Dystrophin–glycoprotein complex sequesters Yap to inhibit cardiomyocyte proliferation

Abstract

The regenerative capacity of the adult mammalian heart is limited, because of the reduced ability of cardiomyocytes to progress through mitosis1. Endogenous cardiomyocytes have regenerative capacity at birth but this capacity is lost postnatally, with subsequent organ growth occurring through cardiomyocyte hypertrophy2,3. The Hippo pathway, a conserved kinase cascade, inhibits cardiomyocyte proliferation in the developing heart to control heart size and prevents regeneration in the adult heart4,5. The dystrophin–glycoprotein complex (DGC), a multicomponent transmembrane complex linking the actin cytoskeleton to extracellular matrix, is essential for cardiomyocyte homeostasis. DGC deficiency in humans results in muscular dystrophy, including the lethal Duchenne muscular dystrophy. Here we show that the DGC component dystroglycan 1 (Dag1) directly binds to the Hippo pathway effector Yap to inhibit cardiomyocyte proliferation in mice. The Yap–Dag1 interaction was enhanced by Hippo-induced Yap phosphorylation, revealing a connection between Hippo pathway function and the DGC. After injury, Hippo-deficient postnatal mouse hearts maintained organ size control by repairing the defect with correct dimensions, whereas postnatal hearts deficient in both Hippo and the DGC showed cardiomyocyte overproliferation at the injury site. In the hearts of mature Mdx mice (which have a point mutation in Dmd)—a model of Duchenne muscular dystrophy—Hippo deficiency protected against overload-induced heart failure.

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Figure 1: Combined loss of the DGC and Hippo pathway in the injured heart.
Figure 2: Yap subcellular localization and expression of downstream targets in Hippo-deficient Mdx cardiomyocytes after apical resection.
Figure 3: Suppression of Mdx cardiomyopathy by Hippo deletion in a pressure overload model.
Figure 4: Yap binding to the DGC.

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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.

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Authors

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 to James F. Martin.

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The authors declare no competing financial interests.

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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 Cardiomyocyte alignment after apical resection in P8 mouse hearts.

ao, 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.

ae, 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. fm, 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. nr, 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.

ae, 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.

an, Flow cytometry analysis of isolated nuclei after TAC surgery. al, 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. ad, PCM1+ population was gated and plots show EdU incorporation. eh, Histogram showing DAPI intensity in PCM1+ population and discrimination between 2N, 4N, and >4N population. il, Histogram showing DAPI intensity in PCM1+, EdU+ population. m, n, Quantification of PCM1+, EdU+ nuclei in >2N–4N (m) and >4N (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. ov, 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.

ad, 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. ek, 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. mt, 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.

ah, 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. jq, 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. ad, Staining for Salv in Mdx hearts transfected with AAV9–GFP (a, b) or AAV9–Salv (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. fi, 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 siSalv, siDmd, 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. bd, 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.

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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

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