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

N-末端（1-600）和中央（2000-2500）与淋巴结受体（RyR）有关的多域、与增生性超热（MH）、碱性氨基酸多态性嗜热（CPVT）和血管炎原性右血管炎2型血管炎。有强有力的证据可以提出，这些地区之间的域间互动在渠道监管机制中发挥了一个重要作用。最近，我们报道了丹罗琳，一种用于治疗MH的特殊药物，预防了不正常Ca^<2+>通过对MU RyRI中N终端和中央域之间的有效互作用的纠正。在此，我们研究了六甲苯对Ca^<2+>释放RyR 2在人类CPVT相关RyR 2 ^<R24745/+> knock-in （KI）老鼠模型的影响，以识别六甲苯绑定区域我们筛选了几种重组片段（-600氨基酸溶液）对应地将不同区域覆盖到N终端区域的N终端区域。 ... More 1.什么是可互换的域？Dantrolene发现RyR 2使用石英晶体微测量技术（一种高度敏感的质量测量技术）将其特别绑定到其领域601-620。心电图在KI鼠（n=6）和野生型（WT）鼠（n=6）中监测，注射肾上腺素（1.0毫克/千克）前和后监测或跑步机。在KI老鼠双方向性静脉曲张（VT）在注射肾上腺素或运动后很容易被诱导，但不是在WT老鼠中。在7天内用丹罗琳处理K1老鼠，在剩余的明显退化的大量前肠道契约，VT没有被注射肾上腺素或锻炼所诱导。在来自KI米、Ca^<2+>火花（SpF; s^<-1>·100μm^<-1>：15.8±0.6， p<0.01）和延迟介导后Ca^<2+> transient （DAD-CaT）经常出现在异环酚添加剂后，与来自WT米的那些（SpF： 0.8±0.1）。两SpF和DAD-CaT在KI中发现了老鼠被1.0 μM丹曲烯抑制（SpF： 1.5±0.2， p<0.01）.In cardiomyocytes from KI mice， Ca' spark（SpF ; s1900 prif 15.8±0.6， p<0.01）和延迟的后极化介导的Ca' transient（DAD-CaT）在添加了异丙醇后经常出现，与WT老鼠的那些（SpF ： 0.8±0.1）比较。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融合， dantrolene，通过与N终端可变域的相互作用，似乎正确地通过RyR 2突变导致的超敏感通道门控，抑制了diastolic Ca^<2+>火花， DAD，然后是lethal arrhythmia。Less（低）

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