Abstract| Volume 23, ISSUE 10, SUPPLEMENT , S81, October 2017

Protein Kinase a Activation Reduces Calmodulin-Ryanodine Affinity in R176Q/+ and R2474S/+ Knock-in Mouse

      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. To further understand the pathophysiology of RyR2 channel pore mutation on calmodulin (CaM)-RyR2 affinity in 3 distinct CPVT mouse models. We tested the hypothesis that RyR2 channel pore mutation increases Ca leak by reduced CaM affinity in distinct knock-in (KI) mouse models with CPVT-associated RyR2 mutation (R176Q/+, R2474S/+, and R4496C/+). Ventricular cardiomyocytes were isolated from hearts of 3 KI and wild-type (WT) mice. Fluorescence resonance energy transfer between donor-F-FKBP (FK506-binding protein 12.6) and acceptor-F-CaM was measured to assess CaM-RyR2 binding in saponin-permeabilized myocytes by using confocal microscopy. Under resting conditions (without cAMP), CaM-RyR2 affinity was unaltered in WT and all KI. Under stress conditions (with cAMP), CaM-RyR2 affinity was unchanged in R4496C/+ KI and WT, but reduced in R176Q/+ and R2474S/+. 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.
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