ANT1巯基亚硝基化修饰在病理性心肌肥厚中的作用研究

Pathological myocardial hypertrophy is an independent risk factor for cardiovascular disease such as heart failure, while the mechanism of myocardial hypertrophy is still not elucidated yet. Adenine nucleotide translocator 1(ANT1), is an important mitochondrial protein that plays a crucial role in regulating the mitochondria functional and energy metabolism of myocardial. ANT1 S-nitrosylation can be induced by exogenous nitric oxide. Whether the S-nitrosylation of ANT1 mediates myocardial hypertrophy remains unclearly. Our preliminary experiments have confirmed that ANT1 S-nitrosylation were significantly upregulated in angiotensin II-treated cardiomyocytes and myocardial tissues isolated from mice 4 weeks after aorta constriction operation, meanwhile, the protein level of nitrosoglutathione reductase (GSNOR) was downregulated. These data suggested that ANT1 S-nitrosylation may play an important role in myocardial hypertrophy. Therefore, the aim of the project is to validate the hypothesis: GSNOR-mediated ANT1 S-nitrosylation may influence mitochondrial function, then regulate myocardial energy metabolism, oxidative stress and apoptosis to participate in the process of myocardial hypertrophy in vivo and in vitro. The significance of our research is to discover the novel mechanism of myocardial hypertrophy and provide more unambiguous therapeutic targets for prevention or treatment of cardiovascular diseases.

病理性心肌肥厚可明显增加心衰等心血管疾病的发生，其发病机制未阐明。腺嘌呤核苷酸转运体1（ANT1）作为一种重要的线粒体蛋白在心肌线粒体功能及能量代谢中起重要作用。外源性给予一氧化氮可引起ANT1发生巯基亚硝基化修饰，但此种修饰是否参与心肌肥厚尚无报道。我们的预实验结果显示在血管紧张素II处理的心肌细胞及行主动脉缩窄术后4周小鼠的心肌组织中ANT1的巯基亚硝基化水平明显增高，且亚硝基谷胱甘肽还原酶（GSNOR）蛋白表达下降，提示ANT1巯基亚硝化修饰在心肌肥厚中可能发挥着重要的调控作用。因此本项目拟在离体细胞及整体动物上，联合基因敲除、过表达及特异性位点突变方法，验证科学假说：致病理性心肌肥厚因子可通过影响GSNOR调节ANT1巯基亚硝基化修饰水平，进而调控心肌线粒体功能、能量代谢、氧化应激及凋亡，促进心肌肥厚的发生发展，揭示心肌肥厚新机制，为心肌肥厚的防治提供新的干预靶点和思路。

心肌肥厚是心肌缺血、心肌梗死、心力衰竭等多种心血管疾病的独立危险因素，发生机制不清。线粒体功能障碍是心肌肥厚的重要原因。ANT1是线粒体能量代谢和结构维持的重要开关。然而，ANT1的巯基亚硝基化修饰是否通过改变线粒体功能参与调节心肌肥厚尚不清楚。我们发现与对照组相比，心肌肥厚病人心脏组织、自发性高血压大鼠和TAC小鼠心肌肥厚模型中ANT1的巯基亚硝基化修饰水平显著升高；在Ang II刺激的心肌细胞肥大模型中，ANT1的巯基亚硝基化修饰水平也显著升高。过表达GSNOR降低ANT1的巯基亚硝基化修饰水平可显著减轻心肌肥厚。质谱筛选了ANT1巯基亚硝基化修饰位点Cys160，并在293细胞中得到进一步证实。Cys160位点突变显著降低Ang II和TAC诱导的细胞和小鼠心肌肥厚。机制上，ANT1的巯基亚硝基化修饰诱导线粒体功能障碍，降低膜电位，增加线粒体氧化应激，下调线粒体自噬，激活坏死信号通路。过表达线粒体自噬蛋白PARKIN可抑制下游坏死信号的激活。因此，我们的结果提示ANT1的巯基亚硝基化修饰通过引起线粒体功能障碍，下调线粒体自噬进而激活坏死通路促进心肌肥厚。该项研究为心肌肥厚的治疗提供更有效的药物靶点和治疗前景，具有崭新的理论价值和潜在的应用价值。