Selective RyR2 inhibition reduces arrhythmia susceptibility in human cardiac slices

Ryanodine receptor 2 (RyR2) hyperactivity is frequently observed in structural heart disease (SHD), commonly caused by ischemic heart disease. This aberrant Ca²⁺ release promotes irregular electrical activity and life-threatening arrhythmias. Our previous work demonstrated that the pan-ryanodine receptor (RyR) inhibitor dantrolene can reverse arrhythmogenic substrates, but its lack of RyR2 selectivity limits its therapeutic potential. The unnatural verticilide enantiomer (ent-verticilide) was identified as a selective RyR2 inhibitor, showing promising specificity without activating skeletal muscle RyR1. This study evaluated the antiarrhythmic potential of a selective RyR2 antagonist, ent-(+)-verticilide (ent-Vert), in human ventricular slices. Right and left ventricular slices were prepared from human hearts not designated for transplantation. Pseudo-electrocardiograms (ECG) tracked premature ventricular contraction (PVC) incidence, and optical mapping identified arrhythmogenic substrates. Baseline optical recordings were obtained before treatment with isoproterenol (Iso) (250 nM) and caffeine (200 µM) to induce Ca² ⁺ leak and arrhythmogenic activity, followed by sequential ent-Vert application (1 and 3 µM). The effects of ent-Vert alone were tested with baseline recordings followed by a single 3 µM dose. Iso and caffeine increased PVC incidence in both right (RV) and left ventricular (LV) slices, but ent-Vert (1 and 3 µM) significantly reduced it. Iso and caffeine also shortened action potential duration (APD) in both slice types, but ent-Vert, after Iso and caffeine or alone (under control conditions), did not significantly change APD. In conclusion, ent-Vert suppressed arrhythmic triggers and reduced arrhythmogenic substrates in human ventricular slices, highlighting its potential as a targeted antiarrhythmic therapy for SHD. KEY POINTS: RyR2 hyperactivity is a major driver of arrhythmias in structural heart disease, producing abnormal Ca² ⁺ release and premature ventricular contractions. The RyR2-selective inhibitor ent-Vert was evaluated in human RV and LV slices to determine its antiarrhythmic potential. β-Adrenergic and RyR2 activation with Iso + caffeine induced Ca² ⁺ leak, ectopic beats and shortened APD. Sequential ent-Vert application (1 and 3 µM) significantly suppressed ectopy incidence in a dose-dependent manner. ent-Vert did not alter APD or conduction velocity under control or stimulated conditions, demonstrating that it suppresses arrhythmic triggers without disrupting normal cardiac electrophysiology.