Tateishi Hiroki, Yano Masafumi, Mochizuki Mamoru, Suetomi Takeshi, Ono Makoto, Xu Xiaojuan, Uchinoumi Hitoshi, Okuda Shinichi, Oda Tetsuro, Kobayashi Shigeki, Yamamoto Takeshi, Ikeda Yasuhiro, Ohkusa Tomoko, Ikemoto Noriaki, Matsuzaki Masunori
Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan.
Cardiovasc Res. 2009 Feb 15;81(3):536-45. doi: 10.1093/cvr/cvn303. Epub 2008 Nov 7.
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.
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.
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.
已知一种与心脏2型兰尼碱受体（RyR2）的甘氨酸（2460）-脯氨酸（2495）区域匹配的结构域肽（DP），即DPc10，由于其干扰了N端（1 - 600）和中央（2000 - 2500）结构域的正常相互作用（即结构域解链），可模拟与儿茶酚胺能多形性室性心动过速（CPVT）相关的通道功能障碍。我们使用DPc10和另外两个具有不同突变位点的DP，研究了衰竭心脏中异常钙（Ca2 +）循环的潜在机制。
从犬左心室肌肉中分离出肌浆网（SR）囊泡和心肌细胞，用于Ca2 +泄漏和钙火花检测。使用分别对应于RyR2的163 - 195和4090 - 4123区域的DP（分别为DP163 - 195和DP4090 - 4123）作为定点载体，用乙酸甲基香豆素（MCA）对SR的RyR2部分进行荧光标记。两种DP均介导了RyR2的特异性MCA荧光标记。将任一DP添加到用MCA标记的SR中均诱导了结构域解链，这可通过结合的MCA对大尺寸荧光猝灭剂的可及性增加来证明。衰竭心肌细胞中的SR Ca2 +泄漏和Ca2 +火花频率（SpF）均显著增加。将DP163 - 195或DP4090 - 4123引入正常SR或心肌细胞后，Ca2 +泄漏和SpF均增加到与衰竭心肌细胞相当的水平。K201（JTV519）抑制了DP163 - 195诱导的所有效应（结构域解链以及Ca2 +泄漏和SpF增加）或衰竭心肌细胞中的效应，但未抑制DP4090 - 4123诱导的效应。
N端和中央结构域之间的结构域间相互作用缺陷会诱导舒张期Ca2 +泄漏，导致心力衰竭和致命性心律失常。CPVT中所见的C端区域突变似乎与上述N端和中央结构域间相互作用无关，尽管同样会诱导自发性Ca2 +泄漏。
