迷走神经调控肥胖所致心肌重构的线粒体稳态失衡机制研究

Cardiac remodeling induced by obesity is the important risk factor in heart failure. Mitochondrial homeostasis is of great significance for preserving cardiac function and metabolic balance，which might related to cardiac remodeling. Our previous study indicated that the cardiac function of hyperlipidemia rats could be improved by enhancing vagal activity, but there is not yet systematic mechanism study about link of vagal regulation with mitochondrial homeostasis and cardiac remodeling induced by obesity. This project will establish cardiac remodeling induced by obesity combined with vagal nerve measurement and cholinergic drug intervention to: 1. investigate the effect of vagal nerve on epicardial fat phenotype transformation and lipotoxicity induced mitochondrial oxidative damage in cardiac remodeling by using microdialysis and immunohistochemistry; 2.disclose the effect of vagal nerve on mitochondrial homeostasis in cardiac remodeling model induced by obesity and related signal pathway. We are going to a) explore the effect of cholinergic drug on mitochondrial shaping proteins Drp1, OPA1 and mitofilin/Mic10/Sam50 complex by co-immunoprecipitation and siRNA and further clarify the effect of mitochondrial dynamics and cristae remodeling on cardiac mitochondrial structure homeostasis. b) measure the effect of DNA methylation, mtUPR and mitochondrial-endoplasmic reticulum interactions on cardiac mitochondrial function homeostasis signal pathway by bisulfite sequencing, gas chromatograph-mass spectrometer and Co-immunoprecipitation. Our study may provide pharmacological interventions targeting on anti-metabolic cardiovascular disease and explore new mitochondria-related drug targets.

肥胖所致心肌重构是心衰发生发展的重要危险因素，线粒体对维持心脏功能和代谢具关键作用。我们前期研究发现增强迷走活性能改善高血脂大鼠心脏功能，但迷走神经如何调控肥胖所致心肌重构及线粒体机制尚不清。本项目拟建立肥胖诱导心肌重构模型结合检测迷走活性并给予拟胆碱药物等干预：①用微透析和免疫组化等方法观察脂毒性及线粒体氧化损伤对心肌重构的影响及迷走对脂肪表型转化的调控；②探讨迷走调控肥胖所致心肌重构的线粒体稳态机制，包括：a)用siRNA和免疫共沉淀等检测线粒体动力学和嵴塑形蛋白Drp1、OPA1和mitofilin/Mic10/Sam50复合物变化，探究迷走调节线粒体结构稳态的药靶；b)用重亚硫酸盐测序、气-质联用、免疫共沉淀等技术检测DNA甲基化、mtUPR和线粒体-内质网交互，探讨迷走调节线粒体功能稳态的信号通路。本项目有望为防治代谢性心血管病提供新药理学干预措施和线粒体药物靶点。

肥胖导致的心肌重构是诱导心衰的关键因素，线粒体稳态失衡可能是肥胖导致心肌重构的关键病理环节和新兴治疗靶点。改善迷走神经可发挥显著的抗心肌重构和线粒体保护作用，但迷走神经对肥胖所致心肌代谢紊乱、对线粒体稳态的作用缺乏系统研究。本项目以线粒体稳态为核心，从整体、组织、细胞、分子等水平，通过建立肥胖SD大鼠、肥胖C57BL/6J小鼠、棕榈酸酯（PA）诱导的原代心肌细胞等多种心肌重构疾病模型，运用小动物超声心动、无线遥测技术、全基因组重亚硫酸盐测序、免疫组化、免疫共沉淀、siRNA等分子生物学技术，结合功能及形态学检测，给予拟胆碱药物探讨迷走神经对肥胖所致心肌重构的作用及线粒体稳态相关机制。本项目：1.揭示迷走神经对脂毒性和脂肪表型转化的调控：胆碱酯酶抑制剂吡斯的明可通过减少脂质沉积，促进白色脂肪组织棕色化和增加棕色脂肪组织的活化从而抑制肥胖所致的心室重构，改善心脏功能。2.阐明活化迷走神经调节线粒体未折叠蛋白反应（UPRmt）的分子机制：迷走神经递质乙酰胆碱（ACh）的前体物质胆碱可通过激活SIRT3-AMPK增加UPRmt、改善线粒体的形态和功能；促进酮体生成并增加心肌对酮体和脂肪酸的利用，加强心肌能量供给，改善心肌能量代谢重构减轻压力超负荷和Ang II诱导的心肌损伤。3.探究迷走调节线粒体结构稳态的药靶：吡斯的明/ACh通过活化迷走调节线粒体塑形蛋白（分裂蛋白p-Drp1、Fis1，融合蛋白OPA1、Mfn1/2以及嵴相关复合物mitofilin/Sam50/CHCHD3）改善线粒体嵴结构，减轻肥胖小鼠心肌重构及PA诱导的心肌细胞肥大。4.揭示了胆碱对心肌重构过程中基因表达的表观遗传修饰作用：胆碱通过恢复DNA甲基化和调节2-OG/TET信号通路改善SHR大鼠心脏功能，减轻心肌重构。5.吡斯的明通过调节线粒体融合/分裂蛋白表达以及线粒体呼吸链复合物蛋白表达，改善线粒体稳态减轻糖尿病小鼠心肌重构。6.探讨了ACh对线粒体-脂滴交互作用的调节机制：ACh可能通过促进脂解和激活PLIN5介导的脂滴-线粒体相互作用来改善棕榈酸酯诱导的线粒体功能障碍和心肌细胞凋亡。本项目表明增强迷走神经活性可通过改善线粒体结构和功能稳态从而减轻肥胖所致的心肌重构。本研究结果可为防治代谢性心血管病提供新药理学干预措施和线粒体药物靶点。