调控TLR4/NF-κB信号通路重建心肌细胞线粒体动力学平衡治疗扩张型心肌病的机制研究

The mitochondria are arranged in a filamentous network and highly dynamic structure in cardiomyocytes. The imbalance of mitochondrial fusion and fission may play a pivotal role in the pathological process of dialated cardiomyopathy, but the actual mechanism is still unclear. Our preliminary study indicated that innate immunity was activated in the pathogenesis of dilated cardiomyopathy. Inhibition of Toll-like receptor 4 (TLR4) with LPS-RS ultrapure significantly improves cardiac function by down-regulation of calcineurin-mediated activation of mitochondrial fission program in cardiomyopathic mice. It is logical to assume that regulation of TLR4/NF-κB pathway by targeting calcineurin and dynamin-related protein-1 (Drp1) to rebuild the balance of mitochondrial dynamics can improve cardiac function in dilated cardiomyopathy. In this study, we screen the unknown target genes of transcription factor NF-κB by chromatin immunoprecipitation assay (ChIP) and confirm NF-κB-mediated calcineurin gene transcription with luciferase reporter gene assay. To elucidate the inner mechanism, small interfering RNA expression constructs targeting calcineurin will be used to regulate mitochondrial fission in vitro. The systematic study will contribute to not only clarify the pathophysiological mechanism of dilated cardiomyopathy at the molecular, cellular and in vivo level, but also provide novel strategies for clinical treatment.

心肌细胞具有独特的线粒体动力学特征，线粒体融合-分裂失衡在扩张型心肌病的病理进程中的作用机制尚未阐明。我们前期研究发现，扩张型心肌病进展阶段固有免疫处于持续激活状态，应用TLR4拮抗剂LPS-RS可以下调心肌细胞钙调磷酸酶基因表达，从而抑制线粒体过度分裂，改善扩张型心肌病小鼠的心功能。在此基础上我们提出假说：抑制TLR4/NF-κB信号通路，下调心肌细胞钙调磷酸酶基因表达，通过抑制Drp1去磷酸化，重建线粒体动力学平衡可以治疗扩张型心肌病。本项目拟运用染色质免疫沉淀技术筛选NF-κB的靶基因，荧光素酶报告基因系统验证NF-κB激活促进钙调磷酸酶基因转录，RNA干扰技术证实钙调磷酸酶介导Drp1去磷酸化是调控线粒体分裂的关键环节。本课题从分子、细胞和个体水平上开展整合性研究，相关结果有助于进一步揭示扩张型心肌病的病理生理机制，为临床治疗提供新思路和理论依据。

心肌细胞具有独特的线粒体动力学特征，线粒体融合-分裂失衡在扩张型心肌病的病理进程中的作用机制尚未阐明。通过本课题研究，我们发现TLR4激活促进小鼠自身免疫性心肌炎向扩张型心肌病进展，主要通过上调炎症因子以及氧化应激介导线粒体蛋白酶OMA1激活及YME1L失调，最终导致线粒体促融合分子OPA1功能障碍，出现线粒体动力学失平衡，抑制TLR4/NF-κB信号通路，重建心肌细胞线粒体动力学平衡可以改善扩张型心肌病小鼠的心功能。本课题从分子、细胞和个体水平上开展整合性研究，相关结果有助于进一步揭示扩张型心肌病的病理生理机制，为临床防治提供新思路和理论依据。本项目共培养2名博士研究生，课题负责人以第一作者或通讯作者共发表5篇SCI论文。目前正在进行的拓展实验可以作为预实验结果，未来期望在该领域再次获得国家自然科学基金面上项目的资助。