Investigating CDK9 inhibitor treatment during the innate inflammatory and regenerative response in a zebrafish model of cardiac injury

Abstract

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.