期刊
CARDIOVASCULAR RESEARCH
卷 81, 期 3, 页码 536-545出版社
OXFORD UNIV PRESS
DOI: 10.1093/cvr/cvn303
关键词
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资金
- Ministry of Education in Japan [18390234, 20390226, 18590777, 20590868, 18591706, 20591805, 19209030]
- National Heart, Lung and Blood Institutes [HL072841]
- Grants-in-Aid for Scientific Research [20591805, 19209030, 18390234, 20390226, 20590868, 18590777, 18591706] Funding Source: KAKEN
A domain peptide (DP) matching the Gly(2460)-Pro(2495) region of the cardiac type-2 ryanodine receptor (RyR2), DPc10, is known to mimic channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), owing to its interference in a normal interaction of the N-terminal (1-600) and central (2000-2500) domains (viz. domain unzipping). Using DPc10 and two other DPs harboring different mutation sites, we investigated the underlying mechanism of abnormal Ca2+ cycling in failing hearts. Sarcoplasmic reticulum (SR) vesicles and cardiomyocytes were isolated from dog left ventricular muscles for Ca2+ leak and spark assays. The RyR2 moiety of the SR was fluorescently labelled with methylcoumarin acetate (MCA) using DPs corresponding to the 163-195 and 4090-4123 regions of RyR2 (DP163-195 and DP4090-4123, respectively) as site-directed carriers. Both DPs mediated a specific MCA fluorescence labelling of RyR2. Addition of either DP to the MCA-labelled SR induced domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. Both SR Ca2+ leak and Ca2+ spark frequency (SpF) were markedly increased in failing cardiomyocytes. Upon introduction of DP163-195 or DP4090-4123 into normal SR or cardiomyocytes, both Ca2+ leak and SpF increased to the levels comparable with those of failing myocytes. K201 (JTV519) suppressed all of the effects induced by DP163-195 (domain unzipping and increased Ca2+ leak and SpF) or those in failing cardiomyocytes, but did not suppress the effects induced by DP4090-4123. Defective inter-domain interaction between N-terminal and central domains induces diastolic Ca2+ leak, leading to heart failure and lethal arrhythmia. Mutation at the C-terminal region seen in CPVT does not seem to communicate with the aforementioned N-terminal and central inter-domain interaction, although spontaneous Ca2+ leak is similarly induced.
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