ERBB2 drives YAP activation and EMT-like processes during cardiac regeneration

Abstract

Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2–Neuregulin and Hippo–YAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelial–mesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.

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Fig. 1: ERBB2 induces cardiac regeneration in a heart failure model that involves EMT-like processes.
Fig. 2: YAP is activated downstream of ERBB2 signalling in CMs.
Fig. 3: The Hippo pathway is activated in OE hearts.
Fig. 4: YAP is required for ERBB2-related cardiac phenotypes.
Fig. 5: ERBB2 alters the mechanical state of CMs, enhancing the interaction of YAP with cytoskeletal and nuclear-envelope components.
Fig. 6: YAP phosphorylation on S274 and S352 is required for mitosis and occurs downstream of ERK.

Data availability

The RNA-seq data were deposited in the Gene Expression Omnibus under accession code GSE144391. The mass spectrometry data have been deposited in ProteomeXchange under accession code PXD020731. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This study has been supported by grants to E.T. from the European Research Council (ERC StG grant no. 281289, CM turnover, and ERC AdG grant no. 788194, CardHeal), ERA-CVD CARDIO-PRO, EU Horizon 2020 research and innovation programme REANIMA, the U.S.–Israel Binational Science Foundation (BSF; to both E.T. and J.F.M.), the Israel Science Foundation (ISF), Foundation Leducq Transatlantic Network of Excellence and Minerva foundation, with funding from the Federal German Ministry for Education and Research. We thank the Benoziyo Endowment Fund for the Advancement of Science, Head of the Yad Abraham Research Center for Cancer Diagnostics and Therapy, Zuckerman STEM Leadership Program, Dr. Dvora and Haim Teitelbaum Endowment Fund and Daniel S. Shapiro Cardiovascular Research Fund. This work was supported by grants from the National Institutes of Health (grant nos HL127717, HL130804 and HL118761 (J.F.M.)), Vivian L. Smith Foundation (J.F.M.) and State of Texas funding (J.F.M.). We thank O. Singer for help with the AAV preparations, G. Friedlander for RNA-seq analysis and input, and N. Priel for teaching ‘trackmate’ software usage.

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Contributions

A.A. and E.T. conceived and designed the experiments. A.A., with help from A.Shakked, carried out most of the experiments and analysed the data. Specifically, A.Shakked helped with the animal studies, tissue culture work, RT–qPCR and cloning of YAP mutants into AAV viruses and all associated work and analysis. K.B.U. helped with the animal studies, RNA-seq preparation and analysis. A.Savidor and Y.L. performed the proteomic analysis. A.G. helped with the image analysis. D.K. and D.L. performed the myocardial infarction and echocardiographic analysis. O-Y.R. helped with the functional gelatin-degradation assays and migration time-lapse microscopy. Y.M. helped with the YAP5SA heart sections. J.D. provided custom-made and pYAP S274 antibody. B.G. and J.F.M. contributed to the planning and discussion of the project. E.T. supervised the entire project. A.A. and E.T. wrote the manuscript with contributions from all of the authors.

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Correspondence to Eldad Tzahor.

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Extended data

Extended Data Fig. 1 Transient ERBB2 activation in injured hearts (HF model) promotes functional improvement.

a, % Scar quantification for the 3WPMI time point (relates to Fig. 1a, orange arrow) for adult WT (n=5) and OE (n=4) hearts, p=0.180. Statistics were determined by two-tailed t-test. (b-d) Cardiac parameters derived from echocardiographic analysis. Green window represents duration of ERBB2 activation. b, Left ventricular anterior wall (LVAW) thickness, p<0.0001. c, Stroke volume, p=0.0002 (d) Cardiac output, p=0.04, in WT (n=16), OE (n=11) and Sham (n=5) hearts. Statistics, one-way ANOVA followed by Tukey’s multiple comparisons test. Data represent mean ± s.e.m. For statistical source data, see Source Data Extended Data Figure 1. Source data

Extended Data Fig. 2 EMT hallmarks are upregulated in OE hearts.

a, Analysis of bulk RNA- seq of adult WT–MI (n=4) and OE–MI (n=4) hearts using GSEA hallmark module (relates to Fig. 1h). Bar plot depict the normalized enrichment scores (NES). b-e, RNA- seq derived heat map of the indicated genes belonging to the EMT hallmark (as shown in (a) and Fig. 1h). Genes were annotated as (b) ECM composition (c) ECM modulators (d) Transcription factors and (e) Transmembranal and secreted proteins. FC to the right indicates OE/WT fold change of the detected transcripts by RNA- seq using a threshold FC≥1.5 and p adjusted ≤0.05. Statistics were determined by DESeq2, and adjusted for multiple testing using the procedure of Benjamini and Hochberg. f, Representative image in adult WT and OE hearts. Scale bar, 50 µm.

Extended Data Fig. 3 ERBB2 activation results in reduced CM connectivity and sarcomeric organization, partially depending on YAP.

a, Representative IF images and (b) Representative Haematoxylin & Eosin stain for adult WT, OE and OE-KD heart sections. Scale bar 50µm. c, Quantification of normalized sarcomeric fluorescence intensity for WT (n=3), OE (n=3) and OE-KD (n=3) adult hearts, p=0.047. d, Quantification of normalized interstitial gaps between CMs, for WT (n=3), OE (n=3) and OE-KD (n=3) adult hearts. WT compared to OE, p=0.0018. OE compared to OE-KD, p=0.007. e, Representative IF images of WT (n=3), OE (n=3) and OE-KD (n=3) adult hearts. Scale bar 100µm. Insets highlight separate channels as indicated, white and yellow arrow heads point to connections between CMs with intact and mispatterened CDH2, correspondingly, at the termini of CMs. Inset Scale bar 10µm. Data represent mean ± s.e.m. All figure statistics were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. For statistical source data, see Source Data Extended Data Figure 3. Source data

Extended Data Fig. 4 YAP Co-IP–MS binding partners.

a, Heat map of YAP enrichment in Yap Co-IP and IgG reactions for WT (n=4) and OE (n=4) adult hearts, as described in Fig. 5a. FC indicates fold change between IP and IgG reactios. b, Heat map of differential YAP binding partners between WT (n=4) and OE (n=4) adult hearts. FC indicates fold change between OE to WT IP reactions of the detected protein by Mass Spec. All displayed results are of statistical significance p ≤ 0.05 between OE and WT IP reactions, which were non-significantly different between OE and WT IgG reactions (similar background). All figure statistics were determined by two-tailed t-test.

Extended Data Fig. 5 Phosphorylation of YAP on S274 and S352 is prevalent in OE hearts and peaks during mitosis.

a, WB analysis of YAP on the soluble fraction of adult WT/OE hearts. b, WB analysis of pYAP S274 on the soluble fraction of adult WT/OE hearts. c, WB analysis of pYAP S352 on the soluble fraction of adult WT/OE hearts. d, WB analysis of YAP on whole cell lysates (includes cytoskeletal components underrepresented in soluble extracts) of adult WT/OE hearts. Blue and red arrow heads point to the usual (“upper”) and below 63kDa (“lower”) bands. e, YAP IP (and IgG) from WT/OE hearts blotted for YAP and pYAP S274. Purple circles represent cut-out bands analysed by Mass-Spec in (f). f, Peptide-spectrum match (#PSM) for YAP analysed by Mass-Spec of (e), an experiment performed once. g, h, Diagram of murine YAP (YAP2L, 488aa) (Plus Myc-DDK tag, as purchased) in (g) and human YAP (YAP2L, 504aa) in (h). Domains indicated above, Hippo phosphorylation sites denoted in grey, and S274/S352 (S289/367 in human) phosphorylation sites circled in red. i, j, Representative images of P7 WT/OE cardiac cultures. Scale bar 50µm. WT and OE insets highlight with an arrow a non-CM, and a CM, at metaphase with peaking pYAP S274/S352 stain. Scale bar 10 µm. k, Quantification of P7 OE Aurkb+ CMs (blue curve) and pYAP S352 mitotic localization (orange curve) at stages of mitosis. Lower panel shows pYAP S352-Aurkb mitotic overlap. n=2274 CMs, from 3 OE hearts. l, Representative image at metaphase of P7 OE cardiac cultures Scale bar 50µm. m, Representative image of embryonic WT E16.5 heart sections. Scale bar 50µm. Inset arrows point to CMs in metaphase. Inset scale bar 10µm. n, Representative image of OE and 5SA adult hearts. White, yellow arrows point to metaphase, pre-metaphase CMs, correspondingly. Scale bar, 10µm. Uncropped blots in panels a, b, c, d, e and numerical source data in panel k are provided in Source Data Extended Data Figure 5. Source data

Extended Data Fig. 6 ERK interacts with YAP and drives YAP S274 phopshorylation and morphological changes in ERBB2-OE CMs.

a, b, Representative IF images of P7 WT/OE cardiac cultures upon vehicle (DMSO) or ERK-i treatment. Scale bar 50µm. ce, quantification of morphological features of circularity (p), Axial ratio (q), and solidity, (r) upon vehicle (DMSO) or ERK-i (n=127) treatment, of P7 WT (n=123) or OE (n=126) CMs derived from 4 WT and 4 OE P7 hearts. For circularity, WT compared to OE, p=0.0220, OE compared to OE-ERK-i, p=0.0498. For axial ratio, WT compared to OE, p=0.0029, OE compared to OE-ERK-i, p=0.0199. For solidity, WT compared to OE, p=0.0008. OE compared to OE-ERK-i, p=0.0001. f, Quantification of PLA foci in adult cardiac sections, with individual corresponding technical controls for WT (n=6) and OE (n=6) hearts. Data represent mean ± s.e.m. All figure statistics were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. () if P ≤0.05, () if P <0.01, () if P <0.001 and () if P <0.0001. For statistical source data, see Source Data Extended Data Figure 6. Source data

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Supplementary Video 1

Time-lapse movie (96 h) of a P7 WT cardiac culture showing CMs tagged with tdTomato fluorescent protein and non-CMs in phase. n = 6 independent experiments.

Supplementary Video 2

Time-lapse movie (96 h) of a P7 OE cardiac culture showing CMs tagged with tdTomato fluorescent protein and non-CMs in phase. n = 4 independent experiments.

Supplementary Table

Table of supplementary information regarding the primer sequences and antibodies used in the study.

Source data

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Aharonov, A., Shakked, A., Umansky, K.B. et al. ERBB2 drives YAP activation and EMT-like processes during cardiac regeneration. Nat Cell Biol 22, 1346–1356 (2020). https://doi.org/10.1038/s41556-020-00588-4

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