Limits...
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.

Show MeSH

Related in: MedlinePlus

Epicardial ablation and responses to resection injurya,tcf21:nucEGFP or tcf21:NTR; tcf21:nucEGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz, before collection of ventricles at 7 (n = 8 animals per group) and 30 dpa (n = 11). tcf21:NTR; tcf21:nucEGFP wounds had less epicardial cells at 7 dpa and comparable occupancy by 30 dpa compared to tcf21:nucEGFP wounds. White dashed lines, wound edge. b, Quantification of EGFP+ epicardial cells at 7 and 30 dpa with respect to wound edge lengths from (a). NS, not significant; Mann-Whitney Rank Sum Test. c, tcf21:NTR clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. MLCK+ cells indicating myofibroblasts had comparable presence in both groups at 14 dpa (n = 7 for each group). d,tcf21:NTR; fli1a:EGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. Epicardial ablation led to lower vascular density in 30 dpa wounds compared with controls. Red dashed lines, wound edge. e, Quantification of EGFP+ vascular endothelial pixel area in wounds of tcf21:NTR; fli1a:EGFP zebrafish treated with Mtz (n = 12) or vehicle (n = 6), or of fli1a:EGFP zebrafish treated with Mtz (n = 6), with respect to the wound edge lengths. Student's two-tailed t-test. f, By 60 dpa, 55 days after epicardial ablation protocols, muscularization (top) and wound collagen deposition (bottom) were grossly normal (n = 23). Brackets, area of regeneration. Yellow dashed lines in (a, c, d), approximate amputation plane. Scale bars, 50 μm. Error bars, s.d.
© Copyright Policy - permission
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494087&req=5

Figure 5: Epicardial ablation and responses to resection injurya,tcf21:nucEGFP or tcf21:NTR; tcf21:nucEGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz, before collection of ventricles at 7 (n = 8 animals per group) and 30 dpa (n = 11). tcf21:NTR; tcf21:nucEGFP wounds had less epicardial cells at 7 dpa and comparable occupancy by 30 dpa compared to tcf21:nucEGFP wounds. White dashed lines, wound edge. b, Quantification of EGFP+ epicardial cells at 7 and 30 dpa with respect to wound edge lengths from (a). NS, not significant; Mann-Whitney Rank Sum Test. c, tcf21:NTR clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. MLCK+ cells indicating myofibroblasts had comparable presence in both groups at 14 dpa (n = 7 for each group). d,tcf21:NTR; fli1a:EGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. Epicardial ablation led to lower vascular density in 30 dpa wounds compared with controls. Red dashed lines, wound edge. e, Quantification of EGFP+ vascular endothelial pixel area in wounds of tcf21:NTR; fli1a:EGFP zebrafish treated with Mtz (n = 12) or vehicle (n = 6), or of fli1a:EGFP zebrafish treated with Mtz (n = 6), with respect to the wound edge lengths. Student's two-tailed t-test. f, By 60 dpa, 55 days after epicardial ablation protocols, muscularization (top) and wound collagen deposition (bottom) were grossly normal (n = 23). Brackets, area of regeneration. Yellow dashed lines in (a, c, d), approximate amputation plane. Scale bars, 50 μm. Error bars, s.d.

Mentions: To determine whether epicardial depletion impacts the well-documented capacity of the zebrafish heart to regenerate13, we transiently incubated tcf21:NTR zebrafish with Mtz after resection of the ventricular apex. Mtz treatment reduced epicardial cell number in the 7 days post-amputation (dpa) injury site by ~45%, while reducing cardiomyocyte proliferation indices by ~33% (Fig. 1c, d, Extended Data Figs. 1a, b and 3c). Myofibroblasts were represented similarly in vehicle- and Mtz-treated clutchmates by 14 dpa (Extended Data Fig. 1c). Injured ventricles of Mtz-treated animals displayed reduced vascularization and muscularization by 30 dpa (Fig. 1e and Extended Data Fig. 1d, e), associated with fibrin and collagen retention (Fig. 1e). By 60 dpa, ventricles from Mtz-treated animals consistently showed normal muscularization and a large complement of tcf21-positive cells, along with minor collagen deposits (Extended Data Fig. 1f). Thus, depletion of epicardial tissue inhibits cardiomyocyte proliferation and vascularization after resection injury, reducing the efficacy of heart regeneration.


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

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

Epicardial ablation and responses to resection injurya,tcf21:nucEGFP or tcf21:NTR; tcf21:nucEGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz, before collection of ventricles at 7 (n = 8 animals per group) and 30 dpa (n = 11). tcf21:NTR; tcf21:nucEGFP wounds had less epicardial cells at 7 dpa and comparable occupancy by 30 dpa compared to tcf21:nucEGFP wounds. White dashed lines, wound edge. b, Quantification of EGFP+ epicardial cells at 7 and 30 dpa with respect to wound edge lengths from (a). NS, not significant; Mann-Whitney Rank Sum Test. c, tcf21:NTR clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. MLCK+ cells indicating myofibroblasts had comparable presence in both groups at 14 dpa (n = 7 for each group). d,tcf21:NTR; fli1a:EGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. Epicardial ablation led to lower vascular density in 30 dpa wounds compared with controls. Red dashed lines, wound edge. e, Quantification of EGFP+ vascular endothelial pixel area in wounds of tcf21:NTR; fli1a:EGFP zebrafish treated with Mtz (n = 12) or vehicle (n = 6), or of fli1a:EGFP zebrafish treated with Mtz (n = 6), with respect to the wound edge lengths. Student's two-tailed t-test. f, By 60 dpa, 55 days after epicardial ablation protocols, muscularization (top) and wound collagen deposition (bottom) were grossly normal (n = 23). Brackets, area of regeneration. Yellow dashed lines in (a, c, d), approximate amputation plane. Scale bars, 50 μm. Error bars, s.d.
© Copyright Policy - permission
Related In: Results  -  Collection

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

Figure 5: Epicardial ablation and responses to resection injurya,tcf21:nucEGFP or tcf21:NTR; tcf21:nucEGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz, before collection of ventricles at 7 (n = 8 animals per group) and 30 dpa (n = 11). tcf21:NTR; tcf21:nucEGFP wounds had less epicardial cells at 7 dpa and comparable occupancy by 30 dpa compared to tcf21:nucEGFP wounds. White dashed lines, wound edge. b, Quantification of EGFP+ epicardial cells at 7 and 30 dpa with respect to wound edge lengths from (a). NS, not significant; Mann-Whitney Rank Sum Test. c, tcf21:NTR clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. MLCK+ cells indicating myofibroblasts had comparable presence in both groups at 14 dpa (n = 7 for each group). d,tcf21:NTR; fli1a:EGFP clutchmates underwent resection injury and were treated for 3 days with 1 mM Mtz or vehicle with random separation into two groups for treatment. Epicardial ablation led to lower vascular density in 30 dpa wounds compared with controls. Red dashed lines, wound edge. e, Quantification of EGFP+ vascular endothelial pixel area in wounds of tcf21:NTR; fli1a:EGFP zebrafish treated with Mtz (n = 12) or vehicle (n = 6), or of fli1a:EGFP zebrafish treated with Mtz (n = 6), with respect to the wound edge lengths. Student's two-tailed t-test. f, By 60 dpa, 55 days after epicardial ablation protocols, muscularization (top) and wound collagen deposition (bottom) were grossly normal (n = 23). Brackets, area of regeneration. Yellow dashed lines in (a, c, d), approximate amputation plane. Scale bars, 50 μm. Error bars, s.d.
Mentions: To determine whether epicardial depletion impacts the well-documented capacity of the zebrafish heart to regenerate13, we transiently incubated tcf21:NTR zebrafish with Mtz after resection of the ventricular apex. Mtz treatment reduced epicardial cell number in the 7 days post-amputation (dpa) injury site by ~45%, while reducing cardiomyocyte proliferation indices by ~33% (Fig. 1c, d, Extended Data Figs. 1a, b and 3c). Myofibroblasts were represented similarly in vehicle- and Mtz-treated clutchmates by 14 dpa (Extended Data Fig. 1c). Injured ventricles of Mtz-treated animals displayed reduced vascularization and muscularization by 30 dpa (Fig. 1e and Extended Data Fig. 1d, e), associated with fibrin and collagen retention (Fig. 1e). By 60 dpa, ventricles from Mtz-treated animals consistently showed normal muscularization and a large complement of tcf21-positive cells, along with minor collagen deposits (Extended Data Fig. 1f). Thus, depletion of epicardial tissue inhibits cardiomyocyte proliferation and vascularization after resection injury, reducing the efficacy of heart regeneration.

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