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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-末端（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2型致心律失常性右心室心肌病相关的突变。有强有力的证据表明，这些区域之间的域间相互作用在通道调节机制中起着重要作用。最近，我们报道了一种治疗MH的特异性药物丹曲林，通过纠正MU RyRI内N-末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的Ca^2+渗漏。在此，我们在人CPVT相关RyR 2 ^<R24745/+>基因敲入（KI）小鼠模型中检测了丹曲林对RyR 2的Ca^<2+>释放功能的影响。 ...更多信息 0，其中包括上述可变域。使用石英晶体微量天平技术（一种高灵敏度的质量测量技术）发现丹曲林特异性结合RyR 2的结构域601-620。在注射肾上腺素（1.0 mg/kg）或跑步机运动之前和之后，监测KI小鼠（n = 6）和野生型（WT）小鼠（n=6）的ECG。在KI小鼠中，注射肾上腺素或运动后容易诱发双向室性心动过速（VT），而在WT小鼠中则不然。丹曲林预处理7天的K1小鼠，静息时室性早搏的数量显著减少，注射肾上腺素或运动均未诱导VT。在KI小鼠心肌细胞中，<-1><-1>加入异丙肾上腺素后，与WT小鼠（SpF：0.8±0.1）相比，经常观察到Ca^2+火花（SpF：s^ ·100μm^：15.8±0.6，p<0.01）和延迟后去极化介导的Ca^2+瞬变（DAD-CaT）。1.0 μM丹曲林可抑制KI小鼠的SpF和DAD-CaT（SpF：1.5±0.2，p<0.01）。与野生型小鼠相比（SpF：0.8 ± 0.1），在加入异丙肾上腺素后，经常观察到迟发性后去极化介导的钙瞬变（DAD-CaT）。KI小鼠中观察到的SpF和DAD-CaT均被1.0 μ M丹曲林抑制（SpF：1.5 ±0.2，p<0.01）。结论：丹曲林通过与N端可变结构域相互作用，纠正RyR 2突变引起的超敏通道门控，从而抑制舒张期Ca^2+火花、DAD，进而抑制致死性心律失常。少