Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jun 2;534(7605):119-23.
doi: 10.1038/nature17959. Epub 2016 May 25.

Pitx2 promotes heart repair by activating the antioxidant response after cardiac injury

Ge Tao  1 , Peter C Kahr  1 , Yuka Morikawa  2 , Min Zhang  1 , Mahdis Rahmani  2 , Todd R Heallen  2 , Lele Li  1 , Zhao Sun  3 , Eric N Olson  4 , Brad A Amendt  3 , James F Martin  1   2   5   6
Affiliations

Pitx2 promotes heart repair by activating the antioxidant response after cardiac injury

Ge Tao et al. Nature. .

Abstract

Myocardial infarction results in compromised myocardial function and heart failure owing to insufficient cardiomyocyte self-renewal. Unlike many vertebrates, mammalian hearts have only a transient neonatal renewal capacity. Reactivating primitive reparative ability in the mature mammalian heart requires knowledge of the mechanisms that promote early heart repair. By testing an established Hippo-deficient heart regeneration mouse model for factors that promote renewal, here we show that the expression of Pitx2 is induced in injured, Hippo-deficient ventricles. Pitx2-deficient neonatal mouse hearts failed to repair after apex resection, whereas adult mouse cardiomyocytes with Pitx2 gain-of-function efficiently regenerated after myocardial infarction. Genomic analyses indicated that Pitx2 activated genes encoding electron transport chain components and reactive oxygen species scavengers. A subset of Pitx2 target genes was cooperatively regulated with the Hippo pathway effector Yap. Furthermore, Nrf2, a regulator of the antioxidant response, directly regulated the expression and subcellular localization of Pitx2. Pitx2 mutant myocardium had increased levels of reactive oxygen species, while antioxidant supplementation suppressed the Pitx2 loss-of-function phenotype. These findings reveal a genetic pathway activated by tissue damage that is essential for cardiac repair.

PubMed Disclaimer

Figures

Extended Data Figure 1
Extended Data Figure 1
Pitx2 is required in neonatal myocardial regeneration after LAD-O. (a) Serial trichrome images of control (Pitx2f/f), MCKcre;Pitx2f/f, and Mhccre-Ert;Pitx2f/f 21 days after LAD-O at P2. (b) Percentage of fibrotic left ventricular myocardium quantified at 3 weeks post-LAD-O, n=4. (c, d) Ejection fraction (c) and fractional shortening (d) of LAD-O and sham hearts. L, left ventricle; R, right ventricle. Mean ± S.E.M.; Statistical test, (c–d) one-way ANOVA plus Bonferroni post-test; (b) Mann-Whitney. *, p<0.05; NS, not significant.
Extended Data Figure 2
Extended Data Figure 2
Pitx2 promotes myocardial regeneration after apex resection at P8. (a) Schematic of Pitx2-expressing construct (Pitx2Gof). (b–d) Pitx2Gof was crossed with Mhccre-Ert strain to generate Mhccre-Ert/+;pitx2Gof (Pitx2OE), after Tamoxifen treatment, qPCR (b, n=4), western (c, d, n=3) showed the over-expression of Pitx2 in Pitx2OE ventricles. (e–f) Trichrome-stained cross sections from 13 weeks old sham hearts of control (e) and Pitx2OE (f), with tamoxifen administrated at 7–8 weeks old. (g) Heart weight over body weight ratio of adult sham and LAD-O hearts. (h–j) Apex resection of Pitx2OE (i) and control (Mhccre-Ert/+) (h) hearts at P8 followed by trichrome staining at 28 DPR, scar area was quantified in j. (k, l) Echocardiography showed ejection fraction (k) and fractional shortening (l) at 28 DPR. (m–o) EdU labeling of Pitx2OE (n) and control (m) apical area, 8 days after P8 resection, sections were stained for cTnT (green), EdU (yellow), and DAPI (blue). Arrow indicates EdU-labeled cardiomyocytes, with quantification in o, n=4. Mean ± S.E.M.; Statistical test, (g, k, l) one-way ANOVA plus Bonferroni post-test; (b, d, j, o) Mann-Whitney. *, p<0.05; NS, not significant.
Extended Data Figure 3
Extended Data Figure 3
Pitx2 is required for Hippo deficient heart regeneration. (a) Schematic study plan for figure 3a–e. (b–e) Trichrome-stained apical areas of control (b), SalvCKO (c) and DKO (d) hearts 21 days after P8 apex resection. Scar area was quantified in e. (f) Heart weight to body weight ratio of sham hearts at 28 days after tamoxifen administration. N number, see Methods. Mean ± S.E.M.; Statistical test, (e, f) Mann-Whitney. *, p<0.05; NS, not significant.
Extended Data Figure 4
Extended Data Figure 4
Co-occurrence of Pitx2 and Tead DNA-binding motifs in fetal heart enhancers. (a) Consensus Pitx2 and Tead motifs. (b) Pitx2 and Tead motif co-occurrence in fetal heart Dnase I Hypersensitive (DHS) peaks. (c) Aggregate plot of H3K4me1 in fetal heart ChIP-Seq reads within 6 kb range of DHS peaks. (d) Heat-map of fetal heart H3K4me1 ChIP-Seq or input read density in 6 kb regions of DHS peaks. DHS peaks were centered on Pitx2 motif, Tead motif, Pitx2-Tead motifs, or randomly selected. The read density was in log2 scale; negative values, blue color; positive values, yellow color.
Extended Data Figure 5
Extended Data Figure 5
Generation of GST-tagged proteins and interaction between Pitx2 and Yap in vivo. (a) The mouse Pitx2a, Pitx2c, and truncated proteins were purified and run on 10% SDS-gel, coomassie blue staining showed the GST fusion protein band with correct size (marked by *). (b) Coomassie blue staining of the purified GST-Yap, Yap cut by prescission protease and pure Yap protein. (c) Co-IP of Flag in Pitx2flag ventricles at 5 DPR, and blotting of Yap, Pitx2, and Flag.
Extended Data Figure 6
Extended Data Figure 6
Nuclei-shuttling of Nrf2 is independent of Pitx2. (a) Blotting of Pitx2, α-Tubulin, and TATA-binding protein (TBP) of P19 cell fraction after H2O2, with or without Nrf2 siRNA treatment. (b) Immunofluorescent staining of Nrf2 (green) in P19 and P19-Pitx2KO cells after vehicle or H2O2 treatment. DAPI, blue. Scale bar, 50 μm. (c) The ratio of cells with nuclear Nrf2 over total cell number, n=6. (d) Blotting of α-Tubulin and TBP to show cell fraction of P19 cells used in (e). (e) Co-IP Nrf2 from nuclear and cytoplasmic fraction of P19 cells after vehicle or H2O2 treatment, blotting shows Nrf2 and Pitx2. (f–h) 4 DPMI control (C57BL6) (f) and Nrf2nu/nu (g) cross-sections stained for Pitx2 (red), cTnT (green), and DAPI (blue), with the ratio of cardiomyocytes with nuclei-localized Pitx2 quantified in h, n=4. Arrows, Pitx2+ cardiomyocyte. Mean ± S.E.M.; Statistical test, (c) one-way ANOVA plus Bonferroni post-test; (h) Mann-Whitney. *, p<0.05; NS, not significant.
Extended Data Figure 7
Extended Data Figure 7
Nrf2 is required for neonatal myocardial regeneration. (a) Trichrome images of Nrf2nu/nu and control heart (C57BL6) at 21 days after P2 LAD-O, along with sham controls. (b, c) Ejection fraction (b) and fractional shortening (c) of LAD-O and sham hearts. Mean ± S.E.M.; Statistical test, (b, c) Mann-Whitney. *, p<0.05; NS, not significant.
Extended Data Figure 8
Extended Data Figure 8
Yap and Nrf2 are essential for Pitx2-induced myocardial regeneration. (a–d) Trichrome staining showing apical scarring of different groups at 28 DPR, apex resection was performed at P8. (e) Quantification of scar area, n=4. Mean ± S.E.M.; Statistical test, (e) Mann-Whitney. *, p<0.05 compared to other three groups; NS, not significant.
Extended Data Figure 9
Extended Data Figure 9
Pitx2 regulates antioxidant scavenger genes. (a) Overall change of genes in Pitx2CKO compared to control. (b) Up-regulated genes in 5 DPR control over wild type sham heart (n=480) overlaid with down-regulated genes in 5 DPR Pitx2CKO over 5 DPR control heart (n=1002). (c) GO-analysis of genes up-regulated (left) and down-regulated (right) in Pitx2CKO ventricles over controls at 5 DPR. (d) GO-analysis of genes up-regulated (right) and down-regulated (left) in 5 DPR control ventricles over age matching sham hearts. (e) ChIP-qPCR confirming the binding of Pitx2 to the regulatory regions of target genes, n=4. (f) qPCR detecting Pitx2 and antioxidant genes in wild type and pitx2nu/nu embryonic stem cells after vehicle or H2O2 treatment, n=4. (g) qPCR of antioxidant genes in P19 cells after doxorubicin or H2O2 treatment, n=5. (h) qPCR of Pitx2 in P19 cells after doxorubicin or H2O2 treatment, n=5. Mean ± S.E.M.; Statistical test, (e–h) Mann-Whitney. *, p<0.05; ***, p<0.001; NS, not significant.
Extended Data Figure 10
Extended Data Figure 10
Mechanism model of Pitx2, Nrf2, and Yap responding to oxidative stress. When oxidative stress is low, Nrf2 is sequestered in cytoplasm by its degradation complex (Cul3, Keap1), and Pitx2 stays either in cytoplasm or at low expression level. When redox balance is disturbed by ROS, Nrf2 breaks away from degradation complex, and enter nuclei to up-regulate Pitx2, Nrf2 also binds cytoplasmic Pitx2 and shuttle it to nuclei, where Pitx2 and Yap co-regulate their common targets including critical antioxidant genes. In wild type adult mouse heart, active Yap is maintained at a low level, even after ischemic injury, thus not able to repair myocardium efficiently. When Pitx2 is over-expressed in cardiomyocytes, sufficient amount of Pitx2 will cooperate with low level of resident active Yap to induce the expression of beneficial antioxidant scavengers in a synergetic pattern, rendering protection to injured myocardium. Red arrow, supported by in vitro evidence; Blue arrows, supported by in vivo evidence.
Figure 1
Figure 1
Pitx2 is induced in injured myocardial. (a–c) Border zone of SalvCKO (b) and control (a) hearts stained for Pitx2 (green), cTnT (red), and DAPI (blue) at 10 day-post-MI, with Pitx2+ cardiomyocyte ratio quantified in c, n=4. (d) Pitx2 expression showed by RNA-Seq, P, postnatal day. (e) Western blot of Flag and a-Tubulin in 5 DPR Pitx2flag ventricles, resected at P1. (f) Nrf2 directly binds to Pitx2 enhancer after LAD-O. The heart specific enhancers are marked by H3K27ac ChIP-Seq. red bar, Nrf2 binding element. (g) DHS-Seq and chromatin state tracks of fetal and adult human heart tissue. Orange color indicates active enhancer regions. (h) qPCR showed knocking-down of Nrf2 by siRNA in P19 cells, n=4. (i) qPCR of Pitx2 in P19 cells with siRNA targeting Nrf2, and Nrf2nu/nu heart, compared to controls, n=4. Mean ± S.E.M.; Statistical test, (c) one-way ANOVA plus Bonferroni post-test; (i, right part) Mann-Whitney; (h, i left part) see Methods; *, p<0.05; NS, not significant.
Figure 2
Figure 2
Pitx2 is required and sufficient to promote myocardial regeneration. (a–c) Trichrome-stained Pitx2f/f (a) and Pitx2CKO (b) apex at 21 DPR, with scar size quantified in c. (d, e) Echocardiography showed the ejection fraction (d) and fractional shorting (e) at 21 DPR. (f–h) 5 DPR Pitx2f/f (f) and Pitx2CKO (g) apical sections stained for EdU (yellow), cTnT (red), and DAPI (blue). Arrow, EdU+ cardiomyocyte. Cardiomyocyte proliferative ratio was quantified in h, n=4. (i) Serial transverse heart sections at 5 weeks post-LAD-O, performed at 8weeks. (j) Percentage of fibrotic left ventricular myocardium quantified at 5 weeks post-LAD-O, n=5. Scale bar, 1mm. (k, l) Ejection fraction (k) and fractional shortening (l) of LAD-O and sham hearts. Mean ± S.E.M.; Statistical test, (d, e) one-way ANOVA plus Bonferroni post-test; (c, h, j–l) Mann-Whitney; *, p<0.05; NS, not significant.
Figure 3
Figure 3
Pitx2 interacts with Yap in regenerating hearts, and its nuclear shuttling requires Nrf2. (a–d) Trichrome-stained control (Salvf/f;Pitx2f/f) (a), SalvCKO (b) and DKO (c) sections at 28 days after P8 LAD-O with scar size quantification (d), n=4. (e) Echocardiography showed ejection fraction. (f) Diagram of GST-Pitx2 constructs. (g) GST-Pitx2 pull-down assay. Yap was detected by Western blotting. (h–i) Immunofluorescent staining of Pitx2 (green) and DAPI (blue) in P19 cells after vehicle or H2O2 treatment, with control siRNA or siRNA targeting Nrf2. Arrow, cytoplasmic staining; arrowhead, nuclear staining. The ratio of cells with nucleus-localized Pitx2 over total cell number is quantified in i. (j) Co-IP of Flag in 5 DPR Pitx2flag ventricles, resected at P1, blotting of Nrf2 and Pitx2. Mean ± S.E.M.; Statistical test, (e) one-way ANOVA plus Bonferroni post-test; (d, i) Mann-Whitney; *, p<0.05; NS, not significant.
Figure 4
Figure 4
Pitx2 regulates redox balance in neonatal myocardium. (a) Sankey diagram shows direct target genes of Pitx2 from overlaying ChIP-Seq and RNA-Seq profiles. A subset of genes is further branched according to promoter binding activity of Pitx2 and Yap. Gene ontology analysis is performed on 1002 down-regulated genes in Pitx2CKO ventricles at 5 days after P1 resection. The GO terms are listed by significance in descending order. The branches indicate the source of genes for each term. OXPHOS, oxidative phosphorylation. (b) Heat map highlights mitochondrial genes directly targeted by Pitx2. Red bar, genes co-regulated by Pitx2 and Yap; orange, direct binding of Pitx2 on promoters. (c) Heart specific H3K4me3 ChIP-Seq and Yap ChIP-Seq read distribution with in 2 kb interval of promoter region of Pitx2 direct target genes. The width of the curve indicate 95% confidence interval. (d) ChIP-re-ChIP showing co-occupancy of Pitx2 and Yap at the regulatory regions of common target genes (in b, red bar), n=3. (e–i) ROS staining (green) of apical border zone in Pitx2CKO (f, i) and control (e, h), with fluorescent intensity quantified in g. MF20, red; DAPI, blue, n=4. (j–n) Trichrome at 21DPR showed apical scarring of Pitx2CKO (j, k) and control (l, m) hearts treated with PBS (j, l) or NAC (k, m), with scar area quantified in n, n=5. Mean ± S.E.M.; Statistical test, (n) one-way ANOVA plus Bonferroni post-test; (g) Mann-Whitney; (d) Mann-Whitney and Wilcoxon signed-rank test; *, p<0.05; NS, not significant.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Xin M, Olson EN, Bassel-Duby R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat Rev Mol Cell Biol. 2013;14:529–541. doi: 10.1038/nrm3619. - DOI - PMC - PubMed
    1. Porrello ER, et al. Transient regenerative potential of the neonatal mouse heart. Science. 2011;331:1078–1080. doi:331/6020/1078 [pii] 10.1126/science.1200708. - PMC - PubMed
    1. Itoh K, et al. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 1999;13:76–86. - PMC - PubMed
    1. Heallen T, et al. Hippo signaling impedes adult heart regeneration. Development. 2013;140:4683–4690. - PMC - PubMed
    1. Semina EV, et al. Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nat Genet. 1996;14:392–399. - PubMed

Publication types

MeSH terms