Modelling CPVT in a dish: Characterization of two novel ryanodine receptor mutations using human induced pluripotent stem cell-derived cardiomyocytes

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease exposing afflicted patients to sudden cardiac death. Most CPVT cases have been linked to mutations of the gene encoding ryanodine receptor type 2 (RYR2). RyR2 is a calcium channel located in the membrane of the sarcoplasmic reticulum (SR). It mediates release of calcium stocks from the SR to the cytosol, playing a major role in excitation-contraction coupling. Although several studies have investigated mechanisms underlying CPVT in mice or in vitro models, little has been done concerning functional assessment of CPVT physiopathology in human cardiomyocytes. We aimed at characterizing, at cellular level, CPVT phenotype of patients carrying two new RyR2 mutations. Human induced pluripotent stem cells (hiPSCs) were derived from three CPVT patients. Two patients belonging to the same family carry the R4959Q RyR2 pore mutation. The third patient harbors the Y2476D mutation located in the cytosolic domain of the protein. hiPSCs were differentiated into cardiomyocytes for functional studies. Action potentials were recorded using patch-clamp technique. Calcium handling and contractility were investigated using Ionoptix and CardioExcyte technologies. hiPS-derived cardiomyocytes (hiPS-CMs) from CPVT patients displayed impaired beating properties. Calcium handling properties were also disrupted with changes in calcium transients, accompanied with increased diastolic calcium leak. At last, CPVT hiPS-CMs showed characteristics changes in action potential and arrhythmic events. Coherently with the patients’ phenotype, arrhythmic events were more frequent under beta-adrenergic stress. This work enabled us to characterize two novel CPVT mutations, providing more insight into functional mechanisms involved in this pathology, and further validating the use of human pluripotent stem cells for investigating complex cardiac rhythm disorders.