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Neutrophils and macrophages are crucial effectors and modulators of cardiac repair following myocardial infarction (MI). Sustained neutrophilic inflammation is detrimental for cardiac repair and associated with adverse MI outcomes. An attractive therapeutic strategy to treat acute inflammatory disorders, such as MI, is to resolve infiltrating neutrophils to positively influence downstream reparative mechanisms. CDK9 inhibitor compounds enhance the resolution of neutrophilic inflammation, however, their effects on cardiac repair/regeneration are unknown. Unlike adult mammalian hearts, zebrafish hearts regenerate following injury via cardiomyocyte proliferation. Prolonged neutrophil retention has been shown to impair cardiomyocyte proliferation and myocardial wound regression following cardiac injury in zebrafish. I therefore hypothesised that CDK9 inhibitor treatment enhances the resolution of neutrophilic inflammation following injury and promotes cardiac regeneration in zebrafish. I first refined a larval zebrafish cardiac laser injury model and developed bespoke epifluorescence and 4D heartbeat-synchronised light sheet fluorescence microscopy techniques. I characterised the innate inflammatory response to cardiac injury, specifically examining neutrophil and macrophage migration to the injured heart using high resolution imaging of transgenic reporter fish. Additionally, neutrophil and macrophage migratory responses were compared to the archetypal tail fin injury model. Live in vivo imaging permitted mapping of neutrophil and macrophage migration throughout the zebrafish larvae, from primary hematopoietic sites to the myocardial lesion. I found a conserved sequence of events marked by an early and acute phase of neutrophil recruitment followed by sustained macrophage recruitment. In each injury model I found the innate inflammatory response resolves by reverse migration. I used the characterised zebrafish cardiac injury model to test whether CDK9 inhibitors, AT7519 and Flavopiridol (FVP), resolve neutrophil infiltration and whether this regulates downstream macrophage involvement and cardiac repair/regeneration. AT7519 and FVP were found to enhance the resolution of neutrophilic inflammation by inducing neutrophil reverse migration from the injured heart. While continuous exposure to AT7519 or FVP caused adverse cardiac phenotypes, transient (pulsed) treatment accelerated neutrophil resolution and avoided these effects. Transient treatment with AT7519, but not FVP, augmented TNF polarisation of wound-associated macrophages, in turn enhancing cardiomyocyte number expansion and the rate of myocardial wound closure. Furthermore, I developed a selectivity assay using cdk9-/- knockout mutants that demonstrated AT7519 is a more selective CDK9 inhibitor than FVP. These findings highlight the potential of AT7519 as a promising treatment that resolves neutrophilic inflammation following cardiac injury and promotes cardiomyocyte regeneration. In collaboration with BioAscent Discovery Ltd, I performed an in silico ligand-based screen of a 125k compound library. The chemical structure of AT7519 and five other potent and selective CDK9 inhibitor compounds (AZD4573, LDC000067, iCDK9, NVP-2 and MC180295) were used to perform a 3D similarity search against BioAscent's 125k diversity library, with the aim of identifying novel and efficacious CDK9 inhibitors. For each of the six query compounds, the top 1000 BioAscent compounds were ranked based on 3D similarity score. Common (duplicated) compounds between the six ranked query lists were shortlisted and any compounds that displayed pan-assay interference properties were removed. This yielded a final focussed library of 598 BioAscent compounds. Further work is needed to develop a high throughput in vitro CDK9 inhibition assay to screen the custom library and identify promising candidate compounds for downstream validation and optimisation.

Heart failure causes millions of deaths annually and presents a large global healthcare burden. A regenerative cure for the damaged myocardium, where new muscle forms by proliferation of pre-existing cardiomyocytes, is an attractive therapeutic goal. Adult mammalian cardiomyocytes are terminally-differentiated, only capable of proliferating at a very low rate that is insufficient for a regenerative response after myocardial infarction. However, over the past decade, a transient innate capacity for cardiac regeneration during the early neonatal period has been described in both rodents and swine. Whether such a capacity exists in newborn human infants is unknown. Studying the mechanisms of regenerative potential in neonatal rodents and swine could offer greater insight into heart development in human neonates, and also facilitate discovery of novel targets for human heart disease therapy.Cardiomyocyte maturational processes occur concurrent with loss of heart regenerative potential in early neonatal mice. These maturational processes, and the transcriptional mechanisms regulating them, have been successfully manipulated to induce cardiac regenerative repair in adult mouse hearts after injury. Pigs also possess a similar period of early neonatal heart regenerative capacity as mice. However, the maturational dynamics of cardiomyocyte growth in the postnatal pig heart are not well-defined, despite popularity of swine as large mammal models for cardiac preclinical studies. In Chapter 2 of this dissertation, we describe cardiac maturation in postnatal swine from newborn to adolescent ages. Our results show discordance between time of terminal cardiomyocyte maturation and loss of heart regenerative potential in postnatal swine, dissimilar to rodents. Further, postnatal pig cardiomyocytes are distinct from rodents and humans, exhibiting extensive multinucleation of up to 16 nuclei per cardiomyocyte by 6 postnatal months. These differences hold importance for preclinical cardiac studies in swine, and present opportunities to investigate unique cardiomyocyte maturational processes in a mammalian model.Cardiomyocyte proliferative arrest occurs within the first postnatal week in rodents, coincident with loss of regenerative potential. Downregulation of developmental signaling alongside activation of various cell cycle inhibitory factors occurs during this time. Neonatal cardiomyocyte maturation is thus a complex process with many compensatory mechanisms, characterization of which is essential for targeted proliferative repair without uncontrolled growth in adult diseased hearts. Previous studies identified a potential inhibitory role in cardiomyocyte proliferation for transcriptional co-regulator Btg2 (B-cell translocation gene 2). Btg2, and its close family member Btg1, are well-known in non-cardiac cell lineages for their anti-proliferative functions, but are virtually unstudied in the heart. In Chapter 3 of this dissertation, utilizing in vivo and in vitro rodent models of Btg1 and Btg2 (Btg1/2) depletion, we show that Btg1/2 loss significantly increases neonatal cardiomyocyte mitotic activity. Also, by transcriptome analysis, a pleiotropic role for Btg1/2 in cardiomyocyte cell cycle regulation is implicated. These data suggest a novel regulatory role for Btg1/2 in neonatal rodent cardiomyocyte cell cycle arrest.Together, the studies described in this dissertation promote understanding of cardiomyocyte maturation in postnatal rodents and swine. Our results highlight distinctive differences in cardiomyocyte biology among various mammals, and also enhance overall understanding of mammalian cardiomyocyte cell cycle regulation.

Heart disease leading to cardiovascular failure is a major public health issue in the United States with a considerable burden for the health care system. Despite recent progress to advance stem cell-based therapy for patients, heart failure carries a five-year mortality that rivals most cancers. This proposal describes an approach to control and pattern three distinct stem cell populations derived from the human heart to promote superior repair and regeneration after myocardial infarction.Regenerative capacity of the heart is mediated through multiple distinct populations of stem cell types that are the subject of ongoing intense study. In the past decade, isolation and characterization of c-kit+ cardiac progenitor cells (CPCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) have provided substantial insight to the capabilities of stem cells to rebuild the damaged heart and advance clinical therapy. Clinical trials have proven the efficacy and safety of autologous and allogeneic cell delivery to human patients, yet improvements in cardiac function and reduction in scar tissue remain modest and far below that needed for restoration of normal functional output. The work presented in this dissertation project overcomes these current cell-based limitations by using a novel method for improving myocardial repair: CardioClusters, a three-dimensional microenvironment consisting of CPCs, MSCs and EPCs. The innovation of this project is the creation of CardioClusters with the ability to capitalize upon beneficial attributes of multiple human stem cells from a single human heart providing a clinically relevant translational strategy. Collectively, studies in this dissertation will pave the way for interventional approaches to selectively adapt stem cell behavior and merge beneficial attributes of stem cell populations found within the human heart for prevention of heart failure after cardiomyopathic injury.