Oxidative Stress in Myocardial Remodeling and Failure

描述(由申请人提供)：活性氧和氮物种(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证明，重塑刺激，包括机械应变和GQ激活，通过使用ROS激活小GTP酶RAS，通过特定半胱氨酸的氧化S谷胱甘肽氧化反应，导致心肌细胞肥大；此外，谷氧还蛋白可以防止RAS激活和心肌细胞肥大。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ATPase 2(SERCA2)活性降低。我们的初步数据表明，GQ和压力超负荷导致的心肌衰竭与SERCA2中硫醇和酪氨酸的氧化翻译后修饰(OPTM)有关，OPTM在体外可以调节心肌细胞中SERCA2的活性。因此，在之前的资助期间的工作导致了我们的工作假设，即重塑刺激导致SERCA2的OPTM，导致钙调节失调，从而导致心肌衰竭。目的1通过测试a)特定的SERCA2 OPTM是否由ROS的种类、浓度和暴露时间决定，以及b)特定的ROS诱导的OPTM上调或下调SERCA2的活性，来检验ROS引起OPTM调节SERCA2、钙和收缩功能的假说。利用原代培养的心肌细胞，我们将测量a)浓度、b)持续时间和c)确定的活性氧/氮物种类型对心肌细胞SERCA2活性和OPTM、钙处理和收缩功能的影响。将使用SERCA2的定点突变来测试特定OPTM的作用。目的2通过验证重塑刺激通过氧化剂作用，通过引起SERCA2 OPTM导致钙和收缩功能障碍的假说，将AIM 1中描述的OPTM的作用扩展到心力衰竭疾病模型。我们将在体外用重塑刺激暴露的心肌细胞、培养的心肌细胞或在体的GQ和压力超负荷诱导的心肌衰竭小鼠的心肌细胞中测试这篇论文。我们将测试改变或预防SERCA2 OPTM发生的药物和遗传操作的能力，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SERCA2 OPTM。目的3验证SERCA2 OPTM在压力超负荷所致心力衰竭小鼠体内引起心肌细胞钙和收缩功能障碍的假说。我们将检测转基因操作的能力，a)降低有害的ROS(心肌细胞特异性过氧化氢酶，NOS2基因敲除)的水平，b)将活性硫醇维持在还原状态(谷氧还蛋白1)，或c)修饰可疑的OPTM靶点(SERCA2 C674S敲入)，以抑制或防止特定SERCA2 OPTM的发生，改善心肌功能，并改善体内的病理重塑。与公共健康相关的心力衰竭影响了500多万美国人，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，心肌过度劳累会导致氧化应激。这项拨款旨在了解氧化应激导致心力衰竭的方式，并有可能发现治疗这种疾病的新方法。

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.