Synergistic antiarrhythmic mechanism of INa-L and RyR2 blockade: normalization of pathological calcium cycling and CaMKII signaling

Arrhythmias represent a leading cause of mortality among individuals with cardiovascular diseases. Considering the failure of antiarrhythmic drugs targeting single-ion channels in clinical trials, this study aims to evaluate the pharmacodynamics of various combinations of single-channel blockers to identify safer and more effective therapeutic regimens. The antiarrhythmic effects of inhibitors alone and pairwise combination strategies targeting late sodium current (INa-L), hERG potassium current (IKr), ryanodine receptor 2 (RyR2), and L-type calcium current (ICa-L) inhibitors via a checkerboard dosing approach were evaluated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Comprehensive evaluation based on synergy scoring, efficacy, and toxicity (SynergyFinder) was performed to identify the preferred combination with high efficacy and low toxicity. The effectiveness of the preferred combination was further validated in isolated perfused guinea pig hearts. In addition, an analysis of the effects of the preferred combination on calcium sparks and Ca2+/calmodulin-dependent protein kinase II (CaMKII) was conducted. A combination regimen of the INa-L inhibitor GS967 and the RyR2 inhibitor Dantrolene, which exhibited highly effective, low-toxicity synergistic antiarrhythmic properties, was identified through checkerboard high-throughput screening. An alternative combination of the INa-L blocker Eleclazine and the RyR2 inhibitor TMDJ-035 showed similar therapeutic efficacy, indicating that the effectiveness of the preferred combination stems from target-mediated effects. The combination further demonstrated significant efficacy in multiple hiPSC-CM arrhythmia models and an ex vivo guinea pig heart reperfusion arrhythmia model. Mechanistic studies revealed that the dual-target inhibition strategy corrected pathological calcium sparks and calcium cycling dysfunction, downregulated the CaMKII-RyR2 phosphorylation cascade, thereby restoring electrical signaling. The combination of INa-L and RyR2 inhibitors exerts safer and more effective antiarrhythmic effects. This combinational therapeutic strategy provides a theoretical basis for developing novel antiarrhythmic regimens based on dual-target regulation, demonstrating significant translational medical potential and warranting further clinical evaluation.