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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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Ex vivo grafts and epicardial regenerationa-c, (a) Schematic of the experimental design. Epicardial cells transplanted at the base of a an epicardially ablated host regenerated toward the apex regardless of basal (b) (n = 25, behavior 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 toward the apex, not the base (n = 13; all samples). e, (Top) Following epicardial ablation, the host BA 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; behavior seen in all samples). f, (Left) Following ex vivo epicardial ablation in a host tcf21:NTR ventricle, the host BA was replaced with a donor tcf21:nucEGFP BA. (Right) The host ventricular surface contained different amounts of EGFP+ nuclei in these ventricles (n = 3; behavior seen in all samples). Red dashed lines in (b-f), epicardium or epicardial leading edge. White dashed lines in (b-f), ventricle. Yellow dashed lines in (e, f), donor apex (d) or BA (f). Scale bars, 50 μm.
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Figure 9: Ex vivo grafts and epicardial regenerationa-c, (a) Schematic of the experimental design. Epicardial cells transplanted at the base of a an epicardially ablated host regenerated toward the apex regardless of basal (b) (n = 25, behavior 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 toward the apex, not the base (n = 13; all samples). e, (Top) Following epicardial ablation, the host BA 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; behavior seen in all samples). f, (Left) Following ex vivo epicardial ablation in a host tcf21:NTR ventricle, the host BA was replaced with a donor tcf21:nucEGFP BA. (Right) The host ventricular surface contained different amounts of EGFP+ nuclei in these ventricles (n = 3; behavior seen in all samples). Red dashed lines in (b-f), epicardium or epicardial leading edge. White dashed lines in (b-f), ventricle. Yellow dashed lines in (e, f), donor apex (d) or BA (f). Scale bars, 50 μm.

Mentions: To identify possible intrinsic differences in epicardial cells from different ventricular regions, we examined behaviors of basal or apical epicardial tissue patches transplanted to ablated ventricles. In these experiments, transplanted cells of either origin consistently repopulated the ventricular surface in a base-to-apex direction after transplantation (Extended Data Fig. 5a-d), revealing no proliferative bias in ventricular epicardial cells that could explain the directional flow of regeneration. To assess potential extrinsic influences on epicardial regeneration, we removed the atrium or BA from its attachment at the ventricular base prior to epicardial ablation. Atrial extirpation did not noticeably affect regeneration of ventricular epicardium (Fig. 2b and Supplementary Video 2). By contrast, removal of outflow tissue blocked epicardial cell recovery, an arrest that persisted for at least two weeks (Fig. 2c, d and data not shown). To test whether this arrest was solely a consequence of mechanical tissue disruption, we ablated the epicardium after host BA removal, before grafting a non-transgenic BA to the ventricular base 2 days later. In most of these tissue recombination procedures (13 of 21), host tcf21:nucEGFP+ epicardium regenerated to cover the ventricle (Fig. 3a). This effect was not observed when a portion of donor ventricular apex was inverted and transplanted to the host ventricular base (Extended Data Fig. 5e). Complementary grafting experiments indicated that BA could contribute epicardial cells to the ventricular surface, as a potential supplement to expansion of the ventricular epicardial cell pool (Extended Data Fig. 5f). Thus, our experiments indicate that outflow tissue provides an essential interaction for regeneration from existing ventricular epicardial cells.


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

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

Ex vivo grafts and epicardial regenerationa-c, (a) Schematic of the experimental design. Epicardial cells transplanted at the base of a an epicardially ablated host regenerated toward the apex regardless of basal (b) (n = 25, behavior 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 toward the apex, not the base (n = 13; all samples). e, (Top) Following epicardial ablation, the host BA 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; behavior seen in all samples). f, (Left) Following ex vivo epicardial ablation in a host tcf21:NTR ventricle, the host BA was replaced with a donor tcf21:nucEGFP BA. (Right) The host ventricular surface contained different amounts of EGFP+ nuclei in these ventricles (n = 3; behavior seen in all samples). Red dashed lines in (b-f), epicardium or epicardial leading edge. White dashed lines in (b-f), ventricle. Yellow dashed lines in (e, f), donor apex (d) or BA (f). Scale bars, 50 μm.
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Related In: Results  -  Collection

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Figure 9: Ex vivo grafts and epicardial regenerationa-c, (a) Schematic of the experimental design. Epicardial cells transplanted at the base of a an epicardially ablated host regenerated toward the apex regardless of basal (b) (n = 25, behavior 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 toward the apex, not the base (n = 13; all samples). e, (Top) Following epicardial ablation, the host BA 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; behavior seen in all samples). f, (Left) Following ex vivo epicardial ablation in a host tcf21:NTR ventricle, the host BA was replaced with a donor tcf21:nucEGFP BA. (Right) The host ventricular surface contained different amounts of EGFP+ nuclei in these ventricles (n = 3; behavior seen in all samples). Red dashed lines in (b-f), epicardium or epicardial leading edge. White dashed lines in (b-f), ventricle. Yellow dashed lines in (e, f), donor apex (d) or BA (f). Scale bars, 50 μm.
Mentions: To identify possible intrinsic differences in epicardial cells from different ventricular regions, we examined behaviors of basal or apical epicardial tissue patches transplanted to ablated ventricles. In these experiments, transplanted cells of either origin consistently repopulated the ventricular surface in a base-to-apex direction after transplantation (Extended Data Fig. 5a-d), revealing no proliferative bias in ventricular epicardial cells that could explain the directional flow of regeneration. To assess potential extrinsic influences on epicardial regeneration, we removed the atrium or BA from its attachment at the ventricular base prior to epicardial ablation. Atrial extirpation did not noticeably affect regeneration of ventricular epicardium (Fig. 2b and Supplementary Video 2). By contrast, removal of outflow tissue blocked epicardial cell recovery, an arrest that persisted for at least two weeks (Fig. 2c, d and data not shown). To test whether this arrest was solely a consequence of mechanical tissue disruption, we ablated the epicardium after host BA removal, before grafting a non-transgenic BA to the ventricular base 2 days later. In most of these tissue recombination procedures (13 of 21), host tcf21:nucEGFP+ epicardium regenerated to cover the ventricle (Fig. 3a). This effect was not observed when a portion of donor ventricular apex was inverted and transplanted to the host ventricular base (Extended Data Fig. 5e). Complementary grafting experiments indicated that BA could contribute epicardial cells to the ventricular surface, as a potential supplement to expansion of the ventricular epicardial cell pool (Extended Data Fig. 5f). Thus, our experiments indicate that outflow tissue provides an essential interaction for regeneration from existing ventricular epicardial cells.

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