The extracellular matrix protein agrin promotes heart regeneration in mice

Abstract

The adult mammalian heart is non-regenerative owing to the post-mitotic nature of cardiomyocytes. The neonatal mouse heart can regenerate, but only during the first week of life. Here we show that changes in the composition of the extracellular matrix during this week can affect cardiomyocyte growth and differentiation in mice. We identify agrin, a component of neonatal extracellular matrix, as required for the full regenerative capacity of neonatal mouse hearts. In vitro, recombinant agrin promotes the division of cardiomyocytes that are derived from mouse and human induced pluripotent stem cells through a mechanism that involves the disassembly of the dystrophin–glycoprotein complex, and Yap- and ERK-mediated signalling. In vivo, a single administration of agrin promotes cardiac regeneration in adult mice after myocardial infarction, although the degree of cardiomyocyte proliferation observed in this model suggests that there are additional therapeutic mechanisms. Together, our results uncover a new inducer of mammalian heart regeneration and highlight fundamental roles of the extracellular matrix in cardiac repair.

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Figure 1: Identification of agrin in a screen for mouse cardiac ECM-mediated cardiomyocyte proliferation.
Figure 2: Agrin delays neonatal cardiomyocyte maturation and is required for P1 cardiac regeneration following surgical resection.
Figure 3: Agrin induces cardiac regeneration in adult mice.
Figure 4: Agrin promotes cardiomyocyte proliferation through Dag1, ERK and Yap signalling.
Figure 5: Agrin promotes proliferation and attenuates maturation of human iPSC-CMs.

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Acknowledgements

This work was supported by grants to E.T. from the European Research Council, Israel Science Foundation and the Britain Israel Research and Academic Exchange (BIRAX). E.T., J.F.M. and N.B. are supported by Foundation LeDucq and NIH funding. We thank R. Burgess for providing the Agrnfl/fl mice. We thank Y. Levin, head of de Botton Institute for Protein Profiling, The Nancy and Stephen Grand Israel National Center for Personalized Medicine, for protein profiling. We thank E.T. and I.S. laboratory members, A. Navon, K. Yaniv and P Riley for fruitful discussions, O. Goresh and B. Siani for animal husbandry, and N. Akron, D. Gorelik, R. Levine, S. Goldsmith, C. Raanan and M. Osin for genotyping and histology.

Author information

E.B., with help from A.G., performed most of the experiments. Y.E.M. and S.C. performed glycerol-gradient experiments. I.Y.S. and N.B. performed 3D iPSC–CM culture experiments, E.B., K.B.U., D.K., J.L. and J.F.M. performed in vivo animal experiments, O.Y. performed in vitro experiments, D.R.B. performed western blots, D.R. and R.S. performed hiPS–CM experiments, Y.U. and I.S. performed in situ zymography. E.T. supervised the project and with E.B. wrote the manuscript.

Correspondence to Eldad Tzahor.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks K. Yutzey and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 P1 and P7 ECM explants contain increased gelatinase activity.

a, b, Cell removal assessment of heart sections by DAPI or scanning electron microscopy. Scale bars, 50 μm (a) and 20 μm (b). c, A schematic diagram of the in situ zymography assay. d, Immunofluorescence evaluation of Col1, Col4 and gelatin degradation in response to P1 and P7 ECM. e, Quantification of the in situ zymography assay. n = 2 samples.

Extended Data Figure 2 Mass spectrometry results and validation.

a, Venn diagram representing the LC–MS results. b, qPCR analysis of genes obtained from the LC–MS in P1 and P7 whole-heart lysates. n = 3 P1 hearts and 3 P7 hearts. Source data

Extended Data Figure 3 P1 and P8 cardiac cell separation.

qPCR of mRNA from P1 and P8 heart lysates. qPCR analysis of six cell populations (fibroblasts, non-fibroblasts, cardiomyocytes, non-cardiomyocytes, endothelial cells, non-endothelial cells) for CD90 (also known as Thy1, a fibroblast marker), Myh6 (a cardiomyocyte marker) and Pecam1 (Pecam, an endothelial cell marker) (for P1, n = 4 cardiomyocyte, 4 non-cardiomyocyte, 4 fibroblast, 4 non-fibroblasts, 7 endothelial cell, 7 non-endothelial cell samples and for P8 n = 2 cardiomyocyte and 2 non-cardiomyocyte samples). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; statistical significance was calculated using a one-tailed t-test.

Extended Data Figure 4 Agrin induces cardiomyocyte proliferation in vitro.

ad, Immunofluorescence evaluation of P1 and P7 cardiomyocytes (cTnT+ or Myh6CretdTomato+, see Methods) cell number (a), cell-cycle activity (Ki67; b), mitosis (pH3; c) or cytokinesis (Aurkb, also known as Aim1; d) in response to agrin dosage in vitro. White arrowheads indicate proliferating cells. Analysis of five individual wells per treatment (a); n = 1,855 P1 and 1,328 P7 cardiomyocytes pooled from the analysis of three P1 and four P7 samples (b); n = 11,450 P1 and 13,469 P7 cardiomyocytes pooled from the analysis of four P1 and six P7 samples (c); n = 7,971 P1 and 3,856 P7 cardiomyocytes pooled from the analysis of three P1 and three P7 samples (d). Scale bars, 30 μm (b) and 100 μm (c). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; statistical significance was calculated using an ANOVA followed by Dunnett’s post hoc test relative to the control group(ac) or using a one-tailed t-test (d).

Extended Data Figure 5 Agrin-cKO cardiac characterization.

a, Tnni3:Tnni1 protein ratio in P8 agrin-cKO and wild-type mice. n = 4 wild-type and 7 cKO mice. b, Immunofluorescence analysis and Pearsons’s correlation coefficent analysis of P14 T-tubules by Cav3 colocalizion with α-actinin labelled z-lines. n = 5 wild-type and 6 cKO samples. c, Immunofluorescence analysis of WGA membrane staining in P1 wild-type and agrin-cKO depicting changes in cell size. n = 2 wild-type and 3 cKO samples. d, qPCR analysis of pathological hypertrophic marker Acta1 in P1 wild-type and agrin-cKO heart lysates. n = 21 wild-type and 26 cKO samples. e, f, Serial echocardiographic measurements of ejection fraction (EF) and wall thickness of 1-, 3- and 14-month-old agrin cKO and wild-type mice. n = 8 mice per group at 1 month, 4 wild-type and 5 cKO at 3 months and 4 wild-type and 3 cKO at 14 months. g, Heart to body weight ratio of 1-, 3- and 14-month-old agrin-cKO and wild-type mice. n = 9 wild-type and 4 cKO at 1 month, 4 wild-type and 5 cKO at 3 months and 6 wild-type and 5 cKO at 14 months. h, Histological sections of 1- and 3-month-old wild-type and agrin-cKO stained with Masson’s trichrome. i, Scheme of P1 heart resection. j, Representative images of agrin-cKO and wild-type mice 28 days after resection at P1. k, Functional cardiac recovery measurement (cardiac output), 28 days after resection. n = 5 wild-type and 3 cKO mice (j, k). Scale bars, 20 μm (b), 10 μm (c) and 1 mm (h). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01; Statistical significance was calculated using a one-tailed t-test.

Extended Data Figure 6 Agrin induces cardiac regeneration in juvenile mice.

a, A schematic diagram depicting LAD ligation in juvenile and adult mice. b, c, In vivo evaluation of cardiomyocytes cell-cycle re-entry by Ki67 (b) or Aurkb (c) in heart sections seven days after myocardial infarction. n = 1,934 cardiomyocytes pooled from the analysis of three PBS-treated mice and three agrin-treated mice (b); n = 1,450 cardiomyocytes pooled from the analysis of three PBS- and six agrin-treated samples (c). d, Scar quantification based on Masson’s trichrome staining of heart sections of PBS- and agrin-treated juvenile mice. Representative pictures are provided. n = 4 PBS- and 8 agrin-treated mice. eg, Serial echocardiographic measurements of ejection fraction (EF), fractional shortening (FS) and wall thickness of uninjured and injured PBS- and agrin-treated juvenile mice following myocardial infarction, according to the schema in a. n = 3 uninjured, 3 PBS- and 3 agrin-treated mice at day 13; and n = 4 uninjured, 5 PBS- and 9 agrin-treated mice at day 43. Scale bars, 10 μm (b), 20 μm (c) and 1 mm (d). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; statistical significance was calculated using a one-tailed t-test(bd, g) or ANOVA followed by Dunnett’s post hoc test relative to the control group (e, f).

Extended Data Figure 7 Pharmacokinetics of agrin injection in injured adult hearts.

Mice were subjected to LAD ligation (as shown in Fig. 4a), and received either PBS or agrin treatment. Hearts were collected at respective time points after treatment, and were subjected to protein extraction and anti-His-tag immunopreciptation using Ni-NTA resin. a, Representative western blot of agrin from relevant immunoprecipitation samples. Anti-tubulin western blot of respective total extracts, as immunoprecipitation loading control is shown at the bottom. b, The kinetics of recombinant agrin up to 96 h after treatment. The amount of agrin is presented as a percentage of the first time point (t = 0), and normalized according to tubulin. n = 3 repeats.

Extended Data Figure 8 Dag1 expression in P1 and P7 whole hearts and cardiac cell separation.

a, qPCR of Dag1 mRNA from P1 and P7 heart lysates. n = 5 P1 and 3 P7 samples. b, qPCR analysis of Dag1 mRNA in distinct cardiac populations from P1 mice (fibroblasts, non-fibroblasts, cardiomyocytes, non-cardiomyocytes, endothelial cells, non-endothelial cells). n = 4 cardiomyocyte, 4 non-cardiomyocyte, 4 fibroblast, 4 non-fibroblast and 7 endothelial and 7 non-endothelial cell samples. c, Serial immunofluorecence counting of Myh6-lineage-derived tdTomato-labelled cardiomyocytes treated with ouabain and digoxin which inhibit Na+K+ pumps. Quantification was made using acuman (see Methods). n = 31 control, 16 agrin-, 11 ouabain- and 11 digoxin-treated individual wells per treatment. d, quantification of cardiomyocyte sarcomeric status by cTnT and Myh6-linage immunofluorescence analysis of P7 agrin-treated cardiomyocytes cultured in vitro for three days. n = 2,222 cardiomyocytes pooled from the analysis of three samples per group. Representative images of cardiomyocytes with intact, partially disassembled and severely disassembled sarcomeres are provided. Scale bars, 40 μm. Data are presented as mean ± s.e.m. *P < 0.05, ***P < 0.001; statistical significance was calculated using a one-tailed t-test (a, b, d) or ANOVA followed by Dunnett’s post hoc test relative to control group (c).

Extended Data Figure 9 A model for the agrin–DGC–Yap signalling axis in cardiomyocyte maturation and cardiac regeneration.

Agrin triggers mild cardiomyocyte dedifferentiation and proliferation by modulation of DGC integrity and signalling. During neonatal stages agrin suppresses the maturation of the DGC. At P7, agrin levels are reduced and ECM–DGC interaction through Dag1 promotes cardiomyocyte differentiation and maturation. Yap is tethered to the DGC upon cardiomyocyte maturation while upon agrin treatment, it translocates to the nucleus to facilitate cardiomyocyte cell-cycle re-entry.

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Bassat, E., Mutlak, Y., Genzelinakh, A. et al. The extracellular matrix protein agrin promotes heart regeneration in mice. Nature 547, 179–184 (2017). https://doi.org/10.1038/nature22978

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