Data_Sheet_5_Downregulation of Uncoupling Protein 2(UCP2) Mediated by MicroRNA-762 Confers Cardioprotection and Participates in the Regulation of Dynamic Mitochondrial Homeostasis of Dynamin Related Protein1 (DRP1) After Myocardial Infarction in Mice.zip

Acute myocardial infarction (MI) is one of the leading causes of death in the world, and its pathophysiological mechanisms have not been fully elucidated. The purpose of this study was to investigate the role and mechanism of uncoupling protein 2 (UCP2) after MI in mouse heart. Here, we examined the expression and role of UCP2 in mouse heart 4 weeks after MI. The expression of UCP2 was detected by RT-PCR and western blotting. Cardiac function, myocardial fibrosis, and cardiomyocyte apoptosis were assessed by echocardiography and immunohistochemistry. Phosphatase dynamin-related protein1 (P-DRP1) and myocardial fibrosis-related proteins were measured. Cardiomyocytes were exposed to hypoxia for 6 h to mimic the model of MI. Mdivi, an inhibitor of P-DRP1, was used to inhibit DRP1-dependent mitochondrial fission. Mitochondrial superoxide, membrane potential, oxygen consumption rate, and cardiomyocyte apoptosis were detected after hypoxia. It is shown mitochondrial superoxide, membrane potential, oxygen consumption rate, and cardiomyocyte apoptosis were dependent on the level of P-DRP1. UCP2 overexpression reduced cardiomyocyte apoptosis (fibrosis), improved cardiac function and inhibit the phosphorylation of DRP1 and the ratio of P-DRP1/DRP1. However, inhibition of DRP1 by mdivi did not further reduce cell apoptosis rate and cardiac function in UCP2 overexpression group. In addition, bioinformatics analysis, luciferase activity, and western blot assay proved UCP2 was a direct target gene of microRNA-762, a up-regulated microRNA after MI. In conclusion, UCP2 plays a protective role after MI and the mechanism is involved in microRNA-762 upstream and DRP1-dependent mitochondrial fission downstream.

急性心肌梗死（Acute Myocardial Infarction, MI）是全球主要致死病因之一，其病理生理机制尚未完全阐明。本研究旨在探讨解偶联蛋白2（Uncoupling Protein 2, UCP2）在小鼠心肌梗死后心脏中的作用及相关机制。本研究检测了MI后4周小鼠心肌组织中UCP2的表达与功能：通过逆转录聚合酶链反应（RT-PCR）与蛋白质印迹法（Western Blotting）检测UCP2的表达水平；采用超声心动图与免疫组织化学法评估心功能、心肌纤维化程度及心肌细胞凋亡情况，并检测磷酸化动力相关蛋白1（P-DRP1）及心肌纤维化相关蛋白的表达水平。将心肌细胞置于缺氧环境中培养6小时以模拟心肌梗死体外模型，使用P-DRP1抑制剂Mdivi抑制DRP1依赖的线粒体分裂过程，随后检测缺氧处理后线粒体超氧化物水平、线粒体膜电位、细胞耗氧率及心肌细胞凋亡情况。结果显示，线粒体超氧化物水平、线粒体膜电位、细胞耗氧率及心肌细胞凋亡均与P-DRP1的表达水平密切相关。UCP2过表达可降低心肌细胞凋亡（及心肌纤维化）、改善心脏功能，并抑制DRP1的磷酸化及P-DRP1/DRP1的比值。然而，在UCP2过表达组中，使用Mdivi抑制DRP1并未能进一步降低细胞凋亡率或改善心功能。此外，生物信息学分析、荧光素酶活性实验及蛋白质印迹实验证实，UCP2是微小RNA-762（microRNA-762）的直接靶基因，该微小RNA在心肌梗死后呈上调表达。综上，UCP2在心肌梗死后发挥保护性作用，其机制涉及上游的microRNA-762调控及下游的DRP1依赖的线粒体分裂通路。