Channel Activity of Cardiac Ryanodine Receptors (RyR2) Determines Potency and Efficacy of Flecainide and R-Propafenone against Arrhythmogenic Calcium Waves in Ventricular Cardiomyocytes.

Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca2+ waves and prevents catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans. We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca2+ waves in cardiomyocytes. Using confocal microscopy, we studied Ca2+ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models (Casq2-/-, RyR2-R4496C+/-), wild-type (c57bl/6, WT) mice, and WT rabbits (New Zealand white rabbits). Consistent with increased RyR2 activity, Ca2+ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca2+ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca2+ waves with significantly higher potency (lower IC50) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca2+ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca2+ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT

Ca²⁺ sparks and waves in permeabilized myocytes from WT, RyR2-R4496C and Casq2^-/- mice under control (VEH) conditions.

<p>A. Averaged spark frequency, full duration at half maximun (FDHM), spark mass and spark-mediated leak measured for each one of the groups studied. ***<i>P</i><0.001, n = 35–60 cells, N = 2–3 mice/group. Results expressed as mean±SEM. B. Representative line-scans (LS) showing sparks registered in permeabilized WT, RyR2-R4496C, and Casq2^<b>-/-</b> myocytes in internal solution (IS) containing Fluo 4 pentapotassium salt ([EGTA]_IS = 0.4 mM, estimated free [Ca^<b>2+</b>]_IS = 0.04 μM (Maxchelator)). C. Averaged amplitude, frequency and propagation speed of Ca^<b>2+</b> waves measured in WT, RyR2-R4496C and Casq2^<b>-/-</b> permeabilized myocytes. *P<0.05, **P<0.01. n = 30–60 cells, N = 2 mice/group. D. Representative LS of Ca^<b>2+</b> waves from permeabilized WT, RyR2-4496C mutant cells and Casq2^<b>-/-</b> myocytes in internal solution containing Fluo-4 pentapotassium salt ([EGTA]_IS = 0.05 mM, estimated free [Ca^<b>2+</b>]_IS = 3.9 μM (Maxchelator)).</p

Effect of RPROP and TET on Ca²⁺ sparks and waves in permeabilized rabbit ventricular myocytes.

<p>A. Bar representation (left side of the panel) of averaged Ca^<b>2+</b> spark parameters (amplitude, frequency and mass) measured in permeabilized rabbit ventricular myocytes under control conditions (VEH) and in the presence of RPROP 10 μM and TET 50 μM. **P<0.01. n = 30–40 cells, N = 3 rabbits/condition tested. B Representative LS for each condition. C CRCs for RPROP and TET for Ca^<b>2+</b> wave parameters. n = 30 cells, N = 3 rabbits/condition tested. D. Representative LS for VEH, RPROP 25 μM and TET 25 μM.</p

Caffeine increases potency of flecainide (FLEC) against Ca²⁺ waves.

<p>A. Representative LS of Ca^<b>2+</b> waves in permeabilized WT myocytes, WT cells sensitized with 150 μM caffeine (WT+CAFF 150μM) and Casq2^<b>-/-</b> myocytes under control condition (VEH) and in the presence of FLEC 10 μM. B. CRCs for wave parameters (incidence, amplitude, frequency, and propagation speed) as a function of the concentration of FLEC (in μM) in WT, WT+CAFF 150μM, and Casq2^<b>-/-</b> cells. FLEC effect on Ca^<b>2+</b> wave parameters is dose-dependent, with a strongest effect on Ca^<b>2+</b> waves in Casq2^<b>-/-</b> myocytes. FLEC effects on Ca^<b>2+</b> waves in WT cells are partially restored by sensitizing the cells with CAFF 150 μM. Mean±SE for each concentration and each parameter. For each group: n = 20 cells, N = 3 mice/condition tested.</p

Potency (IC₅₀, expressed in μM) and efficacy (defined as maximum drug effect measured at 100 μM) of all the drugs tested in the different mouse groups.

<p>N = 20–25 cells/condition tested. All measurements are relative to measurements obtained in cells exposed to VEH.</p><p>Potency (IC₅₀, expressed in μM) and efficacy (defined as maximum drug effect measured at 100 μM) of all the drugs tested in the different mouse groups.</p

Effects of flecainide (FLEC) on Ca²⁺ wave parameters from permeabilized WT, RyR2-R4496C, and Casq2^-/- myocytes.

<p>A. Representative LS (left side of the panel) obtained for WT, RyR2-R4496C, and Casq2^<b>-/-</b> myocytes, and their corresponding averaged space LS (right side of the same panel) under control condition (VEH) and in the presence of flecainide (FLEC) 10 μM. B. Concentration-response curves (CRCs) for wave incidence, amplitude, frequency, and propagation speed as a function of the concentration of FLEC (in μM) in WT, RyR2-4496C mutant cells, and Casq2^<b>-/-</b> myocytes. Results are expressed as mean±SE for each concentration and each parameter. For each group studied: n = 20 cells, N = 3 mice/ condition tested.</p