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. 2012 Feb;295(2):234-48.
doi: 10.1002/ar.21492. Epub 2011 Nov 18.

The giant danio (D. aequipinnatus) as a model of cardiac remodeling and regeneration

Pascal J Lafontant  1 , Alan R Burns, Jamie A Grivas, Mary A Lesch, Tanmoy D Lala, Sean P Reuter, Loren J Field, Tyler D Frounfelter
Affiliations

The giant danio (D. aequipinnatus) as a model of cardiac remodeling and regeneration

Pascal J Lafontant et al. Anat Rec (Hoboken). 2012 Feb.

Abstract

The paucity of mammalian adult cardiac myocytes (CM) proliferation following myocardial infarction (MI) and the remodeling of the necrotic tissue that ensues, result in non-regenerative repair. In contrast, zebrafish (ZF) can regenerate after an apical resection or cryoinjury of the heart. There is considerable interest in models where regeneration proceeds in the presence of necrotic tissue. We have developed and characterized a cautery injury model in the giant danio (GD), a species closely related to ZF, where necrotic tissue remains part of the ventricle, yet regeneration occurs. By light and transmission electron microscopy (TEM), we have documented four temporally overlapping processes: (1) a robust inflammatory response analogous to that observed in MI, (2) concomitant proliferation of epicardial cells leading to wound closure, (3) resorption of necrotic tissue and its replacement by granulation tissue, and (4) regeneration of the myocardial tissue driven by 5-EDU and [(3) H]thymidine incorporating CMs. In conclusion, our data suggest that the GD possesses robust repair mechanisms in the ventricle and can serve as an important model of cardiac inflammation, remodeling and regeneration.

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Figures

Figure 1
Figure 1
Regeneration of GD ventricle following cautery injury. Gross morphology of GD heart observed on a dissecting microscope of (A) control ventricle, and (B) cauterized ventricle at day 3 with a clearly visible clot (b). (C) Regression of the clot at day 7, (D) day 14, (E) day 21, (F) day 45 after the injury. (G) TTZ stained ventricle showing non-viable tissue in apical portion of the ventricle at 24 hr. (H) Estimation of the volume of ventricle occupied by non-viable tissue from day one to day 60 after the injury. Representative Masson’s trichrome stained sagittal sections of (I) control heart, (J) 7 day heart illustrating the presence of a clot and concomitant loss of myocytic tissue in the injured area (arrow), and (K) the progressive reconstitution of myocardial structure (arrowhead) at 14 days and (L) 60 days. (M) Quantification of the size of the injured area with its component connective tissue as they are replaced by of the myocardial tissue. (Scale bars, 200 um)
Figure 2
Figure 2
Characterization of the injury induced by cauterization in plastic sections and by transmission electron microscopy. (A) Montage of a toluidine blue stained 2-um plastic-embedded section of an injured GD ventricle at day 3 post-injury demonstrating a well defined area of injury with loss of myocytes (arrow). (B) Transmission electron micrograph of well organized trabeculated myocytes with well organized sarcomeres, Z-bands, and dense area of mitochondria in a region distal from the injury. (C) Ultrastructure within the injured area showing complete loss of myocyte structure and the presence of crenated nucleated red blood cells contributing to the clot. (D) Ultrastructure of myocytes at the border zone of the injury showing disorganized and lower density of sarcomere closer to the injured area (lower half oh panel), and myocytes with higher sarcomeric density and organization (upper half of panel). (E) Toluidine blue stained section of a heart 7 days after the injury showing a loss of the structural characteristics of the compact and spongy heart showing, and (F) an uninjured heart with well defined compact and the dense trabeculated spongy myocardium. (G) Reconstitution of the compact heart is coupled with the reappearance of a spongy heart of lesser trabecular density at 14 days.
Figure 3
Figure 3
Inflammation in the injured and regenerating GD heart. Myeloperoxidase immune reactivity (black, arrow) in control heart (A, E higher magnification) section counterstained with eosin (orange). Marked presence of immunoreactive cells at 24 hr (B, F higher magnification), 14 days (C, G higher magnification), decreasing at 45 days (D, H higher magnification). Representative TEM of an heterophilic granulocyte (I, arrow) with cigar-shaped granule at 3 days, and an heterophilic granulocyte (J, top arrow), monocyte (J, bottom arrow), and macrophages (K, arrows) at 14 days in injured area. (L) Kinetics of MPO-positive cells infiltrating the injured GD heart. (Scale bar A–D, 200 um; scale bar E–H, 50 um, scale bar TEM, 2 um)
Figure 4
Figure 4
Collagen accumulation and resorption during GD ventricular remodeling following cauterization. (A) Trichrome stained heart of GD showing minimal presence of collagen (blue fibrils) in the non-injured heart. Accumulation of collagen increases at (B) 7 days, (C) 14 days, and decreases (D) at 45 days. (E, F) Collagen fibril bundles (arrowheads) running in multiple directions adjacent to fibroblasts (Fb) and myocytes (M) within injured GD ventricle at 14 days. (G) Quantification of aniline blue stained collagen fibrils in trichrome stained section in injured and non-injured areas, and (H) in injured area normalized to ventricular area. (Scale bar A–D, 50 um; scale bar E–F, 1 um)
Figure 5
Figure 5
Angiogenic response in the cauterized GD ventricle. (A) B.S. lectin-FITC staining in control heart outlines small vessels in the compact heart. Staining is absent (B) 24 hours following injury but is present in diffused clusters within the wound at 14 days (C). Scattered lectin staining persists at (D) 21 days within the wound, and is regressed to outline the regenerated endocardium and small vessels of the regenerated compact heart at 45 days (E). Hoechst staining of cell nuclei in the control section (F), show increase cell nuclei at (G) 24 hours, (H) 14days, (I) 21 days, and returning to control level at (J) 45 days. (K, L, M, N, O) overlay of A and F, B and G, C and H, D and I, E and J. (P) Representative TEM of a small capillary-like vessel (Cap) with a circulating red blood cell (RBC) in the wound at 14 days, and (Q) another vessel (Cap) with a circulating leukocyte (Leu). (Scale bar A–O, 50 um, scale bar TEM, 2 um)
Figure 6
Figure 6
Epicardial cells and cardiac myocytes support GD heart regeneration. MEF immunoreactivity (A) and EdU incorporating cells (B) in the regenerating area at 14 days, and overlay of MEF and EdU incorporation showing numerous MEF+/EdU+ cells (C, arrow) indicating cardiac myocyte cell cycle activation. Immunoreactivity of PCNA in regenerating GD heart section (D) counterstained with eosin at 14 days. Higher magnification image of the compact myocardium (E) showing marked PCNA immunoreactivity, and original spongy area bordering the proximal part of the injury (F) with few PCNA immunoreactive cells. [3H]Thymidine incorporation (silver grains) in toluidine blue stained section at 7 days showing epicardial cell cycle activation (G, arrow), and cells in the evolving connective tissue (H, I). [3H]Thymidine incorporation in cells of the regenerated compact heart (J, K, arrow) and the regenerating spongy heart cardiac myocytes (L, arrow) at 14 days. (Scale bars, 50 um).
Figure 7
Figure 7
Cardiac myocytes ultrastructure in regenerating myocardium. (A), TEM of myocardium in area remote from the site of injury at 14 days with myocytes containing well organized and aligned sarcomeres, and well ordered Z bands. (B), TEM of myocardium in the injured and regenerating are containing myocytes with less well orgazined sarcomeric components and few Z-bands (Scale bar, 1um for A and B).
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