CaN-NFAT信号转导通路介导心肌肥厚心律失常的机制及益气活血中药干预研究

Left ventricular hypertrophy has the potential death of the electrophysiological mechanisms of arrhythmias. Recent studies have shown that CaN-NFAT signaling pathway is the new targets and hot spots of left ventricular hypertrophy arrhythmia, CaN- NFAT and CaMKII signal pathway play the great role in intracellular calcium regulation and regulation of myocardial hypertrophy arrhythmia. This study aimed to evaluate thoracic aortic coarctation induced cardiac hypertrophy models and the intraperitoneal injection of isoproterenol epinephrine arrhythmia model in vivo, and ET induced hypertrophy of myocardial cell model and electrical stimulation induced arrhythmia model, on the cellular level, using molecular biology , cell transfection, fluorescence immunoassay, and patch clamp and confocal laser technology ,we study wenxinkeli of antiarrhythmic Yiqihuoxue Chinese medicine to regulate CaN-NFAT cell signal transduction pathway related proteins, after CaN-NFAT is mediated by CaMKII, how will L-Ca channel current and intracellular , Ca2+ transients as well as early and late after depolarization change, on the other hand, we want to clarify if Yiqihuoxue Chinese medicine of the anti-hypertrophic arrhythmias can affect the pathway of CaN-of NFAT expression of the key links, inhibit hypertrophy progress and reduce the arrhythmia; and I want to study what are the relationships between CaN-NFAT and CaMKII signal pathway.

左室肥厚具有发生潜在性、致死性心律失常的电生理机制。最新研究证明，CaN-NFAT信号转导通路是左室肥厚心律失常的新靶点和热点，它和CaMKII信号通路，在胞内钙调控作用的发挥中居于中心地位，并且是调控心肌肥厚心律失常发生、发展的关键性途径。本研究拟通过在整体动物上建立胸主动脉缩窄诱导心肌肥厚模型和腹腔注射异丙肾上腺素心律失常模型，细胞水平上建立ET诱导肥厚心肌细胞模型和电刺激诱发心律失常模型，采用分子生物学、细胞转染、荧光免疫与膜片钳与激光共聚焦结合技术，研究已上市的抗心律失常益气活血中药稳心颗粒对CaN-NFAT细胞信号转导通路相关蛋白和CaMKII介导后及L－Ca通道电流和胞内钙瞬变以及早期和晚期后除极发生率变化，进一步明确益气活血抗肥厚心律失常中药是否能够影响CaN-NFAT通路关键环节的表达，进而抑制肥厚进展，减少心律失常的发生，阐明CaN-NFAT和CaMKII的相互作用关系。

实验采用激光共聚焦测细胞骨架蛋白观察细胞肥厚情况，运用全细胞膜片钳技术测定各组细胞ICa-L的变化并进行门控机制研究，免疫荧光激光共聚焦显微镜观察CaMKⅡ、CaN和NFATc4蛋白表达强度和核转移情况，以及分子生物学方法研究Ca2＋- CaM - CaMKⅡ与CaN – NFAT 以及MyD88炎症通路相关蛋白表达量的变化。结果显示，AngⅡ能够激活心肌H9C2细胞CaMKⅡ、CaN-NFAT和MyD88炎症通路，增加通路各蛋白表达，造成心肌肥厚。在沉默siCaMKⅡ心肌H9C2细胞中，AngⅡ诱导心肌肥厚时CaMKⅡ、CaN-NFAT和MyD88炎症通路被抑制，心肌肥厚程度减轻。在CsA干预的心肌H9C2细胞中，AngⅡ诱导心肌肥厚时CaN-NFAT信号转导通路被抑制，但是CaMKⅡ和MyD88炎症通路有被激活的趋势，心肌肥厚程度有所增加。稳心颗粒在AngⅡ诱导的肥厚心肌H9C2细胞模型中，能抑制CaMKⅡ、CaN-NFAT和MyD88炎症通路参与心肌肥厚相关蛋白的表达，起到一定的改善心肌肥厚的作用。本研究无论从动物在体还是细胞离体的心肌肥厚病理条件下，稳心颗粒都能抑制和延缓心肌组织重构和电重构的发生发展，对心肌肥厚起到改善的作用。研究也首次探讨了稳心颗粒发挥作用的分子机制，通过抑制Ca2＋- CaM - CaMKⅡ和CaN-NFAT钙调控相关信号转导通路以及MyD88炎症通路，起到一定的改善心肌肥厚的作用，这可能是稳心颗粒抗心律失常一个重要的作用机制。