Howard A. Rockman - US grants

Heart failure is an abnormality of myocardial cell function resulting in impaired ventricular performance. The functional defect is unknown, but may be related to alterations in calcium (ca++) handling. Evidence suggests that the altered functional properties of the sarcoplasmic reticulum (SR) are in part related to a defect in the SR Ca++ ATPase pump as shown by the decreased expression of mRNA encoding the SR Ca++ ATPase gene in failing hearts. Recently transgenic technology has emerged as a valuable tool to study tissue specific and inducible gene expression in vivo. The goal of this project is to produce transgenic mice which overexpress cardiac SR Ca++ ATPase and apply it to a murine model of cardiac failure induced by pulmonary artery banding (PAB) that I have developed, to determine if SR Ca++ ATPase overexpression in heart failure can rescue the cardiac dysfunction. The present study proposes the following specific aims: 1) To characterize the in vivo murine model of compensated and decompensated heart failure produced by PA banding with respect to; a) The heart failure phenotype; hemodynamics, organ weights, and in vivo contractile function by digital contrast ventriculography. b) Genetic markers (expression of SR Ca++ ATPase, ANF, MLC-2 mRNA. 2) To produce transgenic mice using a MLC-2/SR Ca++ ATPase construct to overexpress SR Ca++ ATPase selectively in the ventricle. 3) To determine if overexpression of SR Ca++ ATPase can diminish the functional abnormality in failing hearts, the PAB model will be applied to transgenic mice which harbor the MLC-2\SR Ca++ ATPase transgene and characterized as in specific aim 1. 4) To assess myocardial calcium homeostasis in compensated and decompensated heart failure; Calcium transients in isolated papillary muscle from ventricles of normal and failing hearts which overexpress SR Ca++ ATPase will be determined.

DESCRIPTION (provided by applicant): ß-arrestins were initially discovered to desensitize GPCR signaling in response to agonist stimulation. However, it is now appreciated that ß-arrestins can also transduce multiple effector pathways independent of G-protein signaling when receptors are stimulated by certain ligands, a concept known as biased signaling. The proposed mechanism for this signaling bias is based on the barcode hypothesis where unbiased and ß-arrestin-biased ligands impart distinct patterns of receptor phosphorylation by specific GPCR kinases (GRKs), thus converting a distinct ligand stabilized receptor conformation into selective ß-arrestin function and downstream signaling. In this proposal we plan to discover new mechanistic insights for the precise molecular details that underlie ß-arrestin biased signaling and determine how this may influence cardiac function. We will use a mass spectrometry phosphoproteomic based approach to determine whether Carvedilol induces a unique phosphorylation bar code of the ß1AR compared to the balance agonist isoproterenol, and will translate these findings in vivo by using mice conditionally lacking ß-arrestin in differnt cell types of the heart and determine the effect of ß-arrestin bias on the transcriptional signatue and cardiac function under normal and injured conditions. We propose the following specific aims: Aim 1: Determine the specific phosphorylation bar code on the c-terminal tail of ß1AR responsible for G?i dependent ß-arrestin-biased signaling. Aim 2: Determine the mechanism of ß1AR-biased signaling in cardiac fibroblasts. Aim 3: Determine the cardiac cell type responsible for ß-arrestin signaling that promotes cardiac fibrosis in response to injury. Aim 4: Determine th cell-type specific RNA signature of wild type and conditionally deleted ß-arrestin1 and 2 KO mice in response to the biased ligand carvedilol and after cardiac injury.

The mechanisms responsible for the transition from stable compensated hypertrophy to myocardial dysfunction are not well understood. Several hypotheses have been suggested to be responsible for the progression of myocardial dysfunction including: alterations in Ca++ availability, abnormalities in the myocyte cytoskeleton such as microtubular polymerization, decreased volume fraction of cardiomyocyte myofibrils, and aberrations in beta-adrenergic receptor (beta-AR) signaling. In this regard, an important characteristic of human heart failure is diminished beta-AR number (receptor down regulation) and impaired beta-AR function (receptor uncoupling). Recent data suggest a step-wise increase in plasma norepinephrine levels in individuals from normal to asymptomatic left ventricular (LV) dysfunction and to symptomatic LV failure. Thus high levels of circulating catecholamines early in the transition from stable cardiac hypertrophy to Lv dysfunction, may account, in part, for the observed loss in beta-AR function. it therefore becomes apparent that abnormalities in beta-AR signaling are potential key targets, that if corrected, may interrupt the gradual deterioration of myocardial function in the clinical course of heart failure. The overall goal of this project is to use mouse genetics to specifically inhibit betaARK or to chronically enhance myocyte contractility during the transition from stable hypertrophy to decompensated dilated cardiomyopathy followed by a comprehensive biochemical, molecular and physiological analysis of beta-AR signaling and cardiac function. Studies are proposed to test the hypothesis that beta-adrenergic receptor desensitization early in the time course of a cardiomyopathy contributes to the progressive deterioration in contractile function, and that correction of the gene defect using mouse genetics can modify or even ameliorate the disease phenotype.

There is increasing evidence that elevated levels of the beta-adrenergic receptor kinase1 (betaARK1) contribute to impaired catecholamine responsiveness observed in heart failure. Using a gene targeted mouse model of heart failure (MLP-/-), we have recently shown that reversal of betaAR desensitization achieved through betaARK1 inhibition, could lead to a marked improvement in cardiac function. The overall hypothesis of this proposal is that increased beta1AR phosphorylation, as a consequence of elevated betaARK1 levels, not only contributes to, but is a primary cause of heart failure. The following specific aims are proposed 1) To determine the role beta1AR phosphorylation and betaARK1 play in the development of the failing heart and whether high levels of circulating catecholamines are required for the pathogenesis of heart failure; MLP(-/-) mice will be crossed with betaARK1 heterozygous knockout mice and dopamine-beta-hydroxylase knockout mice. 2) To determine whether the salutary effects of betaARK1 inhibition represent a ubiquitous pathologic mechanism; another model of severe cardiac dysfunction and premature death achieved through cardiac targeted overexpression of calsequestrin will be crossed into transgenic mice overexpressing the betaARK inhibitor 3) To determine whether betaARK1 phosphorylation of the beta1AR is a mechanism for the pathogenesis of the failing heart, the carboxyl terminal phosphorylation sites for betaARK1 will be mutated by site directed mutagenesis and tested both in vitro and in vivo for the capacity to prevent phosphorylation and desensitization of beta1AR's 4) To determine whether beta1AR desensitization by betaARK1 is a primary pathologic mechanism for the development of heart failure; beta1AR phosphorylation mutant mice will be crossed into the MLP (-/-) heart failure mouse. Using an integrative approach with novel gene targeting strategies and in-depth physiological analysis of gene targeted mice, we will directly test the hypothesis that desensitization of beta1AR's, mediated through heightened betaARK1 levels, play a causative role in the pathogenesis of the failing heart. The collaborations with the other R01's should provide a unique opportunity to understand the relationships between betaAR signaling, E-C coupling and myocyte cell survival.

DESCRIPTION (Adapted from the applicant's abstract) The goal of this proposal is to develop and experimentally validate a high throughput phenotype screening procedure, that when linked to a ethylnitrosourea (ENU) mutagenesis or a random gene-targeted program will: (1) detect mutations in genes previously unrecognized as affecting hypertension; and (2) detect mutations in genes previously unrecognized as affecting cardiac hypertrophy and cardiomyopathy. In designing the screen these investigators note that ENU-induced single base pair changes and random targeted genes will much more likely be loss of function mutations than gain of function mutations. Furthermore, the screen must be able to detect random mutations in heterozygotes, preferably prior to weaning and it must be effective regardless of whether the mutations affect blood pressure and cardiac growth in a positive or negative direction. They propose two novel approaches to identify important phenotypes from a mutagenesis screen: (1) use of surrogate markers; and (2) use of sensitizing mutants. They do not expect that a single surrogate will be adequate to detect mutations in all the presently known systems affecting blood pressure and cardiac growth. However, they postulate that a combination of surrogates will be able to detect a large proportion of mutations known to affect the primary phenotypes, i.e. blood pressure and cardiac growth. Accordingly they propose the following Specific Aims: (1) To identify surrogate markers that can reproducibly detect genes affecting hypertension or cardiac hypertrophy by systematically characterizing available adult heterozygous knock out and transgenic mice with either primary phenotype. Candidate surrogate markers will be evaluated by assaying multiple tissues using; (a) DNA microarrays; (b) real time RT-PCR; (c) activity of essential regulatory kinases; (d) urine cGMP and osmolality; and (e) plasma peptide hormones. (2) To validate whether surrogate markers identified in Specific Aim #1 can distinguish 3-week-old heterozygotes from wild type litter mates in a simulated ENU experiment. Heterozygous knock out and transgenic mice will be bred to wild-type B6 mice to produce F1 pups that are genetically identical except for the presence or absence of the mutation. Surrogate markers will be measured in multiple tissues from pre-weaned F1 progeny. (3) To test the novel strategy of using sensitizing mutations to enhance the resolution of the phenotypic screen. Surrogate marker(s) will be assayed in pre-weaned F1 test progeny generated by mating known heterozygous mutants with sensitizing mice that already have abnormal blood pressure or cardiac growth. By using this strategy, they expect to move the surrogate marker(s) into a range where mutations that either increase or decrease the phenotype can be detected. (4) To develop a high throughput method for detecting plasma peptide hormones and to refine in a high throughput mode, the surrogate marker assays used in specific aims #1, 2, and 3. (End of Abstract.)

DESCRIPTION (Applicant's Abstract) Duke University Medical Center has been engaged in the training of clinicians and scientists in the area of cardiovascular diseases -for over 40 years. Much of this success can be attributed to 25 years of funding of the Cardiovascular Training Program Grant. This application is a competitive renewal for our Cardiovascular Training Program Grant, which was last funded in 1996. The cardiovascular training program represents the integrated efforts of the Departments of Medicine, Surgery, Cell Biology, Pediatrics, Pharmacology, Engineering, and Genetics to train postdoctoral cardiovascular physician-scientists. This will be accomplished by providing a multidisciplinary faculty and environment to trainees in order to foster and develop excellence in biomedical research related to the cardiovascular system. The underlying philosophy of this program will be to integrate training and research by creating an environment that facilitates the natural linkages among programs and departments throughout the University. An extended period of research (2 years or more) will be provided to trainees under the mentorship of program faculty who will closely supervise and direct the academic career development of the trainee. This program will provide an important mechanism for supporting trainees during a critical period of postgraduate medical training and provide an environment conducive to a research-oriented career. During this time, trainees will be "protected" from clinical responsibilities and immersed in research to obtain the background and experience necessary to establish themselves an independent investigators in academic institutions. The individual mentor will provide skills such as experimental design, data analysis, and manuscript and grant preparation during the training period. The goal of this program is to continue this history of productivity by creating an environment that will ensure that Duke will continue to contribute to the development of young investigators capable of pursuing successful research careers in cardiovascular fields. The current application will describe the modifications and improvements that have been recently implemented and will discuss Duke"s ongoing commitment to train the next generation of cardiovascular researchers. The training program emphasizes the training of physician-scientists, and to this end, approximately 90% of the trainees will be MDs or MD/PhDs.

DESCRIPTION (provided by applicant): Heart Failure is a clinical syndrome characterized by progressive ventricular dilatation, depressed cardiac function and premature death. Importantly, variation in the development of heart failure and in the long-term survival irrespective of etiology indicates that additional unidentified genetic factors play a significant role in the phenotypic expression. Unfortunately, these modifier genes have been recalcitrant to direct identification in human populations. In this regard, genetic studies in animals models of disease can identify candidate modifier genes and provide an insight on the genetic interactions that cause phenotypic variation in the humans. The goal of this proposal is to identify genetic modifiers of human heart failure. To accomplish this goal we will use a well-characterized murine model of heart failure created by the overexpression of a calsequestrin (CSQ) transgene to simulate a monogenetic disorder of inherited dilated cardiomyopathy. The phenotype of the CSQ model demonstrates many of the "hallmark" features of dilated cardiomyopathy including progressive cardiac dysfunction, shortened lifespan and abnormalities in beta-adrenergic receptor signaling. Although the model is not one caused by a natural occurring single-gene mutation in humans, it is one that recapitulates the human heart failure phenotype and importantly, is highly dependent on the genetic background. Accordingly, the goal of this project is to use the CSQ mouse to identify modifier genes that contribute to the severity of heart failure in the human population. The following specific aims are proposed: 1) to identify modifier genes in the CSQ mouse model of heart failure that confer susceptibility to premature death. Quantitative Trait Loci mapping has already shown strong linkage of survival to a narrow region on chromosome 2. 2) to identify modifier genes in the CSQ mouse model of heart failure that delay susceptibility to both cardiac dysfunction and premature death. Quantitative Trait Loci mapping has already shown strong linkage of cardiac function and survival to a broad region on chromosome 3 and 8. 3) to test whether modifier genes influence phenotypic variation in human heart failure, we will examine whether genetic polymorphisms identified in the mouse correlate with outcome in patient populations with heart failure. Genetic epidemiology will be performed in collaboration with the Duke Center for Human Genetics.

DESCRIPTION (provided by applicant): Left ventricular hypertrophy (LVH) with heart failure and arteriolopathy of renal vessels are cardinal manifestations of target organ damage in chronic hypertension. We hypothesize that genes predisposing to target organ damage in hypertension operate independently of blood pressure. To identify genetic factors that predispose to target organ damage, we will take advantage of transgenic mouse models that develop LVH and nephrosclerosis with striking similarities to the pathology of human hypertension, but in the absence of elevated blood pressure. We propose the following specific aims: (1) To identify modifier genes that accelerates the severity of LVH and heart failure in a transgenic mouse model. The calsequestrin (CSQ) transgenic mouse model recapitulates many of the features of hypertensive cardiomyopathy. Our preliminary studies have identified a strong modifier locus conferring reduced survival that we have linked to a narrow region on mouse chromosome 2. (2) To identify modifier genes a transgenic mouse model of heart failure that prolong survival without improving cardiac function. In preliminary experiments, backcrossing of the CSQ transgene onto a "resistant" background produces a broad prolongation of survival. (3) To identify modifier genes that confer susceptibility to nephrosclerosis in angiotensin receptor-deficient mice. We have identified strong genetic modifiers that predispose angiotensin receptor-deficient mice to develop a severe renal phenotype resembling hypertensive nephrosclerosis in humans, yet they in the absence of hypertension. Preliminary analysis shows phenotypic segregation suggesting susceptibility to two recessive loci with strong modifying effects. Genomic micro-satellite analysis will be used to map the disease modifying loci and minimal candidate regions. Modifying genes will then be identified using standard positional cloning techniques. Finally, we will test whether the sequence polymorphisms contained within these loci alter susceptibility to target organ injury. The scope of this proposal provides a unique opportunity to develop this program as a unit to maximize efficiency and sharing of resources, taking full advantage of the complementing expertise of the investigative group. Our long-term goal is to identify genes that confer susceptibility to heart and kidney injury in hypertension.

DESCRIPTION (provided by applicant): The central theme of this PPG is to elucidate the molecular intersection of novel adrenergic receptor signaling pathways involved in the physiological and pathological growth of the heart. The ultimate goal is to identify novel molecules and pathways that have the potential to become therapeutic targets in the treatment of heart failure with a theme centered on signaling mechanisms of adrenergic receptors. The experimental organization is crafted such that the specific aims for each project address both basic molecular mechanisms of G-protein-coupled receptor signaling using in vitro and cell culture methods, and the translation of these fundamental concepts into relevant in vivo models of hypertrophy and heart failure. Since each project contains both basic and translational components, we are uniquely positioned in this PPG to take basic discoveries made at the bench and move them into clinically relevant in vivo models of heart failure. This well focused PPG will be led by project leaders who have had a long history of collaboration, as demonstrated by numerous co-authored publications and collaborative grants. The history of close collaboration between the project leaders has resulted in thematically integrated projects, ideally suited for a PPG. We propose 4 projects that each address a unique aspect of adrenergic signaling and will be directed by project leaders that are distinguished scientists in their field. The themes for each of the 4 projects are: Project 1 (Rockman) will study novel aspects of phosphoinositide 3-kinase (PI3K) in beta-adrenergic receptor (beta-AR) internalization; Project 2 (Koch) will study novel aspects of the beta-AR kinase and its regulation of myocardial beta-AR signaling; Project 3 (Stamler and Lefkowitz) will study a new paradigm for beta-AR signaling through regulation by nitric oxide; and Project 4 (Williams and Rosenberg) will study novel aspects of cardiac calcium signaling regulated by G-protein-coupled receptor (GPCR) stimulation. We propose two scientific cores that are integral to the success of the program by providing both small animal and large animal expertise where the novel concepts identified at the bench can be tested as potential therapeutic targets in large animal models of heart failure. We believe this highly synergistic program combines uniquely talented investigators with cutting edge molecular and in vivo animal model methodologies, which will lead to novel discoveries in adrenergic receptor signaling and potential new therapeutic avenues in heart failure.

Beta-adrenergic receptor (betaAR) signaling plays an important role in the regulation of cardiac performance. BetaAR downregulation and desensitization resulting in reduced responsiveness to catecholamines are hallmarks of the failing heart. In human heart failure, diminished receptor number (downregulation) and impaired coupling (desensitization) of betaARs result in reduced responsiveness to catecholamines. The process that initiates desensitization of the receptor begins with the phosphorylation of betaARs by beta-adrenergic receptor kinase1 (betaARK1), followed by the binding of beta-arrestin, which together inhibits coupling to G protein and loss of effector (adenylyl cyclase) signaling. The beta-arrestin receptor complex is then targeted to the clathrin-coated pits for endocytosis through a process involving interaction of beta-arrestin with AP-2 and clathrin. Recently we have shown that betaARK1 interacts with the lipid kinase, phosphoinositide 3-kinase (PI3K), to form a cytosolic complex. We have shown that the betaARK/PI3K interaction is critical for the agonist-mediated internalization of betaARs and if the betaARK/PI3K interaction is disrupted in vivo, betaARs do not undergo downregulation and desensitization with chronic catecholamine stimulation. Importantly, PI3K contains both lipid kinase and protein kinase activity. Although we have shown that the lipid products of PI3K are important for receptor internalization, it is not yet known whether the protein kinase activity of PI3K is necessary for betaAR endocytosis and activation of downstream signaling pathways. Since receptor-localized PI3K plays a critical role in betaAR internalization, knowledge of the distinct roles of the protein and lipid kinase activities in the process of betaAR sequestration, resensitization and signaling would contribute to a better understanding of the regulation of betaAR function in the pathogenesis of heart failure. Our Central Hypothesis is that downregulation of cardiac betaARs, in vivo, can be prevented through inhibition of the local generation of D-3 phosphatidylinositols by PI3K within the receptor complex and preserving betaAR function in the failing heart will lead to an amelioration of heart failure in both small and large animal models of cardiac failure. We will test the hypothesis that the protein kinase activity of PI3K is required for betaAR internalization and downregulation, and that betaAR function will be normal in hearts displaying a "physiological" hypertrophic phenotype compared to hearts that show a "pathological" hypertrophic phenotype. Specific Aims (1) To determine the molecular mechanisms of PI3K-mediated betaAR internalization by studying PI3K mutants that lack either protein kinase activity or lipid kinase activity; (2) To determine whether overexpression of the minimal peptide of PI3K (PIK domain peptide, PIKdp), which acts as a dominant negative in vitro, will prevent betaAR downregulation in vivo and rescue mouse models of heart failure: a) chronic pressure overload (transverse aortic constriction, TAC), b) Calsequestrin overexpression X PIKdp, c) muscle LIM protein knock out X PIKdp; (3) To determine whether overexpression of PI3Kdp in vivo by adenoviral-mediated gene transfer to the heart will prevent betaAR downregulation and rescue two large animal models of cardiac failure: a) AdenoPIKdp injected into a rabbit infarct model, b) the pacing pig heart failure model; (4) To determine whether the mechanism that causes transition from hypertrophy to heart failure is due to the type of load on the heart (exercise vs. pressure) or the chronicity of the load. Chronic TAC will be compared to intermittent TAC and two exercise protocols in mice, together with a comprehensive evaluation of the developing cardiac phenotype.

phosphatidylinositol 3 kinase; enzyme activity; beta adrenergic receptor; ventricular hypertrophy; pathologic process; heart failure; phenotype; nonhuman therapy evaluation; gene therapy; myocardial infarction; receptor binding; calcium binding protein; phosphorylation; disease /disorder model; intracardiac pressure; genetically modified animals; laboratory mouse; laboratory rabbit;

[unreadable] DESCRIPTION (provided by applicant): Genetic approaches, preferentially in model systems that both allow accurate measurement of heart function as well as efficient genetic screening, are necessary for substantial progress in identifying the genetic basis of heart failure. In proposal, we will test the hypothesis that the powerful mutagen, N-ethyl-N- nitrosourea (ENU), mutagenesis in mice, and gene-deletion screens in Drosophila, will lead to the discovery of novel disease-causing and disease-modifying genes for human heart failure. In order to test these hypotheses, we have performed a recessive mutagenesis screen in adult mice at 8 and 16 weeks of age using non-invasive echocardiography to screen for abnormalities in cardiac function and have already identified a heritable region on chromosome 1 that maps to a cardiomyopathic phenotype. To complement the mouse studies we propose to use the fly to identify novel cardiomyopathic genes. Drosophila genetics provides more than 19,000 molecularly-defined P-elements inserted throughout the Drosophila genome that facilitate the generation of high-density genomic coverage. We have developed an innovative approach to phenotype cardiac function in adult awake Drosophila and have identified a P-element mutation in the short gastrulation (sog) gene that results in dilated cardiomyopathy. Based on our preliminary findings, we propose that mouse and Drosophila genetics can identify novel genes and mechanisms that are responsible for human dilated cardiomyopathies. Accordingly, we propose the following specific aims: Aim 1: To identify novel genes causing cardiomyopathy using an ENU phenotype-driven recessive screen in adult mice. We propose to map the disease causing gene located on chromosome 1 in the ENU family with abnormal cardiac function. Aim 2: To investigate the biochemical and genetic mechanisms through which mutations in the dpp/BMP signaling pathway lead to dilated cardiomyopathy in adult Drosophila. Genetic complementation experiments will be performed in Drosophila to prove that sog deficiency causes cardiomyopathy. Aim 3: To test whether decreased antagonism of the BMP pathway in mice, will result in a cardiomyopathic phenotype under conditions of pressure overload. TAG experiments will be performed in knock out mice deficient in the endogenous mammalian BMP antagonists chordin and noggin. Aim 4: To identify novel genes causing cardiomyopathy by performing a genome-wide screen of deletion mutants in Drosophila. Flies heterozygous for PiggyBac derived deletion mutants from the Exelixis collection will be screened for cardiomyopathy using optical coherence tomography followed by fine mapping of the disease-causing gene(s). Thus, these four integrated aims will harness the power of mouse and Drosophila genetics to identify and evaluate novel candidate genes for their role in cardiomyopathy. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The overall goal of this SCCOR proposal is to elucidate the roles of innate and adaptive immunity in the pathogenesis of chronic lung disease. The SCCOR proposal consists of four interrelated research projects that are focused on two chronic lung diseases: asthma and bronchiolitis obliterans syndrome (BOS) with a common underlying theme of epithelial injury, inflammation, repair and fibroproliferation. The central hypothesis to be tested is that chronic lung disease occurs as a consequence of destructive or maladaptive host responses to common environmental insults that challenge the lung. The fundamental roles of innate and adaptive host responses are to recognize invading antigens, pathogens or altered self components with the purpose of eradicating the offending agents and restoring tissue integrity. While resolution can occur when the host response is normal, an exogenous insult cannot be contained when a critical host factor is inactivated, dysregulated, or becomes dysfunctional. Such maladaptive host responses lead to chronic lung disease. The project specific hypotheses within this SCCOR proposal are: Project 1: Surfactant proteins SP-A and SP-D, which are produced by both alveolar and airway cells, interact with cells of both the adaptive and innate immune systems to coordinately maximize defense against inhaled allergens and that cause and exacerbate asthma, while minimizing an over exuberant immune response that could result in inflammation, tissue damage and chronic lung disease. Project 2: Interleukin 13 (IL-13) modulates airway fibroblast function in human asthma via increased expression of platelet-derived growth factor (PDGF), an adaptive host response, and subsequent airway remodeling via fibroblast proliferation, collagen expression and decreased elastin expression. Project 3: Activation of innate immunity through toll like receptors (TLRs) in the transplanted lung promotes the adaptive alloimmune response leading to acute rejection and BOS. Project 4: Innate immune mechanisms regulate chronic inflammation and tissue remodeling and specifically, host hyaluronan and TLR interactions are critical components of the injury and repair response in non-infectious lung injury, BOS and chronic asthma. These studies will contribute to our understanding of normal and altered host responses on lung structure and function and will provide a basis for investigation and development of new therapies for the treatment of chronic lung diseases. (End of Abstract) INDIVIDUAL PROJECTS AND CORE UNITS PROJECT 1. Immunoprotective Effects of Surfactant Proteins in Asthma (Wright, Jo R.) DESCRIPTION (provided by applicant): Although pulmonary surfactant has been traditionally viewed as a surface tension reducing substance, recent studies demonstrate that it also functions in host defense. Two surfactant proteins, SP-A and SP-D, are members of a family of innate immune proteins known as collectins that bind pathogens and facilitate their clearance by immune cells. SP-A and SP-D also regulate a variety of immune cell functions. The overall hypothesis to be tested in this proposal is that SP-A and SP-D. which are synthesized and secreted by both alveolar and airway cells, interact with cells of both the adaptive and innate immune systems to coordinatelv maximize defense against inhaled allergens that cause and exacerbate asthma, while minimizing an over exuberant immune response that could result in persistent inflammation, tissue damage and chronic lung disease. We propose to evaluate the roles of SP-A and SP-D in regulating functions of two immune cells that play a role in asthma pathogenesis: dendritic cells and T-lymphocytes. Preliminary studies show that SP-D enhances antigen uptake and presentation by dendritic cells, that SP-A and SP-D inhibit lymphocyte proliferation, modulate production of regulatory and inflammatory cvtokines by dendritic cells and that SP-A null mice have enhanced susceptibility to lung injury and allergic inflammation. Our hypothesis is also supported by published studies showing that SP-A and SP-D inhibit allergen-induced lymphocyte proliferation and histamine release by immune cells from asthmatic children and by studies showing that SP-D null mice are more susceptible to allergic inflammation. Four aims are proposed. Aim 1 will determine the mechanisms by which SP-A and SP-D and their receptors, including toll like receptors (TLRs), regulate dendritic cell function. Studies will be conducted in vitro with isolated cells and in vivo with mice. Aim 2 will investigate the mechanism by which SP-A and SP-D regulate lymphocyte activation and whether SP-A and SP-D directly or indirectly (via dendritic cells) affect T-cell proliferation and polarization to a TH1 or Tn2 phenotype. Aim 3 is to investigate the role of SP-A and SP-D in the pathogenesis of inflammatory lung disease using mouse models of asthma and chronic allergic inflammation in collectin null mice. Aim 4 is to compare characterize levels of SP-A and SP-D in lavage fluid from asthmatics and normals. These studies will provide information about the role of SP-A and SP-D in regulating the functions of two important cells of the adaptive immune system and contribute to our understanding of the role of SPA and SP-D inflammatory lung diseases. This project investigates the role of TLRs in chronic lung disease in conjunction with Projects 2, 3 and 4. In addition, patient samples from Project 2 will be analyzed. The project will interact with all the Cores. (End of Abstract)

Blood capillaries; CRISP; Capillaries; Capillary; Capillary, Unspecified; Computer Retrieval of Information on Scientific Projects Database; Funding; Grant; Institution; Investigators; Knockout Mice; Mammals, Mice; Measures; Mice; Mice, Knock-out; Mice, Knockout; Murine; Mus; Myocardial perfusion; NIH; National Institutes of Health; National Institutes of Health (U.S.); Null Mouse; Research; Research Personnel; Research Resources; Researchers; Resources; Source; United States National Institutes of Health; capillary; density

Beta-adrenergic receptor (betaAR) signaling plays an important role in the regulation of cardiac performance. BetaAR downregulation and desensitization resulting in reduced responsiveness to catecholamines are hallmarks of the failing heart. In human heart failure, diminished receptor number (downregulation) and impaired coupling (desensitization) of betaARs result in reduced responsiveness to catecholamines. The process that initiates desensitization of the receptor begins with the phosphorylation of betaARs by beta-adrenergic receptor kinase1 (betaARK1), followed by the binding of beta-arrestin, which together inhibits coupling to G protein and loss of effector (adenylyl cyclase) signaling. The beta-arrestin receptor complex is then targeted to the clathrin-coated pits for endocytosis through a process involving interaction of beta-arrestin with AP-2 and clathrin. Recently we have shown that betaARK1 interacts with the lipid kinase, phosphoinositide 3-kinase (PI3K), to form a cytosolic complex. We have shown that the betaARK/PI3K interaction is critical for the agonist-mediated internalization of betaARs and if the betaARK/PI3K interaction is disrupted in vivo, betaARs do not undergo downregulation and desensitization with chronic catecholamine stimulation. Importantly, PI3K contains both lipid kinase and protein kinase activity. Although we have shown that the lipid products of PI3K are important for receptor internalization, it is not yet known whether the protein kinase activity of PI3K is necessary for betaAR endocytosis and activation of downstream signaling pathways. Since receptor-localized PI3K plays a critical role in betaAR internalization, knowledge of the distinct roles of the protein and lipid kinase activities in the process of betaAR sequestration, resensitization and signaling would contribute to a better understanding of the regulation of betaAR function in the pathogenesis of heart failure. Our Central Hypothesis is that downregulation of cardiac betaARs, in vivo, can be prevented through inhibition of the local generation of D-3 phosphatidylinositols by PI3K within the receptor complex and preserving betaAR function in the failing heart will lead to an amelioration of heart failure in both small and large animal models of cardiac failure. We will test the hypothesis that the protein kinase activity of PI3K is required for betaAR internalization and downregulation, and that betaAR function will be normal in hearts displaying a "physiological" hypertrophic phenotype compared to hearts that show a "pathological" hypertrophic phenotype. Specific Aims (1) To determine the molecular mechanisms of PI3K-mediated betaAR internalization by studying PI3K mutants that lack either protein kinase activity or lipid kinase activity; (2) To determine whether overexpression of the minimal peptide of PI3K (PIK domain peptide, PIKdp), which acts as a dominant negative in vitro, will prevent betaAR downregulation in vivo and rescue mouse models of heart failure: a) chronic pressure overload (transverse aortic constriction, TAC), b) Calsequestrin overexpression X PIKdp, c) muscle LIM protein knock out X PIKdp; (3) To determine whether overexpression of PI3Kdp in vivo by adenoviral-mediated gene transfer to the heart will prevent betaAR downregulation and rescue two large animal models of cardiac failure: a) AdenoPIKdp injected into a rabbit infarct model, b) the pacing pig heart failure model; (4) To determine whether the mechanism that causes transition from hypertrophy to heart failure is due to the type of load on the heart (exercise vs. pressure) or the chronicity of the load. Chronic TAC will be compared to intermittent TAC and two exercise protocols in mice, together with a comprehensive evaluation of the developing cardiac phenotype.

1. Core Functions: The Administrative Core will be headed by Dr. Rockman who serves as the overall Program Director of this PPG. Administrative responsibilities of the Program Director include: 1. Following and implementing all guidelines for Program Project Grant support, including administering the budget in accordance with all NIH policies and recommendations of the NHLBI. 2. Monitor all Project and Core budgets in conjunction with Ms. Jennifer White, the PPG Administrative Manager. This will include preparation and planning of annual budgets and expenses. 3. Organize and lead the regular meetings of the Internal and External advisory committees. 4. Allocate travel funds among investigators of the PPG. The goal of the Program Project is to prioritize travel money to investigators presenting research at National meetings specifically related to the theme of the PPG. 5. Overall scientific coordination of the research objectives of the PPG, including plotting strategies for new ideas and research directions related to the central theme and potentially phasing out unproductive projects. This will be done in concert with input from both the Internal and External advisory committees. 6. Conduct and lead the weekly research data meetings that will be held for investigators, fellows and students involved in the different program Projects and Cores. 7. Work with the Administrative Manager on preparing annual reports and renewal applications to the NIH. The day-to-day operations of the Administrative Core will be handled by Ms. Jennifer White, who will serve as the Administrative Manager. She currently is the Grants Manager for Dr. Rockman and is quite adept at administrating all aspects of a large number of grants and funding sources. She will also assist Dr. Rockman with the following administrative duties associated: 1. Monitoring the financial status of the Program Project Grant and reconciling monthly reports from the Duke University Accounting Office, which will include resolving any accounting discrepancies and project expenditures. 2. Work with Dr. Rockman to prepare monthly and yearly financial reports. 3. Work with individual lab managers' form the various Projects and Cores to assure compliance on budgeting and ordering supplies, animals and reagents. As outlined in the Program Project Introduction, our organizational plan is that individual Projects and Cores will be responsible for daily and weekly ordering of supplies specific for the PPG but they will (on a monthly basis) prepare and submit updated expense reports to Ms. White for her reconciliation of the overall budgets and expenditure reports. 4. Work with Dr. Rockman on preparing the annual report and renewal applications to the NIH. 5. Organize all aspects including travel and meeting itinerary of the yearly visits of the External Advisory Committee.

Abstract B-arrestins are multifunctional proteins that are recruited to G protein-coupled receptors (GCPRs) following agonist stimulation. While the classical role of (3-arrestin is to mediate receptor desensitization, work by investigators of this PPG have recently shown that P-arrestin can stimulate signaling in the absence of classical G protein activation. The existence of B-arrestin-mediated signaling independent of G proteins requires that receptors adopt multiple active conformations or ligand selective states. The ability of unique ligand-receptor conformations to promote preferential B-arrestin signaling is an emerging concept known as biased signaling. The molecular mechanisms that underlie p-an-estin-biased signaling for the p-adrenergic receptor of (PAR), and its physiological consequences in the heart, are not known. In this proposal, we will test the hypothesis that mutant p i - and P2 can be engineered that will selectively stimulate p-arrestinbiased signaling independent of G protein activation, and that p-arrestin-biased signaling will promote cardiomyocyte cell survival to limit the development of heart failure in response to pathological stimuli. Accordingly, the specific aims of the study are: Aim 1: To engineerBp1 AR mutants that show selective bias for p-arrestin recruitment. Aim 2: To identify the mechanism of activation and signaling pathways activated by P1AR and B2AR mutants in the absence of G protein activation. Aim 3: To test in adult cardiomyocytes whether p-arrestin-biasedBP2AR TYY and B1 AR mutants activate cardioprotective signaling in response to agonist stimulation and ischemia. Aim 4: To test in vivo whether the B-arrestin-biased Bp2AR TYY and pi AR mutant activities cardioprotective pathways under conditions of pathological stress. By exploring these aims, we will define the pathways by which G protein-Independent activation of BARs may lead to stimulation of cardioprotective signaling. If our hypothesis is correct, we will show that ligandstimulated PARS, which selectively activate B-arrestin signaling pathways, are cardioprotecitve. Since, by definition, the administration of a ligand that does not stimulate G protein signaling is B-blackade, we will have demonstrated proof-of concept for the development of an entirely novel class of receptor blockers. We believe these data will provide considerable impetus for the development of novel p-arrestin-biased therapeutic agents to treat human heart failure.

SUMMARY OF WORK: Core A ? Administrative Core Administrative organizations, grants management, travel for advisory board, scheduling of data meetings.

SUMMARY OF WORK: Project 1: ?-Arrestin Biased Mechanosensitive Angiotensin Receptor Signaling Mechanotransduction plays an important role in pathophysiological processes such as vascular constriction, cardiac hypertrophy, and fluid balance through osmoregulation of thirst and salt appetite. It is now appreciated that the angiotensin-II type-1 receptor (AT1R) can function as a mechanosensor in a number of cell types and does not require the ligand angiotensin II (AngII). We have recently discovered that AT1Rs when acting as mechanosensors selectively engage ?-arrestin to induce cellular signaling. During the past PPG funding period, together with Drs. Lefkowitz, Stamler and Koch, we have studied ?-arrestin-mediated signaling in the absence of G protein activation and have elucidated the scientific basis for the concept that we now term ?biased GPCR signaling. The overall objective of this project is to determine the molecular and structural basis for the transduction of AT1R signals in response to changes in membrane stretch and biased ligands, and to understand the physiological consequence of AT1Rs as mechanosensors as it relates to the regulation of thirst and salt appetite, critically important in the treatment of patients with heart failure. Our central hypothesis, is that mechanical stretch allosterically modulates the AT1R to induce a unique receptor conformation that allows for the phosphorylation of specific GRK sites on the C-terminal tail of the receptor. Phosphorylation of select amino acid residues on the c-tail of the AT1R defines a ?bar-code? that promotes the recruitment of ?-arrestin to form an AT1R-?-arrestin complex that results in a unique pattern of cellular signaling known as biased signaling. We further hypothesize that AT1Rs in cells in the brain that are involved in osmoregulation respond to osmotic stretch by activating ?-arrestin-biased AT1R signaling as a homeostatic mechanism to maintain salt and water balance in the body.