Characterization of N-terminal RYR2 variants outside CPVT1 hotspot regions using patient iPSCs reveal pathogenesis and therapeutic potential

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac channelopathy causing ventricular tachycardia following adrenergic stimulation. Pathogenic variants in RYR2-encoded ryanodine receptor 2 (RYR2) cause CPVT1 and cluster into domains I–IV, with the most N-terminal domain involving residues 77–466. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated for RYR2-F13L, -L14P, -R15P, and -R176Q variants. Isogenic control iPSCs were generated using CRISPR-Cas9/PiggyBac. Fluo-4 Ca²⁺ imaging assessed Ca²⁺ handling with/without isoproterenol (ISO), nadolol (Nad), and flecainide (Flec) treatment. CPVT1 iPSC-CMs displayed increased Ca²⁺ sparking and Ca²⁺ transient amplitude following ISO compared with control. Combined Nad treatment/ISO stimulation reduced Ca²⁺ amplitude and sparking in variant iPSC-CMs. Molecular dynamic simulations visualized the structural role of these variants. We provide the first functional evidence that these most proximal N-terminal localizing variants alter calcium handling similar to CPVT1. These variants are located at the N-terminal domain and the central domain interface and could destabilize the RYR2 channel promoting Ca²⁺ leak-triggered arrhythmias.