ERBB2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation

Abstract

The murine neonatal heart can regenerate after injury through cardiomyocyte (CM) proliferation, although this capacity markedly diminishes after the first week of life. Neuregulin-1 (NRG1) administration has been proposed as a strategy to promote cardiac regeneration. Here, using loss- and gain-of-function genetic tools, we explore the role of the NRG1 co-receptor ERBB2 in cardiac regeneration. NRG1-induced CM proliferation diminished one week after birth owing to a reduction in ERBB2 expression. CM-specific Erbb2 knockout revealed that ERBB2 is required for CM proliferation at embryonic/neonatal stages. Induction of a constitutively active ERBB2 (caERBB2) in neonatal, juvenile and adult CMs resulted in cardiomegaly, characterized by extensive CM hypertrophy, dedifferentiation and proliferation, differentially mediated by ERK, AKT and GSK3β/β-catenin signalling pathways. Transient induction of caERBB2 following myocardial infarction triggered CM dedifferentiation and proliferation followed by redifferentiation and regeneration. Thus, ERBB2 is both necessary for CM proliferation and sufficient to reactivate postnatal CM proliferative and regenerative potentials.

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Figure 1: NRG1-induced CM cell-cycle progression is diminished during the first week of postnatal life, in correlation with reduced ERBB2 levels.
Figure 2: ERBB2 is required for NRG1-induced CM proliferation.
Figure 3: Postnatal activation of ERBB2 signalling leads to cardiomegaly.
Figure 4: Postnatal activation of ERBB2 signalling promotes extensive CM proliferation and hypertrophy in vitro and in vivo.
Figure 5: Postnatal activation of ERBB2 signalling promotes CM dedifferentiation.
Figure 6: ERK, AKT and GSK3β/β-catenin differentially mediate ERBB2-induced CM proliferation, dedifferentiation and hypertrophy.
Figure 7: Transient ERBB2 induction triggers CM dedifferentiation, proliferation and cardiac regeneration following injury in juvenile and adult stages.
Figure 8: ERBB2 signalling triggers robust CM dedifferentiation and proliferation and reactivates a dormant postnatal regenerative window in mice.

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Acknowledgements

This work was supported by grants to E.T. from the European Research Council, Israel Science Foundation, the Louis and Fannie Tolz Collaborative Research Project, Estate of Jack Gitlitz and to R.P.H. from the National Health and Medical Research Council (NHMRC) of Australia (573705; 573732) and the Australian Research Council Special Initiative in Stem Cell Science (Stem Cells Australia). We thank C. Birchmeier (MDC, Germany) for providing the Erbb2flox and Erbb2LacZ mice, I. Biton for helping with the MRI measurements, and D. Sawyer (Vanderbilt University, USA), R. Graham (Victor Chang Cardiac Research Institute, Australia), A. Aronheim (Technion, Israel) and K. Yaniv (Weizmann Institute of Science, Israel) for fruitful discussions. We also thank H. Sadek and E. Olson (UT Southwestern Medical Center, USA) for teaching us the neonatal cardiac regenerative model and L. Field (Indiana University School of Medicine, USA) for sharing unpublished data.

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Contributions

G.D’U. and E.T. conceived and designed the experiments. G.D’U. with help from A.A. carried out most of the experiments and analysed the data. M. Lauriola and S.C. performed some western blots and helped with animal studies. D.K. performed myocardial infarction experiment in adult mice. Y.Y-R. performed time-lapse imaging. K.W. and T.K. assisted with myocardial infarction experiment in juvenile mice. E.B., D.R. and O.Y. assisted with gene expression analysis. M. Lysenko performed MRI analysis. O.B. helped with interpretation of histological slides. J.H. helped with the interpretation of echocardiographic analysis. R.S. contributed to the planning and progression of the project. J.L., Y.Y. and M.N. supervised the experiments done by their laboratory members, and E.T. supervised the entire project. R.P.H. provided conceptual inputs and G.D’U., R.P.H. and E.T. wrote the manuscript with editing contributions from all of the authors.

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Correspondence to Eldad Tzahor.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 4 Erbb2-cKO mice display enlarged heart with compensatory hypertrophy.

(a) Western Blot analysis of ERBB2 protein levels in P0 ctrl and Erbb2-cKO heart lysates; (b) Pictures of P0 ctrl and Erbb2-cKO hearts obtained by binocular microscope (LV, left ventricle; RV, right ventricle, LA, left atrium, RA, right atrium); scale bar, 1 mm; (c) Heart weight to body weight ratio in P0 ctrl and Erbb2-cKO mice (n = 22 mice); (d) Picture of P0 ctrl and Erbb2-cKO mice, displaying a pale skin colour before death; scale bar 1 cm; (e) Body weight of P0 ctrl and Erbb2-cKO mice (n = 51 mice); (f) CM size (area) quantification based on immunofluorescence analysis of cardiac Troponin T (cTnT) of P0 ctrl and Erbb2-cKO cells cultured in vitro for 5 days (n = 836 CMs pooled from the analysis of 6 mice); (gh) RT-PCR analysis of hypertrophic markers Nppa, Nppb, Acta1 and β/α-MHC in P0 ctrl and Erbb2-cKO heart lysates (n = 8 mice in g; n = 15 in h). In all panels numerical data are presented as mean + s.e.m.; statistical significance was calculated using two-tailed unpaired Student’s t-test in c and eh; results are marked with one asterisk () if P < 0.05, two () if P < 0.01, three () if P < 0.001 and four () if P < 0.0001; statistics source data can be found in Supplementary Table 1.

Supplementary Figure 5 Generation and analysis of CM restricted overexpression of a constitutively active Erbb2.

(a) Schematic diagram of the cross-breeding to generate CM restricted overexpression of a constitutively active Erbb2 (caErbb2); (b) Kinetics of ERBB2 protein levels in ctrl and neonatal caErbb2 heart lysates following DOX withdrawal according to scheme in Fig. 3a; (ce) Pictures of hearts isolated from ctrl and caErbb2 mice; images were obtained by binocular microscope; Scale bar 1 mm; (fh) Echocardiographic measurements of cardiac ejection fraction (EF) and fractional shortening (FS) in ctrl and caErbb2 mice (n = 14 mice in f; n = 14 mice in g; n = 20 mice in h). In all panels neonatal, juvenile and adult caErbb2 mice were generated according to the schema in Fig. 3a, b. Numerical data are presented as mean + s.e.m.; statistical significance was calculated using two-tailed unpaired Student’s t-test in fh; results are marked with one asterisk () if P < 0.05, two () if P < 0.01, three () if P < 0.001 and four () if P < 0.0001; statistics source data can be found in Supplementary Table 1.

Supplementary Figure 6 in vivo induction of ERBB2 signalling promotes CM dedifferentiation, proliferation and hypertrophy.

(a) CM cross-sectional area evaluation by WGA immunofluorescence analysis of P56 ctrl and adult caErbb2 left ventricular (LV) heart sections (n = 306 CMs pooled from the analysis of 6 mice); representative pictures are provided; Scale bar 10 μm; (b) Calculation of CM number in P56 ctrl and adult caErbb2 hearts (n = 6 mice); (cd) Hematoxilin and Eosin (H&E) histological analysis of LV heart sections in in ctrl and caErbb2 mice; Scale bar 25 μm; (ef) in vivo CM sarcomeric status evaluation by immunofluorescence analysis of cardiac Troponin T (cTnT) in ctrl and caErbb2 LV heart sections; Images were obtained using a spinning-disk confocal microscope; Scale bar 5 μm 0(g) WB analysis of cKIT protein levels in P7 ctrl and neonatal caErbb2 heart lysates. In all panels neonatal, juvenile and adult caErbb2 mice were generated according to the schema in Fig. 3a, b. Numerical data are presented as mean + s.e.m.; statistical significance was calculated using two-tailed unpaired Student’s t-test in a,b; results are marked with one asterisk () if P < 0.05, two () if P < 0.01, three () if P < 0.001 and four () if P < 0.0001; statistics source data can be found in Supplementary Table 1.

Supplementary Figure 7 Analysis of mediators and modulators of caERBB2-induced CM dedifferentiation, proliferation and hypertrophy.

(a) CM sarcomeric status evaluation based on cardiac Troponin T (cTnT) immunofluorescence analysis in P7 ctrl and neonatal caErbb2 heart cells treated in vitro for 2 days with pharmacological inhibitors of ERK and AKT (ERK-i and AKT-i); sarcomeric status was scored according to the criteria presented in Fig. 5c (n = 466 CMs pooled from the analysis of 24 samples); (b) in vivo immunofluorescence analysis of β-Catenin and sarcomeric Troponin T (cTnT) in LV heart sections of P7 ctrl and neonatal caErbb2 mice; images were obtained using a spinning-disk confocal microscope; Scale bar 10 μm (c) in vitro immunofluorescence analysis of β-Catenin and Troponin T (cTnT) in P7 neonatal caErbb2 heart cells treated with pharmacological inhibitors of GSK3β or β-Catenin (GSK3β-i and β-Catenin-i); Scale bar 60 μm; (df) P7 ctrl and neonatal caErbb2 heart cells were treated with p38 inhibitor (p38-i), FGF1 or Periostin in vitro for 2 days; CM were identified by Troponin T (cTnT) staining and analysed for (d) cell-cycle re-entry (Ki67), (e) dedifferentiation marker RUNX1 and (f) size by immunofluorescence analysis (n = 6013 CMs pooled from the analysis of 48 samples in d; n = 4217 CMs pooled from the analysis of 43 samples in e; n = 484 CMs pooled from the analysis of 17 samples in f). In all panels numerical data are presented as mean + s.e.m.; statistical significance was calculated using one-way ANOVA followed by Tukey’s test in a and df; results are marked with one asterisk () if P < 0.05, two () if P < 0.01, three () if P < 0.001 and four () if P < 0.0001; statistics source data can be found in Supplementary Table 1.

Supplementary Figure 8 Transient caErbb2 induces transient CM dedifferentiation and neovascularization following heart injury.

(a) Hematoxylin and eosin (H&E) histological analysis of remote zone of LV heart sections of ctrl and adult transient caErbb2 mice 1-month-post-MI (P70) and 2-months-post-MI (P98, one-month after caErbb2 signal termination) following myocardial infarction at P42 according to the schema in Fig. 7b; Scale bar 50 μm; (b) Angiogenesis evaluation by immunofluorescence analysis of CD34 in heart sections of 3-weeks-post-MI (P63) ctrl and adult caErbb2 following myocardial infarction at P42 according to the scheme in Fig. 7b; heart sections were co-stained with antibodies to cardiac Troponin T (cTnT) for identification of CMs (n = 50 fields pooled form the analysis of 6 mice); numerical data are presented as mean + s.e.m.; statistical significance was calculated using two-tailed unpaired Student’s t-test; results are marked with one asterisk () if P < 0.05; Representative pictures are provided; Scale bar 30 μm. statistics source data can be found in Supplementary Table 1.

Supplementary Figure 9 Uncropped images of immunoblots displayed in Figs 1g, 6a, 6e

Supplementary Table 1 Statistic data source.
Supplementary Table 2 List of primers used for mouse genotyping.
Supplementary Table 3 List of primers used for real-time PCR experiments.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1045 kb)

Cardiac MRI of a control mouse.

4 chamber cardiac MRI of P22 ctrl mouse. (MOV 2077 kb)

Cardiac MRI of caErbb2 mouse.

4 chamber cardiac MRI of P22 juvenile caErbb2 mouse generated according to the schema in Fig. 3b. (MOV 2917 kb)

Time-lapse movie of karyokinesis plus cytokinesis (cell division) in mono-nucleated caErbb2 CMs.

This is a representative time-lapse video of P7 caErbb2 mono-nucleated CMs performing karyokinesis plus cytokinesis (cell division). Heart cells were isolated from P7 caErbb2mice, cultured in vitro for 48 h, then labelled with TMRE to identify CMs (see ‘Methods’ section for further details) and imaged for 12 h at 10 min intervals; Scale bar 30 μm. (AVI 401 kb)

Time-lapse movie of karyokinesis with no cytokinesis (bi-nucleation) in mono-nucleated caErbb2 mice CMs.

This is a representative time-lapse video of P7 caErbb2 mono-nucleated CMs performing karyokinesis but not cytokinesis (bi-nucleation). Heart cells were isolated from P7 neonatal caErbb2m mice, cultured in vitro for 48 h, then labelled with TMRE to identify CMs (see ‘Methods’ section for further details) and imaged for 12 h at 10 min intervals; Scale bar 30 μm. (AVI 566 kb)

Time-lapse movie of cytokinesis in bi-nucleated caErbb2 mice CMs.

This is a representative time-lapse video of P7 caErbb2 bi-nucleated CMs performing cytokinesis. Heart cells were isolated from P7 neonatal caErbb2mice, cultured in vitro for 48 h, then labelled with TMRE to identify CMs (see ‘Methods’ section for further details) and imaged for 12 h at 10 min intervals; Scale bar 30 μm. (AVI 556 kb)

Time-lapse movie of CMs isolated from P7 ctrl mice.

This is a representative time-lapse video of P7 ctrl CMs, showing no events of karyokinesis and cytokinesis. Heart cells were isolated from P7 ctrl mice, cultured in vitro for 48 h, then labelled with TMRE to identify CMs (see ‘Methods’ section for further details) and imaged for 12 h at 10 min intervals; Scale bar 30 μm. (AVI 265 kb)

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D’Uva, G., Aharonov, A., Lauriola, M. et al. ERBB2 triggers mammalian heart regeneration by promoting cardiomyocyte dedifferentiation and proliferation. Nat Cell Biol 17, 627–638 (2015). https://doi.org/10.1038/ncb3149

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