/S) hearts have low λ relative to WT; flecainide then increases λ in RyR2(S/S) but decreases λ in WT.

Conclusions: Flecainide (1 μm) rescues the RyR2-P2328S atrial arrhythmogenic phenotype by restoring compromised INa and λ, changes recently attributed to increased sarcoplasmic reticular Ca(2+) release. This contrasts with the increased arrhythmic incidence and reduced INa and λ with flecainide in WT.

Keywords: CPVT; Na+ currents; atrial arrhythmia; conduction velocity; flecainide; ryanodine receptor.

Figure 1

Conduction velocity analysis. A representative MEA recording is displayed as a set of individual traces obtained at each electrode site in the centre panel. Panels (a–d) illustrate the data analysis in which (a) the local activation time (LAT) is determined from the maximum negative dV/dt (arrowhead) of atrial electrograms at each recording site. (b) LATs from four neighbouring recording sites are used to derive (c) the conduction velocity vector for each 2 × 2 square of electrodes and then visually inspected (d) to ensure the absence of wavefront collision/splitting.

Figure 2

Contrasting actions of flecainide on arrhythmic incidence in RyR2^S/S and WT. (a) Illustration of the S1S2 stimulation protocol, consisting of repeated cycles of 8 S1 stimuli, each followed by a single extrasystolic S2 stimulus imposed at successively shorter S1S2 intervals. The first and last few cycles of the protocol are shown, with the intervening cycles omitted (dashed lines). The protocol was terminated when an S2 either failed to elicit an AP, as observed by a missing atrial electrogram, or produced an arrhythmia. Thus, panel (a) depicts the penultimate stimulus cycle, whose S2 stimulus successfully elicited conducting electrical activity (a), followed by the final cycle that induced either arrhythmia or refractoriness. Typical traces obtained from (b) WT and (c) RyR2^S/S before (i) and following (ii) introduction of 1 μm flecainide were obtained from the last stimulus cycle whose S2 stimulus successfully elicited electrical activity (left panels) and the final cycle which induced either arrhythmia or refractoriness (right panels) as described above. The filled arrowheads indicate timings of regular (S1) stimulation, and the filled arrows indicate the resulting S1 atrial electrogram. The open arrowheads indicate the timing of the extrasystolic (S2) stimuli, and the open arrows indicate the resulting S2 atrial electrogram. The arrowheads are directly below the stimulus artefact, and the arrows are directly above the resulting atrial electrogram. Note that atrial electrogram conduction from the point of stimulation to the point of recording is slow relative to conduction of the stimulus artefact, such that the S2 stimulus artefacts can appear within the preceding S1 waveform at the recording site despite occurring after the atrial electrogram at the stimulus site. Panel (d) depicts the results of applying the PES protocol to 10 WT and 17 RyR2^S/S hearts to assess the incidence of arrhythmic events normalized to the number of hearts studied in each group. * denotes a difference (P < 0.05) at 0 and 1 μm flecainide within a genotype. ^† denotes a difference (P < 0.05) between RyR2^S/S and WT genotypes at the same flecainide concentration.

Figure 3

Paradoxical actions of flecainide on I_Na activation in RyR2^S/S and WTatria. Currents in response to depolarizing steps increased in 10 mV increments from 20 to 120 mV in voltage-clamped WT (a, n = 7) and RyR2^S/S (b, n = 6) left atria in the presence of 0, 1 and 5 μm flecainide. Currents in response to an 80 mV depolarizing step under control conditions and in the presence of the specific RyR blocker dantrolene (10 μm) are shown in the inset. The current–voltage relationships were fitted to Boltzmann functions for WT (c, left panel) and RyR2^S/S (d, left panel) in the presence of 0, 1 and 5 μm flecainide. The right panels in (c) and (d) compare the maximum peak currents before and following withdrawal of flecainide. *denotes significant effects of flecainide or dantrolene. ^†denotes significant differences between RyR2^S/S and WT genotypes at the same flecainide concentration.

Figure 4

Paradoxical actions of flecainide on I_Na inactivation in RyR2^S/S and WT. Currents in response to successively incremented pre-pulse voltages from 0 to 90 mV, and finally 95 mV, followed by a test voltage excursion of 100 mV in voltage-clamped WT (a, n = 7) and RyR2^S/S (b, n = 6) left atria. (c, d) The dependence of peak I_Na upon pre-pulse voltage excursion fitted to Boltzmann functions for WT (c) and RyR2^S/S (d) in the presence of 0, 1 and 5 μm flecainide. These experiments employed the same atria as in the experiments depicted in Figure3.

Figure 5

Paradoxical actions of flecainide on conduction velocities in RyR2^S/S and WT. Three-dimensional representations of local activation times (LATs) each accompanied by matrices representing the calculated velocity vectors in WT (a, n = 15) and RyR2^S/S hearts (b, n = 14) in 0, 1 and 5 μm flecainide. Mean (±SEM) epicardial conduction velocities for WT (clear bars) and RyR2^S/S (black bars) in 0, 1, 5 and following subsequent return to 0 μm flecainide during regular 6.67, 8 and 10 Hz pacing (c). *denotes a difference arising from use of 1 μm flecainide within a genotype compared to the respective control (0 μm flecainide). ^†denotes a difference between RyR2^S/S and WT genotypes with the same concentrations of flecainide. In each case, single, double and triple symbols denote P < 0.05, P < 0.01 and P < 0.001 respectively.

Figure 6

Paradoxical actions of flecainide on AERP in RyR^S/S and WT. Individual paired and mean (±SEM) AERPs in 0 and 1 μm flecainide for WT (n = 9) and RyR2^S/S (n = 17) hearts. ** denotes a difference (P < 0.01) arising from use of 1 μm flecainide within a genotype compared to the respective control (0 μm flecainide).

Figure 7

Paradoxical actions of flecainide on CV, AERP and λ and their correlations with arrhythmic incidence. Left panels: comparison of CV (a), AERP (b) and λ (c) in WT (open bars, n = 8) and RyR2^S/S (filled bars, n = 10) hearts in 0 and 1 μm flecainide. These are correlated with incidences of atrial tachyarrhythmias (AT) (a–c, right panels). * denotes a difference arising from use of 1 μm flecainide within a genotype compared to the respective control (0 μm flecainide). ^† denotes a difference between RyR2^S/S and WT genotypes under the same concentration of flecainide. In each case, single, double and triple symbols denote P < 0.05, P < 0.01 and P < 0.001 respectively.