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

Abstract

The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signaling-deficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex, a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near the scar tissue of injured Hippo signaling-deficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation, after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling. Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to mechanical changes associated with heart injury to promote repair.

Figure 1. Integrated genomic analysis for identifying Yap target genes

(A) Motif analysis for enriched Yap ChIP-Seq peaks (total number = 35,412 from 2 independent biological replicates). De novo motifs and their best matches are shown. (B) The density of Tead binding motifs within a 500-bp distance of the Yap ChIP-Seq peaks is shown. (C) Overlay of genes with increased expression in Salv CKO mouse hearts (total number = 1,706 from 2 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 over-representation 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–H) qRTPCR 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.

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

(A–D) Genome browser view of Yap ChIP-Seq enriched peaks for the labeled genes. Alignments are shown for stage-specific H3K27Ac ChIP-Seq, DHS, and 4C anchor points (for Ctnna3 and Sgcd). For 4C, “vp” denotes viewpoint and “enh” denotes enhancer. Grey 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. (E) Quantification of P7 heart H3K27Ac ChIP-Seq reads in the 6-kb range around Yap ChIP-Seq peaks. N=2 biologic replicates. (F) Luciferase reporter assay data showing that Yap/Tead co-activated 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 co-transfected with Yap and TEAD expression vectors. ***p<0.001 (Mann-Whitney), n.s. not significant. Error bars are standard deviations. Activity was normalized to that of cells expressing the pGL3 vector.

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

LAD occlusion was performed on Myh6-Cre^Ert; mTmG (control) and Myh6-Cre^Ert; Salv^fx/fx; mTmG (Salv CKO) mice, and hearts were collected at 1, 4, 10, and 15 days post myocardial infarction (dpmi). (A, 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. Bars=50 μm. (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). *P<0.05. (D–F) Aurora B kinase immunostaining served as a proxy of cytokinesis in control (D) and Salv CKO (E, F) mouse hearts at 10 dpmi. Arrow shows staining in a non-cardiomyocyte cell, and arrowheads show staining in cardiomyocytes. Bars=20 μm. (G, H): Quantification of Aurora B kinase (G) and TUNEL immunostaining (H) (N=3 mice for each genotype and treatment). **P<0.01. (I–M) Yap localization in border zone of control (I, J) and Salv CKO mouse hearts (K, L) at 10 dpmi. Arrowheads show nuclear localized Yap. Bars=50 μm. Quantification (M) 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. (N) Gene expression of Yap downstream target genes were quantified with qPCR in border zones from heart tissues after myocardial infarction (N=3 biologic replicates). **P<0.01, ***P<0.001

Figure 4. Cardiomyocyte morphology and cytoskeleton rearrangement during adult heart regeneration

(A to F) LAD occlusion was performed on the hearts of Myh6-Cre^Ert; mTmG (control) and Myh6-Cre^Ert; Salv^fx/fx; mTmG (Salv CKO) mice. Control (A–C) and Salv CKO (D–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 Supplemental fig. S6A-F. Arrows show cardiomyocytes with sarcomere disassembly. Arrowheads show cardiomyocyte protrusion. 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, I) and Salv CKO (J, 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. Bars=25 μm. Control (L, M) and Salv CKO (N, 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). Bars=25 μm. (P to W) Control (P, Q) and Salv CKO (R, 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. Bars=20 μm. Control (T, U) and Salv CKO (V, W) mouse heart sections at 10 dpmi were stained for FAK (N=2 mice per genotype). Arrowheads show increased Cofilin staining in border zone cardiomyocytes. Bars=20 μm.

Figure 5. Cardiomyocyte migration through collagen

(A to E) Collagen migration assays were performed with P8 cardiomyocytes from control (Myh6-Cre^Ert; mTmG )(A, B) or Salv CKO (Myh6-Cre^Ert; Salv^fx/fx; mTmG) mouse hearts (C, D). Cardiomyocyte lineage was visualized with eGFP and non-cardiomyocytes were visualized with mTomato. Whole gel view (A, C). Arrowheads show migrated cardiomyocytes. Bars=250 μm. High-magnification images of the area with eGFP positive cells (B, D). Bars=50 μm. Quantification (E) 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–K) P19 cell migration in collagen gel after siRNA treatment with the labeled siRNA. Bars=250 μm. (L) Quantification of migrated cells after each treatment. Cells were treated with either siRNA or Yap inhibitor verteporfin (VP). n=3 biological replicates for all groups.

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

(A, B) LADO and sham surgery were performed in control and Salv CKO mouse hearts at P8, and heart samples were collected at 4 dpmi. Delta-sarcoglycan (Sgcd) mRNA was detected in control and Salv CKO mouse hearts by using qRT-PCR and was normalized to 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). Sgcd band intensities were quantified and normalized to those of alpha-tubulin. ***P<0.001, remaining column comparisons were non-significant (n.s.) (C) Luciferase 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–G) Dystophin glycoprotein complex (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 2 additional control and mutant apexes are shown in fig. S9. Bars=500 μm. Quantification (F) of the scar size at 21 dpr 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. Echocardiography analysis (G) 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 n.s.

Figure 7. Regulation of cardiomyocyte protrusion by the dystrophin complex

(A–D) Yap localization in border zone cardiomyocytes of B6/10 control (A, B) and Mdx-B6/10 (C, D) mouse hearts at 4 days post resection (dpr). Arrows show nuclear localized Yap. Bars=25 μm. (E) Quantification of nuclear Yap in border zone cardiomyocytes at 4 dpr (N=3 mice per genotype). *P<0.05. (F–H) DGC is required for cardiomyocyte protrusion. Sections of the border zone of B6/10 control (F, 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). For (F) and (H), bars=50 μm; for (G), bars=25 μm. (I–K) Collagen gel assay for P1 B10 (I, J) and Mdx-B10 (K) hearts. (J) is a higher magnification image of (I). For (I) and (K), bars=100 μm; for (J), bars=20 μm. (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–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). Bars=250 μm. (Q) Quantification of migrated cells after each treatment. N=3 biological replicates for all groups. *P<0.01.