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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 regeneration after ventricular resectiona, Hearts were removed from tcf21:NTR; tcf21:nucEGFP fish immediately after ventricular resection injuries, followed by 24 hours of Mtz and a 24-hour 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; behavior 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. b, Hearts were removed from tcf21:nucEGFP clutchmates immediately after apical resection injury and cultured ex vivo, before random separation two treatment groups. Epicardial cells covered the wound area by 3 dpa (n = 11; behavior seen in all samples), unless treated with CyA (n = 26; failed coverage in 20 of 26 ventricles). Red dashed lines in (a), ventricle. White dashed lines in (a, b), apical wounds. Scale bars, 50 μm.
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Figure 8: Epicardial regeneration after ventricular resectiona, Hearts were removed from tcf21:NTR; tcf21:nucEGFP fish immediately after ventricular resection injuries, followed by 24 hours of Mtz and a 24-hour 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; behavior 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. b, Hearts were removed from tcf21:nucEGFP clutchmates immediately after apical resection injury and cultured ex vivo, before random separation two treatment groups. Epicardial cells covered the wound area by 3 dpa (n = 11; behavior seen in all samples), unless treated with CyA (n = 26; failed coverage in 20 of 26 ventricles). Red dashed lines in (a), ventricle. White dashed lines in (a, b), apical wounds. Scale bars, 50 μm.

Mentions: To expand our range of experimental manipulations, we refined protocols such that freshly dissected hearts contracted for several weeks ex vivo (Supplementary Video 1)14,15. When Mtz was added transiently to culture medium for one day, ventricular epicardial cells were potently ablated. Epicardial layers of the atrium and the BA (alternatively referred to as outflow tract) were less effectively depleted (Fig. 2a), likely due to differential expression of the NTR transgene among cardiac chambers (Extended Data Fig. 3b). Daily imaging of these hearts confirmed observations from in vivo experiments, demonstrating regeneration of the epicardium from base to apex that is typically completed in 2 weeks (Fig. 2a). Hearts from animals given partial ventricular resections injuries in vivo showed a similar pattern of epicardial regeneration after ex vivo ablation (Extended Data Fig. 4a). Cardiac muscle regeneration was ineffective in explanted hearts in our experiments. Increases in cell number occurred concomitantly with movement across the myocardial surface during epicardial regeneration, with spared epicardial cell patches away from the leading edge eventually incorporated into the sheet (Fig. 2a).


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

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

Epicardial regeneration after ventricular resectiona, Hearts were removed from tcf21:NTR; tcf21:nucEGFP fish immediately after ventricular resection injuries, followed by 24 hours of Mtz and a 24-hour 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; behavior 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. b, Hearts were removed from tcf21:nucEGFP clutchmates immediately after apical resection injury and cultured ex vivo, before random separation two treatment groups. Epicardial cells covered the wound area by 3 dpa (n = 11; behavior seen in all samples), unless treated with CyA (n = 26; failed coverage in 20 of 26 ventricles). Red dashed lines in (a), ventricle. White dashed lines in (a, b), apical wounds. Scale bars, 50 μm.
© Copyright Policy - permission
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494087&req=5

Figure 8: Epicardial regeneration after ventricular resectiona, Hearts were removed from tcf21:NTR; tcf21:nucEGFP fish immediately after ventricular resection injuries, followed by 24 hours of Mtz and a 24-hour 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; behavior 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. b, Hearts were removed from tcf21:nucEGFP clutchmates immediately after apical resection injury and cultured ex vivo, before random separation two treatment groups. Epicardial cells covered the wound area by 3 dpa (n = 11; behavior seen in all samples), unless treated with CyA (n = 26; failed coverage in 20 of 26 ventricles). Red dashed lines in (a), ventricle. White dashed lines in (a, b), apical wounds. Scale bars, 50 μm.
Mentions: To expand our range of experimental manipulations, we refined protocols such that freshly dissected hearts contracted for several weeks ex vivo (Supplementary Video 1)14,15. When Mtz was added transiently to culture medium for one day, ventricular epicardial cells were potently ablated. Epicardial layers of the atrium and the BA (alternatively referred to as outflow tract) were less effectively depleted (Fig. 2a), likely due to differential expression of the NTR transgene among cardiac chambers (Extended Data Fig. 3b). Daily imaging of these hearts confirmed observations from in vivo experiments, demonstrating regeneration of the epicardium from base to apex that is typically completed in 2 weeks (Fig. 2a). Hearts from animals given partial ventricular resections injuries in vivo showed a similar pattern of epicardial regeneration after ex vivo ablation (Extended Data Fig. 4a). Cardiac muscle regeneration was ineffective in explanted hearts in our experiments. Increases in cell number occurred concomitantly with movement across the myocardial surface during epicardial regeneration, with spared epicardial cell patches away from the leading edge eventually incorporated into the sheet (Fig. 2a).

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