Abstract
While the adult human heart has very limited regenerative potential, the adult zebrafish heart can fully regenerate after 20% ventricular resection. Although previous reports suggest that developmental signaling pathways such as FGF and PDGF are reused in adult heart regeneration, the underlying intracellular mechanisms remain largely unknown. Here we show that H2O2 acts as a novel epicardial and myocardial signal to prime the heart for regeneration in adult zebrafish. Live imaging of intact hearts revealed highly localized H2O2 (∼30 μM) production in the epicardium and adjacent compact myocardium at the resection site. Decreasing H2O2 formation with the Duox inhibitors diphenyleneiodonium (DPI) or apocynin, or scavenging H2O2 by catalase overexpression markedly impaired cardiac regeneration while exogenous H2O2 rescued the inhibitory effects of DPI on cardiac regeneration, indicating that H2O2 is an essential and sufficient signal in this process. Mechanistically, elevated H2O2 destabilized the redox-sensitive phosphatase Dusp6 and hence increased the phosphorylation of Erk1/2. The Dusp6 inhibitor BCI achieved similar pro-regenerative effects while transgenic overexpression of dusp6 impaired cardiac regeneration. H2O2 plays a dual role in recruiting immune cells and promoting heart regeneration through two relatively independent pathways. We conclude that H2O2 potentially generated from Duox/Nox2 promotes heart regeneration in zebrafish by unleashing MAP kinase signaling through a derepression mechanism involving Dusp6.
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Acknowledgements
We thank Drs RP Xiao, Y Rao, JR Peng, C Wei, K Ouyang, YM Wang, ZL Huang and JC Luo for critical comments on the manuscript, Dr IC Bruce for language editing, Dr Yingchun Hu for help on transmission electromicroscopy, and the Pathology Core of Institute of Molecular Medicine for histology. We also thank Drs J Wang and KD Poss for their kind help in performing in situ hybridization with duox probes on Tg(tcf21:nucEGFP) ventricles and Drs M Tsang, ZL Wen, T Evans, ZX Wang, LF Luo, GC Burns, J Chen, CM Cao and F Serluca for generous gifts of protocols, plasmid clones and/or transgenic zebrafish lines. This work was supported by the National Basic Research Program of China (2012CB944501, 2010CB529503 and 2013CB531200) and the National Natural Science Foundation of China (31271549, 31130067, 31000644 and 31221002).
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Supplementary information
Supplementary information, Figure S1 (download PDF )
Strand-specific RNA-Seq of sham and 7 dpa adult hearts. (PDF 132 kb)
Supplementary information, Figure S2 (download PDF )
Scatter plot showing expression values of Ensembl genes in sham and 7dpa hearts. (PDF 90 kb)
Supplementary information, Figure S3 (download PDF )
Duox expression during heart regeneration. (PDF 251 kb)
Supplementary information, Figure S4 (download PDF )
The sense duox probe 1 detected no signals during heart regeneration. (PDF 178 kb)
Supplementary information, Figure S5 (download PDF )
Duox expression in the wound area during heart regeneration. (PDF 230 kb)
Supplementary information, Figure S6 (download PDF )
Partial overlapping of duox with epicardial Tg(tcf21:nucEGFP) during heart regeneration. (PDF 42 kb)
Supplementary information, Figure S7 (download PDF )
Nox2, but not nox1, nox4 and nox5, was expressed during heart regeneration. (PDF 466 kb)
Supplementary information, Figure S8 (download PDF )
Ex vivo cultured zebrafish hearts are functionally intact. (PDF 87 kb)
Supplementary information, Figure S9 (download PDF )
(A-G) Ex vivo calibration of Hyper F488/F405 ratio as a function of ambient H2O2 concentration. (PDF 299 kb)
Supplementary information, Figure S10 (download PDF )
Exogenous H2O2 do not stimulate myocyte proliferation in adult hearts. (PDF 50 kb)
Supplementary information, Figure S11 (download PDF )
H2O2 rescues heart regeneration in the presence of DPI. (PDF 566 kb)
Supplementary information, Figure S12 (download PDF )
DPI irreversibly inhibited heart regeneration. (PDF 347 kb)
Supplementary information, Figure S13 (download PDF )
Exogenous H2O2 rescued, while transgenic over-expression of catalase-DsRed suppressed, embryonic heart regeneration. (PDF 219 kb)
Supplementary information, Figure S14 (download PDF )
H2O2 penetrate freely into zebrafish cardiac tissue. (PDF 98 kb)
Supplementary information, Figure S15 (download PDF )
Inhibiting H2O2 signaling has no effect on cardiac sarcomere disassembly during heart regeneration. (PDF 903 kb)
Supplementary information, Figure S16 (download PDF )
pErk are induced in non-myocardium during heart regeneration. (PDF 130 kb)
Supplementary information, Figure S17 (download PDF )
Total Erk mRNA are slightly induced during heart regeneration. (PDF 40 kb)
Supplementary information, Figure S18 (download PDF )
Both pErk and Dusp6 are induced in the epicardium during heart regeneration. (PDF 360 kb)
Supplementary information, Figure S19 (download PDF )
Dusp6 expression after ventricular resection. (PDF 371 kb)
Supplementary information, Figure S20 (download PDF )
Dusp6 are induced primarily in non-myocardium during heart regeneration. (PDF 333 kb)
Supplementary information, Figure S21 (download PDF )
Inhibiting pErk by U0126 decreases Dusp6 expression during heart regeneration. (PDF 160 kb)
Supplementary information, Figure S22 (download PDF )
H2O2 is essential for coronary vessel formation during heart regeneration. (PDF 206 kb)
Supplementary information, Figure S23 (download PDF )
BCI does not stimulate myocyte proliferation in adult hearts. (PDF 42 kb)
Supplementary information, Figure S24 (download PDF )
BCI rescues heart regeneration when H2O2 signal is defective. (PDF 582 kb)
Supplementary information, Figure S25 (download PDF )
Quantification of DPI or Apo effects on Lcp1+ leukocytes during heart regeneration in Figure 6. (PDF 66 kb)
Supplementary information, Figure S26 (download PDF )
H2O2, but not dusp6, is required for Tg(coro1a:EGFP) leukocyte recruitment during heart regeneration. (PDF 603 kb)
Supplementary information, Table S1 (download XLSX )
Up-regulated genes in 7 dpa hearts by RNA-Seq. (XLSX 98 kb)
Supplementary information, Table S2 (download XLSX )
The KEGG enrichment pathways in 7 dpa hearts. (XLSX 9 kb)
Supplementary information, Table S3 (download XLSX )
The immune system disease genes enriched in 7 dpa hearts. (XLSX 10 kb)
Supplementary information, Table S4 (download XLSX )
A number of ROS genes are up- or down-regulated in 7 dpa hearts. (XLSX 17 kb)
Supplementary information, Table S5 (download PDF )
Gene-specific primer sequences (PDF 14 kb)
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Han, P., Zhou, XH., Chang, N. et al. Hydrogen peroxide primes heart regeneration with a derepression mechanism. Cell Res 24, 1091–1107 (2014). https://doi.org/10.1038/cr.2014.108
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DOI: https://doi.org/10.1038/cr.2014.108
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