Investigating the role of macrophages in larval zebrafish heart regeneration

Abstract

Acute myocardial infarction occurs when a coronary artery becomes occluded following atherosclerotic plaque rupture. Consequently, the myocardium suffers ischemic cell death, manifesting as a region of necrotic cardiomyocytes in the left ventricle. Unfortunately, the human heart is unable to replace these lost cardiomyocytes, and this can lead to heart failure. In contrast, zebrafish can completely regenerate their hearts after cardiac injury via dedifferentiation and proliferation of surviving cardiomyocytes. Macrophages are a required cell type for successful zebrafish heart regeneration and so may offer a roadmap to the optimisation of human myocardial repair. However, macrophages exhibit highly plastic and heterogenous phenotypes and are thought to influence healing through a variety of means.

To accelerate the study of macrophages in zebrafish heart regeneration, our laboratory has developed a larval zebrafish model of cardiac regeneration. The model uses a laser to induce cardiac injury in 3 days post fertilisation larvae which recover in just 48 hours, as opposed to 2 months in adults. Larval zebrafish are also small and transparent, facilitating unparalleled in vivo imaging. Together, these qualities facilitate rapid and detailed investigation into both heart regeneration and the role of macrophages in heart regeneration. In this study I have therefore performed detailed characterisations of larval zebrafish heart regeneration, macrophage recruitment and the role of macrophages in larval zebrafish heart regeneration.

I hypothesised that macrophages would be required for the regeneration of the injured larval heart. To test this hypothesis, I first used a combination of whole larva live imaging and photoconversion-based lineage tracing to map macrophage migration from primary haematopoietic sites to the injured heart. These data showed macrophages to be recruited first from the pericardium and then the caudal haematopoietic tissue, preferentially using the abluminal wall of blood vessels to migrate to the site of injury. I next used macrophage-less irf8-/- larvae and metronidazole-nitroreductase macrophage ablation to test the requirement for macrophages for several important regenerative processes. My data showed macrophages to be required for wound debridement and cardiomyocyte proliferation but not structural and functional recovery of the heart. Using in vivo imaging and a variety of pharmacological and recombinant protein interventions, I found epicardial Vegfaa to be required for cardiomyocyte proliferation and macrophages to be required for early epicardial expansion. Finally, I showed Vegfaa to increase endocardial notch signalling, which is known to drive cardiomyocyte proliferation, thus offering a mechanism for how macrophages may influence this process. These findings reveal a previously unrecognised role of macrophages in epicardial activation, providing a novel target for beneficial therapeutic intervention in the future.