CPVT1 point mutations in RyR2 S5 and S6 segments and their Ca2+ signaling consequence.

Precise activation of cardiac ryanodine receptor (RyR2) by small influx of Ca2+ during the action potential triggers the release of SR Ca2+ that activates contraction, a process known as Ca2+-induced Ca2+ release (CICR). Missense mutations in RyR2 often cause aberrant and unregulated Ca2+ releases that are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), often lethal arrhythmias. Here using CRISPR/Cas9 gene editing in human induced pluripotent stem cells (hiPSCs), we extended our previous studies to include two new arrhythmogenic mutations one, R4822H, located in S5-S6 transmembrane luminal loop near RyR2 selective filter and the other, L4865V, located on S6 segment. TIRF-imaging of voltage-clamped mutant myocytes showed that ICa and caffeine-triggered cytosolic Ca2+ rise (Fura-2 signal) or ER-GCaMP6 SR Ca2+ release signals were significantly suppressed in R4822H but not in L4865V myocytes. Spontaneous Ca2+ transients, however, persisted in both mutant lines activating both Fura-2 and ER-GCaMP6 Ca2+ transients in L4865V cells, but only Fura-2 Ca2+ transients in R4822H mutant. Spontaneous Ca2+ sparks igniting frequencies were similar in both mutants, but spark durations were significantly shorter. Although both of these mutations are located at S5 and S6 transmembrane regions of RyR2, their phenotypes diverge markedly. L4865V mutant does not show suppressed E-C coupling function, while R4822H mutant has completely suppressed CICR suggesting that the spontaneous beating in R4822H mutant results from remodeling of dormant Ca2+ signaling pathway expressed in hiPSCCMs.