心肌缺血的内源性保护新机制：Nox4-NAD+/NADH信号调节线粒体稳态

Ischemic heart disease is a worldwide life-threatening disease, which requires a complete elucidation of its pathogenesis in order to develop effective treatment. Data from our previous "gain- and loss-of-function" study demonstrated that transgenic mice with cardiac-specific overexpression of wild-type Nox4 (Tg-Nox4) and catalytically inactive Nox4 (DN-Nox4) both exhibited increased myocardial ischemia/reperfusion (MI/R) injury, suggesting a dual role of Nox4 in aggravating I/R damage and providing endogenous cardioprotection (Circulation,2010; Abstract). However, It remains unknown how Nox4 becomes a cardiac protector against I/R injury and what the underlying mechanism is. Our further preliminary study showed that Nox4 could specifically affect celluar NAD+/NADH, the capacity of the NADH shuttle located in the mitochondrial membrane as well as the mitochondrial energy reserve, implying a potential role of Nox4 in maintaining homeostasis of mitochondrial structure and function. Based on our previous findings, the current study is aimed at exploring the role of "Nox4-NAD+/NADH" signaling in Nox4-mediated mitochondrial protection in MI/R. The clarification of this endogenous cardioprotective mechanism may hopefully inspire new therapeutic pathways and drug targets for future treatment of ischemic heart disease.

缺血性心脏病严重威胁人类健康，彻底阐明其发病机制并寻求有效的治疗措施是医学界的重要课题。我们前期发现，心肌特异性NAD(P)H氧化酶亚型4（Nox4）过表达小鼠（TG-Nox4）和显性缺失突变型小鼠（DN-Nox4）的心脏缺血/再灌注（MI/R）损伤均显著加重，该"gain- and loss-of-function"研究提示Nox4可能发挥加重损伤和保护心肌的双重效应（Circulation,2010; Abstract）。然而，Nox4为何可以保护缺血心肌、其机制是什么？这些问题尚不清楚。我们进一步的预实验表明，Nox4可特异性影响心肌细胞NAD+/NADH稳态水平、线粒体膜NADH转运体活性及线粒体能量储备，提示Nox4可调控线粒体稳态。本项目拟在前期基础上，研究Nox4-NAD+/NADH信号在MI/R线粒体损伤修复中的关键作用及机制，期望为临床防治冠心病提供可能的新策略和新靶点。

阐明心肌缺血再灌注损伤的机制，寻找有效的心肌保护靶点，是目前心血管病防治领域中亟待解决的重要问题。活性氧自由基(Reactive Oxygen Species, ROS)及其介导的氧化应激被公认为心脏缺血再灌注（MI/R）损伤的重要机制之一，然而迄今为止国际上围绕抗氧化治疗的临床试验结果却并不乐观。线粒体电子传递链酶系和NAD(P)H氧化酶系[NAD(P)H oxidase，简称Nox]是心肌细胞内与MI/R损伤关系最为密切的两大ROS供体。.我们前期构建心肌特异性Nox4过表达（Transgenic overexpression, TG-Nox4）及显性缺失突变型（Dominant negative, DN-Nox4）转基因小鼠模型，并首次应用于心肌缺血再灌注损伤的研究，发现Nox4过表达可导致ROS生成增加、MI/R损伤加剧，但Nox4抑制也可加重MI/R损伤。该“gain- and loss-of-function”研究结果提示Nox4可能发挥加重损伤和保护心肌的双重效应。我们在后续预实验中发现，位于胞膜的Nox4与线粒体虽处于细胞内不同的亚结构区域，但Nox4的表达和活性改变不仅可以影响线粒体的ROS生成，而且可以改变线粒体能量储备。据此我们提出“Nox4除介导氧化应激外，还可能参与调控心肌线粒体功能与能量代谢”这一假设。.本课题主要发现如下：(1) H/R可升高H9C2心肌细胞Nox4蛋白水平，降低细胞活性，加重凋亡、氧化应激及线粒体功能障碍，下调Nox4后H9C2心肌细胞H/R损伤不减反增，提示内源性Nox4在H/R损伤中发挥心肌保护作用。(2) 其保护机制可能是通过上调NAD+/NADH比值、增加Sirt3的表达、去乙酰化激活PGC1α进而减轻线粒体氧化损伤和能量合成障碍来实现的。本课题从“心肌Nox4-NAD+/NADH对线粒体的稳态调控”这一全新角度，阐释了内源性Nox4对线粒体ROS生成及能量代谢功能的调控作用和机制，在此基础上研究心肌两大ROS供体系统的互联机制在心血管生理功能维系及介导缺血再灌注损伤中的意义，为心肌缺血时线粒体的损伤修复机制提供了新的认识，并为临床心脏缺血性损伤的抗氧化治疗的靶向性和特异性提供了重要的临床指导。.