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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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Larval epicardial development and regenerationa, tcf21:nucEGFP or tcf21:NTR; tcf21:nucEGFP larval clutchmates were treated with 10 mM Mtz from 6 hpf to 54 hpf, and then imaged at different times from 3 to 5 dpf. tcf21:nucEGFP larvae show normal ventricular epicardial coverage at 3 dpf, while tcf21:NTR; tcf21:nucEGFP coverage is sparse. tcf21:NTR; tcf21:nucEGFP larvae with confirmed full ablation were imaged from 3 to 5 dpf, covering first the ventricular base and then the apex. Three different extents of regeneration at 5 dpf are shown: classes I, greater than two-thirds coverage; II, one-third to two-thirds coverage; and III, some cells but less than one-third coverage. b, A subset of tcf21:NTR; tcf21:nucEGFP larvae with confirmed full ablations were randomly separated and treated with vehicle or CyA, which limited regeneration in most cases (class IV, no ventricular epicardial cells). c, Quantification of extents of regeneration from experiments in (a) and (b). ***P < 0.001; Chi-square test; n = 54 embryos for vehicle, 51 for CyA. d, Epicardial morphogenesis visualized in tcf21:nucEGFP larvae. No epicardial cells are evident at or before 2 days post-fertilization (dpf). By 3 dpf, ventricles contained 17.6 +/- 6 epicardial cells on average (n = 23), whereas 4 dpf larvae contained 45.2 +/- 5.8 cells (n = 21). e, tcf21:nucEGFP larval clutchmates were randomly separated into two groups for treatment with vehicle or 5 μM CyA from 2 to 4 dpf. f, Quantification of ventricular EGFP+ epicardial cells from groups in (e). ***P < 0.001; Student's two-tailed t-test; n = 21 for each group. White dashed lines in (a, b, d, e), ventricle. Boxed areas in (d, e), magnified views. Scale bars, 50 μm. Error bars, s.d.
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Figure 13: Larval epicardial development and regenerationa, tcf21:nucEGFP or tcf21:NTR; tcf21:nucEGFP larval clutchmates were treated with 10 mM Mtz from 6 hpf to 54 hpf, and then imaged at different times from 3 to 5 dpf. tcf21:nucEGFP larvae show normal ventricular epicardial coverage at 3 dpf, while tcf21:NTR; tcf21:nucEGFP coverage is sparse. tcf21:NTR; tcf21:nucEGFP larvae with confirmed full ablation were imaged from 3 to 5 dpf, covering first the ventricular base and then the apex. Three different extents of regeneration at 5 dpf are shown: classes I, greater than two-thirds coverage; II, one-third to two-thirds coverage; and III, some cells but less than one-third coverage. b, A subset of tcf21:NTR; tcf21:nucEGFP larvae with confirmed full ablations were randomly separated and treated with vehicle or CyA, which limited regeneration in most cases (class IV, no ventricular epicardial cells). c, Quantification of extents of regeneration from experiments in (a) and (b). ***P < 0.001; Chi-square test; n = 54 embryos for vehicle, 51 for CyA. d, Epicardial morphogenesis visualized in tcf21:nucEGFP larvae. No epicardial cells are evident at or before 2 days post-fertilization (dpf). By 3 dpf, ventricles contained 17.6 +/- 6 epicardial cells on average (n = 23), whereas 4 dpf larvae contained 45.2 +/- 5.8 cells (n = 21). e, tcf21:nucEGFP larval clutchmates were randomly separated into two groups for treatment with vehicle or 5 μM CyA from 2 to 4 dpf. f, Quantification of ventricular EGFP+ epicardial cells from groups in (e). ***P < 0.001; Student's two-tailed t-test; n = 21 for each group. White dashed lines in (a, b, d, e), ventricle. Boxed areas in (d, e), magnified views. Scale bars, 50 μm. Error bars, s.d.

Mentions: What is the molecular nature of interactions between outflow tract and ventricular epicardium? To address this, we applied a small panel of signaling pathway effectors to epicardially ablated hearts cultured ex vivo. Among several compounds (Extended Data Fig. 7), the Smoothened (Smo) antagonist cyclopamine (CyA) blocked regeneration; yet, regeneration initiated normally after drug washout (Fig. 4a). CyA treatment reduced spontaneous epicardial cell EdU incorporation occurring in the first 2 days of explant culture, suggesting that intact Hh signaling promotes epicardial proliferation (Extended Data Fig. 8a, b). CyA also disrupted in vivo epicardial regeneration, not only in adults but in larvae, an additional developmental setting in which we identified base-to-apex regeneration (Fig. 4b, c and Extended Data Fig. 9a-c). CyA treatment from 2 to 4 dpf also reduced the initial epicardial occupancy of the larval ventricle (Extended Data Fig. 9d-f), indicating that epicardial regeneration recapitulates at least one pathway influential in morphogenesis. Finally, we observed inhibitory effects of CyA on the epicardial proliferative response to muscle resection in vivo, and in coverage of these injuries ex vivo (Extended Data Fig. 4b and Extended Data Fig. 8c, d).


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

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

Larval epicardial development and regenerationa, tcf21:nucEGFP or tcf21:NTR; tcf21:nucEGFP larval clutchmates were treated with 10 mM Mtz from 6 hpf to 54 hpf, and then imaged at different times from 3 to 5 dpf. tcf21:nucEGFP larvae show normal ventricular epicardial coverage at 3 dpf, while tcf21:NTR; tcf21:nucEGFP coverage is sparse. tcf21:NTR; tcf21:nucEGFP larvae with confirmed full ablation were imaged from 3 to 5 dpf, covering first the ventricular base and then the apex. Three different extents of regeneration at 5 dpf are shown: classes I, greater than two-thirds coverage; II, one-third to two-thirds coverage; and III, some cells but less than one-third coverage. b, A subset of tcf21:NTR; tcf21:nucEGFP larvae with confirmed full ablations were randomly separated and treated with vehicle or CyA, which limited regeneration in most cases (class IV, no ventricular epicardial cells). c, Quantification of extents of regeneration from experiments in (a) and (b). ***P < 0.001; Chi-square test; n = 54 embryos for vehicle, 51 for CyA. d, Epicardial morphogenesis visualized in tcf21:nucEGFP larvae. No epicardial cells are evident at or before 2 days post-fertilization (dpf). By 3 dpf, ventricles contained 17.6 +/- 6 epicardial cells on average (n = 23), whereas 4 dpf larvae contained 45.2 +/- 5.8 cells (n = 21). e, tcf21:nucEGFP larval clutchmates were randomly separated into two groups for treatment with vehicle or 5 μM CyA from 2 to 4 dpf. f, Quantification of ventricular EGFP+ epicardial cells from groups in (e). ***P < 0.001; Student's two-tailed t-test; n = 21 for each group. White dashed lines in (a, b, d, e), ventricle. Boxed areas in (d, e), magnified views. Scale bars, 50 μm. Error bars, s.d.
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Figure 13: Larval epicardial development and regenerationa, tcf21:nucEGFP or tcf21:NTR; tcf21:nucEGFP larval clutchmates were treated with 10 mM Mtz from 6 hpf to 54 hpf, and then imaged at different times from 3 to 5 dpf. tcf21:nucEGFP larvae show normal ventricular epicardial coverage at 3 dpf, while tcf21:NTR; tcf21:nucEGFP coverage is sparse. tcf21:NTR; tcf21:nucEGFP larvae with confirmed full ablation were imaged from 3 to 5 dpf, covering first the ventricular base and then the apex. Three different extents of regeneration at 5 dpf are shown: classes I, greater than two-thirds coverage; II, one-third to two-thirds coverage; and III, some cells but less than one-third coverage. b, A subset of tcf21:NTR; tcf21:nucEGFP larvae with confirmed full ablations were randomly separated and treated with vehicle or CyA, which limited regeneration in most cases (class IV, no ventricular epicardial cells). c, Quantification of extents of regeneration from experiments in (a) and (b). ***P < 0.001; Chi-square test; n = 54 embryos for vehicle, 51 for CyA. d, Epicardial morphogenesis visualized in tcf21:nucEGFP larvae. No epicardial cells are evident at or before 2 days post-fertilization (dpf). By 3 dpf, ventricles contained 17.6 +/- 6 epicardial cells on average (n = 23), whereas 4 dpf larvae contained 45.2 +/- 5.8 cells (n = 21). e, tcf21:nucEGFP larval clutchmates were randomly separated into two groups for treatment with vehicle or 5 μM CyA from 2 to 4 dpf. f, Quantification of ventricular EGFP+ epicardial cells from groups in (e). ***P < 0.001; Student's two-tailed t-test; n = 21 for each group. White dashed lines in (a, b, d, e), ventricle. Boxed areas in (d, e), magnified views. Scale bars, 50 μm. Error bars, s.d.
Mentions: What is the molecular nature of interactions between outflow tract and ventricular epicardium? To address this, we applied a small panel of signaling pathway effectors to epicardially ablated hearts cultured ex vivo. Among several compounds (Extended Data Fig. 7), the Smoothened (Smo) antagonist cyclopamine (CyA) blocked regeneration; yet, regeneration initiated normally after drug washout (Fig. 4a). CyA treatment reduced spontaneous epicardial cell EdU incorporation occurring in the first 2 days of explant culture, suggesting that intact Hh signaling promotes epicardial proliferation (Extended Data Fig. 8a, b). CyA also disrupted in vivo epicardial regeneration, not only in adults but in larvae, an additional developmental setting in which we identified base-to-apex regeneration (Fig. 4b, c and Extended Data Fig. 9a-c). CyA treatment from 2 to 4 dpf also reduced the initial epicardial occupancy of the larval ventricle (Extended Data Fig. 9d-f), indicating that epicardial regeneration recapitulates at least one pathway influential in morphogenesis. Finally, we observed inhibitory effects of CyA on the epicardial proliferative response to muscle resection in vivo, and in coverage of these injuries ex vivo (Extended Data Fig. 4b and Extended Data Fig. 8c, d).

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