Ca2+ handling in induced-pluripotent cardiomyocytes from female CPVT patient, and comparison to male

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence Nationale de la Recherche Introduction Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an inherited disease manifested as syncope or sudden death in apparently healthy children or young adults, in the absence of structural cardiac disease. The ryanodine receptor (RyR2) R420Q mutation was identified in a Spanish 14-year-old boy who died suddenly due to emotional stress. We generated human induced-pluripotent (h-iPS) derived cardiomyocytes from two brothers (see Yin’s poster in this meeting). As most of sudden death occurs in males, we had the hypothesis that there might be some sex-dependent differences. Purpose and methods We aim to elucidate the key role of [Ca2+]i in the genesis of cardiac arrhythmia in female RyR2R420Q CPVT patients . We generated here hiPSC-CM from two sisters of the same family, one exhibiting the mutation RyR2R420Q and the other without the mutation used as control. The experiments were conducted at 30-35 days after differentiation into cardiomyocytes. Using confocal microscopy, we assessed intracellular [Ca2+]i handling both spontaneous and during electrical pacing. To record [Ca2+]i transients, h-iPS-CMs were electrically field stimulated by two Pt electrodes at 1Hz. Spontaneous Ca2+ sparks and Ca2+ waves were recorded in quiescent cells, after [Ca2+]i transients recordings. For SR Ca2+ load quantification, h-iPS-CMs were rapidly perfused with 10 mmol/L caffeine. To induce β-adrenergic activation, 100nM isoproterenol (ISO) was added in some experiments. All confocal Ca2+ images analyses were performed by home-made routines using IDL software. Results First, we verify by immunofluorescence the expression of alpha-actinin in the differentiated h-iPS-CMs. Then, by confocal microscopy we found that woman CPVT h-iPS-CMs presented longer cycle length than wt h-iPS-CMs, correlating with the bradycardia observed in CPVT patients. The amplitude of the Ca2+ transients were significantly higher for CPVT h-iPS-CMs compared to wt; however, after ISO perfusion Ca2+ transients did not show a significant increase in CPVT h-iPS-CMs compared to CPVT h-iPS-CMs before ISO. SR Ca2+ load presented no differences between groups but the time decay constant of the caffeine-evoked Ca2+ transients were significantly faster in CPVT h-iPS-CMs whereas it became slower after ISO perfusion. In addition, analysis of Ca2+ pro-arrhythmogenic events were significantly augmented in CPVT h-iPS-CMs compared to wt h-iPS-CMs. After ISO perfusion, pro-arrhythmogenic events in CPVT cells presented no significant difference compared to CPVT cell before ISO perfusion. Although proarrhythmogenic events were similarly higher in both male and female hiPSC-CM, the Ca2+ handling characteristics were slightly different. Conclusion The RyR2R420Q mutation in woman CPVT h-iPS-CMs provides a reliable model to study CPVT in human context.