Dioxin Inhibits Epicardium Development and Regeneration of the Zebrafish Heart

Zebrafish (Danio rerio) are an established vertebrate model for studying heart development, regeneration and cardiotoxicity. Zebrafish embryo-larvae exposed during the temporal window of epicardium development to the aryl hydrocarbon receptor (AHR) agonist 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) exhibit severe heart malformation. Thus, we sought to determine if epicardium development was affected by TCDD exposure. TCDD exposure prevents development of the epicardial progenitors (proepicardium; PE) and subsequent formation of the epicardium. Exposure to TCDD later in development, after the epicardium has formed, does not produce acute cardiac toxicity. However, in adult zebrafish, TCDD exposure prior to ventricular resection prevents cardiac regeneration; the epicardially-derived white epithelium that envelops the blood clot fails to form and cardiomyocyte proliferation is markedly reduced. It is likely that TCDD-induced inhibition of epicardium development and cardiac regeneration occur via a common mechanism. Using the embryonic zebrafish, we have identified sox9b as a downstream AHR target gene in the heart. We find that while sox9b is expressed in the myocardium, it is not expressed in the affected epicardial cells or progenitors. TCDD exposed zebrafish embryos had significantly reduced levels of cardiac sox9b during epicardium development. Furthermore, we found manipulation of sox9b expression could phenocopy most of the effects of TCDD at the heart. Loss of sox9b prevented the formation of epicardial progenitors comprising the PE on the pericardial wall, and prevented the formation and migration of the epicardium around the heart. Zebrafish lacking sox9b showed pericardial edema, heart elongation, reduced blood circulation and lacked endocardial valve cushions and leaflets. Furthermore, sox9b mRNA injection prior to TCDD exposure rescued PE formation, but the epicardium failed to form. Myocardial contractility remained severely affected in sox9b mRNA injected TCDD-treated fish. This led us to investigate the role of myocardial contractility during epicardium formation. Lack of heart contractility resulted in a phenotype analogous to our sox9b rescue experiments; the PE formed but failed to migrate and form the epicardium. These experiments demonstrate myocardial contractility is required for PE cell migration and epicardium formation. Together, zebrafish epicardium development requires sox9b and normal cardiac contractility, which in our model, are severely affected by TCDD.