Flecainide inhibits arrhythmogenic Ca2+ waves by open state block of ryanodine receptor Ca2+ release channels and reduction of Ca2+ spark mass

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin. We recently found that the drug flecainide inhibits RyR2 channels and prevents CPVT in mice and humans. Here we compared the effects of flecainide and tetracaine, a known RyR2 inhibitor ineffective in CPVT myocytes, on arrhythmogenic Ca(2+) waves and elementary sarcoplasmic reticulum (SR) Ca(2+) release events, Ca(2+) sparks. In ventricular myocytes isolated from a CPVT mouse model, flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass. Surprisingly, flecainide significantly increased spark frequency. As a result, flecainide had no significant effect on spark-mediated SR Ca(2+) leak or SR Ca(2+) content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca(2+) leak, resulting in a significantly increased SR Ca(2+) content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca(2+) waves. In lipid bilayers, flecainide inhibited RyR2 channels by open state block, whereas tetracaine primarily prolonged RyR2 closed times. The differential effects of flecainide and tetracaine on sparks and RyR2 gating can explain why flecainide, unlike tetracaine, does not change the balance of SR Ca(2+) fluxes. We suggest that the smaller spark mass contributes to flecainide's antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca(2+) release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca(2+) waves resulting in triggered arrhythmias, such as CPVT.

Fig. 1

Flecainide prevents premature Ca²⁺ waves. (A) Representative examples of cytosolic Ca²⁺ fluorescence recordings from field-stimulated Casq2-/- myocytes loaded with the fluorescent indicator fura2AM. Arrhythmogenic Ca²⁺ waves (arrows) were induced by 1 μM ISO in the bath solution. SR Ca²⁺ content was quantified by rapid caffeine (10 mM) 0.5 Hz pacing train. In vehicle treated cells (VEH), spontaneous Ca²⁺ waves followed each application following the paced beat (|). Pretreatment with flecainide (FLEC, 6 μM) prevented the generation of Ca²⁺ waves during the pacing train. (B) Fraction of Casq2-/- myocytes that exhibit ISO-induced Ca²⁺ waves during the pacing train. VEH n=27, FLEC n=26, ***p<0.001 by Fisher-exact test. (C) Comparison of average rate of Ca²⁺ waves during pacing train. Only myocytes that exhibited Ca²⁺ waves were included in the analysis. VEH n=24, FLEC n=10, *p<0.05, ***p<0.001. (D) Comparison of average SR Ca²⁺ content after the pacing train. VEH n=27, FLEC n=26 myocytes.

Fig. 2

Differential effect of flecainide and tetracaine on Ca²⁺ sparks and SR Ca²⁺ content in intact Casq2-/- myocytes. (A) Representative line scans and individual Ca²⁺ sparks (red box) of quiescent Casq2-/- myocytes loaded with Fluo-4AM. To avoid excessive Ca²⁺ wave generation, ISO concentration was reduced to 0.1 μM in the bath solution. Myocytes were incubated for 10 min with either vehicle (VEH), flecainide (FLEC, 6 μM) or tetracaine (TET, 50 μM) before obtaining the confocal images. Red box: spark plot to the right (B) Representative line scans during rapid caffeine application. The amplitude of the caffeine transient was used as an estimate of SR Ca²⁺ load. (C-F) Comparison of average spark frequency (C), spark mass (D), spark mediated SR Ca²⁺ leak (=spark mass × spark frequency) and SR Ca²⁺ content (F). n=77-97 myocytes per group, *p<0.05, ***p<0.001

Fig. 3

Effects of flecainide or tetracaine on Ca²⁺ waves and the SR Ca²⁺ content in permeabilized rat myocytes (A) Representative integrated line-scan images (left) showing spontaneous Ca²⁺ waves in permeabilized ventricular myocytes during exposure to vehicle (VEH) and after 2 min exposure to either 25 μM flecainide (FLEC) or 50 μM tetracaine (TET). Average data (right) comparing the effects of flecainide and tetracaine on Ca²⁺ wave amplitude and frequency. FLEC n = 9, TET n = 7, **p<0.01, ***p<0.001. (B) Representative integrated line scan images (left) showing spontaneous Ca²⁺ waves in permeabilized ventricular myocytes in the presence of vehicle or 2 min after introduction of 25 μM FLEC. In both cases, the wave frequency was monitored and 10 mM caffeine rapidly applied when a wave would otherwise have occurred (arrowhead). The amplitude of the caffeine-induce Ca²⁺ transient was used as an index of the SR Ca²⁺ content in each case. Average data comparing the effects of flecainide and tetracaine on the SR Ca²⁺ content under these conditions (right). FLEC n = 6, TET n = 6, **p<0.01, ***p<0.001, n.s. = not significant.

Fig. 4

Effects of flecainide and tetracaine on spontaneous Ca²⁺ sparks in permeabilized myocytes (A) Surface plots of raw line scan images obtained from a permeabilized rat ventricular myocyte during spontaneous Ca²⁺ wave generation, in the presence of vehicle (left) or after introduction flecainide (right). Note the increased spark frequency in the myocyte exposed to FLEC. The cell was bathed in weakly Ca²⁺ -buffered solutions (0.05 mM EGTA) approximating to the intracellular milieu, with a free [Ca²⁺] of ∼200 nM. (B) Cumulative data showing the relative changes in Ca²⁺ spark frequency, amplitude, mass, duration and width following introduction of 25 μM flecainide or 50 μM tetracaine. Spark data was obtained in more strongly Ca²⁺ buffered solutions (0.36 mM) to prevent Ca²⁺ wave propagation. **p<0.01, ***p<0.001, n.s. = not significant. 160-341 sparks from n = 25-33 myocytes per group.

Fig. 5

Flecainide and tetracaine block single RyR2 channels by different mechanisms. (A,B) Records are representative examples of single channel activity of RyR2 in lipid bilayers. Control conditions were 1 mM luminal Ca²⁺ (trans bath), 0.1 μM cytoplasmc Ca²⁺ plus 2 mM ATP (cis bath). Bilayer potential was 40 mV (relative to trans bath as ground). Relatively high concentrations of flecainide (FLEC) and tetracaine (TET) were used to better illustrate their differential effect on RyR2 channel gating. (A) Single experiment where flecainide was added to the cytoplasmic bath. Under control conditions, this channel had an open probability (P_o) of 0.25, mean open time (τ_o) of 83 ms and mean closed time (τ_c) of 228 ms. The full duration of typical closed periods is not seen on this time scale. Addition of flecainide introduced short (∼ 1 ms) closures to a substate at ∼20% of the full channel conductance which lead to bursts of short channel openings. (B) Different experiment where tetracaine was added to the bath. Under control conditions, this channel had P_o = 0.11, τ_o = 33 ms and τ_c = 237 ms. Addition of 100 μM tetracaine increased τ_c to 913 ms but had little effect on τ_o (τ_o =27 ms). (C) The effects of flecainide and tetracaine on burst parameters were compared at concentrations that reduced RyR2 Po by approximately 50% (10 μM and 50 μM, respectively). (D-F) Comparison of average burst parameters derived from burst analyses of single channel recordings. The data are expressed as burst properties relative to control before addition of FLEC or TET (control conditions are given in methods). Note that FLEC inhibits RyR2 by reducing burst duration (D) and intraburst Po (E), whereas TET increased the interburst closed duration (F). *p<0.05, **p<0.01, n=10 per group

Fig. 6

Flecainide and tetracaine block single RyR2 channels by different mechanisms. The concentration dependence of the effects of flecainide (●) and tetracaine (■) on the total RyR2 activity within bursts (burst mass). For ease of comparison, drug concentrations are expressed as a ratio of concentration and the IC₅₀ values (flecainide 16 μM, tetracaine 44 μM). Burst mass was calculated from the product of the burst duration (Supplemental Fig. 1C) and the open probability within bursts (Supplemental Fig. 1D) normalized to values obtained in the absence of drug. *p<0.05, **p<0.01, ***p<0.001 compared to control by paired t-test.