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

简介(申请人提供)：心脏病是慢性肾功能衰竭(CRF)患者的主要死因。在接受血液透析治疗的CRF患者中，ROS(活性氧物种)、应激和Na/K-ATPase循环抑制物的增加已被很好地记录下来。此外，Na/K-ATPase是心肌细胞内重要的信号转导元件。在我们前期工作的基础上，我们认为ROS和Na/K-ATPase之间的相互作用激活了多个信号通路，这些信号通路对于调控心肌细胞的生长和基因表达是重要的。此外，ROS和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导致对酶的显著抑制。这种酶的抑制会损害心肌细胞通过Na+/Ca~(2+)交换器排出Na~+，从而~(2+)~(2+)的能力。这无疑是CRF患者发生心脏舒张期功能障碍的重要危险因素。显然，研究ROS如何与Na/K-ATPase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。因此，我们提出了以下三个具体目标来解决这些问题。具体目的I将验证Na/K-ATPase作为ROS受体的假设，以及ROS对Na/K-ATPase的抑制招募并激活Src，导致信号复合体组装并随后激活RAS/MAPK级联。具体目标2将剖析ROS翻译后调节Na/K-ATPase的途径。具体目标3将验证Ras/MAPKs的激活和Na/K-ATPase的抑制调节细胞内钙([Ca+]i)和收缩能力以响应ROS增加的假说，并描述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、共聚焦荧光显微镜等分子生物学技术来严格检验我们的工作假说。我们期待这些基础研究将有助于我们了解Na/K-ATPase、尿毒症心肌病的生物学，并为开发新的治疗方法提供新的信息，以解决CRF患者严重而常见的心脏疾病问题。钙离子和收缩能力对增加的ROS压力的反应，以及Profile 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、共聚焦荧光显微镜等分子生物学技术来严格检验我们的工作假说。我们期待这些基础研究将有助于我们了解Na/K-ATPase、尿毒症心肌病的生物学，并为开发新的治疗方法提供新的信息，以解决CRF患者严重而常见的心脏疾病问题。

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.