Ranolazine stabilizes cardiac ryanodine receptors: a novel mechanism for the suppression of early afterdepolarization and torsades de pointes in long QT type 2

Abstract

Background: Ranolazine (Ran) is known to inhibit multiple targets, including the late Na(+)current, the rapid delayed rectifying K(+)current, the L-type Ca(2+)current, and fatty acid metabolism. Functionally, Ran suppresses early afterdepolarization (EADs) and torsades de pointes (TdP) in drug-induced long QT type 2 (LQT2) presumably by decreasing intracellular [Na(+)](i) and Ca(2+)overload. However, simulations of EADs in LQT2 failed to predict their suppression by Ran.

Objective: To elucidate the mechanism(s) whereby Ran alters cardiac action potentials (APs) and cytosolic Ca(2+)transients and suppresses EADs and TdP in LQT2.

Methods: The known effects of Ran were included in simulations (Shannon and Mahajan models) of rabbit ventricular APs and Ca(2+)transients in control and LQT2 models and compared with experimental optical mapping data from Langendorff rabbit hearts treated with E4031 (0.5 μM) to block the rapid delayed rectifying K(+)current. Direct effects of Ran on cardiac ryanodine receptors (RyR2) were investigated in single channels and changes in Ca(2+)-dependent high-affinity ryanodine binding.

Results: Ran (10 μM) alone prolonged action potential durations (206 ± 4.6 to 240 ± 7.8 ms; P <0.05); E4031 prolonged action potential durations (204 ± 6 to 546 ± 35 ms; P <0.05) and elicited EADs and TdP that were suppressed by Ran (10 μM; n = 7 of 7 hearts). Simulations (Shannon but not Mahajan model) closely reproduced experimental data except for EAD suppression by Ran. Ran reduced open probability (P(o)) of RyR2 (half maximal inhibitory concentration = 10 ± 3 μM; n = 7) in bilayers and shifted half maximal effective concentration for Ca(2+)-dependent ryanodine binding from 0.42 ± 0.02 to 0.64 ± 0.02 μM with 30 μM Ran.

Conclusions: Ran reduces P(o) of RyR2, desensitizes Ca(2+)-dependent RyR2 activation, and inhibits Ca(i) oscillations, which represents a novel mechanism for its suppression of EADs and TdP.

Figure 1. Ran Suppresses EADs and TdP in LQT2

Left Panel: Vm (blue) and Ca_i (red) measured from the same site at the base of the heart. A: Control AP and Ca_iT with the heart was paced at 2 s CL B: 10 min after E4031 (0.5µm) C: 10 min after E4013 plus Ran (10µM) Right Panel: The two drugs were added in the reverse order. A’: Control, 15 min after Ran B’: 10 min after Ran plus E4031 C’: 5 min after washout of Ran but with E4031 D’: Washout of Ran prolonged APD and unmasked EADs due to the presence of E4031

Figure 2. Comparison of mathematical models with experimental data

A: APs (top) and Ca_iT (bottom) derived from Shannon (a) and Mahajan (b) models and optical signals from rabbit hearts (c) at different CLs (500 (black), 1,000 (blue) and 2,000 ms (red)). B: Quantitative comparison of APD₉₀ (a), Ca_iT₇₅ (b), Ca_iT rise-time (c) and peak-Ca_iT (d) between mathematical models (Shannon, red; Mahajan, green) and experimental data (blue). C: Predicted APs at 2 s CL by the Shannon model (a); control in blue and LQT2 in red and by the Mahajan model (b); control in blue and LQT2 in red. LQT2 simulation (see Methods) produced EADs with Shannon but not Mahajan.

Figure 3. Simulation I - Antiarrhythmic effect of Ranolazine

Steady state APs and Ca_iTs for the last four beats in a train of 75 pulses at 2 s CL under LQT2 condition (black); under LQT2 plus 10µM Ran (blue) as described in ‘Supplement’. Ran failed to suppress EADs.

Figure 4. Effects of Ranolazine on P_o of RyR2 and Ca²⁺-dependent [³H]ryanodine binding

A: Characteristic single channel fluctuations following fusion of cardiac SR vesicles to a planar bilayer as a function of [Ran]. P_o was measured in the presence of 50µM Ca²⁺ to maintain a highly active channel (i.e. P_o ~0.5) and was averaged over 2 min of continuous recordings. c = closed, o = open state. B: Normalized P_o±SE vs. [Ran], n=7. C: Ryanodine-binding vs. free [Ca²⁺] (Ca²⁺-selective electrode). Ryanodine binding was measured ± 30 M Ran with SR vesicles (0.5 mg/ml), data are average ± SE (n=4).

Figure 5. Simulated effect of Ranolazine on APs and Ca_iTs

Top traces: Steady state APs and Ca_iTs at 500 ms CL from the Shannon model at control (blue), 5µM (black) and 10µM (red) Ran. Bottom traces: APs and Ca_iTs shown at faster sweep speed.

Figure 6. Simulation II– Various [Ran] in LQT2 model

APs and Ca_iTs from the Shannon model showing the last four beats from a train of 75 pulses at 2 s CL (black), with LQT2 (red), LQT2 plus 5µM Ran (blue) and LQT2 with 10µM Ran (green). EADs persisted with 5µM but not 10µM Ran.

Figure 7. Antiarrhythmic effect of Ranolazine when RyR2 is inhibited

APs and Ca_iTs (Shannon model) showing the last four beats after 75 pulses at 2-s CL with LQT2 (black); with LQT2 and 10µM Ran but without (blue) and with RyR2 inhibition (red).