TRX1调控DRP1的S-亚硝基化在冠状动脉微栓塞后心肌细胞焦亡中的作用及机制研究

Coronary microembolization has been an independent risk factor for poor clinical outcomes in patients with coronary heart disease, however, the mechanisms underlying coronary microembolization-induced myocardial injury remain elusive. It has been reported that the reactive oxygen species released by damaged mitochondria play an important role in activating NLRP3 inflammasome, which priming pyroptosis. Dynamin-related protein 1 (DRP1) has been demonstrated to be modified by S-nitrosylation, which has an impact on mitochondrial dynamics. According to our previous research, mitochondria dysfunction after coronary microembolization induces reactive oxygen species accumulation and NLRP3 inflammasome-dependent pyroptosis, however, how this process primes remains unknown. Our previous proteomics analysis depicted several energy metabolic abnormalities in coronary microembolization mice and the expression changes of DRP1 and thioredoxin 1 (TRX1), raising the conjecture that TRX1 might affect mitochondrial function and pyroptosis via regulating the post translation modification of DRP1 in a de-nitrosylation manner. In the present study, we aim to discover the mechanisms underlying TRX1-regulated pyroptosis, using the TRX1-transgenic mice, primary culture of neonate rat myocardial cells and HEK293T cells to observe the alterations of mitochondrial function and pyroptosis after the gain-of-function and loss-of function of TRX1. Our study will shed light on novel pharmacological targets for treatment of coronary microembolization.

冠状动脉微栓塞（CME）是冠心病患者不良预后的独立危险因素，但其导致心肌损伤的机制尚不明确。研究发现线粒体功能紊乱释放的活性氧是激活NLRP3炎症小体的重要因素，后者可导致细胞焦亡。动力相关蛋白1（DRP1）受S-亚硝基化修饰调控，进而影响线粒体动力学。我们前期研究发现CME后心肌线粒体结构和功能紊乱，活性氧爆发，NLRP3炎症小体显著激活，导致心肌焦亡，然而这一过程的具体机制尚不明确。蛋白质组学分析显示CME后心肌能量代谢相关通路异常，伴随DRP1和硫氧还蛋白1（TRX1）表达改变，我们推测TRX1依靠其脱S-亚硝基作用调控DRP1翻译后修饰影响线粒体功能和活性氧产生，进而影响细胞焦亡。我们拟构建TRX1过表达小鼠，使用原代心肌细胞、HEK293T细胞，观察TRX1表达变化对线粒体功能和细胞焦亡的影响，明确TRX1调控CME后心肌细胞焦亡的机制，为CME后心肌损伤的防治提供新思路。

在冠脉介入术（PCI）后，有1/3左右的患者存在持续的心肌损伤，且50-75% PCI围手术期心肌损伤（PMI）与冠状动脉微栓塞（CME）相关。CME是冠心病患者不良预后的独立危险因素，但其导致心肌损伤的机制尚不明确。研究发现活性氧是激活NLRP3炎症小体的重要因素，后者可导致细胞焦亡。近年来，蛋白质巯基亚硝基化修饰已经受到了越来越广泛的关注，蛋白质的巯基亚硝基化修饰参与多种心血管疾病的调节，如心律失常、心肌梗死、心肌细胞凋亡等。心肌细胞蛋白质S-亚硝基化修饰是否参与CME导致的心肌损伤尚无相关研究。本课题首先使用RNA-seq技术分析PMI患者PBMC基因表达情况，发现PMI患者PBMC中NLRP3炎症小体显著激活。CME小鼠通过诱导心肌细胞NLRP3炎症小体依赖的细胞焦亡，介导心肌损伤和心功能损害。进一步采用生物素转化和链霉亲和素富集方法获取CME小鼠心肌组织发生S-亚硝基化修饰的所有蛋白，质谱分析检测出789个蛋白，生物信息学分析这些蛋白富集在三羧酸循环中，其中ACO2差异明显。进一步鉴定出ACO2第126位半胱氨酸发生S-亚硝基化修饰。构建ACO2位点突变的腺相关病毒和腺病毒，分别转染小鼠和体外细胞，发现ACO2的S-亚硝基化修饰在CME诱导心肌损伤中起到重要作用。CME后心肌组织脱S-亚硝基化酶TRX1显著下降，进一步研究表明TRX1调控ACO2的S-亚硝基化修饰。研究结果可为CME的治疗拓展新方向。