ECSIT蛋白及其泛素化修饰在心肌缺血/再灌注损伤中的作用机制研究

ECSIT (an Evolutionarily Conserved Signaling Intermediate in Toll pathways), an important intermediate in the Toll/IL-1 signal transduction pathway, has been demonstrated to play a key modulatory role in the induction of innate immune and inflammatory responses, oxidative stress, and mitochondrial function. We and others have previously reported that TLRs/IL-1R-mediated signaling pathways play a critical role in myocardial ischemia/reperfusion (I/R) injury. However, the role of ECSIT in myocardial I/R injury has not been investigated. Our preliminary data showed that myocardial I/R significantly increased ubiquitination of ECSIT and decreased ECSIT localized to the inner mitochondrial membrane in the myocardium. In addition, we found that myocardial I/R significantly caused a decrease in the interaction between ECSIT and complex I assembly chaperone NDUFAF1 in the mitochondrial of I/R myocardium. In contrast, increased expression of mitochondrial ECSIT ameliorates hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. Collectively, our data demonstrated that ECSIT is required for correct complex I assembly and/or stability and for mitochondrial function. Importantly, ECSIT may represent a link between mitochondrial function and immune response. Based on our novel finding, we hypothesize that ECSIT plays a central role in the pathophysiologic mechanisms of myocardial I/R injury. To critically evaluate our hypothesis, we will determine the role of ECSIT and its localization to mitochondria in the pathogenesis of myocardial I/R injury using transgenic mice that overexpress ECSIT targeted to mitochondria. Furthermore, we will examine the effect of ECSIT ubiquitination in its function during myocardial I/R injury by transfection of the myocardium with an adenovirus expressing ECSIT ubiquitination sites mutant immediately after induction of myocardial I/R injury in mice. Cardiac function and myocardial morphologic changes will be measured after myocardial I/R injury. We will also examine the effect of ECSIT and its ubiquitination on mitochondrial function, oxidative stress, and activation of TLRs/IL-1R-mediated signaling pathways using both in vivo myocardial I/R injury of mice and in vitro cardiomyocytes H/R injury models that have been established in our laboratory. These studies will generate novel data for understanding the signaling molecular sensor that is critical for myocardial I/R injury. It may be possible to apply this find in a practical fashion to identify new and novel therapeutic approaches to prevent or manage myocardial I/R injury.

ECSIT是TLRs/IL-1R信号通路中高度保守的信号分子，对免疫炎症、氧化应激和线粒体功能具有调控作用。我们和其他实验室已报道TLRs/IL-1R信号在心肌缺血/再灌注（I/R）损伤中具有重要作用，但ECSIT是否在心肌I/R损伤中发挥作用尚不清楚。预实验发现：在I/R损伤心肌组织中ECSIT泛素化增加，定位于线粒体内膜的ECSIT减少，ECSIT与NDUFAF1结合下降；转染增加线粒体ECSIT的表达，可抑制缺氧/复氧诱导的心肌细胞损伤。我们推测ECSIT作为关键信号分子可能在心肌I/R损伤的发病机制中起着重要作用。为此，本项目拟从整体动物与体外细胞两个方面，采用线粒体靶向过表达ECSIT转基因小鼠、ECSIT泛素化位点突变体心肌局部转染等方法，阐释ECSIT及其泛素化修饰，尤其是明确ECSIT的线粒体定位在心肌I/R损伤中的整体功能作用和调控机制，为临床防治心肌I/R损伤提供线索。

ECSIT 是 TLRs/IL-1R 信号通路中高度保守的信号分子，对免疫炎症、氧化应激和线粒体功能具有调控作用。TLRs/IL-1R 信号在心肌缺血/再灌注（I/R）损伤中具有重要作用，但 ECSIT 是否在心肌 I/R 损伤中发挥作用尚不清楚。我们提出假说：ECSIT可能作为调控免疫炎症信号、氧化应激和线粒体功能内在关联的关键节点分子在心肌I/R损伤的发生发展中发挥重要作用，其机制可能主要与ECSIT的泛素化修饰和线粒体定位的变化及其对线粒体复合物Ⅰ的组装和功能的影响相关。本项目动物与细胞两个水平，进行了以下研究：①相关性分析：小鼠心肌I/R 模型，发现ECSIT的泛素化修饰和线粒体定位与I/R 心肌损伤正相关；②线粒体定位的ECSIT蛋白在心肌I/R损伤中的作用与机制：动物实验，采用otclECSIT转基因小鼠及I/R 模型发现，线粒体靶向过表达ECSIT可减轻I/R所致心肌结构损伤并改善心功能；采用adv-ECSIT心肌局部感染增加心肌局部ECSIT蛋白表达，发现过表达ECSIT定位于线粒体，且可减轻I/R所致的心肌梗死。细胞实验，增加ECSIT线粒体定位表达可以明显改善H/R诱导的心肌细胞线粒体超微结构变化及线粒体功能损伤；过表达ECSIT可以明显改善H/R诱导的细胞凋亡；机制与线粒体内过表达ECSIT，增强H/R后心肌细胞线粒体内ECSIT与NDUFAF1交互作用、减少ROS产生有关。③ECSIT蛋白泛素化修饰在其调控心肌I/R损伤中的作用与机制：动物实验，心肌局部感染adv-ECSIT K372A可以降低I/R损伤心肌组织ECSIT的泛素化修饰, 且可显著减少I/R后心肌梗死面积、减少心肌细胞凋亡、减轻早期炎症反应，并改善心功能；细胞实验，原代心肌细胞感染adv-ECSIT K372A，可以明显促进ECSIT的线粒体定位，抑制ECSIT的线粒体泛素化修饰，且可以改善H/R诱导的心肌细胞凋亡，抑制心肌细胞H/R损伤诱导巨噬细胞迁移，机制与ECSIT泛素化修饰调控NF-κB信号有关。综上，证实ECSIT蛋白，尤其是ECSIT蛋白的线粒体定位与泛素化修饰在心肌I/R损伤发病机制中具有重要作用，并揭示ECSIT蛋白在心肌I/R损伤中的调控作用与其影响I/R诱导的线粒体结构功能紊乱、氧化应激和炎症反应过度激活相关；为临床防治心肌 I/R 损伤提供线索。