CPVT-Associated Mutation P.G357S-RYR2 Promotes a Gain of Function in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes (iPS-CM)

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a severe arrhythmogenic disease characterized by adrenergically-induced ventricular tachycardia associated to sudden cardiac death. Recently, a mutation in type-2 ryanodine receptor (RYR2), p.G357S_RYR2, has been associated to CPVT in a large family from the Canary Islands. We were able to obtain skin biopsies from heterozygous and homozygous donors and further derive iPS-CM from those samples. The aim of this study was to evaluate the additive effect of mutated alleles in RYR2 function. We generated iPS-CM using a heparin-based differentiation protocol followed by glucose starvation and hormonal-maturation treatment. We used cells derived from one heterozygous (HT) and one homozygous (HM) patient, and from a non-carrier healthy donor used as control (CTRL). Calcium imaging techniques were employed to assess RYR2 caffeine sensitivity in basal and β-adrenergic stress conditions (100nM isoproterenol). Peak amplitude of calcium transients elicited with 10 mM caffeine was higher in both HT and HM cells respect to CTRL cells (in ΔF/F0: HT=2.67±0.04, HM=2.52±0.16, CTRL=2.22±0.05). In dose-response studies, performed in basal conditions, both HT and HM cells showed a similar caffeine sensitivity, which was increased respect to CTRL cells (KdCTRL=2.53±0.34mM KdHT=1.67±0.19mM and KdHM=1.72±0.10mM). When these experiments were repeated in the presence of isoproterenol, we observed an augmented response in CTRL cells. However, neither HT nor HM cells showed any further increase in their response to caffeine, respect to basal conditions (KdCTRL=1.38±0.06mM, KdHT=1.68±0.09mM and KdHM=1.40±0.12mM). Our present results show that both HT and HM cells present a similar gain of function respect to CTRL, in spite of their different allelic composition. This suggest that for p.G357S_RYR2 one mutated allele is enough to produce a gain of function in RYR2 with no further additive effect.