Introduction and Hypothesis: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited stress-induced arrhythmogenic disease caused by mutations in cardiac ryanodine receptor (RyR2). These mutations cluster in 3 regions of the RyR2 mutable regions (N-terminal, central, and channel pore). Diastolic calcium (Ca) leak via RyR2 can cause lethal arrhythmia such as CPVT. We have reported conformational unzipping within RyR2 reduces calmodulin (CaM)-RyR2 affinity in a central CPVT mouse (R2474S/+). To further understand the pathophysiology of RyR2 mutation on CaM-RyR2 affinity in 3 distinct (N-terminal, central, and channel pore) CPVT knock-in (KI) mice. We tested the hypothesis that RyR2 mutation cause Ca leak by reduced CaM affinity in 3 distinct CPVT KI mice (R176Q/+, R2474S/+, and R4496C/+).
Methods and results: Ventricular myocytes was isolated from hearts of 3 KI and wild-type (WT) mice. Fluorescence resonance energy transfer (FRET) between donor-F-FKBP (FK506-binding protein) and acceptor-F-CaM or acceptor-F-DPc10 was measured to assess CaM-RyR2 binding or DPc10 accessibility (DPc10-RyR2 binding), respectively in saponin-permeabilized myocytes by using confocal microscopy. Under resting conditions (without cAMP), no change in CaM-RyR2 and FKBP12.6-RyR2 affinity in WT and all KI. Under stress conditions (with cAMP), CaM-RyR2 affinity was unchanged in R4496C/+ KI and WT, but reduced in R176Q/+ (-22%, n=12, p<0.05) and R2474S/+ KI (-48%, n=21, p<0.05). FKBP12.6-RyR2 affinity was unchanged in all KI and WT. PKA activation did not enhance the access of F-DPc10 (conformational unzipping within RyR2) in R4496C/+ KI, but in R2474S/+ KI.
Conclusions: These results suggest that reduced CaM-RyR2 affinity cause Ca leak in N-terminal or central CPVT mutation, but channel pore mutation of RyR2 cause Ca leak independent of FKBP12.6-RyR2 and CaM-RyR2 affinity. These findings not only provide novel understanding of the effects of RyR2 channel pore mutation and CaM-RyR2 binding in CPVT, but also serve to advance the knowledge of potential therapeutic targets for this life-threatening arrhythmia.