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Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signalling.

Wang J, Cao J, Dickson AL, Poss KD - Nature (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.

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 inflammation. 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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Epicardial ablation and regenerationa, Adult zebrafish heart. OFT, outflow tract. b,tcf21:NTR; tcf21:nucEGFP adults were incubated with Mtz or vehicle, and hearts collected by random sampling at 3, 7, or 14 days post-incubation (dpi). Proportion of total animals with indicated phenotype is in lower right corner. All 3 dpi ventricles showed major ablation, averaging ~90% loss. c, (Left) Schematic for tests of epicardial ablation on muscle regeneration. (Right) Ventricular cardiomyocyte proliferation at 7 dpa. Brackets, injury site. Arrowheads, proliferating cardiomyocytes. d, Quantified PCNA+ cardiomyocyte indices in injury sites in experiments from (c). ***P < 0.001; Mann-Whitney Rank Sum test; n = 18 (wt) and 19 (tcf21:NTR) animals from two experiments. e, Section images of ventricles at 30 dpa, assessed for muscle recovery (MHC) and scar indicators (fibrin, collagen). One of 11 tcf21:nucEGFP and 8 of 12 tcf21:NTR; tcf21:nucEGFP ventricles showed myocardial gaps. Dashed line, approximate resection plane. **P < 0.01; Fisher Irwin exact test. f, Quantified EGFP+ nuclei from experiments in (b). ***P < 0.001; Student's two-tailed t-test. g, (Left) CreER-based strategy for permanent labeling of tcf21+ progeny. (Right) Section images of lineage-labeled EGFP+ epicardial progeny through 14 dpi, indicating derivation from pre-existing epicardium. Arrows at 3 dpi, EGFP+ cells spared by epicardial ablation. h, Quantified EGFP+ cells from experiments in (g). ***P < 0.001; Student's two-tailed t-test; n = 10 (vehicle, 3 dpi), 13 (Mtz, 3 dpi), and 15 (Mtz, 14 dpi). White dashed lines in (b), ventricle. Insets in (c, g), high magnifications of boxed areas. Scale bars, 50 μm. Error bars, s.d.
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Figure 1: Epicardial ablation and regenerationa, Adult zebrafish heart. OFT, outflow tract. b,tcf21:NTR; tcf21:nucEGFP adults were incubated with Mtz or vehicle, and hearts collected by random sampling at 3, 7, or 14 days post-incubation (dpi). Proportion of total animals with indicated phenotype is in lower right corner. All 3 dpi ventricles showed major ablation, averaging ~90% loss. c, (Left) Schematic for tests of epicardial ablation on muscle regeneration. (Right) Ventricular cardiomyocyte proliferation at 7 dpa. Brackets, injury site. Arrowheads, proliferating cardiomyocytes. d, Quantified PCNA+ cardiomyocyte indices in injury sites in experiments from (c). ***P < 0.001; Mann-Whitney Rank Sum test; n = 18 (wt) and 19 (tcf21:NTR) animals from two experiments. e, Section images of ventricles at 30 dpa, assessed for muscle recovery (MHC) and scar indicators (fibrin, collagen). One of 11 tcf21:nucEGFP and 8 of 12 tcf21:NTR; tcf21:nucEGFP ventricles showed myocardial gaps. Dashed line, approximate resection plane. **P < 0.01; Fisher Irwin exact test. f, Quantified EGFP+ nuclei from experiments in (b). ***P < 0.001; Student's two-tailed t-test. g, (Left) CreER-based strategy for permanent labeling of tcf21+ progeny. (Right) Section images of lineage-labeled EGFP+ epicardial progeny through 14 dpi, indicating derivation from pre-existing epicardium. Arrows at 3 dpi, EGFP+ cells spared by epicardial ablation. h, Quantified EGFP+ cells from experiments in (g). ***P < 0.001; Student's two-tailed t-test; n = 10 (vehicle, 3 dpi), 13 (Mtz, 3 dpi), and 15 (Mtz, 14 dpi). White dashed lines in (b), ventricle. Insets in (c, g), high magnifications of boxed areas. Scale bars, 50 μm. Error bars, s.d.

Mentions: To assess homeostatic properties of the epicardium, we employed an inducible cell ablation system in adult zebrafish. Targeted expression of bacterial Nitroreductase (NTR) depletes specific cell types via conversion of a non-toxic substrate metronidazole (Mtz) to a cytotoxin10-12. We used tcf21 regulatory sequences, which in zebrafish drive the most widespread epicardial expression of known DNA elements2, to create an NTR transgenic line for lesioning this tissue without direct myocardial damage. After treatment of adult tcf21:NTR; tcf21:nucEGFP animals with Mtz, ~90% of EGFP+ epicardial nuclei on average were ablated from the ventricular surface in large patches (Fig. 1a, b, f).


Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signalling.

Wang J, Cao J, Dickson AL, Poss KD - Nature (2015)

Epicardial ablation and regenerationa, Adult zebrafish heart. OFT, outflow tract. b,tcf21:NTR; tcf21:nucEGFP adults were incubated with Mtz or vehicle, and hearts collected by random sampling at 3, 7, or 14 days post-incubation (dpi). Proportion of total animals with indicated phenotype is in lower right corner. All 3 dpi ventricles showed major ablation, averaging ~90% loss. c, (Left) Schematic for tests of epicardial ablation on muscle regeneration. (Right) Ventricular cardiomyocyte proliferation at 7 dpa. Brackets, injury site. Arrowheads, proliferating cardiomyocytes. d, Quantified PCNA+ cardiomyocyte indices in injury sites in experiments from (c). ***P < 0.001; Mann-Whitney Rank Sum test; n = 18 (wt) and 19 (tcf21:NTR) animals from two experiments. e, Section images of ventricles at 30 dpa, assessed for muscle recovery (MHC) and scar indicators (fibrin, collagen). One of 11 tcf21:nucEGFP and 8 of 12 tcf21:NTR; tcf21:nucEGFP ventricles showed myocardial gaps. Dashed line, approximate resection plane. **P < 0.01; Fisher Irwin exact test. f, Quantified EGFP+ nuclei from experiments in (b). ***P < 0.001; Student's two-tailed t-test. g, (Left) CreER-based strategy for permanent labeling of tcf21+ progeny. (Right) Section images of lineage-labeled EGFP+ epicardial progeny through 14 dpi, indicating derivation from pre-existing epicardium. Arrows at 3 dpi, EGFP+ cells spared by epicardial ablation. h, Quantified EGFP+ cells from experiments in (g). ***P < 0.001; Student's two-tailed t-test; n = 10 (vehicle, 3 dpi), 13 (Mtz, 3 dpi), and 15 (Mtz, 14 dpi). White dashed lines in (b), ventricle. Insets in (c, g), high magnifications of boxed areas. Scale bars, 50 μm. Error bars, s.d.
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Figure 1: Epicardial ablation and regenerationa, Adult zebrafish heart. OFT, outflow tract. b,tcf21:NTR; tcf21:nucEGFP adults were incubated with Mtz or vehicle, and hearts collected by random sampling at 3, 7, or 14 days post-incubation (dpi). Proportion of total animals with indicated phenotype is in lower right corner. All 3 dpi ventricles showed major ablation, averaging ~90% loss. c, (Left) Schematic for tests of epicardial ablation on muscle regeneration. (Right) Ventricular cardiomyocyte proliferation at 7 dpa. Brackets, injury site. Arrowheads, proliferating cardiomyocytes. d, Quantified PCNA+ cardiomyocyte indices in injury sites in experiments from (c). ***P < 0.001; Mann-Whitney Rank Sum test; n = 18 (wt) and 19 (tcf21:NTR) animals from two experiments. e, Section images of ventricles at 30 dpa, assessed for muscle recovery (MHC) and scar indicators (fibrin, collagen). One of 11 tcf21:nucEGFP and 8 of 12 tcf21:NTR; tcf21:nucEGFP ventricles showed myocardial gaps. Dashed line, approximate resection plane. **P < 0.01; Fisher Irwin exact test. f, Quantified EGFP+ nuclei from experiments in (b). ***P < 0.001; Student's two-tailed t-test. g, (Left) CreER-based strategy for permanent labeling of tcf21+ progeny. (Right) Section images of lineage-labeled EGFP+ epicardial progeny through 14 dpi, indicating derivation from pre-existing epicardium. Arrows at 3 dpi, EGFP+ cells spared by epicardial ablation. h, Quantified EGFP+ cells from experiments in (g). ***P < 0.001; Student's two-tailed t-test; n = 10 (vehicle, 3 dpi), 13 (Mtz, 3 dpi), and 15 (Mtz, 14 dpi). White dashed lines in (b), ventricle. Insets in (c, g), high magnifications of boxed areas. Scale bars, 50 μm. Error bars, s.d.
Mentions: To assess homeostatic properties of the epicardium, we employed an inducible cell ablation system in adult zebrafish. Targeted expression of bacterial Nitroreductase (NTR) depletes specific cell types via conversion of a non-toxic substrate metronidazole (Mtz) to a cytotoxin10-12. We used tcf21 regulatory sequences, which in zebrafish drive the most widespread epicardial expression of known DNA elements2, to create an NTR transgenic line for lesioning this tissue without direct myocardial damage. After treatment of adult tcf21:NTR; tcf21:nucEGFP animals with Mtz, ~90% of EGFP+ epicardial nuclei on average were ablated from the ventricular surface in large patches (Fig. 1a, b, f).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.

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 inflammation. 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.

Show MeSH
Related in: MedlinePlus