Elucidation of molecular mechanism and treatment for abnormal ryanodine receptor in patients with malignant hyperthermia and lethal arrhythmia

恶性高热致死性心律失常患者兰尼碱受体异常的分子机制阐明及治疗

基本信息

批准号：
18591706
负责人：
KOBAYASHI Shigeki
金额：
$ 2.52万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591706/
关键词：
internal medicine heart failure ryanodine receptor dantrolene arrhythmia

项目摘要

The N-terminal (1-600) and central (2000-2500) domains of the ryanodine receptor (RyR), harbor many mutations associated with malignant hyperthermia (MH), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular cardiomyopathy type 2. There is strong evidence to suggest that inter-domain interaction between these regions plays an important role in the mechanism of channel regulation. Recently we reported that dantrolene, a specific drug for the treatment of MH, prevented abnormal Ca^<2+> leak by the correction of the defective inter-domain interaction between N-terminal and central domains within MU RyRI. Here, we examined the effect of dantrolene on the Ca^<2+> release function of RyR2 in human CPVT-associated RyR2^<R24745/+> knock-in (KI) mice model.To identify the dantrolene-binding region we screened several recombinant fragments (-600 amino acid residues) corresponding to various regions covering the area from the N-terminus to residue 275 … More 0, which include the aforementioned mutable domains. Dantrolene was found to specifically bind to the domain 601-620 of RyR2 using a quartz crystal microbalance technique (a highly sensitive mass-measuring technique). ECG was monitored in KI mice (n=6) and wild-type (WT) mice (n=6), before and after injection of epinephrine (1.0 mg/kg) or exercise by treadmill. In KI mice bi-directional ventricular tachycardia (VT) was easily induced after injection of epinephrine or exercise, but not in WT mice. In K1 mice pretreated with dantrolene for 7 days, number of premature ventricular contractions at rest significantly decreased, and VT was not induced by injection of epinephrine or exercise. In cardiomyocytes from KI mice, Ca^<2+> spark (SpF; s^<-1>・100μm^<-1>:15.8±0.6, p<0.01) and delayed afterdepolarization-mediated Ca^<2+> transient (DAD-CaT) were frequently seen after addition of isoproterenol, compared to those from WT mice (SpF: 0.8±0.1). Both SpF and DAD-CaT seen in KI mice were inhibited by 1.0 μM dantrolene (SpF: 1.5±0.2, p<0.01).In cardiomyocytes from KI mice, Ca' spark (SpF ; s1900prif 15.8±0.6, p<0.01) and delayed afterdepolarization-mediated Ca' transient(DAD-CaT) were frequently seen after addition of isoproterenol, compared to those from WT mice (SpF : 0.8±0.1). Both SpF and DAD-CaT seen in KI mice were inhibited by 1.0 p.M dantrolene (SpF : I.5±0.2, p<0.01). n conclusion, dantrolene, by interacting with N-terminal mutable domain, seems to correct the hypersensitized channel gating caused by RyR2 mutation, thereby inhibiting diastolic Ca^<2+> sparks, DAD, and then lethal arrhythmia. Less

兰尼碱受体 (RyR) 的 N 端 (1-600) 和中央 (2000-2500) 结构域含有许多与恶性高热 (MH)、儿茶酚胺能多形性室性心动过速 (CPVT) 和致心律失常性右心室心肌病类型相关的突变2. 有强有力的证据表明这些区域之间的域间相互作用在该机制中发挥着重要作用最近我们报道了用于治疗 MH 的特异性药物丹曲林通过纠正 MU RyRI 内 N 端和中心结构域之间有缺陷的结构域间相互作用来防止异常 Ca^2+ 泄漏。，我们在人 CPVT 相关的 RyR2^<R24745/+> 敲入 (KI) 小鼠模型中检测了丹曲林对 RyR2 Ca^2+ 释放功能的影响。我们筛选了几个重组片段（-600 个氨基酸残基），对应于覆盖从 N 末端到残基 275 … More 0 的区域，其中包括上述的可变结构域。使用石英晶体微天平技术（一种高灵敏度质量测量技术）对 KI 小鼠 (n=6) 的 RyR2 结构域 601-620 进行监测。和野生型（WT）小鼠（n=6），注射肾上腺素（1.0 mg/kg）或跑步机运动前后，KI小鼠在注射肾上腺素或跑步机后很容易诱发双向室性心动过速（VT）。在用丹曲林预处理 7 天的 K1 小鼠中，静息时室性早搏的次数显着减少，并且注射丹曲林不会诱发 VT。在 KI 小鼠的心肌细胞中，Ca^<2+> 火花 (SpF; s^<-1>·100μm^<-1>:15.8±0.6, p<0.01) 和延迟后去极化介导的 Ca^<与 WT 小鼠 (SpF: KI 小鼠中观察到的 SpF 和 DAD-CaT 均被 1.0 μM 丹曲林抑制（SpF：1.5±0.2，p<0.01）。在 KI 小鼠的心肌细胞中，Ca' 火花（SpF；s1900prif 15.8±0.6， p<0.01) 和延迟后除极介导的 Ca'与 WT 小鼠相比，添加异丙肾上腺素后经常出现瞬态 (DAD-CaT) (SpF：0.8±0.1)。KI 小鼠中出现的 SpF 和 DAD-CaT 均被 1.0 p.M 丹曲林抑制 (SpF：I.5)。 ±0.2，p<0.01) 结论是，丹曲林通过与 N 端可变结构域相互作用，似乎可以纠正RyR2突变引起的超敏通道门控，从而抑制舒张期Ca^2+火花、DAD，进而导致致命性心律失常。