The zebrafish heart regenerates after cryoinjury-induced myocardial infarction

Abstract

Background: In humans, myocardial infarction is characterized by irreversible loss of heart tissue, which becomes replaced with a fibrous scar. By contrast, teleost fish and urodele amphibians are capable of heart regeneration after a partial amputation. However, due to the lack of a suitable infarct model, it is not known how these animals respond to myocardial infarction.

Results: Here, we have established a heart infarct model in zebrafish using cryoinjury. In contrast to the common method of partial resection, cryoinjury results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts. As in mammals, the initial stages of the injury response include thrombosis, accumulation of fibroblasts and collagen deposition. However, at later stages, cardiac cells can enter the cell cycle and invade the infarct area in zebrafish. In the subsequent two months, fibrotic scar tissue is progressively eliminated by cell apoptosis and becomes replaced with a new myocardium, resulting in scarless regeneration. We show that tissue remodeling at the myocardial-infarct border zone is associated with accumulation of Vimentin-positive fibroblasts and with expression of an extracellular matrix protein Tenascin-C. Electrocardiogram analysis demonstrated that the reconstitution of the cardiac muscle leads to the restoration of the heart function.

Conclusions: We developed a new cryoinjury model to induce myocardial infarction in zebrafish. Although the initial stages following cryoinjury resemble typical healing in mammals, the zebrafish heart is capable of structural and functional regeneration. Understanding the key healing processes after myocardial infarction in zebrafish may result in identification of the barriers to efficient cardiac regeneration in mammals.

Figure 1

Cyroinjury compared to resection. (A) After amputation of the ventricular apex (upper panel), the damaged myocardium is restricted to the narrow layer of cells above the amputation plane. A blood clot fills up the missing tissue below the plane of amputation. After cryoinjury (lower panel), a large portion of the damaged apoptotic myocardium (red) remains integrated with the functioning organ. (B) A photograph of the cryoinjury procedure. A small incision of the zebrafish chest is opened with help of forceps. The cryoprobe is placed in contact with the ventricle.

Figure 2

Massive cell apoptosis distinguishes cryoinjury from ventricular resection. (A and C) AFOG (Acid Fuchsin Orange-G) staining labels healthy muscle cells (light orange), damaged cells with plasma (dark orange), and collagen (blue). (B and D) Immunostaining with Tropomyosin (TPM) for cardiac muscle (blue), and nuclear MEF-2 as a marker for healthy cardiomyocytes (red). Apoptotic cells are detected by the TUNEL assay (green). (B' and D') Higher magnifications of the framed area in the left panels. (A-B) Adjacent longitudinal sections of the heart one day after ventricular resection. (A) The ventricle (v) comprises a narrow stripe of damaged cardiomyocytes (dark orange) just above the amputation plane (dashed line). Underneath the amputation plane a blood clot (bc) seals the wound; a, atrium; ba, bulbus arteriosus. (B-B') A narrow layer of damaged TPM-positive myocardium above the amputation plane (dashed line) display enhanced apoptosis and reduced MEF-2 expression (arrowheads). (C-D) Adjacent cross sections of the heart one day after cryoinjury. (C) The ventricle encompasses a large disk-shaped damage (encircled with dashed line). (D-D') This region contains abundant apoptotic TPM-positive cardiomyocytes that downregulate MEF-2 expression. Scale bars in (A-B'), 300 μm.

Figure 3

Analysis of the infarct size after cryoinjury. (A-B) Whole hearts of transgenic fish expressing GFP under control of cardiac specific promoter cmlc-2. (A) Uninjured ventricle at 4 days post sham (dps); v, ventricle; va, valve; ba, bulbus arteriosus; scale bar 300 μm. (B) At 4 days post cryoinjury (dpci), a portion of the ventricular wall is devoid of GFP expression, indicating the damaged myocardium (encircled by dashed line). (C) Schematic drawing demonstrating the plane of sectioning of the heart, which we applied in all our analysis. A typical cross-section is shown at the right side of the panel. (D) 7 dpci, a consecutive series of cross-sections of one heart from the top of the ventricle (left upper corner) to the ventricular apex (right bottom corner) labeled to AFOG staining to visualize the healthy myocardium in orange, fibrin in red and collagen in blue. (E-H) Higher magnification of selected images shown in (D). The post-infarct zone (black dashed line) expands from the apex to approximately a half-length of the ventricular wall. In our morphometric analysis, the measurement of the infarct volume was taken from all the sections of six hearts at different time points after injury. v, ventricle; va, valve; ba, bulbus arteriosus; a, atrium.

Figure 4

Scar formation and resorption during healing of myocardial infarction after cryoinjury. (A-F and H) Heart sections stained with AFOG representing the subsequent stages after cryoinjury. (A) At 4 dpci (days post cryoinjury), the damaged myocardium (dark orange) becomes surrounded by non-muscle cells (light gray, denoted by arrow). (B) At 7 dpci, a fibrin layer (red) forms along the inner side of the wound margin that consists of non-myocytes (arrow). The central part of the post-infarct consists of a loose collagen network (blue). (C) At 14 dpci, the edges of the fibrin layer (red) resolve and are replaced by new myocytes (orange arrow). The central part of the post-infarct contains abundant collagen fibers (blue). (D) At 21 dpci, a wall of cardiac tissue surrounds the entire infarct (orange arrows). Fibrin (red) is markedly reduced, while the collagen fibers (blue) persist. (E) At 30 dpci, no fibrin is visible. The collagen-containing area has markedly decreased. (F) At 60 dpci, the infarct scar is nearly completely resolved. Only occasionally, a few collagen fibers are detected (blue arrow). (G) A change of the scar size relative to the entire ventricle at different stages after injury. For measurements, all cross sections of six hearts per time point were analyzed. (H) In control, at 4 dps (days post sham), no fibrin or collagen fibers are present. Scale bar in (A), 300 μm.

Figure 5

Cell apoptosis during the replacement of the scar with a new myocardium. (A-C) Confocal images of quadruple-stained cross-sections of hearts at different time points after cryoinjury. Cardiomyocytes are labeled by the presence of TPM (blue) and MEF-2 (red). All the nuclei are visualized by DAPI (yellow) and the apoptotic cells are detected by the TUNEL assay (green). Dashed lines encircle the scar. (A', B' and C') Higher magnifications of framed area shown in left panels. (A-A') At 4 dpci, a large infarct area is devoid of cardiomyocytes, and it contains abundant apoptotic cells. (B-B') At 14 dpci, numerous non-myocytes located in the infarct zone undergo apoptosis. Arrows indicate new myocardium invading the post-infarct. (C-C') At 30 dpci, cell apoptosis is no longer detected in the remaining post-infarct region. Cardiomyocytes have replaced a large portion of the wound, and only a small area lacks cardiomyocytes. Arrowheads denote a new myocardial wall. Scale bars in (A-A') represent 300 μm.

Figure 6

Enhanced cardiomyocyte proliferation during heart regeneration. (A-D) Confocal images of the heart from transgenic fish expressing nuclear DsRed2Nuc under the control of cmlc-2 cardiomyocyte-specific promoter. Immunostaining for Tropomyosin (blue) labels the myocardium, MCM5 (green) detects mitotic cells and DAPI (yellow in the left panels) marks all the nuclei. Proliferating cardiomyocytes are identified by the co-expression of DsRed2-Nuc and MCM5. Dashed line denotes the post-infarct. (A', B', C' and D') Higher magnifications of the framed area in the left panels. At 4 dpci (A-A'), 7 dpci (B-B'), 14 dpci (C-C') and 30 dpci (D-D'), many proliferating cardiomyocytes are located in the vicinity of the post-infarct. At 4, 7 and 14 dpci, a layer of proliferating non-myocytes surrounds the periphery of the scar (arrows in A, B and C). Bars in (A and A'), 300 μm. (E) Ratios of MCM5/DsRed2-Nuc-positive nuclei relative to DsRed2-Nuc nuclei. (n = 10, 2 representative sections of 5 hearts, *P < 0.01 compared to uncut).

Figure 7

VIM-positive fibroblasts and TNC localize at the border zone between the myocardium and fibrotic tissue. (A, C, E and G) Heart sections immunostained for a cardiac nuclear marker MEF-2 (red) and an intermediate filament marker Vimentin (VIM, green). (B, D, F and H) Heart sections immunostained for a cardiac sarcomeric marker TPM (red) and an extracellular de-adhesive protein Tenascin-C (TNC, green). (A', B', C', D', E', F', G' and H') Higher magnifications of the framed area in the left panels. (A-A' and B-B') In control, at 4 days after sham operation, no significant expression of VIM and TNC can be detected in the ventricle. (C-C' and D-D') At 7 dpci, the scar margin and the interface between the myocardium and post-infarct are highlighted by VIM- and TNC-expressing fibroblasts. (E-E' and F- F') At 14 dpci, protrusions of cardiomyocytes expand along VIM/TNC-expressing cells (arrows). (G-G' and H-H') At 30 dpci, the scar tissue is largely replaced by cardiomyocytes. A new compact myocardial wall (arrowheads) surrounds residual VIM and TNC. Dashed line encircle the post-infarct. Bars in (A-A'), 300 μm.

Figure 8

Electrocardiogram (ECG) analysis following cryoinjury revealed a prolonged duration of the ventricular action potential in scar-containing hearts. An average ECG of uninjured animals (A) and hearts at 7 dpci (B) demonstrates a similar pattern of waves except the prolonged time between the Q and T peaks. (C) Average duration of the QTc-intervals at different time points after cryoinjury (n = 6 fish, *P < 0.01).