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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 cell proliferation without injury and after epicardial ablationa, Limited epicardial cell proliferation on the ventricular surface. tcf21:nucEGFP fish were injected with 10 mM EdU once daily for 3 days and collected one 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 labeling). 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 hours later. Boxed areas in images of whole-mounted hearts show magnified views. Yellow arrows in (a, b), 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 hours later. Red arrows, 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.
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Figure 6: Epicardial cell proliferation without injury and after epicardial ablationa, Limited epicardial cell proliferation on the ventricular surface. tcf21:nucEGFP fish were injected with 10 mM EdU once daily for 3 days and collected one 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 labeling). 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 hours later. Boxed areas in images of whole-mounted hearts show magnified views. Yellow arrows in (a, b), 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 hours later. Red arrows, 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.

Mentions: These experiments suggested a high capacity of epicardial cells to regenerate after major depletion. To test this directly, we examined otherwise uninjured hearts at different times after epicardial ablation. Ventricular epicardial cells typically have a low proliferation index (Extended Data Fig. 2a). However, within 3 days of Mtz treatment (3 dpi), many spared epicardial cells entered the cell cycle (Extended Data Fig. 2b, c). At 7 dpi, ventricles displayed quantifiable epicardial recovery that was more prominent at the chamber base (Fig. 1b). By 14 dpi, and as early as 7 dpi, ventricles were fully covered to their apices with tcf21:nucEGFP+ epicardial cells (Fig. 1b, f). The temporal variation in recovery likely reflects variation in location/pattern of epicardial cells spared by ablation among clutchmates, or in chamber size (Extended Data Fig. 3a). To examine origins of regenerated epicardium, we employed inducible Cre-based genetic fate-mapping to permanently label tcf21-expressing cells and their progeny prior to injury2. Labeling and subsequent fate-mapping experiments indicated that pre-existing epicardial cells, and not a tcf21-negative precursor, are a primary source for regeneration (Fig. 1g, h). In total, these experiments reveal that adult epicardium regenerates after substantial genetic ablation, through a mechanism of expansion by spared epicardial cells.


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

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

Epicardial cell proliferation without injury and after epicardial ablationa, Limited epicardial cell proliferation on the ventricular surface. tcf21:nucEGFP fish were injected with 10 mM EdU once daily for 3 days and collected one 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 labeling). 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 hours later. Boxed areas in images of whole-mounted hearts show magnified views. Yellow arrows in (a, b), 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 hours later. Red arrows, 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.
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Figure 6: Epicardial cell proliferation without injury and after epicardial ablationa, Limited epicardial cell proliferation on the ventricular surface. tcf21:nucEGFP fish were injected with 10 mM EdU once daily for 3 days and collected one 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 labeling). 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 hours later. Boxed areas in images of whole-mounted hearts show magnified views. Yellow arrows in (a, b), 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 hours later. Red arrows, 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.
Mentions: These experiments suggested a high capacity of epicardial cells to regenerate after major depletion. To test this directly, we examined otherwise uninjured hearts at different times after epicardial ablation. Ventricular epicardial cells typically have a low proliferation index (Extended Data Fig. 2a). However, within 3 days of Mtz treatment (3 dpi), many spared epicardial cells entered the cell cycle (Extended Data Fig. 2b, c). At 7 dpi, ventricles displayed quantifiable epicardial recovery that was more prominent at the chamber base (Fig. 1b). By 14 dpi, and as early as 7 dpi, ventricles were fully covered to their apices with tcf21:nucEGFP+ epicardial cells (Fig. 1b, f). The temporal variation in recovery likely reflects variation in location/pattern of epicardial cells spared by ablation among clutchmates, or in chamber size (Extended Data Fig. 3a). To examine origins of regenerated epicardium, we employed inducible Cre-based genetic fate-mapping to permanently label tcf21-expressing cells and their progeny prior to injury2. Labeling and subsequent fate-mapping experiments indicated that pre-existing epicardial cells, and not a tcf21-negative precursor, are a primary source for regeneration (Fig. 1g, h). In total, these experiments reveal that adult epicardium regenerates after substantial genetic ablation, through a mechanism of expansion by spared 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