Mutation in RyR2-FKBP Binding site alters Ca2+ signaling modestly but increases “arrhythmogenesis” in human Stem Cells derived Cardiomyocytes

ABSTRACT Aims: To gain insights into FKBP regulation of cardiac ryanodine receptor (RyR2) and Ca2+ signaling, we introduced the point mutation (N771D-RyR2) corresponding to skeletal mutation (N760D-RyR1) associated with skeletal muscle central core disease (CCD) via CRISPR/Cas9 gene-editing in the RyR2 FKBP binding site expressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Patients inflicted with CCD and other hereditary skeletal muscle diseases often show higher incidence of atrial or ventricular arrhythmias. Methods and Results: Ca2+ imaging of voltage-clamped N771D-RyR2 mutant compared to WT hiPSC-CMs showed: (1) ∼30% suppressed ICa with no significant changes in the gating kinetics of ICa; (2) 29% lower SR Ca2+ content and 33% lower RyR2 Ca2+ leak; (3) higher CICR gain and 30-35% increased efficiency of I Ca -triggered Ca 2± release ; (4) higher incidence of aberrant SR Ca2+ releases, DADs, and Ca2+ sparks; (5) no change in fractional Ca2+-release, action potential morphology, sensitivity to isoproterenol, and sarcomeric FKBP-binding pattern. Conclusions: The more frequent spontaneous Ca2+ releases and longer Ca2+ sparks underlie the increased incidence of DADs and cellular arrhythmogenesis of N771D-RyR2 mutant. The smaller RyR2 Ca 2± leak and SR content results from suppressed I Ca that is compensated by higher CICR gain.