Loren J. Field - US grants

ANF is released into the circulation in response to volume expansion, and elicits numerous physiological effects which act in a combinatorial fashion to reduce blood pressure. The physiological responses of ANF include vasodilation, natriuresis, diuresis, inhibition of the renin-angiotensin- aldosterone axis, inhibition of AVP secretion and modulation of neural activity. While the effects of acute ANF administration are well characterized, the chronic role of the hormone in cardiovascular homeostasis has remained controversial. In an effort to directly address this issue, we have generated a transgenic model system which exhibits chronically elevated levels of ANF in the systemic circulation. These transgenic animals, designated TTR-ANF, have immuno-reactive plasma ANF levels which are approximately 10 fold elevated as compared to their non- transgenic litter mates. Moreover, the TTR-ANF mice have a mean arterial blood pressure (as determined in conscious resting mice with indwelling annulus) of 75.5 plus/minus 9.9 mm Hg as compared to 103.9 plus/minus 2.0 for the non-transgenic litter mates. This study clearly demonstrates that chronically elevated levels of ANF can induce a sustained hypotensive response. In this proposal, we will continue to expand on our transgenic model. Specifically, we will: (1) Further assess the physiological consequences of elevated ANF levels in the TTR-ANF transgenic mice. These studies will include a detailed analysis of kidney function (nephron puncture and micro-catheterization analyses), assessment of baroreceptor gain, characterization of the response of the various ANF receptors, and assessment of the resistance of the transgenic animals to experimentally induced hypertension. (2) Generate transgenic mice in which an "activated" ANF receptor is expressed in the cell types which are responsive to the hormone. Studies by other groups have demonstrated that deletion of the kinsae domain in the ANF-A receptor results in constitutive guanylate cyclase activity. Expression of such a "trans-dominant" ANF receptor can be targeted to specific cell types which are responsive to ANF. This approach will allow us to experimentally establish correlates between specific target tissue activation and physiological response in intact animals. (3) Generate transgenic models with chronically elevated ANF levels in the central nervous system. Numerous studies have shown that ANF cannot breach the blood brain barrier, and that intra-cerebral injection of ANF can elicit discrete physiological responses. We will generate a transgenic model system in which ANF is specifically secreted into the cerebrospinal fluid in order to assess the consequences of chronically elevated hormone levels in the CNS. (4) Generate transgenic animals which do not synthesize ANF. Recent advances in mouse embryology have made it possible to genetically inactivate specific genes in embryonic stem (ES) cells. These cells retain a certain degree of pluripotency, and have been used to generate chimeric animals in which cells that carry the mutated allele have populated the germline. These chimeric animals have subsequently passed the mutation on to progeny mice. We will use this approach to generate animals which fail to express ANF. All of the transgenic models will be subjected to comprehensive molecular and physiological analyses to assess the consequences or altered ANF expression (or the consequences of altered activity of the ANF signal transduction pathway). These experiments will enable us to understand the physiological role that ANF exerts in chronic cardiovascular regulation.

Cardiomyocytes in the adult mammal exhibit little if any capacity to undergo cell division. Consequently cardiomyocyte loss due to injury or disease is irreversible. Identification of the gene products which regulate cardiomyocyte proliferation and terminal differentiation might provide useful molecular targets with which to induce therapeutic myocardial growth in the adult heart. We have used cardiomyocyte cell lines derived from transgenic mouse tumors to identify 3 proteins (p193, p107 and p380) which bind either directly or indirectly to the SV40 Large T-Antigen (T-Ag) oncoprotein. In addition, we have cloned the mouse cDNA and genes from the Tuberous Sclerosis Complex (TSC) 1 and 2 loci: TSC is an autosomal dominant familial cancer which affects a variety of organs including the heart. We hypothesize that these proteins participate in the regulation of cardiomyocyte proliferation and/or terminal differentiation. In support of this, experiments performed during the current funding period have shown that p193 exhibits tumor suppressor activity in NIH-3T3 cells, and that p107 and TSC2 impact upon cardiomyocyte terminal differentiation and proliferation, respectively. In this competitive renewal application, we propose to further test our hypotheses with additional gain and loss of function models. Four Specific Aims are proposed. In Aim number 1, we will generate gain and loss of function transgenic mice to ascertain the role of p193 in normal and pathologic cardiac development, as well as identify the cellular proteins which interact with p193. In Aim 2, we will establish the molecular mechanism by which p107 expression renders the hearts of transgenic mice resistant to isoproterenol-induced hypertrophy, as well as determine if these animals are resistant to other hypertrophic stimuli. In Aim 3 we will isolate and clone p380, a novel myocardial p53 binding protein, and will generate gain and/or loss of function transgenic models to directly test its role in cardiac growth and development. Finally, in Aim 4 we will generate TSC1 deficient ES cells to test the role of this gene product in the regulation of cardiomyocyte proliferation in vitro and in vivo. We will also generate transgenic mice which express TSC2 dominant negative mutants in the myocardium. If the putative regulatory roles for these proteins are confirmed, they may serve as intracellular targets for therapeutic myocardial growth in the adult heart.

ANF is released into the circulation in response to volume expansion, and elicits numerous physiological effects which act in a combinatorial fashion to reduce blood pressure. The physiological responses of ANF include vasodilation, natriuresis, diuresis, inhibition of the renin-angiotensin- aldosterone axis, inhibition of AVP secretion and modulation of neural activity. While the effects of acute ANF administration are well characterized, the chronic role of the hormone in cardiovascular homeostasis has remained controversial. In an effort to directly address this issue, we have generated a transgenic model system which exhibits chronically elevated levels of ANF in the systemic circulation. These transgenic animals, designated TTR-ANF, have immuno-reactive plasma ANF levels which are approximately 10 fold elevated as compared to their non- transgenic litter mates. Moreover, the TTR-ANF mice have a mean arterial blood pressure (as determined in conscious resting mice with indwelling annulus) of 75.5 plus/minus 9.9 mm Hg as compared to 103.9 plus/minus 2.0 for the non-transgenic litter mates. This study clearly demonstrates that chronically elevated levels of ANF can induce a sustained hypotensive response. In this proposal, we will continue to expand on our transgenic model. Specifically, we will: (1) Further assess the physiological consequences of elevated ANF levels in the TTR-ANF transgenic mice. These studies will include a detailed analysis of kidney function (nephron puncture and micro-catheterization analyses), assessment of baroreceptor gain, characterization of the response of the various ANF receptors, and assessment of the resistance of the transgenic animals to experimentally induced hypertension. (2) Generate transgenic mice in which an "activated" ANF receptor is expressed in the cell types which are responsive to the hormone. Studies by other groups have demonstrated that deletion of the kinsae domain in the ANF-A receptor results in constitutive guanylate cyclase activity. Expression of such a "trans-dominant" ANF receptor can be targeted to specific cell types which are responsive to ANF. This approach will allow us to experimentally establish correlates between specific target tissue activation and physiological response in intact animals. (3) Generate transgenic models with chronically elevated ANF levels in the central nervous system. Numerous studies have shown that ANF cannot breach the blood brain barrier, and that intra-cerebral injection of ANF can elicit discrete physiological responses. We will generate a transgenic model system in which ANF is specifically secreted into the cerebrospinal fluid in order to assess the consequences of chronically elevated hormone levels in the CNS. (4) Generate transgenic animals which do not synthesize ANF. Recent advances in mouse embryology have made it possible to genetically inactivate specific genes in embryonic stem (ES) cells. These cells retain a certain degree of pluripotency, and have been used to generate chimeric animals in which cells that carry the mutated allele have populated the germline. These chimeric animals have subsequently passed the mutation on to progeny mice. We will use this approach to generate animals which fail to express ANF. All of the transgenic models will be subjected to comprehensive molecular and physiological analyses to assess the consequences or altered ANF expression (or the consequences of altered activity of the ANF signal transduction pathway). These experiments will enable us to understand the physiological role that ANF exerts in chronic cardiovascular regulation.

Cardiomyocytes in the adult mammal exhibit little if any capacity to undergo cell division. Consequently cardiomyocyte loss due to injury or disease is irreversible. The ability to utilize intracardiac grafting as a means to replace scarred, nonfunctional myocardium in a diseased heart with viable, functional cardiomyocytes would have significant therapeutic value. In addition, long-term delivery of recombinant molecules (as for example cardioprotective proteins, angiogenic factors, inotropic peptides or neurotrophins) via genetically engineered myocyte grafts could be of therapeutic value for individuals with myocardial ischemia or congestive heart failure. The local delivery of such molecules may also significantly reduce the arrhythmogenic potential of compromised myocardium without the complications which arise from systemic delivery. Preliminary experiments from this laboratory have established the feasibility of intra-cardiac grafting using a variety of cell types. These studies demonstrate that the myocardium can serve as a stable platform for cells which have been manipulated in vitro, and suggest that intra-cardiac grafts may provide a useful means for the local, long-term delivery of recombinant molecules to the heart. In this application we propose to extend these findings in an effort to develop models which test the feasibility of utilizing intra-cardiac grafting to effect both myocardial replacement and therapeutic delivery of recombinant molecules. We propose two Specific Aims. They are: (1) To develop models which test the feasibility of intracardiac grafting as a means to effect myocardial repair. (2) Develop models which test the feasibility of intra-cardiac grafting as a means to effect long term delivery of recombinant protein to the myocardium. In each instance, feasibility of the approach will first be established in mice. Once successful, similar grafting procedures will be attempted in dogs, where the in house availability of well established methods to assess both cardiovascular function and arrhythmogenic predilection can be employed to ascertain the functional significance of the grafting maneuver. These experiments will be performed in collaboration with the other components of this SCOR program.

Cardiomyocytes in the adult mammal exhibit little if any capacity to undergo cell division. Consequently cardiomyocyte loss due to injury or disease is irreversible. The ability to utilize intracardiac grafting as a means to replace scarred, nonfunctional myocardium in a diseased heart with viable, functional cardiomyocytes would have significant therapeutic value. Preliminary experiments from this laboratory have established the feasibility of intracardiac grafting using a variety of cell types. These studies demonstrate that the myocardium can serve as a stable platform for cells which have been manipulated in vitro. In this application we propose to extend these findings in an effort to develop models which test the feasibility of utilizing intracardiac grafting to effect myocardial replacement. We propose five Specific Aim with the goal of developing models which test the feasibility of intracardiac grafting as a means to effect myocardial repair. They are: 1: Intracardiac grafting in the mouse heart with fetal ventricular cardiomyocytes derived from mice expressing a cytological "tag" in the myocardium. 2: Intracardiac grafting in the mouse heart with cardiomyocytes derived from murine embryonic stem (ES) cells. 3: Intracardiac grafting in the mouse heart with genetically modified skeletal myoblasts. 4: Intracardiac grafting in the infarcted rat heart with fetal ventricular cardiomyocytes derived from mice expressing a cytological "tag" in the myocardium. 5: Intracardiac grafting in the dystrophic canine heart using dystrophin expressing fetal cardiomyocytes. Together, the proposed experiments should enable us to ascertain the feasibility of employing intracardiac graftings as a means to repair damaged or diseased myocardium. Specific Aims 4 and 5 have the advantag that functional analyses of graft-bearing canine hearts are readily accomplished using established in house methodologies.

DESCRIPTION (Adapted from the applicant's abstract) Cardiomyocyte death is a common feature of many forms of heart disease. Since the myocardium lacks a substantive endogenous regenerative potential, cardiomyocyte death is essentially irreversible. It has recently become apparent that exogenous myocytes can be successfully engrafted into the adult myocardium, thereby increasing the number of cells present in the heart. This procedure may be of considerable therapeutic value if engrafted cells can augment function in a diseased heart. Indeed, strategies aimed at increasing myocyte number was viewed with the highest priority by the NHLBI Special Emphasis Panel on Heart Failure Research and by this RFA. However, several rather formidable issues and obstacles must be addressed before any therapy based on myocyte engraftment can he realized. The five highly integrated Collaborative R01s proposed herein are designed to directly address these issues. A major goal of the proposed studies is to establish the fate of donor cells following engraftment. Particular emphasis is being placed on identifying factor(s) which enhance donor cell viability (Dr. Kedes), and on determining the degree to which donor and host myocytes can interact (Field and Murry). Other studies (Field, Murry, Kedes and Hauschka) will establish the relative merits of a variety of different donor cells (fetal cardiomyocytes, skeletal myo-blasts, ES- and EC-derived cardiomyocytes, and smooth muscle cells). Particular emphasis will be placed on weighing the issue of donor cell availability versus the functional characteristics of their respective grafts. Functional analyses of the engrafted hearts will rely largely on highly sensitive 2D echocardiography (KIoner). These latter studies will also establish to what degree cellular engraftment has a direct versus indirect effect on cardiac function (that is, participation in contractile force generation versus a positive effect on remodeling). The assembled investigators have established track records in relatively new field of cardiac engraftment, and additionally bring a diverse spectrum of experimental expertise which collectively provide a comprehensive battery of molecular, cellular and functional experimental methods. (End of Abstract)

Clinical recovery from myocardial infarction is thwarted, in part, by inability of surviving ventricular myocytes to reconstitute functional cardiac mass through a corresponding, compensatory increase in cell number. This highlights the limited capacity to restore cardiac mass by hypertrophy alone, and deleterious effects associated with hypertrophy that further impair survival. On-going myocyte loss also appears likely as an eventual contributor to end-stage heart failure. Conventional therapies for heart failure are aimed at rescuing jeopardized myocardium, optimizing mechanical load, or augmenting the mechanical performance of surviving myocytes. In principle, strategies to increase the number of functional ventricular myocytes have potential for a clinical benefit. (This theme is among the highest priorities expressed by the NHLBI Special Emphasis Panel on Heart Failure Research and the present RFA.) Three complementary, gene-based approaches have been brought to bear on the problem of cardiac cell number in this Collaborative RO1-transdifferentiation, manipulation of cell cycle constraints, and interference with pathways for programmed cell death (apoptosis). Viral delivery of cardiogenic transcription factors and upstream cardiogenic signals will be explored by Dr. Robert Schwartz. Drs. Michael Schneider and Loren Field will use gain-and loss-of- function mutations to dissect the "post-mitotic" phenotype in vivo, and will use co-precipitation or interaction cloning to isolate the endogenous cardiac proteins affecting cell cycle exit. Dr. Konstantin Galaktionov, an expert on Cdc25, will study molecular regulators of the G2/M transition, a second checkpoint that must be overcome for cell number to be increased. Mechanisms and countermeasures for cardiac apoptosis will be tested by Dr. Doug Mann, with emphasis on dilated cardiomyopathy triggered by overexpression of tumor necrosis factor alpha, and on investigations of human myocardium.

DESCRIPTION (provided by applicant): Cardiomyocytes in the adult mammal exhibit little if any capacity to undergo cell division. Consequently cardiomyocyte loss due to injury or disease is irreversible. Transplantation of donor cells (either cardiomyocytes or stem cells with cardiomyogenic potential) has emerged as a novel way to treat damaged hearts. Recent studies have demonstrated that adult-derived stem cells display a greater degree of plasticity than previously anticipated, and several studies have presented data suggestive of cardiomyogenic differentiation from adult bone marrow-derived stem cells. The experiments proposed in this application will assess the cardiomyogenic potential of three independent bone marrow stem cells, namely Side Population (SP) stem cells, lin-/c-kitHI stem cells, and Multipotent Adult Progenitor Cells (MAPCs). In Aim 1 will determine the capacity of the various bone marrow stem cells to undergo cardiomyogenic differentiation in vitro in response to known growth factors and under co-culture conditions with cardiomyogenic cells. In Aim 2 the undifferentiated stem cells will be transplanted into developing embryos or into normal and injured adult hearts to determine their ability to respond to cardiomyogenic cues during development and following myocardial injury. Aim 3 will determine if cardiomyocytes produced by in vitro differentiation of the bone marrow stem cells can be used for transplantation. Finally, Aims 4 and 5 will determine if genetic interventions which permit the purification and amplification of ES-derived cardiomyocytes can be applied to bone marrow stem cell-derived cardiomyocytes. These experiments will provide the first comparative analyses of the cardiomyogenic potential of multiple bone marrow stem cells in vivo and in vitro, and will also establish the genetic tractability and malleability of the cells. In this regard, the application is responsive to four of the seven focus areas suggested in the RFA. The information obtained from these studies will establish the feasibility and potential utility of bone marrow stem cells as a source of transplant cardiomyocytes for the treatment of cardiovascular disease.

The IUCC Transgenic and Knockout Mouse Core provides services for: (1) the production of transgenic mice (via pronuclear injection of recombinant DNA molecules) and (2) the production of knockout mice (via homologous recombination in ES cells). Core activities are as follows: (1) For transgenic mouse production, investigators provide the DNA construct to the core. The core then harvests single cell embryos, microinjects the embryos with the DNA construct, transplants the microinjected embryos into pseudo-pregnant females, performs Cesarean sections as required. At weaning age, the resulting pups are provided to the investigator for analyses; and (2) For knock-out mouse production, the core provides three services, namely ES cell transfection, blastocysts injections and rapid germ-line breeding. For ES cell transfection, the investigator provides the core with a targeting vector. The core then transfects ES cells with the targeting vector, selects the resulting transfected clones, generates frozen stocks of the clones, and provides the investigator with DNA samples from each of the clones for molecular analyses. For blastocysts injections, the investigator provides (or identifies) the desired ES clone. The core then recovers and amplifies the ES cell line, provides the investigator with cells for additional molecular and cell biologic analyses, generates back-up frozen stocks, injects the cells into blastocysts, transplants the injected blastocysts into pseudo-pregnant females, performs Cesarean sections as required, and culls the resulting chimeric animals. At weaning age, the chimeric pups are either provided to the investigator for analyses or are used for rapid germ-line breeding. For rapid germ line breeding, core personnel rotate female mice with the chimeric males. Plugged females are replaced, until a total of 10 pregnancies are culled. The resulting pups are screened for germ line transmission of the ES cell genome (determined via coat color). Tail biopsies are provided to the investigator for molecular confirmation of germ line transmission. At weaning age, the resulting heterozygous animals are provided to the investigator for analyses. Additionally, core personnel will provide advice concerning construction of transgenic and gene targeting constructs, animal breeding, and maintenance of the resulting mouse colonies. Overall, the core provides transgenic mice to IUCC investigators in a timely and cost-efficient manner.

DESCRIPTION (provided by applicant): Cardiomyocytes in the adult mammal exhibit little if any capacity to undergo cell division. The ability to reactivate cardiomyocyte proliferation in a diseased heart could be of considerable therapeutic value if the newly replicated cells are able to contribute to contractile function. We have recently generated transgenic mice that express D-type cyclins in the heart, in order to determine the effects of forced G1/S transit on cardiomyocyte cell cycle activity. Expression of cyclin D1, D2, or D3 promoted low levels of cardiomyocyte DNA synthesis in adult transgenic hearts. Surprisingly, myocardial infarct and beta-adrenergic stimulation markedly increased the rates of cardiomyocyte DNA synthesis in peri-infarct zone of the ventricle and left atrium, respectively, in transgenic mice that express cyclin D2. These features appear to be specific for cyclin D2, as both cardiac injury and beta adenergic stimulation markedly reduced cardiomyocyte DNA synthesis in transgenic mice expressing cyclin D1 or cyclin D3. Subsequent studies have shown that DNA synthesis can culminate with cardiomyocyte cytokinesis in the cyclin D2 model. Our results indicate a fundamental difference in the capacity of the D-type cyclins to promote cell cycle activity in cardiomyocytes, and also suggest that cyclin D2 might be used to affect myocardial regeneration following injury. We propose four Specific Aims to study the effects of cyclin D2 expression in cardiomyocytes. Aim 1 will characterize the response of ventricular cardiomyocytes to cardiac injury in MHCcycD2 transgenic mice; these experiments will determine the extent to which cell cycle activation can ameliorate muscle loss following myocardial infarction, as well as test the capacity of cyclin D2 to induce cell cycle activity in naive adult ventricular cardiomyocytes. Aim 2 will establish the molecular basis for the differential effects of D-type cyclins in cardiomyocytes; these experiments will test the hypothesis that differential nuclear-to-cytoplasmic trafficking underlies the differential capacity of the D-type cyclins to promote cell cycle activity in response to myocardial injury. Aim 3 will characterize the response of left atrial cardiomyocytes to beta-adrenergic stimulation in MHC-cycD2 transgenic mice; these experiments will establish the degree to which cyclin D2- mediated adult cardiomyocyte cell cycle activity can be driven, as well as characterize the functional activity of replicated atrial cells. The proposed experiments will further delineate the consequences of cyclin D2 expression in the adult heart, and should provide additional information regarding the regulation of the cardiomyocyte cell cycle. Ultimately, these experiments will determine if manipulation of the cyclin pathway can be exploited for the induction of therapeutic myocardial growth in the adult heart.

[unreadable] DESCRIPTION (provided by applicant): The ability to reconstitute lost cardiac mass following myocardial infarction could thus be of considerable therapeutic value. One approach to accomplish this entails the direct transplantation of donor cardiomyocytes or cardiomyogenic stem cells into injured hearts. Recent studies have identified a population of Sca-1+ stem cells isolated from the adult heart that home to the infarct border zone following intravenous infusion after reperfusion injury. After homing, the Sca-1+ cells have at least two fates: some of the cells fuse to infarct border zone cardiomyocytes forming heterokaryons, while others undergo de novo cardiomyogenic differentiation. The net result is that approximately 5% of the cardiomyocytes at the infarct border zone are derived either totally or in part from Sca-1+ stem cells. In this application we will combine the use of Sca-1+ stem cell transplantation with several validated cardioproliferative and/or cardioprotective genetic pathways in an effort to increase the number of Sca-l+ derived cardiomyocytes that reconstitute the heart following myocardial injury. Four Specific Aims are proposed. In Aim 1, we will use cell cycle and apoptosis analyses to establish the replicative and survival characteristics of Sca-1+ derived cardiomyocytes. These somewhat descriptive studies will provide requisite base line parameters for the remaining aims of the application. In Aim 2, we will test the hypothesis that lineage-restricted expression of cell cycle regulatory genes will enhance the ability of Sca-1+ stem cells to reconstitute damaged hearts. These experiments will utilize stem cells derived from mice expressing either the SV40 Large T Antigen oncoprotein, cyclin D2, or a dominant interfering pi93 mutant; all three proteins are able to drive cardiomyocyte cell cycle activity in transgenic animals. In Aim 3, we will test the hypothesis that lineage-restricted expression of cell survival genes will enhance the ability of Sca-1+ stem cells to reconstitute damaged hearts. These studies will utilize transgenes expressing Bcl-2 or a dominant interfering version of p53, both of which bestow cardioprotective phenotypes when expressed in the hearts of transgenic mice. Finally, in Aim 4 we will test the hypothesis that border zone cardiomyocytes derived from Sca-1+ stem cells are able to participate in a functional syncytium with the host myocardium. These studies will utilize two photo molecular excitation laser scanning microscopy to simultaneously monitor intracellular calcium transients in host and Sca-1+ derived cardiomyocytes in intact hearts. The experiments proposed here will use a variety of transgenic mouse resources to determine if genetic modification can enhance the capacity of Sca-1+ derived cardiomyocytes to repopulate myocardial infarcts, and furthermore will determine if the nascent cells are able to participate in a functional syncytium with the surviving host myocardium. Ultimately these approaches might be useful to reconstitute myocardial mass following cardiac injury, as well as deliver beneficial genes to cardiomyocytes at the border zone. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Although the adult mammalian myocardium exhibits a limited ability to undergo regenerative growth, the intrinsic renewal rate is insufficient to reverse pathophysiologic cardiomyocyte loss. The ability to reconstitute lost cardiac mass could thus be of considerable therapeutic value. One approach to accomplish this entails transplantation of donor cardiomyocytes or cardiomyogenic stem cells. This application focuses on cardiac-resident stem cells which express the Stem Cell Factor receptor c-kit. The proposed experiments are based on the recent observation that approximately 3% of the c-kit+ cells isolated from neonatal mouse hearts can give rise to well developed cardiomyocytes when co-cultured with fetal or neonatal cardiomyocytes. These cells appear to be either cardiomyogenic stem cells, or committed progenitor cells which require a heart-like environment to manifest a cardiac phenotype. Similar analyses using adult hearts suggested that the cardiomyogenic activity of c-kit+ cells is markedly limited or completely absent at later stages of development. The studies proposed in this competitive renewal application will establish the utility of cardiomyogenic c-kit+ cells (isolated from early post-natal hearts, from in vitro differentiating embryonic stem cells, and from adult hearts) for cell transplantation-based interventions aimed at repopulating the myocardium. Specific Aim 1 will characterize the developmental profile of cardiomyogenic c-kit+ cells, determine the degree to which the c-kit+ cells can be amplified while maintaining cardiomyogenic potential, and test the hypothesis that these cells are better suited than fetal cardiomyocytes to repopulate the adult myocardium following intra-cardiac transplantation. Specific Aim 2 will test the hypothesis that relaxation of epigenetic tags during prolonged culture will unmask cardiomyogenic potential in adult heart-derived c-kit+ cells. Ultimately these approaches might be useful to reconstitute myocardial mass following cardiac injury.

Increases in cardiac mass during normal development are characterized by proliferation of differentiated cardiomyocytes. After birth there is a dramatic reduction in the rate of cardiomyocyte cell cycle activity, and subsequent increases in cardiac mass occur largely as a consequence of cardiomyocyte hypertrophy. Abnormalities in these developmental processes can give rise to congenital heart defects. Moreover, the absence of substantive postnatal cardiomyocyte cell cycle activity, coupled with cardiomyocyte loss (via apoptosis and/or other mechanisms), contributes significantly to morbidity and mortality in neonatal patients with congestive heart failure. We have generated a number of mouse models which exhibit enhanced cardiomyocyte cell cycle activity during embryonic and postnatal life. We have also generated mouse models that exhibit reduced hypertrophic growth during neonatal life and which are resistant to injury- induced cardiomyocyte apoptosis as a consequence of targeted BmpIO expression. The proposed experiments will use these models to explore the regulation of cardiomyocyte apoptosis and hyperplastic cardiomyocyte growth in the setting of neonatal heart failure. Specific Aim 1a will test the hypothesis that BmpIO expression can exert cardioprotective activity in response to acquired injuries which model childhood heart failure in humans (namely anthracycline cardiotoxicity and viral myocarditis). Specific Aim 1b will test the hypothesis that BmpIO functions via paracrine pathways in the postnatal heart, and will also determine how long ventricular cardiomyocytes remain responsive to cytokine-mediated cardioprotection. Specific Aim 2a will test the hypothesis that inhibition of hypertrophic growth renders postnatal cardiomyocytes more susceptible to cell cycle re-entry. Specific Aim 2b will test the hypothesis that induction of cardiomyocyte cell cycle activity can reverse the adverse consequences of congenital and acquired injuries which give rise to childhood heart failure. The proposed studies will benefit greatly from the organization of the Program Grant application, in that many reagents, techniques and mouse models developed in the other projects will be used here. The ultimate goal of this project is to gain an understanding of how regulation of cardiomyocyte apoptosis and hyperplastic cardiomyocyte growth can be exploited to protect at-risk myocardium, and/or to promote the formation of new heart tissue, in the setting of childhood heart failure.

DESCRIPTION (provided by applicant): Heart failure in children can result from congenital or acquired injury to the myocardium. This Program Project Grant application is focused on studying the origins and treatment of heart failure in the young. The proposed studies will establish how aberrant expression of transcription factors can give rise to morphogenic anomalies that contribute to heart failure (Project 1), establish the degree to which modulation of the ROCK1 pro-apoptotic signal transduction pathway can protect against cardiomyocyte death following injuries which induce childhood heart failure (Project 2), and establish the extent to which cardioprotective cytokine pathways and hyperplastic growth programs can be manipulated to salvage at-risk myocardium and/or promote regeneration of damaged myocardial tissue following injuries which induce childhood heart failure (Project 3). The projects are thematically linked in that they examine how gene expression, cell survival and cell growth pathways are interrelated in the origins of heart failure, and furthermore how manipulation of these pathways can be exploited therapeutically. In addition to this thematic integration, the projects are technically linked in that common methodologies, approaches, and reagents will be utilized, thereby rapidly accelerating the pace of discovery. Moreover, genetic and acquired injury models and/or therapeutic interventions developed in one project will be used for proof-of-concept studies in other projects within the application, thereby accelerating the potential translation of discoveries. It should also be noted that each of the three projects represents an equal and significant contribution of effort from two established investigators having complementary skills and expertise, with one serving as Project Leader and the other as Collaborating Investigator. Thus, the Program Project Grant application will integrate the activities of six laboratories within the Cardiac Developmental Biology Program in the Herman B Wells Center for Pediatric Research with a unifying theme, thereby further enhancing the pace and productivity of research for an important and unmet clinical need, namely the study and treatment of heart failure in children.

DESCRIPTION (provided by applicant): Documentation of cardiomyocyte cell cycle activity and myocardial tissue regeneration typically requires extensive histochemical processing and frequently relies on the use of subjective criteria for cell lineage determination. Although reporter transgenes can greatly assist such analyses, their use also requires considerable tissue processing, and current transgenic reporter systems do not readily permit monitoring of cumulative regenerative growth. The studies proposed in this R21 application will generate transgenic reporter models which can be used to quantitate cardiomyocyte cell cycle activity and cumulative myocardial regeneration with minimal tissue processing. Aim 1 will utilize a cardiomyocyte-restricted promoter to target expression of a fusion between proteins with intrinsic fluorescent activity and proteins which undergo cell cycle- dependent changes in sub-nuclear localization. Cardiomyocyte cell cycle status can be quantitated simply by monitoring the pattern of reporter protein epifluorescence within the nucleus. Aim 2 will develop a tertiary transgenic reporter system to quantitate cumulative de novo myocardial growth in adult hearts. The system will utilize an existing conditional Cre-recombinase transgenic model, in combination with two new conditional transgenes, to permanently activate a nuclear localized EGFP reporter protein in all adult cardiomyocytes undergoing de novo proliferation. Consequently, cumulative myocardial growth resulting from cardiomyocyte proliferation can be determined simply by scoring the number of cells with nuclear EGFP epifluorescence. We will utilize existing transgenic models that exhibit enhanced cardiomyocyte proliferation to validate both reporter gene systems. Once validated, we will develop automated data acquisition and analyses protocols. The reporter transgenes proposed in this application will have the distinct advantage over existing models in that data can be acquired with minimal sample processing (in essence, requiring only tissue fixation and sectioning). Moreover, the systems will permit quantitation of cumulative regenerative growth, which cannot easily be determined with existing models. The use of automated techniques for data acquisition and analysis should permit precise quantitation of low-frequency events, and the use of fluorescent reporters will permit analyses in living tissue.

DESCRIPTION (provided by applicant): Although the adult mammalian myocardium exhibits a limited ability to undergo regenerative growth, the intrinsic renewal rate is insufficient to reverse pathophysiologic cardiomyocyte loss. The ability to reconstitute lost cardiac mass in injured hearts could thus be of considerable therapeutic value. One approach to accomplish this entails inducing cell cycle activity in the surviving cardiomyocytes. Preliminary data indicate that targeted expression of cyclin D2, a key member of the regulatory complex which drives transit through the G1/S cell cycle check-point, is sufficient to induce cardiomyocyte cell cycle activity in adult hearts. Moreover, cyclin D2-induced cell cycle activity can reverse structural damage and restore function following myocardial injury. The experiments proposed in this application will further elucidate the mechanism of cyclin D-mediated cardiomyocyte cell cycle regulation. Specific Aim 1 will test the hypothesis that post-translational modification of the D-type cyclins regulates their ability to mediate regenerative growth following myocardial injury. These experiments will utilize newly generated transgenic mice expressing D-type cyclins carrying the relevant phospho-mimetic and non-phosphorylatable amino acid residue substitutions. Other studies in Aim 1 will test the hypothesis that specific phosphorylation events mediate the interaction of D-type cyclins and the p193/Cul7 E3 ubiquitin ligase, and that blocking this interaction enhances cell cycle activity in injured hearts. Experiments proposed in Specific Aim 2 will further test the hypothesis that cyclin D2-mediated cardiomyocyte cell cycle activation can be used to promote myocardial repair in the adult heart. Initial experiments will utilize a conditional transgenic mouse model to determine if cyclin D2 can induce de novo cardiomyocyte proliferation in adult hearts. Other studies are proposed to determine the degree to which pharmacologic interventions which limit adverse post-injury remodeling are able to benefit long-term, cardiomyocyte cell cycle-induced regeneration. Collectively, these studies will help establish the mechanism by which D-type cyclins regulate cardiomyocyte cell cycle entry, and the degree to which targeted expression of cyclin D2 is able to promote myocardial regeneration. The overall goal is to gain an understanding of how cell cycle regulatory pathways can be manipulation to promote the repair of injured hearts. Identification of such molecular targets may ultimately lead to the development of pharmacologic agents to promote regenerative growth in diseased hearts. PUBLIC HEALTH RELEVANCE: Studies proposed in this application will establish the mechanism by which D-type cyclins regulate cardiomyocyte cell cycle entry, and the degree to which targeted expression of cyclin D2 is able to promote myocardial regeneration. The overall goal is to gain an understanding of how cell cycle regulatory pathways can be manipulation to promote the repair of injured hearts. Ultimately these approaches might be useful to reconstitute myocardial mass in diseased hearts, as for example, following myocardial infarction.

Cardiomyocyte cell cycle induction offers the potential for restoration of myocardial mass, and consequently contractile function, following cardiac injury. Considerable effort has thus been invested studying the degree to which cardiomyocytes can reenter the cell cycle and progress through cytokinesis in normal and injured adult mammalian hearts. Using a transgenic reporter system to identify cardiomyocyte nuclei in tissue sections in conjunction with continuous BrdU infusion, we have developed a digital imaging and analysis system which permits both quantitation and 3D anatomical mapping of cumulative cardiomyocyte S-phase activity across the entire heart. Using this system, we observed discrete clusters of cardiomyocyte S-phase activity in mice with permanent coronary artery ligation. We also observed very high rates of cardiomyocyte S-phase activity in the remote myocardium of mice with ischemia/reperfusion (I/R) injury. The studies proposed in this application will identify the underlying mechanistic basis for the differential cardiomyocyte cell cycle responses observed following myocardial injury. In Specific Aim 1, the variability in cardiomyocyte S-phase induction and cell cycle progression following permanent coronary artery ligation will be established and the resulting data sets will then be used for mathematical modeling with the goal of establish sampling criteria to quantitate total heart cardiomyocyte S-phase activity which takes into account these intrinsic anatomical variations. Other studies will determine if the observed clusters of S-phase activity arise from the clonal expansion of a subset of cardiomyocytes which retain the potential for cell cycle reentry. In Specific Aim 2, I/R injury will be performed in reporter mice maintained in an inbred genetic background to determine if the nature and/or degree of injury are responsible for high levels of cell cycle induction in the remote myocardium. Other studies will utilize informative backcrosses to determine the extent to which modifying genes can impact cardiomyocyte cell cycle reentry following I/R injury. In both Aims, the degree to which the S-phase positive cardiomyocytes progress through the cell cycle will also be quantitated. The proposed experiments will establish a 3D atlas of cardiomyocyte S-phase activity in response to commonly used and clinically relevant injury models, and will establish the degree to which increased levels of cardiomyocyte DNA synthesis contribute to polyploidization, multi-nucleation, and/or cardiomyocyte renewal. In addition, these experiments will characterize the impact of gender, genetic background and mode of injury on the magnitude of cardiomyocyte cell cycle reentry, as well as determine the consequences of natural variation in cardiomyocyte cell cycle activity on cardiac function post-injury. These data will provide useful insight for the development of interventional strategies with which to promote regenerative growth of the heart, as well as provide a comprehensive reference set for studies aimed at inducing cardiomyocyte renewal.

Abstract. The Specific Aim of this proposal is to obtain partial funding for the organization of the 2019 Weinstein Cardiovascular Development Conference, which will be held in Indianapolis, Indiana. The Weinstein Cardiovascular Development Conference originated from a series of annual meetings where Investigators, funded by RFAs on Cardiac Development awarded in 1986, 1988, and 1990, came together for face-to-face interactions under the direction of Dr. Constance Weinstein and her colleagues at the NHLBI. Since its inception, the 3-day Weinstein Conference has been the premier opportunity for basic scientists, clinicians, engineers, and geneticists in the field of cardiovascular development to interact and present innovative, novel data and ideas on various traditional and emerging topics pertinent to cardiogenesis, the understanding of the mechanisms that underlie congenital heart defects, as well as adult disease with congenital origins. This Conference is one-of-a-kind for several reasons: First, the Conference is entirely driven by local and international organizing committees made up of researchers who hold no affiliation with established scientific societies. Second, the Conference is well attended by senior leaders in cardiac biology, many of whom have been attending this Conference throughout their academic careers. Third, the overriding goal of the Weinstein Conference is to expose and highlight the participation of early stage principle investigators, post-doctoral fellows, and pre-doctoral students for the majority of the platform and poster presentations. Fourth, a well- established collaborative environment associated with the Weinstein Conference that encourages dissemination of new, unpublished data, thereby increasing the impact of the scientific content. The objectives of each Conference are to bring together basic scientists, clinicians, engineers, and geneticists working in the areas of cardiovascular developmental and regenerative biology all over the world, to provide an interactive forum for early stage principle investigators, post-doctoral fellows, and pre-doctoral students to present their work and interact and network with senior principle investigators and peers, and to actively support diversity in cardiovascular science. In this proposal, financial support is requested for the 2019 Weinstein Conference to be held in Indianapolis. Funds will be used a) To supplement registration fees for trainees to keep costs at a minimum encouraging robust attendance of early career scientists, b) to provide financial assistance in the form of travel awards for under-represented minorities, and c) to support general conference costs.

Numerous studies have shown that induction of cardiomyocyte cell cycle activity can have a profound beneficial impact on cardiac structure and function following myocardial infarction. It has also been shown that genetic background can impact the intrinsic rate of cardiomyocyte cell cycle activity in mice. We have observed that mice in a DBA/2J genetic background (abbreviated DBA) have very low levels of cardiomyocyte cell cycle activity following myocardial infarction. However, when crossed with C57Bl6/NCR mice (abbreviated NCR), the resulting (DBA x NCR)-F1 animals mice exhibit a marked increase in cardiomyocyte S-phase activity following infarction, indicating the presence of an autosomal gene (or genes) in the NCR background which acts in a dominant manner to facilitate cell cycle re-entry. Analysis of backcross mice established that this gene (or genes) resides in a region of interest (ROI) located on the distal end of chromosome 3. The experiments proposed in Aim 1 will test hypothesis that a single gene within the ROI is responsible for elevated cardiomyocyte S-phase activity post-infarction. Candidate genes within this region will be identified based on expression patterns observed in infarcted DBA vs. NCR hearts as well as by the presence of sequence variants predicted to impact protein structure and/or activity. The candidates will be systematically tested by generating genetically modified animals, subjecting them to myocardial infarction, and then monitoring the level of cardiomyocyte S-phase activity; an induction of cardiomyocyte cell cycle activity would confirm that the candidate gene being tested is responsible for the trait. The experiments proposed in Aim 2 will test the hypothesis that the elevated cell cycle activity encoded by the NCR ROI alleles has a positive impact on the diminished cardiac function and adverse myocardial remodeling which is encountered post-infarction. Congenic mice in a DBA genetic background which retain heterozygosity on the distal end of chromosome 3 and thus carry the NCR allele (or alleles) which is a major contributor to cardiomyocyte S-phase induction will be generated. The mice will then be subjected to myocardial infarction and longitudinal functional analysis. Terminal analyses will include comprehensive hemodynamic measurements as well as assessment of adverse remodeling (cardiomyocyte apoptosis, hypertrophy and myocardial fibrosis); relative improvements in cardiac function and structure would indicate a beneficial effect from the NCR-encoded cell cycle activity following myocardial injury. Ultimately, the identification and validation of genes underlying intrinsic differences in cardiomyocyte cell cycle rates observed in different strains of mice could suggest potential therapeutic targets with which to enhance regenerative growth in injured hearts.