Abstract 5322: Mutation-Linked, Defective Inter-Domain Interaction within the Cardiac Ryanodine Receptor as a Critical Cause of Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)

Abstract

We previously reported that interaction of N-terminal (0–600) and central domains (2000–2500) of the cardiac ryanodine receptor (RyR2), harboring many mutations associated with CPVT, is defective (i.e. domain unzipping) in failing hearts. Here, we examined the pathogenic role of the inter-domain interaction within RyR2 in the mutation-linked channel disorder in human CPVT-associated RyR2 R2474S/+ knock-in (KI) mice model. In all KI mice (6/6), bi-directional ventricular tachycardia was observed during or after exercise with treadmill, but not in wild-type (WT) mice (0/7). Sarcoplasmic reticulum (SR) vesicles were isolated (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460–2495 (DPc10), harboring the same CPVT mutation site as KI mice; R2474S, as a site-directing carrier. Only in KI (but not in WT) mice, partial domain unzipping was taken place at baseline, and further domain unzipping occurred in response to cAMP (1 μM), as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In the saponin-permiabilized KI cardiomyocytes; [Ca 2+ ] was buffered at 30 nM by 0.5 mM EGTA, the relationship curve between the frequency of Ca 2+ sparks (SpF: s −1 ·100μm −1 ; by Rhod- 2) and SR Ca 2+ content {Ca 2+ con ; by caffeine application}, obtained by incremental addition of cAMP (0.1–1 μM), was markedly shifted to the left (towards lower SR Ca 2+ content) compared to WT cardiomyocytes, although RyR2 2808Ser phosphorylation is similarly increased in WT and KI. For instance, at 1 μM of [cAMP], Ca 2+ con decreased by 43 % from WT, although SpF was more increased in KI than WT (KI: 19.6±0.5; WT: 14.7±0.4, p<0.01). Interestingly, addition of DPc10 (50 μM), which was found to induce similar domain unzipping (competing with native domain) in WT as KI (+cAMP), to WT cardiomyocytes reproduced the leftward shift of the SpF-Ca 2+ con curve seen in KI. In conclusion, single point mutation at specific region may causatively induce defective inter-domain interaction between N-terminal and central domains in response to PKA phosphorylation, and in turn enhance the sensitivity of the channel to activation by luminal [Ca 2+ ]: i.e. decreased threshold [Ca 2+ ] L to induce spontaneous Ca 2+ sparks, leading to CPVT.