ZiJian Xie - US grants

Cardiac glycosides, which improve the force of cardiac contraction, have been the most widely used drugs in the therapy of heart failure. Recently, we have found that interactions of cardiac glycoside (.e.g., ouabain) with sarcolemmal NaK-ATPase not only affect contractility, but also generate signals that are transduced to the nucleus, altering the expressions of growth-related genes, and causing myocyte hypertrophy. Because growth abnormalities of myocytes and other heart cells are involved in the development of heart failure, we now propose to extend our initial discoveries along the following lines: In studies of Specific Aim 1, cultured neonatal rat cardiac myocytes will be used to define ouabain- initiated transduction pathways that lead to the transcriptional regulations of two growth-related genes of these myocytes. The two model genes are c-fos and those of skeletal alpha-actin. The studies are designed to clarify the roles of Ca2+ and several protein kinases and transcription factors in these pathways, as suggested by our recent findings. Studies of Specific Aim 2 are designed to identify interactions (cross-talk) between the above ouabain-initiated pathways and the gene regulation pathways of several well-established hypertrophic stimuli; and to determine if such interactions lead to additive, synergistic, or antagonistic effects of ouabain and the other stimuli on myocyte growth. In specific Aim 3, we plan to begin the extension for our studies from cellular level to higher levels of complexity by comparing the growth- related effects of ouabain in neonatal myocytes with those in adult myocytes, and in isolated hearts obtained from normal rats and rats whose hearts are subjected to pressure or volume overload. In studies of Specific Aim 4, ouabain-induced effects on myocytes will be compared with those of low extracellular K+ and antisense-induced down-regulation of Nak-ATPase to determine if the mode of inhibition of NaK-ATPase affects the pathways of cardiac gene regulation. We expect that these basic studies, along with the ongoing studies of others on pathophysiological mechanisms of cardiac hypertrophy, will contribute to the understanding of the processes involved in transit from cardiac hypertrophy to heart failure.

DESCRIPTION (provided by applicant) The purpose of this core remains the centralization of repetitive preparations of several items that are "starting materials" and are used by one or more of the projects. We have found this approach to be efficient and convenient for all concerned. It allows the core technicians to master a small number of difficult tasks; when one is temporarily absent or needs to be replaced the work does not stop; and there is a continuous supply of high quality material available to the users. The common core facility, equipment, and routine supplies, administered and supervised by a research scientist with appropriate expertise, is also quite economical. The preparations to be made by the core are: 1. Primary cultures of neonatal rat cardiac myocytes, and adult rat cardiac myocytes either in suspension or as plated cultures, to be used by Projects 1, 11, and some of the experiments of Project 12 that will be done in Toledo. 2. Preparations of crude Sf-21 insect cell membranes containing the recombinant H+/K+-ATPase to be used by Project 10. 3. Preparation of purified recombinant Src expressed in Sf-9 cells, to be used primarily by Project 10. 4. Preparations of cardiac sarcolemmal membranes from beef and rat heart ventricles, to be used primarily by Project 1. 5. Preparation of purified Na+/K+-ATPase from pig kidney, to be used primarily by Project 11, and to smaller extents by Projects 1 and 10.

mitogen activated protein kinase; sodium potassium exchanging ATPase; genetic regulation; gene expression; cardiac myocytes; enzyme activity; enzyme mechanism; cell age; ouabain; genetic promoter element; representational difference analysis; gel mobility shift assay; laboratory rat; fluorescence spectrometry; transfection; tissue /cell culture;

[unreadable] 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.

DESCRIPTION (provided by applicant): Na/K-ATPase is an energy transducing ion pump. The enzyme serves as a receptor for digitalis drugs in the heart. Our recent work demonstrates that the enzyme is also a signal transducer. Significantly, we have shown that the signal transduction function of the enzyme is not only involved in control of cell growth and gene expression, but also required for ouabain to regulate cardiac calcium and contractility. This application is built upon these novel findings, and is proposed to define the molecular mechanisms by which Na/K-ATPase transduces the ouabain signals. Specifically,we propose three Specific Aims to test the hypothesis that Na/KATPase, when it binds to ouabain or is activated by ouabain, is capable of recruiting and assembling a group of proteins into different signaling platforms that relays the extracellular ouabain signal into different cellular compartments. In Specific Aim 1, we plan to use proteomic approaches to decipher the composition of the proteins that have the potential to interact with the Na/K-ATPase. We shall then use immunoprecipitation and in vitro binding assays to confirm the interactions between the Na/K-ATPase and the identified candidate proteins. Finally, we shall examine how ouabain regulates the phosphorylation of these enzyme-associated proteins. In Specific Aim 2, we shall use in vitro assays to define the domains that mediate the interactions between the Na/K-ATPase and its signaling partners. Depending on the outcomes of these studies, both co-localization imaging and BRET analysis will be performed to test the role of the identified binding domain(s) in ouabain-induced protein interactions. In Specific Aim 3, we plan first to determine the role of protein/protein interaction in ouabain-induced activation of Src. Second, we shall test the hypothesis that caveolins are involved in assembly of the ouabain-activated signaling branches including those leading to the activationof MAPKs and phosphorylation of the L-type calcium channel. We believe that our proposal is highly focused and that the outcomes of our study shall contribute significantly to our understandingof the biology of the Na/K-ATPase as well as the pharmacology of digitalis drugs, which will ultimately aid in the development of novel therapeutic approaches.

DESCRIPTION (provided by applicant): Na/K-ATPase belongs to the family of P-type ATPases and was discovered as an energy transducing ion pump. A major difference between the Na/K-ATPase and other P-type ATPases is its ability to bind cardiotonic steroids (CIS) such as ouabain. While endogenous CTS regulate blood pressure via their effects on vasculature and renal salt handling, the plant-derived CTS have been used as drugs for more than 200 years. Recently, we have demonstrated that the Na/K-ATPase is an important receptor that resides in caveolae and interacts directly with Src, a non-receptor tyrosine kinase. We know now that the effects of low doses of ouabain on many cellular functions are not due to the simple inhibition of the ATPase;rather they require the activation of the Na/K-ATPase/Src receptor complex. In addition, the Na/K-ATPase contains multiple binding motifs (domains) and is capable of bringing Src and other signaling enzymes to their effectors such as ion channels. These findings have led the research field to look at the Na/K-ATPase not only as an ion pump, but also a classical receptor complex. This shift of the paradigm has brought about an important question: which regulatory purposes does this signaling Na/K-ATPase serve in regulation of cellular functions that are relevant to the physiology of endogenous CTS (e.g. blood pressure control)? This application is proposed to bridge this gap by studying the most likely target of this receptor complex, namely the Ca2+-signaling module because changes in intracellular Ca2+ are known to play a key role in regulation of vascular function and renal salt handling. Specifically, we will investigate how the Na/K-ATPase integrates multiple constituents into a functional Ca2+-signaling module in renal epithelial cells. We propose to use a combined biochemical, cellular, genetic and dynamic imaging approach to 1) define the molecular mechanism by which the Na/K-ATPase integrates Src/PLC-y/PKC and IPS receptor into a dynamic Ca2+ signaling module;2) reveal whether disruption of the interaction between the Na/K-ATPase and IPS receptors affects IPS receptor trafficking and ouabain-induced Ca2+ signaling;And 3) identify the plasma membrane channel (s) that interacts with the Na/K-ATPase and is responsible for ouabain-induced Ca2+ influx. These studies will not only relate the newly discovered receptor function of the Na/K-ATPase to renal physiology of CTS, but also provide detailed mechanistic information on the formation of a Ca2+-signaling module that will eventually give us a new target for developing therapeutic approaches to renal and cardiovascular diseases involving dysfunction of intracellular Ca regulation. Calcium is a universal second messenger that plays an essential role in control of kidney and cardiovascular function. Abnormality in intracellular calcium regulation will lead to both kidney and heart diseases such as hypertension. We are using a simple model to dissect the formation of a very important calcium controlling system in kidney cells and to investigate how we can manipulate this system to eventually develop new approaches to prevent renal and cardiovascular diseases.

Digitalis drugs (cardiac glycosides, cardiotonic steroids) have been valuable for the management of heart failure and cardiac arrhythmias. Recent studies have demonstrated that the Na/K-ATPase has a novel receptor function in addition to its well described pumping function;i.e., in response to a ligand-like effect of a digitalis compound, Na/K-ATPase activates protein tyrosine kinases. Specifically, we have shown that Na/K-ATPase directly interacts with Src to form a functional digitalis receptor, and ouabain binding to this receptor stimulates the associated Src kinase. This, in turn, results in the increased protein tyrosine phosphorylation and recruitment of protein kinases and lipid kinases to form a functional signalosome that transmits the ouabain signal to different intracellular compartments. Concomitantly, activation of this receptor also induces endocytosis of the signalosome which may terminate the signaling events, or exert various intracellular effects. Moreover, we have recently mapped the interaction domains between the or subunit of Na/K-ATPase and Src. These interactions illustrate a unique and Na/K-ATPase-specific cellular mechanism of Src regulation. Furthermore, we have been able to target the identified interacting domains, and have developed a cci-specific peptide that disrupts the formation of Na/K-ATPase/Src receptor complex and inhibits Src activity. This application is built upon these new discoveries and preliminary findings, and is aimed to further delineate the molecular interactions that constitute the formation of the Na/K-ATPase/Src receptor complex, and to evaluate the functionality of this receptor in digitalis-activated signal transduction. To accomplish these goals, we propose the following three Specific Aims. First, we will test the hypothesis that the formation of a functional Na/K-ATPase/Src receptor complex requires a pair of interactions involving the Na/K-ATPase a, A-domain/Src SH2 domain, and the a, N-domain/Src kinase domain. Second, we will develop and employ cell permeable Na/K-ATPase-specific Src inhibitors/activators to test the hypothesis that activation of the Na/K-ATPase/Src receptor is responsible for the pharmacological/signaling actions of ouabain in the heart. Finally, we will employ genetically modified animal models to further evaluate the functionality of the Na/K-ATPase/Src receptor complex in the heart. The results of these studies will provide new insights into the molecular mechanism of Na/K-ATPase-mediated signal transduction and digitalis pharmacology. Moreover, with a better understanding of these new cellular signaling mechanisms, new targets for developing effective therapeutic interventions for the treatment or prevention of human diseases, including cardiac dysfunctions, may be established.

DESCRIPTION (provided by applicant): We find that the Na/K-ATPase has an ion pumping-independent receptor function. Specifically, it associates with Src to form a receptor complex. Binding of cardiotonic steroids to this receptor complex activates Src, which, in turn, initiates a series of signaling cascades including the generation of reactive oxygen species (ROS) and the activation of PI3K/Akt pathways in renal and cardiac cells. Moreover, we demonstrate a significant increase in circulating CTS in rat model of uremic cardiomyopathy induced by 5/6 nephrectomy (PNx). Neutralization of this increase in CTS reduced ROS stress and diminished cardiomyopathy characterized by myocyte hypertrophy and cardiac fibrosis. Thus, we hypothesize that chronic stimulation of this newly appreciated receptor mechanism by elevated CTS may be the cause of the cardiac remodeling observed in uremic rats. Conversely, inhibition of the receptor function could attenuate the pathological changes in the target organs under this and other clinical conditions where CTS are elevated. Recently, we have developed several new tools that allow us to further test these hypotheses in vivo. First, we find that the N-terminus (NT) of a1 subunit acts as a dominant negative mutant capable of inhibiting CTS-induced signal transduction. Second, NT transgenic mice, in contrast to their wild-type littermates, are resistant to high salt diet-induced structural damages in the heart and in the kidney. Third, we have developed a peptide antagonist of receptor Na/K-ATPase/Src complex and demonstrated its effectiveness in vitro and in vivo. Finally, we have identified a novel class of small molecule antagonists that prevent ouabain from activating protein kinases in cell cultures. Therefore, we propose the following three specific aims. Aim 1 will test whether that chronic stimulation of the ion pumping-independent receptor function of Na/K-ATPase by CTS could result in pathological changes in the heart and the kidney. Aim 2 will address whether inhibition of Na/K-ATPase- mediated signal transduction by NT or pNaKtide attenuates high salt- and PNx-induced remodeling. In aim 3 we will further characterize the in vitro and in vivo activity of MB5 as a new class of CTS antagonists and test the in vivo effectiveness of MB5 in conferring resistance to CTS-induced organ damage. The completion of these three specific aims would significantly advance our understanding of endogenous CTS and Na/K- ATPase-mediated signal transduction in animal pathophysiology. Moreover, it would provide proof of the concept that antagonists of the receptor Na/K-ATPase can actually reduce renal and cardiac damages. PUBLIC HEALTH RELEVANCE: We have discovered a new receptor mechanism that plays an important role in the pathogenesis of renal and cardiac diseases. The proposed research will further advance our understanding of this receptor mechanism and explore the possibility of using the newly discovered receptor antagonists to prevent renal insufficiency and high salt diet-induced cardiac lesions.