The Role of ROS and Na/K-ATPase in Uremic Cardiomyopathy

描述（由申请人提供）：心脏病是慢性肾衰竭（CRF）患者死亡的主要原因。在接受血液透析治疗的CRF患者中，ROS（活性氧）、应激和Na/ k - atp酶循环抑制剂的增加已经得到了充分的证明。此外，Na/ k - atp酶是心肌细胞中重要的信号转导元件。基于我们之前的工作，我们认为ROS和Na/ k - atp酶之间的相互作用激活了多种信号通路，这些信号通路对心肌细胞生长和基因表达的调节很重要。此外，ROS与其他循环泵抑制剂之间的相互作用可以通过转录和翻译后机制引起酶的显著抑制。这种酶的抑制会损害心肌细胞通过Na+/Ca2+交换器挤出Na+，从而挤出Ca2+的能力。这无疑是慢性肾功能衰竭患者发生心脏舒张功能障碍的一个重要危险因素。显然，研究ROS如何与Na/ k - atp酶相互作用，以及这种相互作用在调节心脏生长、基因表达和心脏收缩功能中的作用是很重要的。为此，我们提出以下三个具体目标：我将测试Na/K-ATPase作为ROS受体的假设，以及ROS对Na/K-ATPase的抑制招募和激活Src，导致信号复合物的组装和随后Ras/MAPK级联的激活。特异性目标2将剖析ROS在翻译后调节Na/ k - atp酶的途径。特异性目的3将验证Ras/MAPKs的激活和Na/ k - atp酶的抑制调节细胞内Ca2+([Ca2+]i)和收缩性以响应增加的ROS应激的假设，并分析ROS诱导的心肌细胞基因表达和蛋白质结构的变化。我们建议结合蛋白质组学、腺病毒介导的基因表达、cDNA表达阵列、代表性差异分析、共聚焦荧光显微镜和其他分子生物学技术来严格检验我们的工作假设。我们期望这些基础研究将有助于我们对Na/ k - atp酶和尿毒症心肌病的生物学理解，并为开发新的治疗方法提供新的信息，以解决CRF患者严重和常见的心脏病问题。Ca2+和收缩力对ROS应激增加的响应，以及ROS诱导心肌细胞基因表达和蛋白质结构的变化。我们建议结合蛋白质组学、腺病毒介导的基因表达、cDNA表达阵列、代表性差异分析、共聚焦荧光显微镜和其他分子生物学技术来严格检验我们的工作假设。我们期望这些基础研究将有助于我们对Na/ k - atp酶和尿毒症心肌病的生物学理解，并为开发新的治疗方法提供新的信息，以解决CRF患者严重和常见的心脏病问题。

DESCRIPTION (provided by applicant): Heart disease is the major cause of death in chronic renal failure (CRF) patients. Increases in ROS (reactive oxygen species), stress and circulating inhibitors of Na/K-ATPase have been well documented in CRF patients treated with hemodialysis. Moreover, Na/K-ATPase is an important signal transduction element in cardiac myocytes. Based on our prior work, we believe that interaction between ROS and Na/K-ATPase activates multiple signaling pathways that are important for regulation of cell growth and gene expression in cardiac myocytes. Further, interaction between ROS and other circulating pump inhibitor can cause a significant inhibition of the enzyme through both transcriptional and post-translational mechanisms. Such inhibition of the enzyme will impair the ability of cardiac myocytes to extrude Na+, thus Ca2+ through Na+/Ca2+ exchanger. This certainly represents an important risk factor for development of diastolic dysfunction of the heart in CRF patients. Clearly, it is important to study how ROS interact with Na/K-ATPase and the roles of such interaction in regulation of cardiac growth, gene expression and cardiac contractile function.We, therefore, proposed the following three specific aims to address these issues. Specific Aim I will test the hypotheses that Na/K-ATPase serves as a receptor for ROS and that inhibition of Na/K-ATPase by ROS recruits and activates Src, resulting in assembly of a signaling complex and subsequent activation of the Ras/MAPK cascade. Specific Aim 2 will dissect pathways by which ROS post-translationally regulate Na/K-ATPase. Specific Aim 3 will test the hypothesis that activation of Ras/MAPKs and inhibition of Na/K-ATPase regulate intracellular Ca2+([Ca2+]i) and contractility in response to increased ROS stress, and profile ROS-induced changes in gene expression and protein structures in cardiac myocytes. We proposed to use a combination of proteomics, adenovirus-mediated gene expression, cDNA expression array, representation difference analysis, confocal fluorescence microscopy, and other molecular biology techniques to critically test our working hypotheses. We expect that these basic investigations will contribute to our understanding of the biology of Na/K-ATPase, uremic cardiomyopathy and provide new information for developing novel therapies addressing the serious and common problem of heart diseases in CRF patients. Ca2+ and contractility in response to increased ROS stress, and profile ROS-induced changes in gene expression and protein structures in cardiac myocytes. We proposed to use a combination of proteomics, adenovirus-mediated gene expression, cDNA expression array, representation difference analysis, confocal fluorescence microscopy, and other molecular biology techniques to critically test our working hypotheses. We expect that these basic investigations will contribute to our understanding of the biology of Na/K-ATPase, uremic cardiomyopathy and provide new information for developing novel therapies addressing the serious and common problem of heart diseases in CRF patients.