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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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Hedgehog ligand expressiona-c, Quantitative RT-PCR revealing shha, ihhb and dhh expression in atrium (a) or ventricle (b) in uninjured hearts and 3 days post-ablation, or in separated ventricular basal (the basal third of the chamber) and apical (the apical third) tissue after ablation (c). Three separate quantitative RT-PCR experiments on pooled tissues were performed, using a total of 90 zebrafish for (a) and (b) experiments, and another 90 fish for (c). shhb and ihha were not detected in these tissues. d, In situ hybridization (ISH) for shha or dhh in wild-type (wt) or tcf21:NTR clutchmate hearts at 3 days post Mtz treatment, indicating expression in outflow tract (OFT) but not ventricle or atrium. OFT of uninjured and epicardially ablated hearts showed comparable shha and dhh signals by ISH, a qualitative/semi-quantitative assay. e, Section of adult shha:EGFP heart, indicating fluorescence in outflow tract tissues. Smooth muscle cells (MLCK, red) and epicardial cells (outer layer) in outflow tract showed clear EGFP signals, while there is no obvious EGFP fluorescence in ventricle and atrium. Valve mesenchyme also displays EGFP fluorescence. Arrowheads, EGFP signals in smooth muscle cells and epicardium. f, Ventricular resection induces shha:EGFP fluorescence in the basal ventricular epicardium at 2 dpa. Arrows, ventricular epicardial fluorescence. White dashed line in (d, e), outflow tract (d) or atrioventricular junction (e). Boxed areas in (d-f), magnified views. Scale bars, 50 μm. Error bars, s.d.
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Figure 14: Hedgehog ligand expressiona-c, Quantitative RT-PCR revealing shha, ihhb and dhh expression in atrium (a) or ventricle (b) in uninjured hearts and 3 days post-ablation, or in separated ventricular basal (the basal third of the chamber) and apical (the apical third) tissue after ablation (c). Three separate quantitative RT-PCR experiments on pooled tissues were performed, using a total of 90 zebrafish for (a) and (b) experiments, and another 90 fish for (c). shhb and ihha were not detected in these tissues. d, In situ hybridization (ISH) for shha or dhh in wild-type (wt) or tcf21:NTR clutchmate hearts at 3 days post Mtz treatment, indicating expression in outflow tract (OFT) but not ventricle or atrium. OFT of uninjured and epicardially ablated hearts showed comparable shha and dhh signals by ISH, a qualitative/semi-quantitative assay. e, Section of adult shha:EGFP heart, indicating fluorescence in outflow tract tissues. Smooth muscle cells (MLCK, red) and epicardial cells (outer layer) in outflow tract showed clear EGFP signals, while there is no obvious EGFP fluorescence in ventricle and atrium. Valve mesenchyme also displays EGFP fluorescence. Arrowheads, EGFP signals in smooth muscle cells and epicardium. f, Ventricular resection induces shha:EGFP fluorescence in the basal ventricular epicardium at 2 dpa. Arrows, ventricular epicardial fluorescence. White dashed line in (d, e), outflow tract (d) or atrioventricular junction (e). Boxed areas in (d-f), magnified views. Scale bars, 50 μm. Error bars, s.d.

Mentions: Smo is an effector for several Hh family ligands, which have potent short-range effects in multiple contexts of embryonic development16-21. Quantitative PCR revealed shha, ihhb and dhh ligand transcripts in adult atrium, ventricle, and BA, where in situ hybridization detected shha and dhh transcript signals in smooth muscle tissue (Extended Data Fig. 10a-d). Epicardial ablation injury boosted BA and ventricular shha levels, as well as levels of ptch1 and gli2a in purified epicardial cells (Fig. 4d, e). Moreover, a shha:EGFP reporter strain visualized shha regulatory sequence-driven fluorescence in smooth muscle and epicardial tissues of the BA (Extended Data Fig. 10e). No additional in situ hybridization or shha:EGFP fluorescence patterns were detectable after epicardial ablation; however, apical resection injury induced fluorescence in ventricular epicardial tissue by 2 dpa (Extended Data Fig. 10d, f). To test whether local Hh ligand delivery is sufficient to substitute for the BA, we removed atrium and BA from cardiac explants, ablated epicardial cells, and applied beads soaked with Shh protein to the exposed ventricular base. Shh-soaked beads stimulated epicardial regeneration (one-half or greater coverage) in 9 of 32 ventricles, whereas this level of recovery never occurred after transplantation of BSA-soaked beads ((0 of 27) ventricles; Fig. 4f). We speculate that these effects of Hh on the epicardial sheet might involve cytoplasmic extensions or a factor transport system22,23. Together, our findings support a model in which Hh ligand from outflow tract, and possibly additional tissues, guides the base-to-apex regeneration of ventricular epicardium.


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

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

Hedgehog ligand expressiona-c, Quantitative RT-PCR revealing shha, ihhb and dhh expression in atrium (a) or ventricle (b) in uninjured hearts and 3 days post-ablation, or in separated ventricular basal (the basal third of the chamber) and apical (the apical third) tissue after ablation (c). Three separate quantitative RT-PCR experiments on pooled tissues were performed, using a total of 90 zebrafish for (a) and (b) experiments, and another 90 fish for (c). shhb and ihha were not detected in these tissues. d, In situ hybridization (ISH) for shha or dhh in wild-type (wt) or tcf21:NTR clutchmate hearts at 3 days post Mtz treatment, indicating expression in outflow tract (OFT) but not ventricle or atrium. OFT of uninjured and epicardially ablated hearts showed comparable shha and dhh signals by ISH, a qualitative/semi-quantitative assay. e, Section of adult shha:EGFP heart, indicating fluorescence in outflow tract tissues. Smooth muscle cells (MLCK, red) and epicardial cells (outer layer) in outflow tract showed clear EGFP signals, while there is no obvious EGFP fluorescence in ventricle and atrium. Valve mesenchyme also displays EGFP fluorescence. Arrowheads, EGFP signals in smooth muscle cells and epicardium. f, Ventricular resection induces shha:EGFP fluorescence in the basal ventricular epicardium at 2 dpa. Arrows, ventricular epicardial fluorescence. White dashed line in (d, e), outflow tract (d) or atrioventricular junction (e). Boxed areas in (d-f), magnified views. Scale bars, 50 μm. Error bars, s.d.
© Copyright Policy - permission
Related In: Results  -  Collection

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Show All Figures
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Figure 14: Hedgehog ligand expressiona-c, Quantitative RT-PCR revealing shha, ihhb and dhh expression in atrium (a) or ventricle (b) in uninjured hearts and 3 days post-ablation, or in separated ventricular basal (the basal third of the chamber) and apical (the apical third) tissue after ablation (c). Three separate quantitative RT-PCR experiments on pooled tissues were performed, using a total of 90 zebrafish for (a) and (b) experiments, and another 90 fish for (c). shhb and ihha were not detected in these tissues. d, In situ hybridization (ISH) for shha or dhh in wild-type (wt) or tcf21:NTR clutchmate hearts at 3 days post Mtz treatment, indicating expression in outflow tract (OFT) but not ventricle or atrium. OFT of uninjured and epicardially ablated hearts showed comparable shha and dhh signals by ISH, a qualitative/semi-quantitative assay. e, Section of adult shha:EGFP heart, indicating fluorescence in outflow tract tissues. Smooth muscle cells (MLCK, red) and epicardial cells (outer layer) in outflow tract showed clear EGFP signals, while there is no obvious EGFP fluorescence in ventricle and atrium. Valve mesenchyme also displays EGFP fluorescence. Arrowheads, EGFP signals in smooth muscle cells and epicardium. f, Ventricular resection induces shha:EGFP fluorescence in the basal ventricular epicardium at 2 dpa. Arrows, ventricular epicardial fluorescence. White dashed line in (d, e), outflow tract (d) or atrioventricular junction (e). Boxed areas in (d-f), magnified views. Scale bars, 50 μm. Error bars, s.d.
Mentions: Smo is an effector for several Hh family ligands, which have potent short-range effects in multiple contexts of embryonic development16-21. Quantitative PCR revealed shha, ihhb and dhh ligand transcripts in adult atrium, ventricle, and BA, where in situ hybridization detected shha and dhh transcript signals in smooth muscle tissue (Extended Data Fig. 10a-d). Epicardial ablation injury boosted BA and ventricular shha levels, as well as levels of ptch1 and gli2a in purified epicardial cells (Fig. 4d, e). Moreover, a shha:EGFP reporter strain visualized shha regulatory sequence-driven fluorescence in smooth muscle and epicardial tissues of the BA (Extended Data Fig. 10e). No additional in situ hybridization or shha:EGFP fluorescence patterns were detectable after epicardial ablation; however, apical resection injury induced fluorescence in ventricular epicardial tissue by 2 dpa (Extended Data Fig. 10d, f). To test whether local Hh ligand delivery is sufficient to substitute for the BA, we removed atrium and BA from cardiac explants, ablated epicardial cells, and applied beads soaked with Shh protein to the exposed ventricular base. Shh-soaked beads stimulated epicardial regeneration (one-half or greater coverage) in 9 of 32 ventricles, whereas this level of recovery never occurred after transplantation of BSA-soaked beads ((0 of 27) ventricles; Fig. 4f). We speculate that these effects of Hh on the epicardial sheet might involve cytoplasmic extensions or a factor transport system22,23. Together, our findings support a model in which Hh ligand from outflow tract, and possibly additional tissues, guides the base-to-apex regeneration of ventricular epicardium.

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