Epicardium Formation as a Sensor in Toxicology

Abstract

Zebrafish (Danio rerio) are an excellent vertebrate model for studying heart development, regeneration and cardiotoxicity. Zebrafish embryos 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 malformations. TCDD exposure prevents both proepicardial organ (PE) and epicardium development. Exposure later in development, after the epicardium has formed, does not produce cardiac toxicity. It is not until the adult zebrafish heart is stimulated to regenerate does TCDD again cause detrimental effects. TCDD exposure prior to ventricular resection prevents cardiac regeneration. It is likely that TCDD-induced inhibition of epicardium development and cardiac regeneration occur via a common mechanism. Here, we describe experiments that focus on the epicardium as a target and sensor of zebrafish heart toxicity.

Keywords: AHR; TCDD; epicardium; heart regeneration; proepicardium.

Figure 1

TCDD exposure causes epicardium detachment from the underlying myocardium in developing mice. Murine dams were exposed to vehicle control (corn oil) or TCDD (5 μg/g) at E7 and embryos were collected at E12 for sectional hematoxylin and eosin staining (n = 3/group). Shown are representative sections of control and TCDD hearts at the epicardium. Black arrows denote epicardial cells and their detachment from myocardium.

Figure 2

VPA exposed zebrafish larvae lack epicardium. Embryos from a zebrafish epicardial reporter line (tcf21:DsRed; [83] were exposed to VPA (0.06 μM), or vehicle (EtOH) shortly following fertilization. The control is shown at left, while the VPA heart is shown at right. The presence or absence of epicardium was assessed with immunohistochemistry at 120 hours post fertilization using an antibody against tcf21, expressed in epicardial cells (Red). Cell nuclei are stained with DAPI shown as blue. Cytoplasmic cmlc2:GFP shown as green outlines the cardiomyocytes. Representative single plane confocal microscopy images (n = 6/group) are shown with anterior to the left (V = ventricle, A = atrium).