Defective domain-domain interactions within the ryanodine receptor as a critical cause of diastolic Ca2+ leak in failing hearts.
OBJECTIVE
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 Ca(2+) cycling in failing hearts.


RESULTS
Sarcoplasmic reticulum (SR) vesicles and cardiomyocytes were isolated from dog left ventricular muscles for Ca(2+) 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 Ca(2+) leak and Ca(2+) spark frequency (SpF) were markedly increased in failing cardiomyocytes. Upon introduction of DP163-195 or DP4090-4123 into normal SR or cardiomyocytes, both Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) leak is similarly induced.
Tateishi
Hiroki
H
Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
Yano
Masafumi
M
Mochizuki
Mamoru
M
Suetomi
Takeshi
T
Ono
Makoto
M
Xu
Xiaojuan
X
Uchinoumi
Hitoshi
H
Okuda
Shinichi
S
Oda
Tetsuro
T
Kobayashi
Shigeki
S
Yamamoto
Takeshi
T
Ikeda
Yasuhiro
Y
Ohkusa
Tomoko
T
Ikemoto
Noriaki
N
Matsuzaki
Masunori
M
D000818:Animals; D001145:Arrhythmias, Cardiac / Q000209:etiology / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002304:Cardiac Pacing, Artificial / Q000009:adverse effects; D004195:Disease Models, Animal; D004285:Dogs; D005456:Fluorescent Dyes; D006333:Heart Failure / Q000209:etiology / Q000378:metabolism*; D006352:Heart Ventricles / Q000378:metabolism; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D022021:Tacrolimus Binding Proteins / Q000378:metabolism; D013997:Time Factors
D005456:Fluorescent Dyes; D019837:Ryanodine Receptor Calcium Release Channel; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B; D002118:Calcium