Cardiovascular Research Advance Access [Accepted Manuscript] published online on November 7, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn303
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Defective domain-domain interactions within the ryanodine receptor as a critical cause of diastolic Ca2+ leak in failing hearts
* Department of Medicine and Clinical Science, Division of Cardiology Yamaguchi University Graduate School of Medicine 1-1-1 Minamikogushi, Ube, Yamaguchi, 755-8505, Japan
Address for correspondence: Masafumi Yano, MD, PhD Department of Medicine and Clinical Science Division of Cardiology Yamaguchi University Graduate School of Medicine 1-1-1 Minamikogushi, Ube, Yamaguchi, 755-8505, JAPAN Tel: +81-836-22-2248 Fax: +81-836-22-2246 Email: yanoma{at}yamaguchi-u.ac.jp
Aims: A domain peptide (DP) matching the Gly2460-Pro2495 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 domain peptides harboring different mutation sites, we investigated the underlying mechanism of abnormal Ca2+ cycling in failing hearts.
Methods: 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 labeled with methylcoumarin acetate (MCA) using domain peptides corresponding to the 163-195 and 4090-4123 regions of RyR2 (DP163-195 and DP4090-4123, respectively) as site-directed carriers.
Results: Both DPs mediated a specific MCA fluorescence labeling of RyR2. Addition of either DP to the MCA-labeled 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.
Conclusions: 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.
Time for primary review: 22 Days
# Boston Biomedical Research Institute, Watertown, Massachusetts 02472
Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115
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