Calcium signaling consequences of RyR2 mutations associated with CPVT1 introduced via CRISPR/Cas9 gene editing in human-induced pluripotent stem cell-derived cardiomyocytes: Comparison of RyR2-R420Q, F2483I, and Q4201R.

BACKGROUNDHuman-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) created from patients with catecholaminergic polymorphic ventricular tachycardia 1 (CPVT1) have been used to study CPVT1 arrhythmia.

OBJECTIVEThe purpose of this study was to evaluate the Ca2+ signaling aberrancies and pharmacological sensitivities of 3 CRISPR/Cas9-introduced CPVT1 mutations located in different molecular domains of ryanodine receptor 2 (RyR2).

METHODSCRISPR/Cas9-engineered hiPSC-CMs carrying RyR2 mutations-R420Q, Q4201R, and F2483I-were voltage clamped, and their electrophysiology, pharmacology, and Ca2+ signaling phenotypes measured using total internal reflection fluorescence microscopy.

RESULTSR420Q and Q4201R mutant hiPSC-CMs exhibit irregular, long-lasting, spatially wandering Ca2+ sparks and aberrant Ca2+ releases similar to F2483I unlike the wild-type myocytes. Large sarcoplasmic reticulum (SR) Ca2+ leaks and smaller SR Ca2+ contents were detected in cells expressing Q4201R and F2483I, but not R420Q. Fractional Ca2+ release and calcium-induced calcium release gain were higher in Q4201R than in R420Q and F2483I hiPSC-CMs. JTV519 was equally effective in suppressing Ca2+ sparks, waves, and SR Ca2+ leaks in hiPSC-CMs derived from all 3 mutant lines. Flecainide and dantrolene similarly suppressed SR Ca2+ leaks, but were less effective in decreasing spark frequency and durations.

CONCLUSIONCRISPR/Cas9 gene editing of hiPSCs provides a novel approach in studying CPVT1-associated RyR2 mutations and suggests that Ca2+-signaling aberrancies and drug sensitivities may vary depending on the mutation site.