Oxidative Stress in Myocardial Remodeling and Failure

 描述(申请人提供)：代谢性心脏病(MHD)是一种常见的心肌病，以线粒体能量受损和血流动力学障碍为特征，目前还没有特殊的治疗方法。MHD患者心肌能量受损和血流动力学障碍的机制尚不清楚。我们发现，肌浆网Ca++-ATPase(SERCA)敲入(SKI)小鼠，其中C674被氧化还原不敏感的丝氨酸取代，可以保护小鼠免受HFHS喂养引起的MHD。这一发现揭示了我们对MHD认识上的差距，因为目前的模型没有解释SERCA的氧化如何导致MHD的发展。越来越多的证据表明，钙离子在调节线粒体功能中起着关键作用，相反，限制线粒体钙离子可能会减少ATP的产生，增加ROS的产生。线粒体Ca++信号的受损可能反映了肌浆网(SR)储存的减少，这是由于通过SERCA的再灌流减少和/或通过兰尼定受体的泄漏。此外，由于Na+/K+-ATPase活性降低和/或肌膜Na+/Ca++交换增加而引起的胞浆[Na]i升高可能通过增加线粒体Na+/Ca++交换器(NCXmito)而增加线粒体Ca++外流，从而损害线粒体的钙信号转导。这些观察结果导致了我们的中心假设，即SERCA的氧化抑制损害了线粒体的Ca++信号，从而导致ATP生成减少和ROS产生增加，这在MHD的发病机制中发挥了关键作用。使用HFHS喂养的小鼠作为MHD的模型，我们将追求3个相互关联的目标来检验这一工作假说，即通过a)防止SERCA的氧化来纠正线粒体Ca++信号的干预，b)减轻细胞内[Na+]i升高的影响或c)清除线粒体ROS将改善MHD患者的心肌能量和血流动力学功能。在目标1中，我们验证了一种假说，即由于硫醇氧化抑制SERCA导致线粒体Ca++信号受损，从而导致MHD能量障碍和过量ROS产生。在目标2中，我们验证了一种假说，即胞浆[Na+]i升高会损害线粒体的钙信号，从而导致能量障碍和MHD患者过量的ROS产生。在目标3中，我们验证了这样的假设，即来自目标1和2的纠正线粒体Ca++信号的干预将减少a)线粒体ROS的产生和b)相关蛋白硫醇的氧化。为实现这些目标，开发了创新的方法。我们将通过31P核磁共振同时评估心脏跳动中的能量学和血流动力学功能，并使用体内传递的基因靶向指示剂测量完整心肌细胞中的线粒体Ca++和ROS。在目标3中，我们使用串联质量标记结合多反应监测MS来评估ROS对硫醇氧化靶标的累积影响并区分干预效果 从所有三个目标。我们认为，拟议的研究将产生对MHD预防和治疗的新的机械性理解和新的方法，从而做出重大贡献。

 DESCRIPTION (provided by applicant): Metabolic heart disease (MHD) is a common cardiomyopathy characterized by impaired mitochondrial energetics and hemodynamic dysfunction for which there are no specific therapies. The mechanism responsible for impaired myocardial energetics and hemodynamic dysfunction in MHD is not known. We found that sarcoplasmic reticulum Ca++-ATPase (SERCA) knock-in (SKI) mice in which C674 is replaced by a redox- insensitive serine are protected from MHD caused by HFHS-feeding. This finding reveals a knowledge gap in our understanding of MHD, as current models do not explain how oxidation of SERCA leads to the development of MHD. Emerging evidence suggests that Ca++ plays a key role in regulating mitochondrial function, and conversely, that limited mitochondrial Ca++ may decrease ATP generation and increase ROS production. Impaired mitochondrial Ca++ signaling may reflect decreased sarcoplasmic reticulum (SR) stores due to decreased refilling via SERCA and/or leak via the ryanodine receptor. In addition, elevated cytosolic [Na]i due to decreased Na+/K+-ATPase activity and/or increased sarcolemmal Na+/Ca++ exchange may impair mitochondrial Ca++ signaling by increasing mitochondrial Ca++ efflux via the mitochondrial Na+/Ca++ exchanger (NCXmito). These observations lead to our central hypothesis that oxidative inhibition of SERCA impairs mitochondrial Ca++ signaling, thereby leading to decreased ATP generation and increased ROS production that play key roles in the pathogenesis of MHD. Using HFHS-fed mice as a model of MHD we will pursue 3 interrelated aims to test the working hypothesis that interventions that correct mitochondrial Ca++ signaling by a) preventing the oxidation of SERCA, b) mitigating the effects of elevated cytosolic [Na+]i or c) scavenging mitochondrial ROS will improve myocardial energetics and hemodynamic function in MHD. In Aim 1 we examine the hypothesis that inhibition of SERCA due to thiol oxidation contributes to impaired mitochondrial Ca++ signaling that causes energetic dysfunction and excess ROS production in MHD. In Aim 2 we examine the hypothesis that elevated cytosolic [Na+]i impairs mitochondrial Ca++ signaling thereby contributing to energetic dysfunction and excess ROS production in MHD. In Aim 3 we examine the hypothesis that interventions from Aims 1 and 2 that correct mitochondrial Ca++ signaling will decrease a) mitochondrial ROS production and b) the related protein thiol oxidation in HFHS-fed mice. Innovative methods were developed to address these aims. We will assess energetics by 31P NMR and hemodynamic function simultaneously in beating hearts, and measure mitochondrial Ca++ and ROS in intact myocytes using genetically-targeted indicators delivered in vivo. In Aim 3 we use Tandem Mass Tags combined with multiple reaction monitoring MS to assess the cumulative effects of ROS on thiol oxidation targets and differentiate the effects of interventions from all 3 aims. The contribution of the proposed research would be significant, in our opinion, by resulting in new mechanistic understanding and new approaches to the prevention and treatment of MHD.