Oxidative Stress in Myocardial Remodeling and Failure

描述（由申请人提供）：活性氧和活性氮（ROS）与心肌衰竭的病理生理有关。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，通过使用ROS激活小GTPase Ras，通过特定半胱氨酸的氧化s -谷胱甘肽化，导致肌细胞肥大；此外，Ras激活和肌细胞肥大可以通过glutaredoxin来预防。心肌衰竭的一个标志是心肌细胞收缩功能障碍，这部分是由于肌浆网钙atp酶2 （SERCA2）活性降低。我们的初步数据显示，Gq和压力过载诱导的心肌衰竭与SERCA2中硫醇和酪氨酸的氧化翻译后修饰（OPTM）有关，并且OPTM可以调节肌细胞中SERCA2的体外活性。因此，在先前拨款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导致钙失调，从而导致心肌衰竭。Aim 1通过测试是否a)特异性SERCA2 OPTM由ROS种类、浓度和暴露时间决定，检验了ROS引起调节SERCA2、钙和收缩功能的OPTM的假设；b)特异性ros诱导的OPTM上调或下调SERCA2活性。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)定义的活性氧/氮种类对心肌细胞SERCA2活性和OPTM，钙处理和收缩功能的影响。特异性OPTM的作用将通过SERCA2位点定向突变进行测试。Aim 2将Aim 1中描述的OPTM的作用扩展到心力衰竭疾病模型，通过测试重塑刺激通过氧化剂作用，通过引起SERCA2 OPTM引起钙和收缩功能障碍的假设。我们将在体外培养的肌细胞或体内Gq和压力过载引起的心肌衰竭小鼠的肌细胞中，对暴露于重塑刺激的肌细胞进行这一理论的验证。我们将测试修饰或预防SERCA2 OPTM发生的药理学和遗传学操作的能力，包括可疑OPTM靶点的位点定向突变，以减轻钙和收缩功能障碍。为了测试我们假设的临床相关性，我们将评估狗和心力衰竭患者心肌中的SERCA2 OPTM。目的3验证了SERCA2 OPTM在体内引起压力过载引起的心肌衰竭小鼠心肌细胞钙和收缩功能障碍的假设。我们将研究转基因操作的能力，a)降低有害ROS（肌细胞特异性过氧化氢酶，NOS2敲除）的水平，b)维持活性硫醇在还原状态（glutaredoxin 1），或c)修饰可疑的OPTM靶点（SERCA2 C674S敲入），以抑制或防止特异性SERCA2 OPTM的发生，改善心肌功能和改善体内病理重塑。心力衰竭影响着超过500万美国人，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，心肌的过度劳累会导致氧化应激。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.