Programmed electrical stimulation in human iPSC-derived cardiomyocytes reveals mechanisms of lethal arrhythmias in Calcium Release Deficiency Syndrome

BackgroundCalcium release deficiency syndrome (CRDS) is a recently described inherited channelopathy caused by loss-of-function variants in RYR2. Clinically, CRDS patients present with lethal ventricular arrhythmias which are not reproduced on exercise stress testing, unlike catecholaminergic polymorphic ventricular tachycardia. A hallmark trigger identified for CRDS mimics a long-burst, long-pause, short-coupled extra-stimulus (LBLPS) programmed electrical stimulation protocol, which was experimentally validated in humans and mouse models. Moreover, application of a long-burst, long-pause (LBLP) protocol alone can induce an abnormal repolarization on the first sinus beat that is unique to CRDS. However, the electrophysiological basis of CRDS in human cardiac tissue, including other triggers, are not fully understood, and whether clinically relevant arrhythmias can be observed in human stem cell models remains unknown. MethodsWe performed electrophysiological and arrhythmia inducibility studies using clinically relevant programmed electrical stimulation protocols in two-dimensional cardiac tissue generated from metabolically matured human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the CRDS variant RyR2-E4146D. High spatiotemporal optical mapping and multielectrode arrays were used for electrophysiological phenotyping. ResultsAt baseline, E4146D+/- monolayers showed no arrhythmias, similar to controls. During rapid pacing, E4146D+/- promoted electrical vulnerability by reducing the threshold for action potential duration (APD) alternans and Ca2+ alternans and increasing the propensity for spatial discordance of alternans. In response to LBLP pacing, E4146D+/- monolayers often demonstrated an abnormal repolarization response characterized by spatially dispersed APD prolongation and large Ca2+ release. Notably, LBLPS pacing produced early-after depolarization (EAD)-driven triggered activity resulting in re-entrant tissue conduction patterns, explaining the short-coupled ectopy driven arrhythmias seen in CRDS patients. Similar arrhythmias were observed when EADs developed during spatially discordant alternans. Lastly, flecainide showed efficacy in suppressing arrhythmia inducibility for the here studied variant. ConclusionsWe developed the first hiPSC model for CRDS which recapitulates clinically observed and inducible arrhythmias. Our model provides novel insights into tissue-level, re-entrant arrhythmias, which are initiated by EADs during electrically vulnerable states in CRDS human cardiac tissue and can be suppressed by flecainide. This model provides the framework for studying other CRDS variants and complex arrhythmias in hiPSC-CMs and establishes a human-based new approach method (NAM) for drug and gene therapy development for CRDS. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABS{blacksquare} We developed the first human stem cell-derived cardiomyocyte (hiPSC-CM) tissue model for calcium release deficiency syndrome (CRDS) which recapitulates its hallmark clinical features, including inducible ventricular arrhythmias with programmed electrical stimulation and post-pacing repolarization abnormalities. {blacksquare}Using genome edited and metabolically matured hiPSC-CMs combined with high spatiotemporal optical mapping, we show that tissue-level arrhythmias are initiated by early-after depolarizations (EADs) which develop during electrically vulnerable states, leading to re-entrant conduction patterns. We comprehensively characterize the features of EAD-induced triggered activity, showing that these ectopic beats promote re-entry through slower conduction velocities and shorter action potential durations. This uncovers how EAD-induced short-coupled ectopy leads to malignant ventricular arrhythmias in CRDS patients, and establishes the phenotype for future hiPSC-CM investigations. {blacksquare}We identified flecainide as an effective agent in suppressing arrhythmias on single cell and tissue levels in hiPSC-CMs for this CRDS variant, reproducing clinical results. What are the clinical implications?{blacksquare} CRDS has only recently been described as a unique channelopathy caused by loss-of-function RYR2 variants, and much of its triggers and mechanisms in human cardiomyocytes remain unclear. The arrhythmias observed are often not related to exercise, and exercise stress testing does not reproduce these abnormalities. No human models exist to date which closely recapitulate the triggers shown to induce tissue-level arrhythmias in patients and mouse models. Our model demonstrates that programmed electrical stimulation, without pharmacological {beta}-adrenergic stimulation, can reliably induce the same arrhythmias seen clinically, enabling accurate disease modeling and drug development. {blacksquare}Combining programmed electrical stimulation in cardiac tissue derived from genome-edited hiPSC-CMs with high spatiotemporal optical mapping is a robust and novel approach to identify the mechanisms of complex, tissue-level arrhythmias which remain underexplored, such as short-coupled ventricular fibrillation, in a patient-specific and translational manner.