Adrenergically-induced Arrhythmias in Induced Pluripotent Stem Cell-derived Cardiomyocytes Generated from CPVT Patients Carrying Mutations in the Ryanodine or Calsequestrin Genes

Atara Novak ^1,2,3 Barad Lili ^1,2,3 Avraham Lorber ⁴ Liron Eldor ⁵ Joseph Itskovitz-Eldor ^1,3,6 Michael Eldar ⁷ Michael Arad ⁷ Ofer Binah ^1,2,3

¹-, The Sohnis Family Stem Cells Center, Haifa
²The Rappaport Family Institute for Research in the Medical Sciences, Technion, Haifa
³Ruth & Bruce Rappaport Faculty of Medicine, Technion, Haifa
⁴Department of Pediatric Cardiology, Rambam Health Care Campus, Haifa
⁵Department of Plastic Surgery, Rambam Health Care Campus, Haifa
⁶Department of Obstetrics and Gynecology, Rambam Health Care Campus, Haifa
⁷Leviev Heart Center, Sheba Medical Center, Tel Hashomer and Sackler School of Medicine, Tel Aviv University, Tel Aviv

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder characterized by episodic syncope and sudden death occurring during exercise or acute emotion in individuals without structural cardiac abnormalities. The disease is caused by abnormal Ca2+ handling resulting from mutations in the RYR2 or the CASQ2 genes. The present study had two aims: (1) to investigate for the first time cardiomyocytes derived from induced Pluripotent Stem Cells (iPSC-CM) generated from dermal fibroblasts obtained from CPVT patients caring the heterozygous R420Q mutation in the RYR2 gene (CPVT-RYR2R420Q-CM). (2) To compare isoproterenol-induced functional derangement of CPVT-RYR2R420Q-CM and iPSC-CM generated from patients carrying the D307H mutation in the CASQ2 gene (CPVT-CASQ2D307H-CM). Our major findings were: (1) while in healthy iPSC-CM β-adrenergic stimulation with isoproterenol only increased the spontaneous firing rate, in CPVT-RYR2R420Q-CM isoproterenol caused delayed afterdepolarizations (DADs) and triggered activity blocked by the β-blocker Bisoprolol. (2) In both CPVT mutations, in the age range of 20-70 day-old embryoid bodies (EBs), the arrhythmogenic phenotype was age-dependent, being more pronounced in cardiomyocytes > 50 day old. We propose that these age-dependent isoproterenol-induced arrhythmias were due do maturational changes in sarcoplasmic reticulum function and in its Ca2+ storage/release capacity. (3) Compared to healthy iPSC-CM, caffeine releases more Ca2+ in CPVT-CASQ2D307H-CM and less Ca2+ in CPVT-RYR2R420Q-CM. Additionally, while healthy iPSC-CM and CPVT-RYR2R420Q-CM demonstrated prompt recovery after caffeine-induced SR Ca2+ release, the CPVT-CASQ2D307H-CMs demonstrated slower recovery. These findings may result from one or more of the following options: (1) Different Ca2+-storing capacity of the mutated iPSC-CM; (2) CPVT-CASQ2D307H-CM have unstable RyR2 channel due to non-functional CASQ2 which causes uncontrollable Ca2+ release; (3) The two mutations have different responsiveness to caffeine. Our results show that the mutated cardiomyocytes can be used to study the electrophysiological mechanisms and the differences between the mutations in RYR2 and CASQ2 underlying CPVT.