A zebrafish heart can fully regenerate after amputation of up to 20% of its ventricle. During this process, newly formed coronary blood vessels revascularize the regenerating tissue. The formation of coronary blood vessels during zebrafish heart regeneration likely recapitulates embryonic coronary vessel development, which involves the activation and proliferation of the epicardium, followed by an epithelial-to-mesenchymal transition. The molecular and cellular mechanisms underlying these processes are not well understood. We examined the role of PDGF signaling in explantderived primary cultured epicardial cells in vitro and in regenerating zebrafish hearts in vivo. We observed that mural and mesenchymal cell markers, including pdgfrβ, are up-regulated in the regenerating hearts. Using a primary culture of epicardial cells derived from heart explants, we found that PDGF signaling is essential for epicardial cell proliferation. PDGF also induces stress fibers and loss of cell-cell contacts of epicardial cells in explant culture. This effect is mediated by Rhoassociated protein kinase. Inhibition of PDGF signaling in vivo impairs epicardial cell proliferation, expression of mesenchymal and mural cell markers, and coronary blood vessel formation. Our data suggest that PDGF signaling plays important roles in epicardial function and coronary vessel formation during heart regeneration in zebrafish.epicardium | mesenchymal cells | mural cells | zebrafish heart regeneration C oronary heart disease is among the leading causes of disability and mortality in the United States and worldwide (1). Scars form in injured human hearts, which results in decreased cardiac performance and the eventual development of heart failure (2). In contrast to humans, zebrafish and newts have remarkable regenerative abilities (3, 4). After 20% resection of the ventricle, zebrafish fully regenerate lost heart tissue (3, 4). During this process, newly formed coronary blood vessels vascularize the regenerating myocardium (5, 6). Expression of the embryonic epicardial markers tbx18 and raldh2 is induced in the epicardium of adult regenerating hearts (5, 6), suggesting that an embryonic gene expression program in the epicardium is activated in response to injury. This activation starts throughout the entire ventricle and gradually becomes localized to the apex. The activated epicardium proliferates from 3 to 7 d postamputation (dpa) (5). A previous study suggested that the activated epicardium undergoes an epithelial-to-mesenchymal transition (EMT) and subsequently contributes to newly formed coronary blood vessels (5). The lineages of the different cell types in blood vessels formed during zebrafish heart regeneration have not yet been conclusively determined.Zebrafish heart regeneration, at least in part, likely recapitulates embryonic heart development. EMT is a key step during heart development in mice and chicks, wherein the epicardium forms epicardium-derived cells (EPDCs), which then differentiate into fibroblasts, smooth muscle cells (7-9)...

The zebrafish (Danio rerio) is an emerging model for cardiovascular research. The zebrafish heart regenerates after 20% ventricular amputation. However, assessment of the physiological responses during heart regeneration has been hampered by the small size of the heart and the necessity of conducting experiments in an aqueous environment. We developed a methodology to monitor a real-time surface electrocardiogram (ECG) by the use of micro-electrodes, signal amplification, and a low pass-filter at a sampling rate of 1 kHz. Wavelet transform was used to further remove ambient noises. Rather than paralyzing the fish, we performed mild sedation by placing the fish in a water bath mixed with MS-222 (tricane methanesulfonate). We recorded distinct P waves for atrial contraction, QRS complexes for ventricular depolarization, and QT intervals for ventricular repolarization prior to, and 2 and 4 days post-amputation (dpa). Sedation reduced the mean fish heart rate from 149 +/- 18 to 90 +/- 17 beats/min. The PR and QRS intervals remained unchanged in response to ventricular apical amputation (n = 6, p > 0.05). Corrected QT intervals (QTc) were shortened 4 dpa (n = 6, p < 0.05). In a parallel study, histology revealed that apical thrombi were replaced with fibrin clots and collagen fibers. Atrial arrhythmia was noted in response to prolonged sedation. Unlike the human counterpart, ventricular tachycardia or fibrillation was not observed in response to ventricular amputation 2 and 4 dpa. Taken together, we demonstrated a minimally invasive methodology to monitor zebrafish heart function, electrical activities, and regeneration in real-time.