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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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Cardiac outflow tract is required for regeneration of adjacent ventricular epicardiuma, (Top) Schematic for epicardial ablation and regeneration in hearts cultured ex vivo. (Bottom) Regeneration occurs in a base-to-apex direction (arrows). Isolated patches (circled by blue dashed lines) do not participate in regeneration until contacted by the leading edge. b, Ventricular epicardium regenerates in the absence of the atrium (n = 19; behavior seen in all samples). Arrows, direction of regeneration. c, d, Ventricular epicardium fails to regenerate in the absence of the BA. Ventricular epicardium showed defective regeneration in these experiments with (c) (n = 6; all samples) or without atrium (d) (n = 14; all samples), even when, in rare cases, many basal epicardial cells were spared (d). Red dashed lines, epicardium or epicardial leading edge. White dashed lines, ventricle. Scale bars, 50 μm.
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Figure 2: Cardiac outflow tract is required for regeneration of adjacent ventricular epicardiuma, (Top) Schematic for epicardial ablation and regeneration in hearts cultured ex vivo. (Bottom) Regeneration occurs in a base-to-apex direction (arrows). Isolated patches (circled by blue dashed lines) do not participate in regeneration until contacted by the leading edge. b, Ventricular epicardium regenerates in the absence of the atrium (n = 19; behavior seen in all samples). Arrows, direction of regeneration. c, d, Ventricular epicardium fails to regenerate in the absence of the BA. Ventricular epicardium showed defective regeneration in these experiments with (c) (n = 6; all samples) or without atrium (d) (n = 14; all samples), even when, in rare cases, many basal epicardial cells were spared (d). Red dashed lines, epicardium or epicardial leading edge. White dashed lines, ventricle. 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)

Cardiac outflow tract is required for regeneration of adjacent ventricular epicardiuma, (Top) Schematic for epicardial ablation and regeneration in hearts cultured ex vivo. (Bottom) Regeneration occurs in a base-to-apex direction (arrows). Isolated patches (circled by blue dashed lines) do not participate in regeneration until contacted by the leading edge. b, Ventricular epicardium regenerates in the absence of the atrium (n = 19; behavior seen in all samples). Arrows, direction of regeneration. c, d, Ventricular epicardium fails to regenerate in the absence of the BA. Ventricular epicardium showed defective regeneration in these experiments with (c) (n = 6; all samples) or without atrium (d) (n = 14; all samples), even when, in rare cases, many basal epicardial cells were spared (d). Red dashed lines, epicardium or epicardial leading edge. White dashed lines, ventricle. 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 2: Cardiac outflow tract is required for regeneration of adjacent ventricular epicardiuma, (Top) Schematic for epicardial ablation and regeneration in hearts cultured ex vivo. (Bottom) Regeneration occurs in a base-to-apex direction (arrows). Isolated patches (circled by blue dashed lines) do not participate in regeneration until contacted by the leading edge. b, Ventricular epicardium regenerates in the absence of the atrium (n = 19; behavior seen in all samples). Arrows, direction of regeneration. c, d, Ventricular epicardium fails to regenerate in the absence of the BA. Ventricular epicardium showed defective regeneration in these experiments with (c) (n = 6; all samples) or without atrium (d) (n = 14; all samples), even when, in rare cases, many basal epicardial cells were spared (d). Red dashed lines, epicardium or epicardial leading edge. White dashed lines, ventricle. 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