Abstract
In response to cardiac damage, a mesothelial tissue layer enveloping the heart called the epicardium is activated to proliferate and accumulate at the injury site. Recent studies have implicated the epicardium in multiple aspects of cardiac repair: as a source of paracrine signals for cardiomyocyte survival or proliferation; a supply of perivascular cells and possibly other cell types such as cardiomyocytes; and as a mediator of inflammation1,2,3,4,5,6,7,8,9. However, the biology and dynamism of the adult epicardium is poorly understood. To investigate this, we created a transgenic line to ablate the epicardial cell population in adult zebrafish. Here we find that genetic depletion of the epicardium after myocardial loss inhibits cardiomyocyte proliferation and delays muscle regeneration. The epicardium vigorously regenerates after its ablation, through proliferation and migration of spared epicardial cells as a sheet to cover the exposed ventricular surface in a wave from the chamber base towards its apex. By reconstituting epicardial regeneration ex vivo, we show that extirpation of the bulbous arteriosus—a distinct, smooth-muscle-rich tissue structure that distributes outflow from the ventricle—prevents epicardial regeneration. Conversely, experimental repositioning of the bulbous arteriosus by tissue recombination initiates epicardial regeneration and can govern its direction. Hedgehog (Hh) ligand is expressed in the bulbous arteriosus, and treatment with a Hh signalling antagonist arrests epicardial regeneration and blunts the epicardial response to muscle injury. Transplantation of Sonic hedgehog (Shh)-soaked beads at the ventricular base stimulates epicardial regeneration after bulbous arteriosus removal, indicating that Hh signalling can substitute for the influence of the outflow tract. Thus, the ventricular epicardium has pronounced regenerative capacity, regulated by the neighbouring cardiac outflow tract and Hh signalling. These findings extend our understanding of tissue interactions during regeneration and have implications for mobilizing epicardial cells for therapeutic heart repair.
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Acknowledgements
We thank J. Burris, N. Lee, A. Dunlap and S. Davies for fish care, and M. Bagnat, B. Hogan, J. Kang and R. Karra for comments on the manuscript. This work was funded by postdoctoral fellowships from the American Heart Association to J.W. and J.C., and grants from the National Institutes of Health (HL081674) and American Federation for Aging Research to K.D.P.
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J.W., J.C. and K.D.P. designed the experimental strategy, analysed data, and prepared the manuscript. J.W. generated the transgenic system for epicardial cell ablation and performed in vivo regeneration experiments and analysis. J.C. developed the ex vivo culture assay and performed ex vivo regeneration experiments and analysis. A.L.D. performed histology and data analysis. All authors commented on the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Epicardial ablation and responses to resection injury.
a, tcf21:nuceGFP or tcf21:NTR; tcf21:nuceGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz, before collection of ventricles at 7 (n = 8 animals per group) and 30 dpa (n = 11). tcf21:NTR; tcf21:nuceGFP wounds had fewer epicardial cells at 7 dpa and comparable occupancy by 30 dpa compared with tcf21:nuceGFP wounds. White dashed lines indicate wound edge. b, Quantification of eGFP+ epicardial cells at 7 and 30 dpa with respect to wound edge lengths from a. NS, not significant; Mann–Whitney rank sum test. c, tcf21:NTR clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. MLCK+ cells, indicating myofibroblasts, had comparable presence in both groups at 14 dpa (n = 7 for each group). d, tcf21:NTR; fli1a:eGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. Epicardial ablation led to lower vascular density in 30 dpa wounds compared with controls. Red dashed lines indicate wound edge. e, Quantification of eGFP+ vascular endothelial pixel area in wounds of tcf21:NTR; fli1a:eGFP zebrafish treated with Mtz (n = 12) or vehicle (n = 6), or of fli1a:eGFP zebrafish treated with Mtz (n = 6), with respect to the wound edge lengths. Student’s two-tailed t-test. f, By 60 dpa, 55 days after epicardial ablation protocols, muscularization (top) and wound collagen deposition (bottom) were grossly normal (n = 23). Brackets indicate area of regeneration. a, c, d, Yellow dashed lines indicate the approximate amputation plane. Scale bars, 50 µm. Error bars indicate s.d.
Extended Data Figure 2 Epicardial cell proliferation without injury and after epicardial ablation.
a, Limited epicardial cell proliferation on the ventricular surface. tcf21:nuceGFP fish were injected with 10 mM EdU once daily for 3 days and collected 1 day after the last injection. 105 ventricular nuceGFP+ cells were assessed for EdU reactivity in 15 animals, from which 608 cells were positive (a 0.6% rate for 4 days EdU labelling). Whole-mount image is shown, and arrows in enlarged boxed area indicate eGFP+EdU+ nuclei. b, tcf21:nuceGFP or tcf21:NTR; tcf21:nuceGFP fish were injected with 10 mM EdU at 3 days post-Mtz treatment, and hearts were collected 4 h later. Boxed areas in images of whole-mounted hearts show magnified views. a, b, Yellow arrows indicate representative eGFP+ (green) EdU+ (red) nuclei. c, fli1a:eGFP or tcf21:NTR; fli1a:eGFP fish were injected with 10 mM EdU at 3 days post-Mtz treatment, and hearts were collected 4 h later. Red arrows indicate representative eGFP+ (green) EdU+ (magenta) endocardial cell nuclei; yellow arrowheads, representative eGFP+ (green) EdU+ (magenta) vascular endothelial cell nuclei; white arrowheads, EdU+ (magenta) nuclei within the ventricular lumen, ostensibly erythrocyte nuclei. Scale bars, 50 µm.
Extended Data Figure 3 Mosaic NTR expression and patterns of spared epicardial cells after ablation.
a, Whole-mounted examples of varied location/pattern of spared epicardial cells in ventricles from tcf21:NTR; tcf21:nuceGFP adult clutchmates 3 days after incubation with 10 mM Mtz. White dashed lines delineate ventricle. b, Differential expression of the NTR transgene among cardiac chambers. In adult tcf21:NTR; tcf21:nuceGFP hearts, eGFP expression is comparable in epicardial tissue covering the atrium, outflow tract (OFT) and ventricle. By contrast, NTR (red, indicated by mCherry) expression is patchy and/or weak in the atrium and outflow tract compared with ventricular expression. c, Section images of ventricles from tcf21:nuceGFP (left) or tcf21:NTR; tcf21:nuceGFP zebrafish (right) treated with 1 mM Mtz (right) for 3 days, and collected 2 days later. Ventricular epicardium was ablated effectively in these experiments. Scale bars, 50 µm.
Extended Data Figure 4 Epicardial regeneration after ventricular resection.
a, Hearts were removed from tcf21:NTR; tcf21:nuceGFP fish immediately after ventricular resection injuries, followed by 24 h of Mtz and a 24 h washout ex vivo. A base-to-apex pattern of epicardial regeneration was observed, in this example covering the apical wound by 11 dpa (n = 18; behaviour seen in all samples). Epicardial coverage of resection injuries in these ablation experiments is delayed compared to ventricles recovering with an intact epicardium (b, top). Yellow boxed area, magnified view of the apical wound. Red dashed lines delineate ventricle. b, Hearts were removed from tcf21:nuceGFP clutchmates immediately after apical resection injury and cultured ex vivo, before random separation into two treatment groups. Epicardial cells covered the wound area by 3 dpa (n = 11; behaviour seen in all samples), unless treated with CyA (n = 26; failed coverage in 20 of 26 ventricles). a, b, White dashed lines indicate apical wounds. Scale bars, 50 µm.
Extended Data Figure 5 Ex vivo grafts and epicardial regeneration.
a, Schematic of the experimental design. b, c, Epicardial cells transplanted at the base of an epicardially ablated host regenerated towards the apex regardless of basal (b) (n = 25, behaviour seen in all samples) or apical (c) (n = 27, all samples) origin. d, Top, epicardial cells from the base of a transgenic donor ventricle were transplanted to the chamber midpoint of an epicardially ablated host ventricle and observed for regeneration. Bottom, transplanted cells eventually migrated towards the apex, not the base (n = 13; all samples). e, Top, after epicardial ablation, the host bulbous arteriosus was replaced with a non-transgenic donor ventricular apex and observed for regeneration. Bottom, ventricular epicardium showed little or no regeneration in these experiments (n = 7; behaviour seen in all samples). f, Left, after ex vivo epicardial ablation in a host tcf21:NTR ventricle, the host bulbous arteriosus was replaced with a donor tcf21:nuceGFP bulbous arteriosus. Right, the host ventricular surface contained different amounts of eGFP+ nuclei in these ventricles (n = 3; behaviour seen in all samples). b–f, Red dashed lines indicate epicardium or epicardial leading edge; white dashed lines delineate ventricle. e, f, Yellow dashed lines indicate donor apex (e) or bulbous arteriosus (f). Scale bars, 50 µm.
Extended Data Figure 6 Context-specific effects of outflow tract on epicardial regeneration.
a, Top, after ex vivo epicardial ablation and bulbous arteriosus removal, ventricles were co-cultured with ten outflow tracts in a transwell assay and observed for regeneration. Bottom, no evidence for epicardial regeneration was observed in these experiments (n = 9; behaviour seen in all samples). b, Left, after ex vivo epicardial ablation and bulbous arteriosus removal, a non-transgenic bulbous arteriosus (labelled as donor OFT) was transplanted to the apex and observed for regeneration. Right, no evidence for regeneration of eGFP+ epicardium from apex to base was observed in these experiments (n = 10; behaviour seen in all samples). Red dashed lines delineate epicardium; white dashed lines delineate ventricle; yellow dashed lines delineate donor outflow tract. Scale bars, 50 µm.
Extended Data Figure 7 Small-scale screen for compounds that inhibit epicardial regeneration.
Ex vivo ablation and regeneration of tcf21:NTR; tcf21:nuceGFP ventricles over 7 days. Mtz was added for 24 h to freshly isolated hearts, washed out, and compounds were added after 2 days (day 0). Hearts were treated with vehicle (n > 10), 10 µM DEAB (n = 5; Sigma-Aldrich), 100 nM LDN193189 (n = 4; Cayman Chemical), 10 µM SU5402 (n = 5; Santa Cruz Biotechnology), 1 µM cyclosporin A (n = 4; Sigma-Aldrich), or 0.1 µg ml−1 FK506 (n = 5; Sigma-Aldrich), in each case showing base-to-apex recovery (behaviour seen in all samples). The dissected hearts were randomly separated into groups for drug treatment. Red dashed lines indicate epicardial leading edge; white dashed lines delineate ventricle. Scale bars, 50 µm.
Extended Data Figure 8 Epicardial proliferation is regulated by Hh signalling.
a, Freshly dissected tcf21:nuceGFP hearts were randomly separated into two groups and cultured for 47 h with vehicle (n = 11) or 5 µM CyA (n = 8). Then, 25 µM EdU was added to the medium for 1 h before collection at 48 h. CyA treatment decreases epicardial cell proliferation ex vivo. Arrows indicate representative eGFP+ (green) EdU+ (red) nuclei. b, Quantification of eGFP+EdU+ nuclei per mm2 on the ventricular surface, from hearts in a. **P < 0.01, Student’s two-tailed t-test. c, tcf21:nuceGFP adult fish were subjected to partial ventricular resection surgery, and randomly separated into two groups for treatment with vehicle (n = 8) or 10 µM CyA (n = 10) from 2 to 3 dpa. Then, 10 mM EdU was injected intraperitoneally 1 h before collection. CyA treatment decreases epicardial cell proliferation in vivo. Arrowheads indicate representative eGFP+ (green) EdU+ (red) nuclei. d, Quantification of eGFP+EdU+ nuclei per mm2 on the ventricular surface, from hearts in c. ***P < 0.001; Mann–Whitney rank sum test. c, Yellow dashed lines indicate resection plane; white dashed lines delineate ventricle. a, c, Boxed areas, magnified views. Scale bars, 50 µm. Error bars indicate s.d.
Extended Data Figure 9 Larval epicardial development and regeneration.
a, tcf21:nuceGFP or tcf21:NTR; tcf21:nuceGFP larval clutchmates were treated with 10 mM Mtz from 6 hpf to 54 hpf, and then imaged at different times from 3 to 5 dpf. tcf21:nuceGFP larvae show normal ventricular epicardial coverage at 3 dpf, while tcf21:NTR; tcf21:nuceGFP coverage is sparse. tcf21:NTR; tcf21:nuceGFP larvae with confirmed full ablation were imaged from 3 to 5 dpf, covering first the ventricular base and then the apex. Three different extents of regeneration at 5 dpf are shown: class I, greater than two-thirds coverage; class II, one-third to two-thirds coverage; and class III, some cells but less than one-third coverage. b, A subset of tcf21:NTR; tcf21:nuceGFP larvae with confirmed full ablations were randomly separated and treated with vehicle or CyA, which limited regeneration in most cases (class IV, no ventricular epicardial cells). c, Quantification of extents of regeneration from experiments in a and b. ***P < 0.001, chi-squared test; n = 54 embryos for vehicle, 51 for CyA. d, Epicardial morphogenesis visualized in tcf21:nuceGFP larvae. No epicardial cells are evident at or before 2 dpf. By 3 dpf, ventricles contained 17.6 ± 6 epicardial cells on average (n = 23), whereas 4 dpf larvae contained 45.2 ± 5.8 cells (n = 21). e, tcf21:nuceGFP larval clutchmates were randomly separated into two groups for treatment with vehicle or 5 µM CyA from 2 to 4 dpf. f, Quantification of ventricular eGFP+ epicardial cells from groups in e. ***P < 0.001, Student’s two-tailed t-test; n = 21 for each group. a, b, d, e, White dashed lines delineate ventricle. d, e, Boxed areas, magnified views. Scale bars, 50 µm. Error bars indicate s.d.
Extended Data Figure 10 Hh ligand expression.
a–c, Quantitative RT–PCR revealing shha, ihhb and dhh expression in atrium (a) or ventricle (b) in uninjured hearts and 3 days post-ablation, or in separated ventricular basal (the basal third of the chamber) and apical (the apical third) tissue after ablation (c). Three separate quantitative RT–PCR experiments on pooled tissues were performed, using a total of 90 zebrafish for experiments shown in a and b, and another 90 fish for c. shhb and ihha were not detected in these tissues. d, In situ hybridization (ISH) for shha or dhh in wild-type (WT) or tcf21:NTR clutchmate hearts at 3 days after Mtz treatment, indicating expression in outflow tract but not ventricle or atrium. Outflow tract of uninjured and epicardially ablated hearts showed comparable shha and dhh signals by ISH, a qualitative/semiquantitative assay. e, Section of adult shha:eGFP heart, indicating fluorescence in outflow tract tissues. Smooth muscle cells (MLCK, red) and epicardial cells (outer layer) in outflow tract showed clear eGFP signals, while there is no obvious eGFP fluorescence in ventricle and atrium. Valve mesenchyme also displays eGFP fluorescence. Arrowheads indicate eGFP signals in smooth muscle cells and epicardium. f, Ventricular resection induces shha:eGFP fluorescence in the basal ventricular epicardium at 2 dpa. Arrows indicate ventricular epicardial fluorescence. d, e, White dashed line indicate outflow tract (d) or atrioventricular junction (e). d–f, Boxed areas, magnified views. Scale bars, 50 µm. Error bars indicate s.d.
Supplementary information
Explanted hearts contract and express epicardial markers under rocking culture conditions
A tcf21:nucEGFP heart that has been in culture for 33 days is shown. Contraction can be observed for several weeks ex vivo. (MP4 11561 kb)
Video of epicardial regeneration
Shown is a ventricle imaged daily over a 14-day period starting from Mtz addition (Day 0), visualized for tcf21:nucEGFP+ cells. (MP4 12437 kb)
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Wang, J., Cao, J., Dickson, A. et al. Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signalling. Nature 522, 226–230 (2015). https://doi.org/10.1038/nature14325
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DOI: https://doi.org/10.1038/nature14325
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