Research ArticleRegeneration

Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice

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Science Signaling  05 May 2015:
Vol. 8, Issue 375, pp. ra41
DOI: 10.1126/scisignal.2005781
  • Fig. 1 Integrated genomic analysis for identifying Yap target genes.

    (A) Motif analysis for enriched Yap ChIP-Seq peaks (total number = 35,412 from two independent biological replicates). De novo motifs and their best matches are shown. (B) The density of Tead-binding motifs within a 500–base pair (bp) distance of the Yap ChIP-Seq peaks is shown. (C) Overlay of genes with increased expression in Salv CKO mouse hearts (total number = 1706 from two mice) and genes annotated from Yap ChIP-Seq peaks (total number = 10,396). (D) Gene ontology analysis of genes with increased expression in Salv CKO mouse hearts and with Yap binding peaks (total number = 928). Enriched terms were calculated by using overrepresentation statistics and measured by using z scores. (E) Heat map of Yap target genes identified by the overlay of microarray and Yap ChIP-Seq genes in the labeled categories. Heat maps show relative expression between Salv CKO and control mouse hearts. (F to H) Quantitative real-time PCR (qRT-PCR) validation of Yap target genes in P8 control (unshaded bar) and P8 Salv CKO (shaded bar) mouse hearts. n = 3 independent biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 2 Preferential expression of Yap target genes in the fetal heart.

    (A to D) Genome browser view of Yap ChIP-Seq enriched peaks for the labeled genes. Alignments are shown for stage-specific H3K27Ac ChIP-Seq, DHS, 4C anchor points [for Ctnna3 and Sgcd, and conservation between human and mouse genomes (cons.)]. For 4C, “vp” denotes viewpoint and “enh” denotes enhancer. Gray blocks show regulatory regions used as luciferase reporters with red lines indicating consensus Tead-binding motif. The y axes of Yap ChIP peaks show the normalized read number. E14.5, embryonic day 14.5. (E) Quantification of P7 heart H3K27Ac ChIP-Seq reads within the 6-kb range around Yap ChIP-Seq peaks. n = 2 biological replicates. (F) Luciferase reporter assay data showing that Yap/Tead coactivated target gene expression through regulatory regions identified in Yap ChIP-Seq. n = 3 independent transfection experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (G) Luciferase reporter assay data of Yap enhancers with or without Tead motifs. n = 3 independent transfection experiments. All luciferase constructs were cotransfected with Yap and Tead expression vectors. ***P < 0.001 (Mann-Whitney), n.s., not significant. Error bars are SD. Activity was normalized to that of cells expressing the pGL3 vector.

  • Fig. 3 DNA synthesis and Yap localization in border zone cardiomyocytes during adult heart regeneration.

    LADO was performed on Myh6-CreErt; mTmG (control) and Myh6-CreErt; Salvfx/fx; mTmG (Salv CKO) mice, and hearts were collected at 1, 4, 10, and 15 days post–myocardial infarction (dpmi). (A and B) De novo DNA synthesis was detected by measuring EdU incorporation in control (A) and Salv CKO (B) mouse hearts at 10 dpmi. Red arrowhead shows EdU-stained nucleus. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (C) Quantification of de novo DNA synthesis in the border zone at 1, 4, 10, and 15 dpmi or in sham mice (n = 3 mice for each genotype and time point). MI, myocardial infarction; CM, cardiomyocytes. *P < 0.05. (D to F) Aurora B kinase immunostaining served as a proxy of cytokinesis in control (D) and Salv CKO (E and F) mouse hearts at 10 dpmi. Arrow shows staining in a noncardiomyocyte cell, and arrowheads show staining in cardiomyocytes. Scale bars, 20 μm. (G and H) Quantification of Aurora B kinase (G) and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) immunostaining (H) (n = 3 mice for each genotype and treatment). **P < 0.01. (I to L) Yap localization in border zone of control (I and J) and Salv CKO mouse hearts (K and L) at 10 dpmi. Arrowheads show nuclear-localized Yap. Scale bars, 50 μm. (M) Quantification of nuclear Yap in border zone cardiomyocytes at 10 dpmi (n = 3 mice for each genotype and treatment). *P < 0.05; remaining column comparisons were nonsignificant. eGFP, enhanced green fluorescent protein; Nuc, nuclear. (N) Gene expression of Yap downstream target genes was quantified with qPCR in border zones from heart tissues after myocardial infarction (n = 3 biological replicates). **P < 0.01; ***P < 0.001.

  • Fig. 4 Cardiomyocyte morphology and cytoskeleton rearrangement during adult heart regeneration.

    (A to D) LADO was performed on the hearts of Myh6-CreErt; mTmG (control) and Myh6-CreErt; Salvfx/fx; mTmG (Salv CKO) mice. Control (A to C) and Salv CKO (D to F) mouse hearts were stained for the cardiomyocyte marker cTnT to visualize morphology of the cardiomyocytes in the border zone at 4, 7, and 10 dpmi (n = 3 mice for each genotype and time point). More images from different hearts of 10-dpmi hearts are shown in fig. S6, A to F. Arrows show cardiomyocytes with sarcomere disassembly. Arrowheads show cardiomyocyte protrusion. Scale bars, 50 μm. (G) Quantification of protrusions. Cardiomyocytes adjacent to the scar were analyzed for length and number of protrusions at 10 dpmi. One hundred cardiomyocytes from each mouse were analyzed (n = 3 mice per genotype). *P < 0.05. (H to O) Control (H and I) and Salv CKO (J and K) mouse heart sections at 10 dpmi were stained for talin (n = 2 mice per genotype). Arrows show increased talin staining in border zone cardiomyocytes. Scale bars, 25 μm. Control (L and M) and Salv CKO (N and O) mouse heart sections at 10 dpmi were stained for the focal adhesion molecule vinculin. Arrowheads show the rearrangement of vinculin in the protruding front of the cardiomyocytes (n = 3 mice per genotype). Scale bars, 25 μm. (P to W) Control (P and Q) and Salv CKO (R and S) mouse heart sections at 10 dpmi were stained for FAK (n = 2 mice per genotype). Arrows show increased FAK staining in border zone cardiomyocytes. Scale bars, 20 μm. Control (T and U) and Salv CKO (V and W) mouse heart sections at 10 dpmi were stained for cofilin (n = 2 mice per genotype). Arrowheads show increased cofilin staining in border zone cardiomyocytes. Scale bars, 20 μm.

  • Fig. 5 Cardiomyocyte migration through collagen.

    (A to D) Collagen migration assays were performed with P8 cardiomyocytes from control (Myh6-CreErt; mTmG) (A and B) or Salv CKO (Myh6-CreErt; Salvfx/fx; mTmG) mouse hearts (C and D). Cardiomyocyte lineage was visualized with eGFP and noncardiomyocytes were visualized with mTomato. (A and C) Whole-gel view. Arrowheads show migrated cardiomyocytes. Scale bars, 250 μm. (B and D) High-magnification images of the area with eGFP-positive cells. Scale bars, 50 μm. (E) Quantification of the number of hearts in which migration was observed (n = 6 hearts per genotype). P = 0.002, control compared to Salv CKO mice. (F to K) P19 cell migration in collagen gel after siRNA treatment with the labeled siRNA. Scale bars, 250 μm. (L) Quantification of migrated cells after each treatment. Cells were treated with either siRNA or the Yap inhibitor verteporfin (VP). n = 3 biological replicates for all groups.

  • Fig. 6 The dystrophic complex is downstream of the Hippo pathway and is required for cardiac regeneration.

    (A and B) LADO and sham surgery were performed in control and Salv CKO mouse hearts at P8, and heart samples were collected at 4 dpmi. Sgcd mRNA was detected in control and Salv CKO mouse hearts by using qRT-PCR and was normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (n = 3 hearts for all groups) (A). Sgcd protein was detected by using Western blot analysis (n = 3 hearts per genotype and treatment) (B). Cnt, control. Sgcd band intensities were quantified and normalized to those of α-tubulin. ***P < 0.001, remaining column comparisons were nonsignificant. (C) Luciferase (luc) assays were performed with P19 embryonal carcinoma cells. Cells were transfected with either the control luciferase reporter, a reporter containing the Sgcd enhancer, or a reporter containing the Sgcd enhancer but lacking the Tead site. Three independent experiments with technical triplicates were performed. *P < 0.05. (D and E) DGC is required for endogenous cardiac regeneration. Representative images of trichrome-stained heart sections from B10 control (D) and mdx-B10 (E) mice subjected to resection of the cardiac apex. Images of two additional control and mutant apexes are shown in fig. S9. Scale bars, 500 μm. (F) Quantification of the scar size at 21 days after resection in B10, (n = 11), B6/10 (n = 4), mdx-B10 (n = 7), and mdx-B6/10 (n = 6) mouse hearts.*P < 0.05; ***P < 0.001. (G) Echocardiographic analysis of control sham (n = 3), control apex resection (n = 7), mdx sham (n = 4), and mdx apex resection (n = 7) mouse hearts 21 days after surgery. ***P < 0.001, remaining column comparisons were nonsignificant.

  • Fig. 7 Regulation of cardiomyocyte protrusion by the dystrophin complex.

    (A to D) Yap localization in border zone cardiomyocytes of B6/10 control (A and B) and mdx-B6/10 (C and D) mouse hearts at 4 days post-resection (dpr). Arrows show nuclear-localized Yap. Scale bars, 25 μm. (E) Quantification of nuclear Yap in border zone cardiomyocytes at 4 dpr (n = 3 mice per genotype). *P < 0.05. (F to H) DGC is required for cardiomyocyte protrusion. Sections of the border zone of B6/10 control (F and G) and mdx-B6/10 (H) mouse hearts were stained with the cardiac marker cTnT at 4 dpr. Arrows show protruding cells at the border zone. (G) is a higher-magnification image of (F). Scale bars, 50 μm (F and H); 25 μm (G). (I to K) Collagen gel assay for P1 B10 (I and J) and mdx-B10 (K) hearts. (J) is a higher magnification image of (I). Scale bars, 100 μm (I and K); 20 μm (J). (L) Quantification of cardiomyocyte migration from B10 control (n = 11) and mdx-B10 (n = 6) mouse hearts. P = 0.035, control compared to mdx. (M to P) P19 cell migration in collagen gel after transfection with the indicated siRNA. Negative control (M), Salv (N), dystrophin (Dmd) (O), and Salv and Dmd combined (P). Scale bars, 250 μm. (Q) Quantification of migrated cells after each treatment. n = 3 biological replicates for all groups. *P < 0.01.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/375/ra41/DC1

    Fig. S1. Microarray and Yap ChIP-Seq reproducibility.

    Fig. S2. Yap target genes are enriched in the fetal heart.

    Fig. S3. Yap/Tead regulatory elements are enriched in fetal heart DHS and Yap binding peaks.

    Fig. S4. Analysis of cell fusion in hearts 21 days after myocardial infarction.

    Fig. S5. Evaluation of cardiomyocyte size in Hippo-deficient hearts.

    Fig. S6. Cardiomyocyte protrusions in Hippo-deficient hearts.

    Fig. S7. Collagen gel migration assay with mitomycin C treatment.

    Fig. S8. Western blot analysis to quantitate sarcoglycan δ and syntrophin β1.

    Fig. S9. Scar formation in the apex resection model in mdx hearts.

    Fig. S10. Cardiomyocyte proliferation in the mdx apex-resected heart.

    Table S1. List of primers used to amplify enhancer elements and location of the Tead motif deletion.

  • Supplementary Materials for:

    Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice

    Yuka Morikawa, Min Zhang, Todd Heallen, John Leach, Ge Tao, Yang Xiao, Yan Bai, Wei Li, James T. Willerson, James F. Martin*

    *Corresponding author. E-mail: jfmartin{at}bcm.edu

    This PDF file includes:

    • Fig. S1. Microarray and Yap ChIP-Seq reproducibility.
    • Fig. S2. Yap target genes are enriched in the fetal heart.
    • Fig. S3. Yap/Tead regulatory elements are enriched in fetal heart DHS and Yap binding peaks.
    • Fig. S4. Analysis of cell fusion in hearts 21 days after myocardial infarction.
    • Fig. S5. Evaluation of cardiomyocyte size in Hippo-deficient hearts.
    • Fig. S6. Cardiomyocyte protrusions in Hippo-deficient hearts.
    • Fig. S7. Collagen gel migration assay with mitomycin C treatment.
    • Fig. S8. Western blot analysis to quantitate sarcoglycan δ and syntrophin β1.
    • Fig. S9. Scar formation in the apex resection model in mdx hearts.
    • Fig. S10. Cardiomyocyte proliferation in the mdx apex-resected heart.
    • Table S1. List of primers used to amplify enhancer elements and location of the Tead motif deletion.

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    Citation: Y. Morikawa, M. Zhang, T. Heallen, J. Leach, G. Tao, Y. Xiao, Y. Bai, W. Li, J. T. Willerson, J. F. Martin, Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice. Sci. Signal. 8, ra41 (2015).

    © 2015 American Association for the Advancement of Science

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