Dystrophin–glycoprotein complex sequesters Yap to inhibit cardiomyocyte proliferation (original) (raw)

References

  1. Xin, M., Olson, E. N. & Bassel-Duby, R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat. Rev. Mol. Cell Biol. 14, 529–541 (2013)
    Article CAS Google Scholar
  2. Porrello, E. R. et al. Transient regenerative potential of the neonatal mouse heart. Science 331, 1078–1080 (2011)
    Article CAS ADS Google Scholar
  3. Bergmann, O. et al. Dynamics of cell generation and turnover in the human heart. Cell 161, 1566–1575 (2015)
    Article CAS Google Scholar
  4. Heallen, T. et al. Hippo signaling impedes adult heart regeneration. Development 140, 4683–4690 (2013)
    Article CAS Google Scholar
  5. Heallen, T. et al. Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size. Science 332, 458–461 (2011)
    Article CAS ADS Google Scholar
  6. Morikawa, Y . et al. Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice. Sci. Signal. 8, ra41 (2015)
    Article Google Scholar
  7. Ryder-Cook, A. S. et al. Localization of the mdx mutation within the mouse dystrophin gene. EMBO J. 7, 3017–3021 (1988)
    Article CAS Google Scholar
  8. Houtchens, G. R., Foster, M. D., Desai, T. A., Morgan, E. F. & Wong, J. Y. Combined effects of microtopography and cyclic strain on vascular smooth muscle cell orientation. J. Biomech. 41, 762–769 (2008)
    Article Google Scholar
  9. van Westering, T. L., Betts, C. A. & Wood, M. J. Current understanding of molecular pathology and treatment of cardiomyopathy in Duchenne muscular dystrophy. Molecules 20, 8823–8855 (2015)
    Article CAS Google Scholar
  10. Kassiri, Z. et al. Combination of tumor necrosis factor-α ablation and matrix metalloproteinase inhibition prevents heart failure after pressure overload in tissue inhibitor of metalloproteinase-3 knock-out mice. Circ. Res. 97, 380–390 (2005)
    Article CAS Google Scholar
  11. Kamogawa, Y. et al. Dystrophin-deficient myocardium is vulnerable to pressure overload in vivo. Cardiovasc. Res. 50, 509–515 (2001)
    Article CAS Google Scholar
  12. Tommasi di Vignano, A., Di Zenzo, G., Sudol, M., Cesareni, G. & Dente, L. Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex. FEBS Lett. 471, 229–234 (2000)
    Article CAS Google Scholar
  13. Zhao, B., Li, L., Tumaneng, K., Wang, C. Y. & Guan, K. L. A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ-TRCP. Genes Dev. 24, 72–85 (2010)
    Article CAS Google Scholar
  14. Li, J. et al. Alpha-catenins control cardiomyocyte proliferation by regulating Yap activity. Circ. Res. 116, 70–79 (2015)
    Article CAS Google Scholar
  15. Halder, G., Dupont, S. & Piccolo, S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat. Rev. Mol. Cell Biol. 13, 591–600 (2012)
    Article CAS Google Scholar
  16. Long, C. et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351, 400–403 (2016)
    Article CAS ADS Google Scholar
  17. Nelson, C. E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351, 403–407 (2016)
    Article CAS ADS Google Scholar
  18. Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016)
    Article CAS ADS Google Scholar
  19. McGreevy, J. W., Hakim, C. H., McIntosh, M. A. & Duan, D. Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy. Dis. Model. Mech. 8, 195–213 (2015)
    Article CAS Google Scholar
  20. Alkass, K. et al. No evidence for cardiomyocyte number expansion in preadolescent mice. Cell 163, 1026–1036 (2015)
    Article CAS Google Scholar
  21. Richardson, G. D. Simultaneous assessment of cardiomyocyte DNA synthesis and ploidy: a method to assist quantification of cardiomyocyte regeneration and turnover. J. Vis. Exp. (111) e53979 ( 2016)

Download references

Acknowledgements

This project was supported in part by an Intellectual and Developmental Disability Research Center grant (1U54 HD083092) from the Eunice Kennedy Shriver National Institute of Child Health & Human Development; the Mouse Phenotyping Core at Baylor College of Medicine with funding from the National Institutes of Health (U54 HG006348); and grants from the National Institutes of Health (DE 023177, HL 127717, HL 130804, and HL 118761 to J.F.M.) and the Vivian L. Smith Foundation (to J.F.M.). J.F.M. was supported by the Transatlantic Network of Excellence Award LeDucq Foundation Transatlantic Networks of Excellence in Cardiovascular Research 14CVD01. T.H. was supported by the American Heart Association Scientist Development Grant (16SDG26460001). We thank N. Stancel of the Texas Heart Institute for editorial assistance.

Author information

Authors and Affiliations

  1. Cardiomyocyte Renewal Laboratory, Texas Heart Institute, Houston, 77030, Texas, USA
    Yuka Morikawa, Todd Heallen, Yang Xiao & James F. Martin
  2. Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, 77030, Texas, USA
    John Leach & James F. Martin
  3. Program in Developmental Biology, Baylor College of Medicine, Houston, 77030, Texas, USA
    James F. Martin
  4. Cardiovascular Research Institute, Baylor College of Medicine, Houston, 77030, Texas, USA
    James F. Martin

Authors

  1. Yuka Morikawa
    You can also search for this author inPubMed Google Scholar
  2. Todd Heallen
    You can also search for this author inPubMed Google Scholar
  3. John Leach
    You can also search for this author inPubMed Google Scholar
  4. Yang Xiao
    You can also search for this author inPubMed Google Scholar
  5. James F. Martin
    You can also search for this author inPubMed Google Scholar

Contributions

J.F.M. and Y.M. conceived the project and designed the experiments. Y.M. performed experiments and analysed data. T.H. performed immunoprecipitation experiments, protein-binding assay and western blotting. J.L. performed cardiac function analysis and AAV9 viral studies. Y.X. performed several immunohistochemical studies. J.F.M., Y.M. and J.L. performed statistical analyses. J.F.M. supervised the project and analysed data. Y.M. and J.F.M. wrote the manuscript. All authors edited and approved the manuscript.

Corresponding author

Correspondence toJames F. Martin.

Ethics declarations

Competing interests

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.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Cardiomyocyte alignment after apical resection in P8 mouse hearts.

a–o, Hearts were collected 21 days, after apical resection was performed in P8 mice. Hearts of control (a, e, i), Mdx (b, f, j), Salv cKO (c, g, k) and Salv;Mdx dKO (d, h, l) mice were stained for cTnT and wheat-germ agglutinin (WGA) for cardiomyocytes and cell membranes, respectively. For control and Mdx hearts, the apex regions above the scar are shown. For Salv cKO and Salv;Mdx dKO hearts, the apex regions that regenerated are shown. m, Sarcomere length in cardiomyocytes was measured. Groups were compared using a one-way ANOVA (n = 3 for each genotype). No statistically significant difference was observed between samples. Data are mean ± s.e.m. n, o, Cardiomyocyte orientation in either the border zone or repaired area. n, A representative image shows how the orientation angles of cardiomyocytes were measured. Angles referenced to the plane of resection were measured for each cardiomyocyte. A total of 50 cardiomyocytes were measured for each sample. o, Histograms showing the distribution of cardiomyocyte orientation angles for each genotype (n = 3 for each genotype; 150 cardiomyocytes total were analysed for each genotype). Variance between genotype groups was compared using an _F_-test.

Extended Data Figure 2 Protrusion formation in border zone cardiomyocytes and migration of postnatal cardiomyocytes after apical resection in mice.

a–e, Apical resection was performed in P8 hearts of control (a), Mdx (b), Salv cKO (c) and Salv;Mdx dKO (d) mice, and hearts were collected four days after resection. Cardiomyocytes were stained for cTnT, and images of the tissue around border zone cardiomyocytes were taken. Dotted lines show the plane of resection. Arrowheads show cardiomyocyte protrusions. e, Quantification of cardiomyocyte protrusions (n = 3 mice for each genotype). Cardiomyocytes adjacent to the scar were analysed for length and number of protrusions four days after resection. One hundred cardiomyocytes from each heart were analysed. Groups were compared using ANOVA with Bonferroni’s multiple comparison test for pairwise comparisons. **P < 0.01. CM, cardiomyocyte. f–m, Immunostaining for vinculin to visualize cytoskeletal rearrangement in border zone cardiomyocytes of control (f, j), Mdx (g, k), Salv cKO (h, l) and Salv;Mdx dKO (i, m) mouse hearts. Cardiomyocytes were labelled with anti-sarcomeric actinin. Arrowheads indicate where the upregulation of vinculin is visible in Salv cKO border zone cardiomyocytes. n–r, Collagen gel assay results for P10 hearts collected from control (n), Mdx (o), Salv cKO (p) and Salv;Mdx dKO mice (q). Gel was stained with DAPI and for cTnT. r, Quantification of hearts in which migration was observed. Groups were compared using the Fisher’s exact test. Control compared to Salv cKO mice, P = 0.0097; Mdx compared to Salv cKO mice, P = 0.0097; Salv cKO compared to Salv;Mdx dKO mice, P = 0.007. Data are mean ± s.e.m.

Extended Data Figure 3 Characterization of mouse hearts after TAC surgery.

a–e, Knockout efficiency in Salv cKO mice. Immunohistochemical analysis of Salv was performed in control (a, c) and Salv cKO (b, d) mouse hearts two weeks after TAC surgery. Cardiomyocytes were labelled with anti-sarcomeric actinin. e, Quantification of Salv intensity (n = 3 mice for each treatment) measured according to pixel intensity (n = 3 for each treatment). f, g, Histology and cell size after TAC surgery. f, Representative images showing trichrome staining of hearts two weeks after TAC surgery in control, Mdx, Salv cKO and Salv;Mdx dKO mice. g, Cardiomyocyte size two weeks after sham or TAC surgery. Cell size was measured in WGA-stained sections using ImageJ software (n = 3 each). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; groups were compared using the Mann–Whitney _U_-test (e) or ANOVA with Bonferroni’s post hoc test for pairwise comparisons (g).

Extended Data Figure 4 Echocardiographic measurements.

a, Colour Doppler echocardiography across the transverse aorta before transverse aortic constriction (pre-TAC, left), and after transverse aortic constriction (post-TAC, right). The site of constriction (TAC) is labelled on the post-TAC image. b, Doppler echocardiography measurement of peak pressure through the site of constriction two weeks after TAC. Sham (n = 9), TAC (n = 7). c, Interventricular septal (IVS) thickness during diastole (IVS.d, left) and systole (IVS.s, right). d, Left ventricular internal diameter (LVID) during diastole (LVID.d, left) and systole (LVID.s, right). e, Left ventricular posterior wall (LVPW) thickness during diastole (LVPW.d, left) and systole (LVPW.s, right). Sham control (n = 4); sham Salv cKO (n = 4); sham Mdx (n = 6); sham Salv;Mdx dKO (n = 12). TAC control (n = 5); TAC Salv cKO (n = 8); TAC Mdx (n = 5); TAC Salv;Mdx dKO (n = 11). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; groups were compared using ANOVA with Tukey’s post hoc test for pairwise comparisons; significant differences with the Mdx group two weeks after TAC are indicated.

Extended Data Figure 5 EdU incorporation analysis after TAC surgery.

a–n, Flow cytometry analysis of isolated nuclei after TAC surgery. a–l, Representative images of flow cytometry analysis of the nuclei isolated from control (a, e, i), Mdx (b, f, j), Salv cKO (c, g, k), and Salv;Mdx dKO (d, h, l) mouse hearts after TAC surgery. a–d, PCM1+ population was gated and plots show EdU incorporation. e–h, Histogram showing DAPI intensity in PCM1+ population and discrimination between 2_N_, 4_N_, and >4_N_ population. i–l, Histogram showing DAPI intensity in PCM1+, EdU+ population. m, n, Quantification of PCM1+, EdU+ nuclei in >2_N_–4_N_ (m) and >4_N_ (n) population (n = 3). Data are mean ± s.e.m. *P < 0.05, **P < 0.01; groups were compared using ANOVA with Tukey’s post hoc test for pairwise comparisons. o–v, Representative images showing EdU staining of heart sections of control (o, r), Mdx (p, s), peri-fibrotic area of Mdx (q, t), Salv cKO (u, x), Salv;Mdx dKO (v, y) and peri-fibrotic area of dKO (w, z) mice after TAC surgery collected after two weeks. Cardiomyocytes were stained for actinin and cells were delineated by WGA. Arrowheads show EdU+ cardiomyocytes. Quantification of EdU+ cardiomyocytes is shown in Fig. 3n. Peri-fibrotic area is defined in Methods.

Extended Data Figure 6 Immunohistochemical analysis of mouse hearts after TAC surgery.

a–d, Representative images for aurora kinase B (AurkB) staining of control (a), Mdx (b), Salv cKO (c) and Salv;Mdx dKO (d) mouse hearts two weeks after TAC surgery. Cardiomyocytes were stained with anti-cTnT antibody. Quantification of AurkB+ cardiomyocytes is shown in Fig. 3o. Arrowheads indicate AurkB+ staining. e–k, Representative images showing Yap staining of control (e, i), Mdx (f, j), Salv cKO (g, k) and Salv;Mdx dKO (h, l) mouse hearts after TAC surgery. Cardiomyocytes were detected by immunostaining for cTnT. Arrowheads point to Yap localized in nuclei. Quantification of cardiomyocytes with nuclear Yap is shown in Fig. 3p. m–t, Representative images for active caspase-3 staining of control (m, q), Mdx (n, r), Salv cKO (o, s) and Salv;Mdx dKO (p, t) mouse hearts one and two weeks after TAC surgery. Cardiomyocytes were stained with anti-cTnT antibody. Arrowheads show active caspase-3+ cardiomyocytes. Quantification of active caspase-3+ cardiomyocytes is shown in Fig. 3q.

Extended Data Figure 7 Immunohistochemical analysis for pYap and vinculin after TAC surgery.

a–h, Representative images showing pYap staining of control (a, e), Mdx (b, f), Salv cKO (c, g) and Salv;Mdx dKO (d, h) mouse hearts after TAC surgery. Cardiomyocytes were detected by immunostaining for cTnT. Arrows indicate pYap in intercalated discs. i, Quantification of pYap levels (n = 3 for each genotype) measured according to pixel intensity. Data are mean ± s.e.m. **P < 0.01; groups were compared using a one-way ANOVA with Tukey’s post hoc test for pairwise comparisons. j–q, Representative images for vinculin staining of control (j, n), Mdx (k, o), Salv cKO (l, p) and Salv;Mdx dKO (m, q) mouse hearts after TAC surgery. Vinculin staining was used to detect cytoskeletal rearrangements in cardiomyocytes after TAC surgery. Sarcomeres were stained with anti-sarcomeric actinin.

Extended Data Figure 8 AAV9-treated Mdx hearts after TAC surgery.

Mouse hearts were collected 11 weeks after TAC surgery. a–d, Staining for Salv in Mdx hearts transfected with AAV9–GFP (a, b) or AAV9–Sal_v (c, d). Cardiomyocytes were stained with actinin. e, Quantification of Salv (n = 5 mice per each treatment) measured according to pixel intensity. Data are mean ± s.e.m. **P < 0.01; groups were compared using the Mann–Whitney U_-test. f–i, Staining for Yap in Mdx mouse hearts transfected with AAV–GFP (f, g) or AAV–_Salv (h, i). Arrowheads point to Yap localized in nuclei. j, k, Representative images showing trichrome staining of Mdx mouse hearts transfected with AAV9–GFP (j) or AAV9–_Salv (k).

Extended Data Figure 9 Immunoprecipitation and subcellular localization studies in C2C12 cells.

a, Knockdown efficiency of the siRNAs used in this study. C2C12 cells were differentiated and treated with si_Salv_, si_Dmd_, or control siRNA for 48 h before collection (n = 3 for each treatment). Data are mean ± s.e.m. *P < 0.05; groups were compared using the Mann–Whitney _U_-test. b–d, Immunoprecipitation was performed using protein extracts of differentiated C2C12 cells with antibodies specific to Yap (b), Flag (c) or Dag1 (d), followed by immunoblotting of indicated proteins. Yap(5SA) or GFP constructs were transfected into C2C12 cells before differentiation (c, d). For gel source data, see Supplementary Fig. 1.

Extended Data Figure 10 Model of interaction between the Hippo pathway and the DGC.

ICD, intercalated disc. Left, regenerative (neonatal) cardiomyocytes. (1) Hippo signalling is low, YAP phosphorylation and YAP binding to the DGC is reduced; (2) YAP–TEAD promote the transcription of target genes including Sgcδ and α-catenin; (3) YAP–TEAD promote DGC assembly by promoting the expression of the core component Sgcδ; (4) The ICD is immature in neonatal cardiomyocytes, Yap promotes the expression of the ICD component α3-catenin. Right, non-regenerative (adult) cardiomyocytes. (1) Hippo signalling is high, YAP phosphorylation and YAP binding to the DGC is increased; (2) YAP–TEAD transcription-activating activity is reduced; (3) The DGC sequesters phosphorylated YAP through an interaction involving the PPxY motif of Dag1; (4) The ICD is mature in adult cardiomyocytes, YAP is incorporated into the ICD independent of Hippo through α-catenin binding.

Supplementary information

Supplementary Figure

This file contains the uncropped scans with size marker indications. (PDF 6941 kb)

PowerPoint slides

Source data

Rights and permissions

About this article

Cite this article

Morikawa, Y., Heallen, T., Leach, J. et al. Dystrophin–glycoprotein complex sequesters Yap to inhibit cardiomyocyte proliferation.Nature 547, 227–231 (2017). https://doi.org/10.1038/nature22979

Download citation