Oxidative Stress in Myocardial Remodeling and Failure

描述（由申请人提供）：活性氧和氮物质（ROS）与心肌衰竭的病理生理学有关。我们的实验室和许多其他实验室已经表明，重塑刺激，包括机械应变和Gq激活，导致肌细胞肥大，通过使用ROS激活小GTdR，Ras，通过氧化S-谷胱甘肽的特定半胱氨酸;而且，Ras激活和肌细胞肥大可以防止谷氧还蛋白。心肌衰竭的一个标志是心肌细胞收缩功能障碍，部分原因是肌浆网钙ATP酶2（SERCA 2）活性降低。我们的初步数据表明，Gq和压力超负荷诱导的心肌衰竭与SERCA 2中巯基和酪氨酸的氧化翻译后修饰（OPTM）有关，并且OPTM可以调节肌细胞中SERCA 2的体外活性。因此，在先前的授权期间的工作导致了我们的工作假设，即重塑刺激引起SERCA 2的OPTM，导致钙调节异常，导致心肌衰竭。目的1通过测试a）特异性SERCA 2 OPTM是否由ROS种类、浓度和暴露持续时间决定;和B）特异性ROS诱导的OPTM是否上调或下调SERCA 2活性，检验ROS引起调节SERCA 2、钙和收缩功能的OPTM的假设。使用原代培养的心肌细胞，我们将测量分级a）浓度、B）持续时间和c）类型的限定活性氧/氮物质对肌细胞SERCA 2活性和OPTM、钙处理和收缩功能的影响。将使用SERCA 2的定点突变来测试特异性OPTM的作用。目的2通过测试以下假设将目的1中描述的OPTM的作用扩展到心力衰竭疾病模型：通过氧化剂起作用的重塑刺激通过引起SERCA 2 OPTM而引起钙和收缩功能障碍。我们将在体外培养的肌细胞或体内Gq和压力超负荷诱导的心肌衰竭小鼠的肌细胞中暴露于重塑刺激的肌细胞中测试这一论文。我们将测试药理学和遗传操作改变或预防SERCA 2 OPTM发生的能力，包括可疑OPTM靶点的定点突变，以缓解钙和收缩功能障碍。为了检验我们的假设的临床相关性，我们将评估患有心力衰竭的狗和人的心肌中的SERCA 2 OPTM。目的3在压力超负荷诱导的心肌衰竭小鼠体内验证SERCA 2 OPTM导致心肌细胞钙和收缩功能障碍的假设。我们将检查转基因操作的能力，a）降低攻击性ROS（肌细胞特异性过氧化氢酶，NOS 2敲除）的水平，B）将反应性硫醇保持在还原状态（谷氧还蛋白1），或c）修饰可疑的OPTM靶点（SERCA 2 C674 S敲入），以抑制或预防特异性SERCA 2 OPTM的发生，改善心肌功能并改善体内病理性重塑。影响超过500万美国人的心力衰竭最常见的原因是高血压或心脏病发作导致心肌过度劳累。现在有证据表明，过度劳累的心肌会导致氧化应激。这项资助旨在了解氧化应激导致心力衰竭的方式，并有可能发现这种情况的新治疗方法。

DESCRIPTION (provided by applicant): Reactive oxygen and nitrogen species (ROS) are implicated in the pathophysiology of myocardial failure. Our laboratory and many others have shown that remodeling stimuli, including mechanical strain and Gq activation, cause myocyte hypertrophy by using ROS to activate the small GTPase, Ras, via the oxidative S-glutathiolation of a specific cysteine; and further, that Ras activation and myocyte hypertrophy can be prevented by glutaredoxin. A hallmark of myocardial failure is cardiac myocyte contractile dysfunction, which is due in part to reduced activity of sarcoplasmic reticulum calcium ATPase 2 (SERCA2). Our preliminary data show that Gq- and pressure overload-induced myocardial failure are associated with oxidative post-translational modifications (OPTM) of thiols and tyrosines in SERCA2, and that OPTM can regulate SERCA2 activity in vitro in myocytes. Thus, work during the prior grant period has led to our working hypothesis that remodeling stimuli cause OPTM of SERCA2 leading to calcium dysregulation that contributes to myocardial failure. Aim 1 examines the hypothesis that ROS cause OPTM that regulate SERCA2, calcium and contractile function, by testing whether a) specific SERCA2 OPTM are determined by the ROS species, concentration and duration of exposure; and b) specific ROS-induced OPTM up- or down- regulate SERCA2 activity. Using cardiac myocytes in primary culture we will measure the effects of graded a) concentrations, b) durations and c) types of defined reactive oxygen / nitrogen species on myocyte SERCA2 activity and OPTM, calcium handling and contractile function. The role of specific OPTM will be tested using site-directed mutations of SERCA2. Aim 2 extends the role of the OPTM delineated in Aim 1 to heart failure disease models by testing the hypothesis that remodeling stimuli, acting via oxidants, cause calcium and contractile dysfunction by causing SERCA2 OPTM. We will test this thesis in myocytes exposed to remodeling stimuli in vitro in cultured myocytes or in vivo in myocytes from mice with Gq- and pressure overload-induced myocardial failure. We will test the ability of pharmacologic and genetic manipulations that modify or prevent the occurrence of SERCA2 OPTM, including site-directed mutation of suspected OPTM targets, to alleviate calcium and contractile dysfunction. To test the clinical relevance of our hypothesis, we will assess SERCA2 OPTM in myocardium from dogs and humans with heart failure. Aim 3 tests the hypothesis that SERCA2 OPTM causes myocyte calcium and contractile dysfunction in vivo in mice with pressure overload-induced myocardial failure. We will examine the ability of transgenic manipulations that, a) reduce the level of offending ROS (myocyte-specific catalase, NOS2 knockout), b) maintain reactive thiols in a reduced state (glutaredoxin 1), or c) modify suspected OPTM targets (SERCA2 C674S knock-in) to inhibit or prevent the occurrence of specific SERCA2 OPTM, improve myocardial function and ameliorate pathological remodeling in vivo. PUBLIC HEALTH RELEVANCE Heart failure, which affects over 5 million Americans, is most often caused by high blood pressure or heart attack leading to overworking of the heart muscle. There is now evidence that overwork of the heart muscle leads to oxidative stress. This grant seeks to understand the ways in which oxidant stress leads to heart failure, and has the potential to discover new treatments for this condition.