William A. Coetzee, DSc - US grants

DESCRIPTION (the applicant's description verbatim): ATP-sensitive (KATP) channels couple energy metabolism and stress to membrane excitability and are highly expressed in heart. Their function in the heart and coronary vasculature has been studied extensively by the use of pharmacological agents that open or block KATP channels. The molecular cloning of subunits of KATP channel revealed that KATP channels consist of pore-forming Kir6 subunits in association with regulatory SUR subunits. There are two Kir6 members (Kir6.1 and Kir6.2) - both are highly expressed in heart. Cardiac KATP channels are believed to consist of Kir6.2/SUR2 complexes. However, the role of Kir6.1 is unknown. The overall goal of this application is to examine the function of Kir6.1 subunits in the cardiovascular system. Since the localization of a protein can be indicative of its function, we will examine the regional, cellular and subcellular distribution patterns of Kir6.1 subunits in cardiac myocytes, the conduction system, in the coronary vasculature and in endothelial cells using immunohistochemistry techniques (we have developed excellent antibodies for this purpose). We show biochemically and electrophysiologically that Kir6.1 and Kir6.2 subunits can co-assemble in heterologous expression systems, suggesting that this may also occur in heart. We will examine whether such co-assembly takes place for native Kir6 proteins. We will also investigate the functional characteristics of heteromeric Kir6 channels using patch clamp techniques. We will generate mice with targeted disruption of the Kir6.1 gene. LoxP sites will be introduced in the Kir6.1 locus and these mice will be crossed with others overexpressing Cre in the heart. We will utilize these mice directly to examine the role of Kir6.1 subunits in the function of heart and vasculature using isolated, Langendorff-perfused heart techniques. Coronary blood flow and -reserve will be measured along with heart function during ischemia, reperfusion and ischemic preconditioning. These studies will provide a framework in which to understand the complexity of KATP channels in the cardiovascular system, in particular the role of Kir6.1 subunits, and will provide molecular insights into the function of KATP channels in animals during normal and pathophysiological conditions.

DESCRIPTION (provided by applicant): The cardiac transient outward current is important for repolarizing the cell membrane during the action potential. As such, factors that modulate this current can have profound effects on excitability and arrhythmogenesis. The molecular components of this current are Kv4 subunits. Early work suggested the existence of accessory subunits that modulate the expression levels and kinetics of Kv4 channels. The recently described KChIP proteins fulfill such a role. We identified another distantly related protein, frequenin (belonging to the same family of Ca2+binding proteins), expressed in human heart, which acts as an additional Kv4 accessory protein. Frequenin co-immunoprecipitates and co-localizes with Kv4 proteins, increases Kv4 membrane trafficking and slows the Kv4 inactivation process. Although initially described as expressed mainly in neurons, we find high frequenin expression levels in mouse heart. We hypothesize that KChIP2 (the prototype KChIP expressed in heart) and frequenin, are important regulators of cardiac Ito expression and function. In a multi-disciplinary approach, we will investigate this hypothesis in three Specific Aims. First, we will examine the role of KChIP2 and frequenin on Ito (and other membrane currents) in mouse cardiac myocytes by examining the Ca2+ sensitivity of Ito, as well as the effects of intervention designed to disrupt the function and expression of these proteins. Our preliminary data suggest that frequenin and KChIPs may interact. This will be investigated in Specific Aim, using co-immunoprecipitation and electrophysiological assays. In Specific Aim 3, we will investigate a proposed mechanism by which frequenin affects Kv4 currents, with the hypothesis that part of its effect on Kv4 trafficking is mediated by an interaction and stimulation of PI4Kbeta, a key regulator of the phosphoinositol pathway. We will investigate this using biochemical, trafficking, molecular biology and electrophysiological assays. The completion of these experiments will be invaluable in identifying the role of a novel accessory protein on the function of an important cardiac repolarizing K+ current. The information gained may in future be invaluable for the rational design of novel anti-arrhythmic agents.

DESCRIPTION (provided by applicant): ATP-sensitive potassium [KATP] channels in the heart muscle and coronary myocytes couple cellular metabolic status to membrane excitability, thereby contributing to the regulation of tissue responses to physiological and pathophysiological stimuli. In the heart muscle, opening of KATP channels participate in the stress response and protect against ischemic episodes. In the coronary vasculature, K(ATP/NDP) channels contribute to the regulation of basal flow as well as responses to metabolic impairment (hypoxic dilatation and ischemic reactive hyperemia). We found glycolytic enzymes to associate with KATP channel subunits, We hypothesize that glycolytic enzymes are integral components of the KATP channel macromolecular complex and that glycolytic enzymes regulate KATP channel activity under physiological and pathophysiological conditions, both in the cardiac myocyte as well as in the coronary smooth muscle and endothelium. In a first Specific Aim, we will investigate the hypothesis that enzymes of the glycolytic complex are associated with the KATP channel. Using co-immunoprecipitation assays we will investigate the specificity of interaction of glycolytic enzymes with individual KATP channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B). Co-immunoprecipitation assays of native proteins will be performed to investigate interactions under physiologically relevant conditions. Protein interactions will also be investigated using advanced proteomic approaches (ICAT &ITRAQ), which has the potential to uncover additional novel KATP channel interacting proteins. In a second Aim, we will examine the hypothesis that physical interaction of glycolytic enzymes with KATP channel subunits is required and that channel modulation occurs because of altered nucleotide levels in the microenvironment of the channel complex. This will be accomplished using mutant KATP channel subunits (lacking interaction with glycolytic enzymes or altered nucleotide sensitivity). In a final Aim, we will investigate the interaction of glycolysis and KATP channels in the context of ischemic protection in cardiac myocytes and the coronary vasculature. To this end, we will utilize our genetic mouse models that express dominant-negative K(ATP) channel subunits specifically in the cardiac myocyte, smooth muscle or endothelium. Our findings may have important implications for understanding the role of KATP channels in the heart and coronary vasculature under physiological and non-pathophysiological conditions. PUBLIC HEALTH RELEVANCE: In many cell types, there is an important link between intracellular energy metabolism and membrane excitability, which affects physiological process as diverse as insulin secretion, control of blood flow and protection of the heart against ischemia. We propose to examine one of the main effectors in this context, namely the ATP-sensitive K+ channel, and how the channel is specifically controlled by the initial step in glucose metabolism, namely glycolysis. Understanding this modality of channel regulation will further our knowledge of the known protective effects of the KATP channel in myocardial ischemia and cardiovascular disease.

DESCRIPTION (provided by applicant): KATP channels are integral to the functions of many different types of cells and tissues. However, even though they were first described almost 25 years ago, the physiological and pathophysiological roles of these channels are only now emerging for most tissues. Electrophysiological and pharmacological methods have allowed for a good understanding of the roles of KATP channels. The molecular identification of KATP channels (Kir6.1, Kir6.2, SUR1, SUR2A and/or SUR2B subunits) has further advanced our knowledge regarding the distribution and function of these channels. The overriding hypothesis to be examined is that physiological and pathophysiological events are subject to the activity of KATP channels that are present in multiple tissues and that synchronous interaction between channel activities in diverse tissue types determines the nature of the physiological or pathophysiological outcome. Conventional pharmacological methods cannot be used to examine this hypothesis in a complex biological setting since these compounds lack required specificity for different classes of KATP channels present in diverse tissue types. Conventional knockout mouse models have been developed for each of the KATP channel subunits and the availability of these animal models has added significantly to our knowledge of the functions of KATP channels in the in-vivo setting. However, since the expression is eliminated in all somatic cells the use of these animals provides challenges when defining the roles of KATP channel subunits in complex biological settings (such as ischemia/reperfusion). To examine the interplay of KATP channels present in multiple tissues and their synchronous interaction in diverse tissue types, new tools are needed in which KATP channels are eliminated only in specific tissues, cell types or subcellular compartments. The purpose of this proposal is therefore to generate four mouse models of conditional knockouts of each of the KATP channel subunits. Given the rich diversity of KATP channels (both in terms to their tissue distribution and molecular composition), a dire need exists to generate conditional knockout mice devoid of each of the KATP channel subunits. The development of these animal models will have a wide ranging impact, not only on our own research, but also on the field in general since these animals generated as part of these studies will allow investigators working in diverse areas (cardiology, endocrinology, neurology, etc) to unravel the tissue specific functions of KATP channel in health and disease. PUBLIC HEALTH RELEVANCE: ATP-sensitive K+ channels are unique in that they transduce intracellular energy metabolism to cellular activity and excitability and they have very important roles on the functions of many different cell types and they help to maintain blood glucose levels, regulate blood flow, participate in the secretion of neurotransmitters and protect against ischemic insults. New challenges have arisen in the study of KATP channel in physiological and pathophysiological context that cannot be addressed using existing tools (even with the available conventional knockout mice), which is the rationale for our proposal to generate new genetically altered mouse models in which KATP channel subunits can be deleted specifically in certain tissue types. These mice, which will be widely shared with the scientific community, will undoubtedly lead to significant breakthroughs in the study of KATP channels in many study areas (in addition to our own research) and this will have a major impact on the fields of biomedical, behavioral and clinical research.

? DESCRIPTION (provided by applicant): Heart disease remains the leading cause of death in the United States and other developed countries. Most deaths are associated with cardiac ischemia and arrhythmias. Sarcolemmal KATP channels in the myocardium open with elevated heart rates and during stress conditions, such as cardiac ischemia. Opening of KATP channels modulate the action potential duration and intracellular Ca2+. As such they have an important role in determining contractility, arrhythmias and electrical conduction. It is well established tht KATP channel opening protects the heart during stress. However, a detailed understanding of the KATP channel function during ischemia, reperfusion and ischemic preconditioning is lacking, which hinders the development of therapeutic strategies. Our preliminary data point to novel subcellular localization patterns of KATP channels within the cardiac myocyte. We further find that myocardial ischemia decreases the surface KATP channel density, which reduces the number of channels that are available for cardioprotection. This proposal is driven by our preliminary observations that a) ischemic preconditioning prevents ischemia-induced internalization of KATP channels and that b) the protective effects of ischemic preconditioning on infarct size are abolished in mice with cardiac-specific knockout of the KATP channel subunit, Kir6.2. We hypothesize that enhancing KATP channel surface density through specific subcellular trafficking pathways is an important element of the protective mechanism of ischemic preconditioning. We will investigate molecular mechanisms that stabilize surface KATP channels (Aim 1), cellular processes that regulate internalization (Aim 2) and potential mechanisms to restore the KATP channel surface density during an ischemic insult (Aim 3). The proposed studies will establish a framework in which to understand novel roles of KATP channels in the heart and will provide molecular insights their cardioprotective function during ischemic pre-conditioning.

? DESCRIPTION (provided by applicant): We are requesting funds for a CytoPatch(tm) 4-Channel System, designed for acquiring high-fidelity patch clamp data at medium to high-throughput. There is currently no new generation automated, high-throughput patch clamp systems (similar to the CytoPatch(tm) System) available at NYU or within New York City. There is a previous-generation, low-throughput planer patch clamp system at NYU School of Medicine, which is an aging system (purchased in 2008), is not available as a shared instrument to all users and has high running costs. Moreover, the use of seal enhancer solutions that contains high concentrations and fluoride and calcium, makes this system unsuitable for many applications and cell types. The CytoPatch(tm) instrument is a new generation recording techniques and can perform simultaneous patch clamp measurements with four individual cells at relatively low cost. The design of the CytoPatch recording quartz chip allows patch clamping in a manner that is much more similar to traditional patch clamping methods, when compared to other planer high- throughput patch clamp systems and dispenses with the need for special recording buffers. End users needs minimal training, the desktop-sized instrument is space saving and the system is easily shared between users. The instrument will be housed in a central location and managed by an experienced cellular electrophysiologist and shared by several NIH-funded major and minor users. NYUSOM Office of Collaborative Science (OCS), will assist to generate a business plan, maintain the service contract and Laboratory Information Management System (LIMS) for scheduling and billing and to generate usage statistics for compliance. The major user group spans several academic departments and represents both basic and translational research interests such as; pediatrics, cardiology, immunology and neurosciences. The major NIH-funded users will have protected use of ~80% of the instrument time whereas minor users will be able to use the remaining instrument time. The availability of this shared instrument will be unique in Manhattan. The requested CytoPatch(tm) 4- Channel automated, high-throughput patch clamp instrument will support the research of several NIH funded researchers at NYU Medical School and NYU Dental School.

SUMMARY Cardiac Na+ channels and KATP channels are generally thought to have very different roles in cardiac electrophysiology. They do, however, share certain characteristics such as being expressed at higher levels at the intercalated disk compared to the lateral membranes and they coexist with desmosomal proteins. Our new data demonstrate that cardiac KATP channels interact with Na+ channels (and with the Na+/K+ ATPase). This proposal will utilize innovate super-resolution microscopy techniques and patch clamping to examine the interaction between Na+ channels and KATP channels. We will identify the membrane subdomains where interaction occurs, the molecular mechanisms responsible for interaction and the functional consequences of interactions. The overall hypotheses is that interaction is particularly prominent at membrane domains at the ICD, that interaction is mediated by specific binding sites in ankyrin-G, and that interaction leads to functional coupling between Na+ channels and KATP channels (via the Na+/K+ ATPase). In a first Aim, we will localize Na+ channels and KATP channels with nanometer precision to membrane subdomains in the lateral membrane (e.g. t-tubules, caveolae, etc.) as well as membrane subdomains at the intercalated disk (e.g. hybrid adhering junctions). A role for ankyrins in targeting channels to these domains will be investigated with siRNA approaches. In a second Aim, we will investigate the molecular mechanisms involved in interaction, testing the hypothesis that Na+ channels and KATP channels bind to similar sites on ankyrins. We will also test the functional consequences of interaction of Na+ channels and KATP channels. These studies will significantly move forward our understanding of Na+ channel and KATP channel and function in the cardiovascular system, and more generally, advance our knowledge on how channel systems in ventricular myocytes physically and functionally interact.

SUMMARY Sarcolemmal ATP-sensitive K+ (KATP) channels are abundantly expressed in the heart. Several groups have now identified a key role for these channels in mediating cardioprotection against ischemic injury and their participation in the protective mechanism of ischemic preconditioning. In the heart there several different subtypes of KATP channels and little is known about the roles during ischemia and reperfusion. Of particular interest are the KATP channel subtypes present in the coronary smooth muscle (SM) and coronary endothelial cells (EC). There is increasing focus on these coronary channels as a target for blood flow regulation and cardioprotection, yet they are relatively poorly understood. The SM and EC KATP channels are distinct from ventricular KATP channels and they are also distinct from each other. A major barrier to our understanding of their respective roles during a complex event such as myocardial ischemia is the lack of currently available resources specifically to study these two channel subtypes. We have generated novel genetic mouse models that can distinguish these subtypes of KATP channels and show with one of these that EC KATP channels strongly participate in myocardial protection during ischemia/reperfusion. The goal of the proposed studies is systematically to examine the role(s) of the EC and SM KATP channel subtypes in the regulation of coronary blood flow, protection during ischemia and the protective response to ischemic preconditioning. We hypothesize that both EC and SM KATP channel subtypes contribute to the regulation of coronary blood flow and cardioprotection, but through distinctly different mechanisms. Using novel and validated conditional knockout mice, we will specifically target EC or SM KATP channel subtypes. The proposed studies have three Aims. In Aim 1, we will investigate the roles of these two coronary KATP channel subtypes in blood flow during ischemia. We will use isolated, pressurized microvessels and isolated, perfused hearts under normal, hypoxic and ischemic conditions. We will additionally investigate the role of EC and SM KATP channels in the myocardial ?no-reflow? phenomenon. Aim 2 will investigate the roles of EC and SM KATP channels in myocardial protection using an in vivo murine I/R model and investigate pathways that regulate infarct development. Aim 3 will investigate the contribution of EC and SM KATP channel subtypes during ischemic preconditioning using an in vivo murine I/R model and cellular assays. We will also examine trafficking of these KATP channel subtypes as a potential protective mechanism and investigate molecular signaling pathways involved. This multi-investigator proposal combines the expertise of three highly established investigators; Dr. Lefer?s extensive expertise with in vivo cardiac ischemia/reperfusion models, and Dr. Coetzee?s track record of studying KATP channels with electrophysiological, biochemical and molecular approaches and Dr. Tinker?s expertise in studying molecular signaling pathways in vascular KATP channels. The proposed studies will provide important molecular insights into the unique functions of coronary KATP channel subtypes under pathophysiological conditions.