迷走刺激及拟胆碱药防治缺血性心脏病的线粒体质量控制研究

Ischemia heart disease (IHD) is the life-threatening cardiovascular disease. Mitochondrial quality control (MQC) is of great significance for the maintenance of life activities. There is not yet systematic study about link of MQC and IHD. Our previous studies have suggested that improved vagal activity ameliorates myocardial and vascular ischemic injury. This project will establish IHD models combined with vagal stimulation and cholinergic drug intervention: ① investigate the effect of improving vagal activity on MQC (mitochondrial fission/ fusion and impaired mitochondrial elimination); ② disclose the molecular mechanisms of vagal nerve regulation of inter-organelle communication. a: explore the effects of improved vagal activity on Grp75/VDAC1/IP3R1 complex to clarify the mitochondria/ endoplasmic reticulum calcium crosstalk identified as drug target of MQC applying electron microscopy and co-immunoprecipitation; b: explore the effects of vagal nerve on mitochondrial unfolded protein response (UPRmt in the mitochondrial matrix and in the intermembrane space) to determine the signal pathway of the communication between mitochondria and nucleus which mediates mitochondrial protein homeostasis using siRNA and laser confocal microscopy. Our research aims to clarify the molecular mechanism underlying improving vagal nerve exerts cardiovascular effects through regulating MQC, explore pharmacological interventions of MQC and novel mitochondrial targeting anti-ischemic drugs.

缺血性心脏病（IHD）是危害人类健康的首要疾病，线粒体质量控制（MQC）对维持生命活动具有重大意义，目前尚未有MQC与IHD防治的系统研究。前期实验发现改善迷走可减轻心血管缺血损伤，本项目将建立IHD模型结合迷走刺激或拟胆碱药干预，1. 探讨改善迷走对MQC（线粒体分裂/融合及受损线粒体识别/清除）的影响；2. 阐明迷走调节对MQC细胞器间交互调节机制。a:应用超微结构分析和免疫共沉淀等技术探讨迷走神经对Grp75/VDAC1/IP3R1复合体的作用，明确线粒体与内质网交互调节钙稳态介导MQC的药物靶点；b:应用siRNA和激光共聚焦等技术探讨迷走神经对线粒体未折叠蛋白反应（线粒体基质/膜间隙UPRmt）及蛋白转位的影响，明确线粒体与细胞核交互调节线粒体蛋白稳态的信号通路。本研究将有助于阐明改善迷走调节MQC启动内源性心血管保护的分子机制，寻求MQC的药理学干预措施和线粒体靶向抗缺血药物的开发。

缺血性心脏病（IHD）是危害人类健康的重要疾病。线粒体质量控制（MQC）是维持线粒体群体结构和功能完整的基础，对细胞生物能量平衡和内环境稳态的维持具重要意义。本项目以MQC为核心，在整体动物、心肌组织、离体血管及细胞水平建立了多种缺血/再灌注（低氧/复氧）模型，运用siRNA、免疫共沉淀等分子生物学技术，结合功能及形态学检测，采用迷走神经刺激（VNS）及递质乙酰胆碱（ACh）和相关药物干预围绕线粒体质量控制进行了相关研究：1.揭示迷走神经刺激对缺血心肌线粒体动力学的调控机制：VNS可通过激活M3AChR/CaMMKKβ/AMPK通路增加线粒体融合蛋白OPA1和Mfn1/2，降低线粒体分裂蛋白Fis1和Drp1，从而改善线粒体动力学，减轻异丙肾上腺素诱导的大鼠心肌缺血。2. 阐明ACh活化线粒体自噬的分子机制：ACh通过清除损伤的线粒体挽救缺血心肌，可通过M2AChR激活低氧/复氧后心肌细胞线粒体自噬，改善自噬活性，恢复PINK1/Parkin由胞浆向线粒体的转位，从而降低细胞死亡、促进细胞存活及改善线粒体形态和功能，发挥抗缺血/再灌注损伤的作用。3. 明确ACh对线粒体未折叠蛋白反应（UPRmt）的作用机制：ACh可通过M3AChR下调低氧/复氧诱导的ROS生成和减轻线粒体核蛋白失衡，从而减轻低氧/复氧诱导的线粒体未折叠蛋白反应，改善线粒体的结构和功能，保护血管内皮细胞。4. 提出ACh调控细胞器交互介导的线粒体钙稳态新机制：ACh可通过M3AChR活化PI3K/Akt/eNOS通路，抑制由内质网向线粒体的钙转运，降低内质网-线粒体钙转运复合物VDAC1/Grp75/IP3R1的蛋白相互作用及连接蛋白Mfn2的表达，从而减轻低氧/复氧诱导的胞浆和线粒体钙超载，抑制线粒体/内质网相关凋亡通路，进而减轻血管内皮细胞死亡。ACh还可通过活化M3AChR/AMPK通路，抑制细胞膜-内质网的复合物（NCX1/TRPC3/IP3R1），进而减轻血管内皮细胞钙超载。5. 吡斯的明可改善压力负荷诱导大鼠心肌肥厚心功能指标异常，与提高迷走神经活性、抑制STIM1/Orai1分子间的交互及所介导的CaN/NFAT3/GATA4信号转导途径的活化有关。本研究结果可为IHD治疗提供与MQC相关新的拟胆碱药物的重要靶点和实验依据。