Mutation in RyR2-FKBP Binding site alters Ca signaling modestly but increases "arrhythmogenesis" in human stem cells derived cardiomyocytes.

Aims: To gain insights into FKBP regulation of cardiac ryanodine receptor (RyR2) and Ca signaling, we introduced the point mutation (N771D-RyR2) corresponding to skeletal muscle mutation (N760D-RyR1) associated with central core disease (CCD) via CRISPR/Cas9 gene-editing in the RyR2 FKBP binding site expressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Patients inflicted with CCD and other hereditary skeletal muscle diseases often show higher incidence of atrial or ventricular arrhythmias.

Methods And Results: Ca imaging of voltage-clamped N771D-RyR2 mutant compared to WT hiPSCCMs showed: (1) ∼30% suppressed I with no significant changes in the gating kinetics of I; (2) 29% lower SR Ca content and 33% lower RyR2 Ca leak; (3) higher CICR gain and 30-35% increased efficiency of I-triggered Carelease; (4) higher incidence of aberrant SR Ca releases, DADs, and Ca sparks; (5) no change in fractional Ca-release, action potential morphology, sensitivity to isoproterenol, and sarcomeric FKBP-binding pattern.

Conclusions: The more frequent spontaneous Ca releases and longer Ca sparks underlie the increased incidence of DADs and cellular arrhythmogenesis of N771D-RyR2 mutant. The smaller RyR2 Caleak and SR content result from suppressed Ithat is compensated by higher CICR gain.