Redox regulation in aging and failing heart

描述（由申请人提供）：还原/氧化（氧化还原）是翻译后修饰控制多种细胞功能的重要机制。线粒体渗漏和NAD(P)H氧化酶（Noxs）等多种来源产生的活性氧（ROS）可氧化信号分子和转录因子。Noxs是主要的酶，负责生产超氧化物（O2-）， ROS的一个组成部分，在不同的亚细胞定位。O2-使含铁硫簇的酶失活，从而释放出游离铁，从而产生高活性的羟基自由基。细胞内产生的O2-迅速转化为H2O2，而O2-也与NO反应生成ONOO-，都起到ROS的作用。尽管质膜相关的Nox（如Nox1和p91phox (Nox2)）的细胞功能已被广泛表征，但Nox4 （Nox在心脏中表达的同工异构体）的细胞功能尚不清楚。虽然心脏中的氧化应激随着衰老和应激以及心力衰竭而增加，但ROS产生的机制和定位尚不清楚。我们的初步研究表明，Nox4在心脏中的表达会因衰老和压力过载而上调。然而，Nox4在氧化应激增加、心脏老化和心力衰竭进展中的作用仍有待阐明。此外，Nox4调控的分子机制（靶点）及其在心脏生长和死亡反应中的作用目前尚不清楚。有趣的是，我们的初步结果表明，抑制Nox4在压力过载时具有保护作用，但在缺血/再灌注时是有害的。该提案的总体假设是，尽管生理水平的Nox4介导某些应激条件下细胞存活所必需的细胞功能，但在衰老和心力衰竭中，Nox4的上调会引起有害影响，例如ROS的产生增加和随后的线粒体功能障碍。具体来说，我们的假设是：1。在衰老和压力过载过程中，Nox4的上调会增加氧化应激、细胞凋亡和心功能障碍，因此是有害的。2. Nox4是心衰期间线粒体氧化应激和线粒体功能障碍的关键介质。3. 生理水平的Nox4可以保护心脏免受缺血/再灌注（I/R）损伤。Nox4在急性缺血期间上调HIF-11发挥重要作用，HIF-11反过来介导糖酵解刺激，从而防止I/R期间心肌细胞死亡。我们将使用新生成的Nox4功能获得和丧失的小鼠模型，包括Nox4转基因小鼠和KO小鼠，蛋白质组学分析和综合生理学研究来解决这些问题。我们的研究结果将阐明Nox4在衰老和应激过程中介导心脏生理和病理功能的作用。公共卫生相关性：蛋白质的氧化和还原是调节蛋白质功能的重要机制。在过去的四年里，我们一直在研究硫氧还蛋白1（一种小抗氧化剂）在心脏中的功能，并证明硫氧还蛋白1是心脏中蛋白质氧化/还原的重要调节剂，并且硫氧还蛋白1通过改变受肥厚刺激氧化的信号分子的半胱氨酸残基来负性调节心脏肥厚。在这个竞争性更新中，我们将扩展这些观察结果，并进一步阐明由心脏细胞内蛋白质的氧化和减少所调节的细胞功能。特别是，我们将专注于NADPH氧化酶4 (Nox4)，一种产生超氧化物的酶，细胞中的主要活性氧。我们的初步结果表明，Nox4在衰老和应激的作用下上调，并介导心脏的生理和病理功能。利用新生成的转基因小鼠模型，结合最新的小鼠生理实验、蛋白质组学方法和核磁共振实验，我们将研究Nox4的心脏功能，并阐明Nox4介导心脏生理和病理功能的分子机制。我们的研究将使我们能够阐明通过氧化和还原对心脏蛋白的修饰如何影响心脏的生理和病理功能。这项研究的结果可能会导致开发一种新的策略，通过靶向特定心脏蛋白的氧化修饰来治疗与衰老相关的心脏病或充血性心力衰竭。

DESCRIPTION (provided by applicant): Reduction/oxidation (redox) is an important mechanism of post-translational modification controlling a wide variety of cellular functions. Reactive oxygen species (ROS) produced from various sources, such as mitochondrial leakage and NAD(P)H oxidases (Noxs), oxidize signaling molecules and transcription factors. Noxs are major enzymes responsible for production of superoxide (O2-), a component of ROS, at various subcellular localizations. O2- inactivates iron-sulfur cluster containing enzymes, thereby liberating free iron, which in turn generates the highly reactive hydroxyl radical. O2- produced in cells is rapidly converted to H2O2, while O2- also reacts with NO to form ONOO- , all of which act as ROS. Although the cellular function of plasma-membrane associated Noxs, such as Nox1 and p91phox (Nox2), has been extensively characterized, that of Nox4, an isoform of Nox expressed in the heart, is not well understood. Although oxidative stress in the heart is increased by aging and stress and during cardiac failure, the mechanism and localization of ROS production are not well understood. Our preliminary studies suggest that expression of Nox4 in the heart is upregulated by aging and pressure overload. However, the contribution of Nox4 to the increased oxidative stress and the progression of cardiac aging and heart failure remains to be elucidated. Furthermore, molecular mechanisms (targets) regulated by Nox4 and their contributions to growth and death responses in the heart are currently unknown. Interestingly, our preliminary results suggest that inhibition of Nox4 is protective during pressure overload but is detrimental during ischemia/reperfusion. The overall hypothesis in this proposal is that although physiological levels of Nox4 mediate cellular functions essential for cell survival under certain stress conditions, upregulation of Nox4 in aging and failing hearts elicits detrimental effects, such as increased production of ROS and subsequent mitochondrial dysfunction. In particular, our specific hypotheses are:1. Upregulation of Nox4 during aging and pressure overload increases oxidative stress, apoptosis and cardiac dysfunction, and is thereby detrimental. 2. Nox4 is a critical mediator of mitochondrial oxidative stress and mitochondrial dysfunction during heart failure. 3. The presence of a physiological level of Nox4 protects the heart from ischemia/reperfusion (I/R) injury. Nox4 plays an essential role in upregulating HIF-11 during acute ischemia, which in turn mediates stimulation of glycolysis, thereby preventing cardiac myocyte death during I/R. We will address these issues using newly generated mouse models of both gain and loss of function of Nox4, including Nox4 transgenic and KO mice, proteomic analyses and integrated physiology studies. Our results will elucidate the role of Nox4 in mediating both physiological and pathological functions in the heart during aging and under stresses. PUBLIC HEALTH RELEVANCE: Oxidation and reduction of proteins are important mechanisms regulating the function of proteins. During the past four years, we have been studying the function of thioredoxin 1, a small anti-oxidant, in the heart and demonstrated that thioredoxin1 is an essential regulator of protein oxidation/reduction in the heart and that thioredoxin1 negatively regulates cardiac hypertrophy by modifying cysteine residues of signaling molecules subjected to oxidation by hypertrophic stimuli. In this competing renewal, we will extend these observations and further elucidate the cellular functions modulated by oxidation and reduction of intracellular proteins in the heart. In particular, we will be focusing on NADPH oxidase 4 (Nox4), an enzyme producing superoxide, major reactive oxygen species in cells. Our preliminary results suggest that Nox4 is upregulated by aging and stress and mediates both physiological and pathological functions in the heart. Using newly generated genetically altered mouse models, together with the state of the art mouse physiology experiments, proteomic methods and NMR experiments, we will investigate the cardiac function of Nox4 and elucidate the molecular mechanisms by which Nox4 mediates both physiological and pathological functions in the heart. Our study will allow us to elucidate how the modification of cardiac proteins by oxidation and reduction affects physiological and pathological functions of the heart. The result obtained from this investigation may lead to development of a novel strategy to treat aging related heart diseases or congestive heart failure in patients by targeting oxidative modifications of specific cardiac proteins.