Abstract 17750: A CPVT Mutation Confers Gain of Function to the Cardiac Ryanodine Receptor Channel. Characterization Using Cardiomyocytes Derived From Patient-Specific Ips Cells
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a primary electrical myocardial disease characterized by adrenergic-induced ventricular tachycardia in young people. Mutations in the cardiac Ryanodine Receptor channel (RyR2) account for an autosomal dominant form of CPVT. We aimed to characterize molecular and cellular mechanisms of CPVT using human induced pluripotent stem cell-derived cardiomyocytes (hIPS-CMs) carrying the RyR2-H2464D (HD) mutation. Episomal reprogramming was used for generating hIPSCs from fibroblast taken from a 19 year old male carrying the HD mutation and from his 44-year old mother as a healthy control. CMs monolayers were obtained by treating hIPSC with CHIR and IWP-4. [3H]Ryanodine binding using homogenates from hIPS-CM monolayers served as an index of RyR2 activity. Ca2+ transients (CaT) were measured by line scan confocal microscopy in hIPS-CM single cells loaded with fluo-4. Optical mapping (OM) of hiPS-CM monolayers loaded with fluo-4, stimulated at 3Hz and treated with isoproterenol (300nM) were used to measure conduction velocity (CV) and CaT patterns. Ca2+-dependence of [3H]ryanodine binding revealed that the HD mutation increases cytosolic Ca2+ sensitivity with respect to control (44±6 vs. 19±3% of maximal binding at pCa 7, respectively). CaT amplitude was decreased in HD with respect to control (1.5±0.2 vs. 2.2±0.4 F/Fo, n=4 and 5, respectively) and time-to-peak was also decreased (101±44 vs. 181±40 ms). However, τ of decay increased in HD respect to control (420±24 vs. 287±26 ms, respectively, p<0.05). Spontaneous calcium release (SCR) events, an index of arrhythmia propensity, were more frequent in HD than control cells (1 SCR/sec vs. 0 SCR/sec, n=4 and 5, respectively). OM revealed that the CV in HD was significantly slower than control (13.6±1.6 vs. 21.9±2.0 cm/s, n=30 and 9, respectively, p<0.05). A fibrillatory pattern after pacing and ISO was found in 5 out of 6 HD monolayers. The HD mutation confers a gain of function to RyR2 channels, suggesting that the increased frequency of SCR in intact HD cells is due to higher Ca2+ sensitivity of RyR2 channels. The remarkable decrease in CV and the fibrillatory pattern in HD monolayers may be linked with the cellular phenotype of the disease.
- © 2013 by American Heart Association, Inc.