Page Anderson - US grants

Developmental changes in cardiac contractility have been attributed to changes in cell structure and function. Our recent observation that the velocity of sarcomere shortening (S) is depressed in the isolated myocyte from the immature rabbit ventricle suggests that S can be used as a probe to explore the basis of developmental changes in contractility. We will test these hypotheses: 1) The developmental increase in S is due to developmental changes in the membrane systems that control activator calcium concentration; 2) S in the immature cell is primarily dependent on transsarcolemmal calcium movement during that contraction while S in the adult cell is primarily dependent on intracellular calcium stores; 3) The developmental acquisition of S dependency on intracellular stores is the result of developmental changes in the amount and orgainzation of the longitudinal, junctional and corbular sarcoplasmic reticulum (SR); 4) The quanitiative characteristics of the restitution of contractility between beats is related to the structure of the SR; 5) The developmental increase in sensitivity of the contractile apparatus to calcium; and 6) The physiologic differences between the rabbit and rat are a result of the corbular SR. At different stages of development, myocardial cells will be isolated from the rabbit and rat ventricle using enzymatic dispersion. Resting and action potentials will be measured. S in the intact cell will be measured in different calcium concentrations, (Ca)o, and contrasted to S or force obtained in the same cell, following detergent skinning. The isolated cell allows the beat-to-beat dependence of S on (Ca)o to be examined. (Ca)o will be changed transiently, e.g. increased during a contraction and then returned to control, and changes in S sought. The pattern of pacing will be altered in constant (Ca)o to discribe how S changes following a contraction. These variations in pacing will be performed also with step changes in (Ca)o. The effects on S of calcium channel blockers and ryanodine, which decreases the availability of calcium from internal stores, will be examined. Cell ultrastructure, e.g. the SR and the glycocalyx, will be examined with electron microscopy and related to the physiologic data. The comparison of the physiologic and ultrastructural properties at different stages of development will allow testing of the components of models of excitation-contraction coupling. By understanding the cellular basis for developmental changes in S, a better basis for therapeutic approaches to the sick child and infant can be established.

The isolated ventricular cell must retain the physiologic characteristics of its native tissue if it is to be useful in studying excitation-contraction coupling. A wide variability in isolated cell size, rest sarcomere length, amount and velocity of sarcomere shortening and action potential has been observed. This proposal seeks to understand this variability by correlating the function of individual cells, as examined by sarcomere motion and rest and action potentials, with the structure of the same cell. Cells will be isolated from the right and left ventricle of the rabbit and rat using enzymatic and mild mechanical dispersion. Rabbit myocardiuim will be studied because it has physiologic properties much more like those of other mammals than those of the rat. The rat will be studied because it has such strikingly different physiologic properties and has been used in many isolated cell investigations. The experiments will make use of the prominent effects induced by altering the rate and pattern of stimulation on contractility and the action potential and the characteristic changes in these effects induced by inotropic agents. Timed changes in extracellular ionic concentrations during rest and contraction will take advantage of the short diffusion distance. The proposal will provide a detailed examination of the physiological and ultrastructural properties of the same cell. This will enable us to assess whether intrinsic in situ differences in cells or the effects of the isolation procedure are the basis of cell-to-cell variability. Sarcomere shortening and action potential characteristics will be related to cell shape, size and ventricular site of origin. Species differences will allow structrue-function correlations to be made. The changes in myocardial performance produced by altering the rate and pattern of stimulation have been used in multicellular preparations to study contractility and to characterize the effects of disease. By producing a detailed quantitative description of the changes in sarcomere shortening velocity induced by altering the pattern of stimulation, this investigation will provide a basis at the cellular level for the force-interval relationship in the intact heart. By assessing the importance of extracellular calcium concentration at different times during activation and rest, this proposal will provide new data for testing models of excitation-contraction coupling. This proposal will provide a basis for future isolated cell investigations of disease-induced derangements in excitation-contraction coupling.

Cardiac development and disease affect cardiac contractility and troponin T (TnT) isoform expression. This study of the molecular basis and functional effects of TnT expression is motivated by results that suggest TnT isoforms and contractility are related. Molecular, morphological, biochemical, and physiological differences will be used to test these hypotheses: 1) Cardiac TnT isoforms modulate the sensitivity of myofilaments to calcium; 2) the heterogeneity in cardiac TnT isoforms modulate the sensitivity of myofilaments to calcium; 2) the heterogeneity in cardiac TnT isoforms has a molecular basis, involving alternative splicing of the primary transcript of a cardiac TnT gene; 3) the sarcomere length dependence of cardiac myofilament sensitivity to calcium is acquired with maturation and is related to TnT isoform expression. Aim 1. The force-pCa relation of fetal and postnatal rabbit myocardium will be measured at different sarcomere lengths. The Hill coefficient and pCa for half-maximal activation (pCa50) will be related to the TnT isoforms in the myofilaments, quantitated on Western blots, to sarcomere length, and to the results of Aims 2 & 4. Aim 2. cDNAs of rabbit cardiac TnT isoforms will be isolated and cloned. The primary structure will be deduced from cDNA sequences. Aim 3. TnT cDNAs will be co-transfected with a neomycin resistance gene into C2C12 mouse skeletal muscle cell myoblasts. Myofilament organization and incorporation of cardiac TnT will be examined with fluorescent and electron immunocytochemistry in differentiating myotubes. Aim 4. The force-pCa relation of control myotubes and those transfected with expression vector alone or with cardiac TnT cDNAs will be measured. The Hill coefficient and pCa50 will be used as in Aim 1. The results of Aims 1-4 will be used to propose structure-function relationships. Aim 5. TnT cDNA will be mutated to alter regions of potential functional importance and used as in Aims 3 & 4. Aim 6. Human cardiac TnT cDNAs from normal and failing human hearts will be isolated and cloned. They will be compared to rabbit cardiac TnT cDNA and used, as in Aims 3 & 4, to test the functional significance of preferential isoform expression in normal and failing hearts. These studies will establish the molecular basis of cardiac TnT isoform expression. These results are important in the understanding of the regulation of cardiac contraction by thin filament protein interaction.

Left ventricular (LV) output increases markedly from fetal to neonatal life. By applying new techniques to the study of the fetus and the neonate, this proposal will test specific hypotheses that clarify the bases of this increase. 1) The Frank-Starling relationship is an important factor in the function of the immature LV. 2) With birth LV end-diastolic volume (LVEDV) increases, enhancing LV stroke volume (SV). 3) The neonatal fall in right ventricular (RV) volume and systolic pressure allow, through ventricular interaction, greater LV filling and ejection for comparable filling pressures. 4) With birth inotropy in increased, in part, through sympathetic stimulation. The chronic instrumentation of the fetal lambs (124-130 days of gestation) includes: an ascending aorta electromagnetic flow probe, ventricular dimension transducers and catheters, LV micromanometer pressure transducer, tracheal catheter, vena caval and pulmonary artery occluders, and atrial pacing electrodes. Three LV dimensions will be used to derive in-vivo EDV, using a prolate ellipsoid model. In-vitro calibration will provide correction factors for the in-vivovivo data. The lambs will be studied in utero and as neonates and the data will be compared. Blood infusions, transient vena caval obstruction, and right atrial and left atrial pacing will be used to obtain the LVEDV-LVEDP and LVSV-LVEDV relationships at different RV end-diastolic dimensions. The effects of RV afterload on the relationships between LVEDV-LVEDP, LVSV-LVEDV, and LV (dP/dt)max-LVEDV will examined by transient constrictions of the main pulmonary artery and by decreasing the inspired oxygen content of the neonate. LV inotropy will be examined with the end-systolic pressure-volume relationship (EES) and post- extrasystolic potentiation (PESP). The effects of preload, inotropy, afterload and heart rate on EES will be established for the fetus. The EES, PESP, and LV output of the fetus and neonate will be compared and the effects of sympathetic blockade examined. Through the application of new techniques to the fetus and neonate, the proposed studies will provide new data upon which it will be possible to formulate the relationships that give rise to the increase in cardiac output following birth. The effects of perinatal diseases on the heart make this information of fundamental importance in the care of the in-utero and newborn infant.

Myocardial contractility is enhanced with development. The proposed studies will test whether increases in cytosolic calcium(Cai) and in the sensitivity of the myofilaments to Ca underlie this enhancement, whether the immature cell depends relatively more on extracellular calcium(Cao), and whether changes in troponin T (TnT) isoform expression with development are correlated with changes in the sensitivity of the myofilaments to Ca. Three preparations obtained from rabbit ventricles at different stages of development will be studied: Calcium-tolerant intact single cells, chemically-skinned single cells, and chemically-skinned multicellular bundles. The membrane-intact isolated cell will be loaded with the fura-2 AM> Sarcomere shortening and changes in the 340/380 fluorescence ration will be measured at various stimulus patterns and perturbations that will allow us to examine the effects of changes in Cai and of different Cao, Nao, and Nao/Cao ratios, with and without the use of a calcium channel blocker. The fluorescence data will be calibrated by measuring the minimum and maximum fluorescence ratios, Rmin and Rmax, of the cell. The effects of age on Cai and extent of it modulation, and their relationship to developmental changes in sarcomere shortening will be examined. Sarcomere shortening waveforms will be recorded from intact cells: the cells will then be chemically skinned and the force-pCa relation determined and compared with that obtained from the multicellular preparation. The effects of development on the relation and on Cai will be studied. The force-pCa relation of chemically-skinned multicellular bundles will be measured and analyzed. Polyacrylamide gel electrophoresis will be used to examine the protein profile of the bundle. The proportion of TnT isoforms expressed in each bundle will be obtained from the gel and will be compared to the force-pCa characteristics of the preparation, pCa50 and the Hill constant. The effect of TnT isoform expression and its changes with development on the force-pCa relation will be analyzed. The proposed studies will provide new data that will allow us to test hypotheses that relate the control of Cai, the Ca sensitivity of the myofilaments, and the troponin T isoforms to the maturational increase in contractility.

Cardiac development and disease affect cardiac contractility and troponin T (TnT) isoform expression. This study of the molecular basis and functional effects of TnT expression is motivated by results that suggest TnT isoforms and contractility are related. Molecular, morphological, biochemical, and physiological differences will be used to test these hypotheses: 1) Cardiac TnT isoforms modulate the sensitivity of myofilaments to calcium; 2) the heterogeneity in cardiac TnT isoforms modulate the sensitivity of myofilaments to calcium; 2) the heterogeneity in cardiac TnT isoforms has a molecular basis, involving alternative splicing of the primary transcript of a cardiac TnT gene; 3) the sarcomere length dependence of cardiac myofilament sensitivity to calcium is acquired with maturation and is related to TnT isoform expression. Aim 1. The force-pCa relation of fetal and postnatal rabbit myocardium will be measured at different sarcomere lengths. The Hill coefficient and pCa for half-maximal activation (pCa50) will be related to the TnT isoforms in the myofilaments, quantitated on Western blots, to sarcomere length, and to the results of Aims 2 & 4. Aim 2. cDNAs of rabbit cardiac TnT isoforms will be isolated and cloned. The primary structure will be deduced from cDNA sequences. Aim 3. TnT cDNAs will be co-transfected with a neomycin resistance gene into C2C12 mouse skeletal muscle cell myoblasts. Myofilament organization and incorporation of cardiac TnT will be examined with fluorescent and electron immunocytochemistry in differentiating myotubes. Aim 4. The force-pCa relation of control myotubes and those transfected with expression vector alone or with cardiac TnT cDNAs will be measured. The Hill coefficient and pCa50 will be used as in Aim 1. The results of Aims 1-4 will be used to propose structure-function relationships. Aim 5. TnT cDNA will be mutated to alter regions of potential functional importance and used as in Aims 3 & 4. Aim 6. Human cardiac TnT cDNAs from normal and failing human hearts will be isolated and cloned. They will be compared to rabbit cardiac TnT cDNA and used, as in Aims 3 & 4, to test the functional significance of preferential isoform expression in normal and failing hearts. These studies will establish the molecular basis of cardiac TnT isoform expression. These results are important in the understanding of the regulation of cardiac contraction by thin filament protein interaction.

During development myocardial proteins and their isoforms undergo marked changes in expression. During that process cardiac contractility also changes. The proposed studies will test hypotheses relate to the effects of development on the mechanisms that control cytosolic calcium concentration ([Ca]i) transients and the response of the myofilaments to [Ca]i. Isoform expression of the L-type calcium channel alpha1 subunit and of troponin T, two proteins central to excitation-contraction coupling and the development acquisition of the sarcoplasmic reticulum (SR) and t-tubule system provide the basis for studying cardiac myocytes from newborn, three week, and adult rabbits. Using the whole-cell voltage clamp preparation and fura-2 fluorescence, we will measure [Ca]i, sarcomere dynamics, and calcium current (ICa). We will examine the effects of development on ICa's contributions to [Ca]i directly and through inducing SR calcium release, and we will correlate those to changes in alpha1 subunit isoform expression using Western blots probed with alpha1 subunit isoform antibodies. The contribution of the SR to [Ca]i as a function of age and alpha1 subunit isoform expression will be determine by measuring SR release of calcium in response to activation by comparing the transients obtained with the SR active and when it is functionally blocked and SR calcium content from the caffeine-induced current. We will determine the force-pCa relation of chemically skinned cells in which, unlike membrane-intact muscle, the myofilament ionic milieu can be controlled tightly in order to test whether myofilament sensitivity to calcium and its dependence on sarcomere length are affected by development. Cells in short term culture will be used to dissect the effects of alpha1 subunit isoforms on ICa and the effects of troponin T isoforms on the sensitivity of myofilaments to calcium. ICa- voltage relations and single channel characteristics will be related to the expression of specific alpha1 subunit isoforms obtained by exposing cells in culture to antisense oligodeoxynucleotides complimentary to alpha1 subunit isoform-specific sequences. We will change troponin T isoform expression in cells in culture by exposing them to antisense oligodeoxynucleotides complimentary to cardiac troponin T isoform specific sequences and will study the effects of such changes on the force-pCa relations of the cells. Studying how development changes myocardial function through altered expression of these proteins important in controlling ICa and the regulation of thick and thin filament interaction will prove valuable in understanding the mechanisms that control [Ca]i and myofilament sensitivity to calcium. The results of these studies will be fundamental to developing rationales for the treatment of patients at all ages with heart disease. For the sick newborn and infant, better care should come through establishing age- dependent therapeutic approaches that are based on knowing how [Ca]i and myofilament response to calcium are controlled at different ages by altered expression of isoform-specific sequences are similar in the human and rabbit.

DESCRIPTION (Adapted from Applicant's Abstract):The aims of this proposal are to determine the role of complement activation, which occurs during cardiopulmonary bypass, on heart and lung function after bypass. The PI has utilized a neonatal piglet model with cardiopulmonary bypass and hypothermia to demonstrate that there is significant activation of various complement pathways resulting in direct injury to myocardium and lungs. They will first determine the effects of blocking the various pathways of complement activation on recovery of heart and lung function after bypass, and in the second part of these studies they will perform similar mechanistic analysis using a neonatal cardiac myocyte model. In subsequent studies they will determine the role of complement activation on cardiac myofilament structure and function and the effects of activation of proteolytic activity on the contractile proteins. In the final phase they will utilize a reconstitution strategy to replace defective troponin proteins to determine if they can restore cardiac function by this approach. Ultimately, the goal would be to induce expression of these proteins to improve cardiac recovery.

DESCRIPTION (Applicant's Description Verbatim): Heart failure is a major cause of premature death and disability in the United States. The basis of abnormal myofilament function in the failing heart is not known. Myofilament function is regulated by troponin, a complex made of three subunits (cTnI, cTnC, cTnT): cTnI inhibits actin-myosin interaction, the binding of Ca2+ to cTnC disinhibits this interaction, and cTnT binds the complex to tropomyosin and is essential for Ca2+ -dependent force development and myofibrillar ATPase activity. This proposal aims to determine the role of the cTnT isoforms in the regulation of myocardial function, and how they affect the failing heart. The function of the cTnT isoforms is not known. We have identified four TnT isoforms in the human heart, whose expression is regulated by development and affected by heart failure. In contrast, the same single isoform of cTnI and cTnC are expressed in the normal and failing adult human heart. We focus on the cTnT isoforms because cTnT isoform expression is correlated with the fall in myofibrillar ATPase activity in the failing human heart. We will test the following: Hypothesis 1(a) cTnT isoforms modulate the binding characteristics of cTnC to Ca2+ in troponin in vitro and the characteristics are further modulated by the incorporation of troponin into the thin filament. Hypothesis 1(b). In myocardium, the cTnT isoforms alter myofilament function by changing the myofilaments' sensitivity to Ca2+ and the sarcomere length-dependence of this sensitivity (and consequently, the Frank-Starling relation). Hypothesis 2. cTnT isoform expression in vivo alters ventricular function in vivo and myofilament function in vitro. Hypothesis 3. cTnT isoform expression in vivo preserves left ventricular function in heart disease. We will use reagents we have recently developed, including recombinant cTnT proteins and transgenic mice overexpressing cTnT isoforms. These will be used to examine the functional role of cTnT isoforms in troponin, the thin filament, isolated myocytes, ventricular bundles and the in vivo heart and to test the effects of cTnI isoform expression of left ventricular structure and function in mouse models of heart disease. These models include constriction of the transverse aorta and over-expression of calsequestrin. Defining the role of cTnT isoforms in the normal and failing heart is expected to provide targets for potential new and effective pharmacological interventions in heart failure.

DESCRIPTION (provided by applicant) Duke University Medical Center and the University of North Carolina at Chapel Hill Medical Center (UNC-CH) will form a Clinical Center in the Pediatric Heart Disease Clinical Research Network. Duke will be the primary site and UNC-CH the subsite. The likely success of this proposed collaboration is supported by the previous and ongoing joint participation by the Centers at multiple levels and over many years. These interactions include collaborations in patient care, post-graduate education, and federally funded clinical research in which Duke and UNC-CH have both been primary and subsites. The longstanding research programs at the centers have studied a broad range of issues relevant to pediatric heart disease, including the pathophysiologic basis of the post-cardiopulmonary bypass (CPB) syndrome, dysrhythmias, and altered ventricular function in patients with congenital cardiac defects and heart failure. The studies have ranged from the contractile proteins to the in vivo heart of the patient and have examined the treatment of dysrhythmias, catheter-based device implantation, and testing the efficacy and safety of pharmacologic drugs. Annually our combined programs have over 11,000 outpatient visits and perform in excess of 600 cardiac catheterizations, 8,000 echocardiograms, 200 electrophysiological interventions/ablations, 150 catheter-based interventions, and 490 pediatric cardiac surgical procedures. The proposed double blind randomized controlled trials are: a long-term study of three years duration, carvedilol efficacy and safety in the Fontan patient with depressed ventricular function; a short-term study of two years duration, the efficacy and safety of methylprednisolone in preventing the post-CPB syndrome in the infant. The primary objective of the carvedilol trial is the Clinical Global Assessment by the physician. In the adult with heart failure, greater sympathetic nervous system activation is associated with worse clinical course. Carvedilol has been found to improve quality of life and decrease mortality in these patients. Our trial will test the hypothesis that carvedilol improves quality of life, clinical course, and ejection fraction in the Fontan patient. Methylprednisolone is widely used and thought to be efficacious in decreasing the morbidity of the post-CPB syndrome. We will test the hypothesis that methylprednisolone decreases PICU stay, maintains normal vascular permeability, prevents renal and hepatic damage, and improves the clinical course. We look forward to participating in the Network and improving the care of infants and children with congenital and acquired heart disease.

DESCRIPTION (provided by applicant): Heart failure is a major cause of premature death and disability in the United States. Adult-derived stem cells could provide a basis for effective therapies. This proposal is based on our finding that a well characterized adult-derived stem cell line (WB-F344), isolated from the adult rat liver, differentiates in vivo in the adult heart into heart cells. We will use the WB-F344 stem cell line and Fischer 344 (F344) rats in the normal heart and left anterior descending (LAD) coronary artery ligation model. We will test:Hypothesis 1. WB-F344 cells engraft in the heart, acquire a structural cardiac phenotype, and differentiate into mature cardiac myocytes in vivo. These processes will be affected by the different host cardiac microenvironments in the two models. We will examine these processes qualitatively and quantitatively. B-galactosidase activity will be used to identify WB-F344-derived myocytes. The commitment to a cardiac lineage and acquisition of a cardiac phenotype will be examined using expression of transcription factors, myofilament proteins, and membrane proteins and the remodeling of anatomical couplings, and their distribution. Hypothesis 2. WB-F344-derived myocytes acquire the functional phenotype of adult cardiac myocytes. These functional properties will be affected by the host cardiac microenvironments in the two models. We will examine the mechanical and electrophysiological properties of isolated single WB-F344-derived myocytes and host myocytes in vitro and their communication with host cells in situ. Hypothesis 3. WB-F344-derived cardiac myocytes affect ventricular function in vivo. Left ventricular dysfunction in the post myocardial infarction heart will be moderated by WB-F344-derived myocytes. The effects of WB-F344 cell engraftment and differentiation on in vivo left ventricular function and size will be examined, using echocardiography and cardiac catheterization. The proposed studies will provide new and important information about the functional properties of stem cell-derived cardiac myocytes and the potential value of stem cell-based approaches to treating heart failure.

DESCRIPTION (provided by applicant) Duke University Medical Center and the University of North Carolina at Chapel Hill Medical Center (UNC-CH) will form a Clinical Center in the Pediatric Heart Disease Clinical Research Network. Duke will be the primary site and UNC-CH the subsite. The likely success of this proposed collaboration is supported by the previous and ongoing joint participation by the Centers at multiple levels and over many years. These interactions include collaborations in patient care, post-graduate education, and federally funded clinical research in which Duke and UNC-CH have both been primary and subsites. The longstanding research programs at the centers have studied a broad range of issues relevant to pediatric heart disease, including the pathophysiologic basis of the post-cardiopulmonary bypass (CPB) syndrome, dysrhythmias, and altered ventricular function in patients with congenital cardiac defects and heart failure. The studies have ranged from the contractile proteins to the in vivo heart of the patient and have examined the treatment of dysrhythmias, catheter-based device implantation, and testing the efficacy and safety of pharmacologic drugs. Annually our combined programs have over 11,000 outpatient visits and perform in excess of 600 cardiac catheterizations, 8,000 echocardiograms, 200 electrophysiological interventions/ablations, 150 catheter-based interventions, and 490 pediatric cardiac surgical procedures. The proposed double blind randomized controlled trials are: a long-term study of three years duration, carvedilol efficacy and safety in the Fontan patient with depressed ventricular function; a short-term study of two years duration, the efficacy and safety of methylprednisolone in preventing the post-CPB syndrome in the infant. The primary objective of the carvedilol trial is the Clinical Global Assessment by the physician. In the adult with heart failure, greater sympathetic nervous system activation is associated with worse clinical course. Carvedilol has been found to improve quality of life and decrease mortality in these patients. Our trial will test the hypothesis that carvedilol improves quality of life, clinical course, and ejection fraction in the Fontan patient. Methylprednisolone is widely used and thought to be efficacious in decreasing the morbidity of the post-CPB syndrome. We will test the hypothesis that methylprednisolone decreases PICU stay, maintains normal vascular permeability, prevents renal and hepatic damage, and improves the clinical course. We look forward to participating in the Network and improving the care of infants and children with congenital and acquired heart disease.

DESCRIPTION (provided by applicant): The overall goal of the proposed fellowship pediatric cardiololgy (sp) training grant program is to develop pediatric fellows into well-trained investigators with the potential to assume leadership roles in the nation's congenital heart disease research agenda. Through this two-year training program they will acquire the ability to perform exceptional clinical and basic science research into the causes, pathological mechanisms, and outcomes of congenital heart disease. Few Pediatric Cardiologists are trained to conduct research and comparatively few pediatric fellowship graduates have careers successfully performing such research, and the need to intensify and expand training programs is clear. At least eight of every 1,000 infants born each year have a heart defect - almost one percent of live-born infants. In fact, congenital heart disease is a leading cause of death during the first year of life. The acute care cost of treating these infants each year, exceeds $400,000,000. This estimate does not include the chronic care costs of clinic visits, medications, and rehabilitation. The program expands the current three-year fellowship training program to four years by adding an additional research year -- either in clinical or basic science research. Furthermore, it enhances the current one by offering trainees access to a wide range of additional Duke research resources and, thus, potential mentors. Examples of such resources include the Duke Clinical Research Institute, the Center for Genome Technology, and the Center for Human Genetics. The overall direction of this program will be provided by Dr. Stephen P. Sanders, a renowned pediatric cardiologist and Program Director of this training program. The multidisciplinary clinical research training program is designed to provide young clinical scientists with the tools and skills needed to perform innovative clinical research, translating and testing basic science discoveries in the clinical arena. Improved therapeutics, realized through the efforts of clinical investigators trained in this program, is the ultimate goal. The basic science research training program is designed to give young investigators the knowledge and skills needed to perform cutting-edge scientific research and succeed in the highly competitive research environment of academic pediatrics. Moreover, one of the most significant goals of the program is to ensure that young physicians will be able to translate, for the benefit of patients, research findings into clinical practice.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Duke University Medical Center, East Carolina University (ECU), and Wake Forest University Health Systems (WFUHS) have formed the North Carolina Consortium (NCC) to participate in Pediatric Heart Disease Network trials and studies. Our three Institutions have collaborated since 1996 in over 48 projects with funding of approximately 56 million dollars. Over 90% of the over 300 patients with congenital heart defects who undergo open heart surgery at our Centers are followed in our clinics annually. Our patient population is unique: our patients are ethnically and racially diverse, poor, and rural. The challenges and unique strengths encountered by our patients are likely to affect outcomes measured in clinical trials. These effects must be identified to generalize the results of PHN clinical trials to encompass the patient population across the United States. We propose a randomized, multi-Institutional, double-blind, placebo-controlled study to evaluate the effect of spironolactone on exercise performance in children who have undergone Fontan surgery: aldosterone blockade and exercise (ABLE). Hypothesis: Spironolactone will improve the exercise performance and quality of life of Fontan patients, 6-21 years of age. Primary objective: Determine the effect of spironolactone on peak oxygen consumption. Secondary Objectives: determine efficacy of spironolactone as measured by 1) quality of life, assessed by validated measures; 2) ventricular filling parameters, assessed by tissue Doppler echocardiography; 3) amount of myocardial scarring assessed by MRI; 4) neurohormonal activation, BNP; biomarkers of collagen synthesis, procollagen I and III peptides; and 5) renin angiotensin aldosterone system polymorphisms. The trial duration is six months. A sample size of 220 per treatment group will be required to achieve 85% statistical power to detect mean differences in peak oxygen consumption between spironolactone and placebo, using a two-sided test without alpha = 0.05, if the expected mean difference in peak oxygen consumption between groups is 2 ml/kg/min and the standard deviation within the group is 7 ml/kg/min. The proposed study is significant and innovative: the results may support a new therapy; it will be the largest randomized study to date of pharmacologic treatment of children with single ventricle physiology; potential mechanisms through which aldosterone antagonism affects these children will be assessed; the role of RAAS polymorphisms in these children's response to pharmacologic therapy will be evaluated. We are also applying for a Clinical Research Skills Development Core. (End of Abstract). [unreadable] [unreadable] [unreadable]