Yap蛋白通过糖酵解依赖的途径在心肌缺血再灌注损伤中发挥的作用及机制研究

Yap (Yes-associated protein) is an important component in Hippo signaling pathway, which is high evolutionarily conserved and regulates cell growth, proliferation and apoptosis. Previously research found that cardiac-specific Yap activation improves survival post myocardial infarction, and glycolysis, which is the major metabolic pathway to protect cardiomyocytes from hypoxic injury, could sustain Yap/Taz activity in kidney cancer cells. However, the role of Yap via glycolytic dependent pathway in myocardial ischemia reperfuion has not been investigation. Our preliminary data showed that low dosage of Poly(I:C) enhanced the glycolysis and promoted proliferation in H9C2 cells and enhanced the nuclear localization of Yap/Taz, and inhibited apoptosis signaling pathway. In addition, we found that treated with a hexokinase inhibitor (2-deoxy-D-glucose, 2-DG) abolished the cardioprotection and the nuclear localization of Yap/Taz of Poly (I:C). Based on our novel findings, we hypothesize that Yap via glycotic pathway plays a central role in the pathophysiologic mechanisms of myocardial I/R injury. To the best of our knowledge, there is no report of glycolysis regulated Yap on myocardial I/R injury. .To critically evaluate our hypothesis, we will determine the role of Yap via glycolytic dependent pathway in the pathogenesis of myocardial I/R injury using transfection of the myocardium with an adenovirus packing with VGLL4 (Adv-VGLL4) ,which inhibites the activity of Yap in the nucleus, one week before induction of myocardial I/R injury in mice, Poly(I:C), inhibitors of glycolysis and ligation of left anterior descending coronary artery (LAD). Myocardial morphologic changes and cardiac function will be measured post myocardial I/R injury. We will also examine the effect of Yap and glycolysis on apoptosis and proliferation using both in vivo myocardial I/R injury of mice and in vitro H9C2 H/R injury models. .These studies will illustrate the signaling molecular sensor that is critical for myocardial I/R injury. It may be possible to apply our finding in a practical fashion to identify new and novel therapeutic approaches to prevent or manage myocardial I/R injury.

Yap是Hippo信号通路中高度保守的信号分子，已有文献报道心肌特异性过表达Yap可增加心梗后存活率；而在肿瘤细胞中，糖酵解可以调控Yap的活性，促进细胞增殖。但Yap是否通过糖酵解依赖的途径在心肌缺血/再灌注（I/R）损伤中发挥作用尚不清楚。预实验发现：低剂量Poly(I:C)可以激活心肌细胞内糖酵解促进细胞增殖，增加Yap/Taz的核定位并抑制凋亡信号通路，而使用2-DG抑制糖酵解后可以取消Poly(I:C)的保护作用和对Yap/Taz核定位的影响。我们推测Yap可能以糖酵解依赖的途径在心肌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，采用抑制Yap活性的Adv-VGLL4心肌局部转染方法、Poly(I:C)和糖酵解抑制剂，结合冠状动脉左前降支结扎术研究糖酵解及其调控的Yap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。

目的：Yap是Hippo信号通路中高度保守的信号分子，核内的Yap调控基因表达，促进细胞增殖和存活信号，并控制细胞命运的决定。早期有文献报道心肌特异性过表达Yap可增加心梗后存活率，近期有研究表明内皮细胞特异性敲除VGLL，可以通过高表达Yap蛋白促进心脏瓣膜过度增殖；此外，在肿瘤细胞中，糖酵解可以调控Yap的活性，促进肿瘤细胞增殖。但Yap是否通过糖酵解依赖的途径在心肌缺血/再灌注（I/R）损伤中发挥作用尚不清楚。.方法：雄性C57BL/6小鼠通过结扎冠状动脉左前降支45分钟再灌注24小时复制心肌梗死模型。采用心肌内局部转染Adv-VGLL4的方法，抑制心肌组织内Yap蛋白的活性；采用2-DG作为糖酵解抑制剂。应用RT-PCR和Western blot方法，分别检测心肌缺血再灌注损伤发生发展过程中心肌组织内的Yap的mRNA和蛋白表达水平，检测糖酵解关键激酶-已糖激酶的蛋白变化水平。心脏功能的变化采用小动物心超检测，采用酸化率检测及乳酸含量检测糖酵解激活程度；心肌细胞增殖采用EDU染色方法了解情况。.结果：与假手术组相比，小鼠心肌I/R损伤后出现明显的心肌梗死并且心肌组织细胞核中Yap/Taz表达明显下降，而在缺血前1小时低剂量腹腔注射给予Poly(I:C）后减少由于I/R所诱导的心肌梗死面积及心功能紊乱，EF%上调22.7%，FS%上调26.5%，同时增加Yap/Taz的核定位。与Poly(I:C)组相比应用2-DG后可以取消Poly(I:C)对心肌I/R损伤后心功能紊乱的保护作用。转染Adv-aYap不仅可以增加心肌细胞缺氧/复氧后Yap蛋白的核定位，而且可以增加H9C2细胞的增殖能力并一定程度的改善由于2-DG所导致的心肌细胞凋亡。糖酵解作为能量代谢的重要途径可以参与调节免疫炎症信号和细胞增殖，在心肌I/R 损伤的发生发展中发挥重要作用，其机制可能通过调节Yap/Taz 蛋白的细胞核内活性从而对心肌细胞增殖和凋亡发挥作用。.结论：本文证明Yap蛋白在糖酵解依赖的途径在心肌I/R 损伤中发挥重要作用，可为探究心肌I/R 损伤及心力衰竭的防治提供新的线索。