乙酰胆碱调控缺血心肌AMPK/PGC-1α介导能量代谢的机制研究

Energy metabolism disorder is ubiquitous and appears to play an essential role in ischemia heart disease (IHD). Traditional medicine therapies on the basis of "hemodynamic" have been disappointing. Metabolic therapy has been used as a new and promising therapeutic approach for IHD. Our previous studies have suggested that autonomic regulation is closely associated with IHD, and improved vagal activity protected ischemic myocardium through regulation of mitochondria. However, the role of energy metabolism remains unclear. In the project, we will establish IHD model combined with vagal nerve transmitter acetylcholine (ACh) treatment. Our project will 1) examine glucose/fatty acid uptake and related transport protein expression and translocation by applying enzyme chemistry and immunofluorescence methods, to clarify the effect and mechanism of ACh on myocardial energy metabolic substrate transport; 2) determine expression of key enzymes involved in fatty acid oxidation and oxidative phosphorylation enzymes and the mitochondrial respiratory enzyme activity by using laser confocol microscopy and molecular biology methods, to discover the target of ACh regulate myocardial mitochondrial -mediated energy metabolism; 3) investigate the effect of AMPK/PGC-1α on myocardial energy substrate selection and mitochondria metabolism by applying siRNA/overexpression technologies, to disclose the signal pathway of ACh regulation of energy metabolism. The project will explore the molecular mechanism of invigorating effects of ACh on energy metabolism and offer a new promising approach for therapy of IHD.

缺血性心脏病（IHD）普遍存在能量紊乱，传统医学基于"血液动力学"的治疗并不理想，代谢治疗成为防治IHD的新尝试。我们前期研究发现自主神经调节与IHD密切相关，改善迷走可通过线粒体调控保护缺血心肌，但对以线粒体为核心的能量代谢还未见报道。本课题拟通过建立IHD模型结合迷走神经递质乙酰胆碱（ACh）干预，应用酶-化学和免疫荧光等检测糖/脂肪酸的摄取及其相应转运蛋白的表达改变/转位，阐明ACh对心肌能量代谢底物胞浆转运的影响及作用机制；采用激光共聚焦和分子生物学等方法检测脂肪酸氧化、氧化磷酸化关键酶表达和线粒体呼吸酶活力，阐明ACh对线粒体介导能量代谢的调节靶点；运用特异性敲除/高表达靶蛋白等技术检测AMPK/PGC-1α对心肌细胞能量底物选择和线粒体代谢过程的影响及内在联系，阐明ACh调节能量代谢的信号通路。本项目旨在探讨ACh优化缺血心肌能量代谢的分子机制，为代谢干预治疗IHD提供新思路。

缺血性心脏病普遍存在着线粒体介导的心肌能量代谢异常。心脏自主神经调节尤其是改善迷走张力对减轻心肌缺血损伤具有至关重要的作用，然而迷走调控对线粒体的直接影响和调节机制并不清楚。本项目通过离体细胞和在体动物两个水平建立缺血性心脏病模型，结合迷走神经递质乙酰胆碱（ACh）或迷走神经刺激（VNS）干预，探讨改善迷走对线粒体调节的心肌保护作用机制。研究发现：1.ACh呈浓度依赖性降低低氧复氧（H/R）后细胞ROS。ACh显著降低H/R后的细胞凋亡，增加ATP水平和线粒体DNA。ACh显著增加SOD1和SOD2的表达和活性，增加PGC-1α表达、降低FoxO3a磷酸化，阿托品消除上述效果。PGC-1α或FoxO3a siRNA均能减弱ACh对SOD2调控。提示ACh可通过FoxO3a/PGC-1α途径增强线粒体和胞浆抗氧化酶活性，减轻H/R诱导的氧化应激损伤。2.ACh呈浓度依赖性促进PINK1和Parkin由胞浆向线粒体转位。ACh抑制细胞和线粒体肿胀，恢复嵴排列， cyt c的胞浆释放，降低caspase 3活性。自噬阻断剂及siRNA抑制PINK1和Parkin蛋白表达，ACh上述作用消失。ACh通过M2受体激活线粒体自噬：ACh升高LC3-Ⅱ/LC3-Ⅰ，降低p62，增加PINK1和Parkin在线粒体的含量，增加Parkin与线粒体共定位增加，M2受体阻断剂和siRNA均抑制ACh的上述作用。提示ACh通过M2受体激活线粒体自噬，改善线粒体形态与功能，抑制病理性线粒体介导的细胞损伤。3.VNS减轻ISO诱导的心肌和线粒体损伤：表现为VNS改善大鼠血流动力学，降低心梗面积和血清心肌酶。减轻线粒体凋亡和结构异常，增加线粒体大小和内部完整性。VNS改善ISO诱导的线粒体动力学蛋白异常，抑制分裂蛋白Drp1和Fis1蛋白表达，减少Fis1线粒体转位；促进线粒体融合蛋白OPA1，Mfn1、Mfn2表达。VNS增加AMPK磷酸化及其底物ACC磷酸化。AMPK抑制剂取消了VNS介导的线粒体分裂/融合蛋白的变化。提示AMPK-ACC信号转导通路参与VNS介导的线粒体保护作用，揭示了VNS调节线粒体动力学的作用靶点。本项目以线粒体介导的能量代谢调节为核心，从氧化应激、线粒体自噬及线粒体动力学等方面探讨了改善迷走对线粒体介导的心肌保护作用机制和信号通路，有助于探索心肌缺血防治的新策略