The remarkable ability of the heart to regenerate has been demonstrated in the zebrafish and giant danio, two fish members of the cyprinid family. Here we use light and electron microscopy to examine the repair response in the heart of another cyprinid, the goldfish (Carassius auretus), following cautery injury to a small portion of its ventricular myocardium. We observed a robust inflammatory response in the first two weeks consisting primarily of infiltrating macrophages, heterophils, and melanomacrophages. These inflammatory cells were identified in the lumen of the spongy heart, within the site of the wound, and attached to endocardial cells adjacent to the site of injury. Marked accumulation of collagen fibers and increased connective tissue were also observed during the first and second week in a transition zone between healthy and injured myocardium as well as in adjacent sub-epicardial regions. The accumulation of collagen and connective tissue however did not persist. The presence of capillaries was also noted in the injured area during repair. The replacement of the cauterized region of the ventricle by myocardial tissue was achieved by 6 weeks. The presence of ethynyl deoxyuridine-positive cardiac myocytes and partially differentiated cardiac myocytes during repair suggest effective cardiac myocyte driven regeneration mechanisms also operate in the injured goldfish heart, and are similar to those observed in zebrafish and giant danio. Our data suggest the ability for cardiac regeneration may be widely conserved among cyprinids.

SummaryLectins are carbohydrate-binding proteins commonly used as biochemical and histochemical tools to study glycoconjugate (glycoproteins, glycolipids) expression patterns in cells, tissues, including mammalian hearts. However, lectins have received little attention in zebrafish (Danio rerio) and giant danio (Devario aequipinnatus) heart studies. Here, we sought to determine the binding patterns of six commonly used lectins-wheat germ agglutinin (WGA), Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin (BS lectin), concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), and Lycopersicon esculentum agglutinin (tomato lectin)-in these hearts. Con A showed broad staining in the myocardium. WGA stained cardiac myocyte borders, with binding markedly stronger in the compact heart and bulbus. BS lectin, which stained giant danio coronaries, was used to measure vascular reconstruction during regeneration. However, BS lectin reacted poorly in zebrafish. RCA I stained the compact heart of both fish. Tomato lectin stained the giant danio, and while low reactivity was seen in the zebrafish ventricle, staining was observed in their transitional cardiac myocytes. In addition, we observed unique staining patterns in the developing zebrafish heart. Lectins' ability to reveal differential glycoconjugate expression in giant danio and zebrafish hearts suggests they can serve as simple but important tools in studies of developing, adult, and regenerating fish hearts. (J Histochem Cytochem 64:687-714, 2016)

The Giant danio (GD) and zebrafish (ZF) are small fish belonging to the diverse family of cyprinids; they both display the remarkable ability for heart regeneration. Our lab has recently described the complex junctional region (JR) at the interface of the compact and spongy myocardial layers of the ZF. However, how these two distinct layers are integrated in the GD has not been studied. Using light, scanning, and transmission electron microscopy (TEM), our study reveals that the GD possesses a complex JR with transitional cardiac myocytes (CM) linking compact heart CM and CM of the spongy heart, and that luminal and abluminal contacts are mediated through discrete membrane appositions and adherens junctions. More importantly we also found a complex network of fibroblasts (FB) with extensive cytoplasmic processes occupying the JR and investing the GD compact heart. To ascertain (FB) function, we treated GD with low dose of the antineoplastic agent Doxorubicin (DOX). Using Masson’s trichrome staining, we observed the accumulation of collagen in the compact heart. In addition, TEM shows increased level of collagen in the JR of treated hearts, as well as increase in FB endoplasmic reticulum and cytoplasmic polyribosomes suggesting a state of activation. We conclude that cardiac FB are resident in the complex JR and compact heart of the GD. Moreover our data suggest they are key to the accumulation of collagen that accompany the DOX‐induced injury response. Grant Funding Source: Supported by FDC‐DePauw University

Doxorubicin (Dox), an antineoplastic anthracycline used in the treatment of childhood leukemia, is associated with late‐onset cardiomyopathy. The mechanisms of Dox‐induced cardiac dysfunction are an area of intense investigations. Using light and transmission electron microscopy (TEM), we studied the effects of Dox on the heart of giant danio (GD), a small fish closely related to zebrafish. We compared short‐term high‐dose to long‐term low‐dose effects of Dox administrations. We found that short‐term (4 days) high‐dose Dox induced little inflammation or collagen accumulation in the GD myocardium. By contrast long‐term (4 weeks) low‐dose Dox induced apoptosis (TUNEL, TEM) and inflammation (MPO, TEM) in the ventricular myocardium. The number of MPO‐positive cells increased by 2 folds and 4 folds in Dox‐treated hearts, at 1 and 4 weeks respectively, with cell cycle activity (Edu) in cardiac myocytes (CM) and increased fibrosis (Trichrome, TEM). Interestingly, we found a spatially‐restricted pattern of fibrosis in the compact heart (CH). Consistent with this finding we observed by TEM that activated fibroblasts in the CH contributed to collagen accumulation. This data suggests that, except for marked cell cycle activation in CM, the GD heart response to Dox treatment appears analogous to that of human. Thus the GD may serve as an important model for the study of Dox‐induced cardiomyopathy.Grant Funding Source: FDC