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Hippo pathway deficiency reverses systolic heart failure after infarction

Nature volume 550, pages 260–264 (2017)Cite this article

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Abstract

Mammalian organs vary widely in regenerative capacity. Poorly regenerative organs, such as the heart are particularly vulnerable to organ failure. Once established, heart failure commonly results in mortality1. The Hippo pathway, a kinase cascade that prevents adult cardiomyocyte proliferation and regeneration2, is upregulated in human heart failure. Here we show that deletion of the Hippo pathway component Salvador (Salv) in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function compared with controls. Using translating ribosomal affinity purification, we isolate cardiomyocyte-specific translating messenger RNA. Hippo-deficient cardiomyocytes have increased expression of proliferative genes and stress response genes, such as the mitochondrial quality control gene, Park2. Genetic studies indicate that Park2 is essential for heart repair, suggesting a requirement for mitochondrial quality control in regenerating myocardium. Gene therapy with a virus encoding Salv short hairpin RNA improves heart function when delivered at the time of infarct or after ischaemic heart failure following myocardial infarction was established. Our findings indicate that the failing heart has a previously unrecognized reparative capacity involving more than cardiomyocyte renewal.

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Figure 1: Activated Hippo signalling in human heart failure.
Figure 2: Reversal of heart failure and cardiomyocyte renewal in SalvCKO mice.
Figure 3: SalvCKO mice activate reparative molecular response to heart failure.
Figure 4: Park2 in heart failure and Salv gene therapy.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (DE023177, HL127717, HL130804, HL118761 (J.F.M.); F31HL136065 (M.C.H.) and 5T32HL007676-23 (J.P.L.)), Vivian L. Smith Foundation (J.F.M.), State of Texas funding (J.F.M. and J.T.W.), LeDucq Foundation Transatlantic Networks of Excellence in Cardiovascular Research (14CVD01) ‘Defining the genomic topology of atrial fibrillation’ (J.F.M.). Supported by Intellectual and Developmental Disabilities Research Center grant number 1U54 HD083092 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development and the Mouse Phenotyping Core at Baylor College of Medicine (U54 HG006348). T.H. was supported by American Heart Association Scientist Development Grant (16SDG26460001). This work was also supported in part by Neuroconnectivity core and Optical Imaging and Vital Microscopy core at Baylor College of Medicine. N. Stancel provided editorial assistance.

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Authors and Affiliations

  1. Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, 77030, Texas, USA

    John P. Leach, Min Zhang & James F. Martin

  2. The Texas Heart Institute, 6770 Bertner Avenue, Houston, 77030, Texas, USA

    Todd Heallen, Mahdis Rahmani, Yuka Morikawa, Ana Segura, James T. Willerson & James F. Martin

  3. Shanghai Children’s Medical Center, Shanghai, 200127, China

    Min Zhang

  4. Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, 77030, Texas, USA

    Matthew C. Hill & James F. Martin

  5. Cardiovascular Research Institute, Baylor College of Medicine, One Baylor Plaza, Houston, 77030, Texas, USA

    James F. Martin

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Contributions

J.P.L., J.T.W., and J.F.M. conceived and designed experiments and interpreted data. J.P.L., T.H., M.Z., M.C.H., Y.M., and M.R. performed experiments. J.P.L. and J.F.M. analysed data and compiled figures. A.S. provided human tissue samples J.P.L., J.T.W., and J.F.M. wrote and edited the manuscript.

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Correspondence to James F. Martin.

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Extended data figures and tables

Extended Data Figure 1 Activated Hippo signalling in human heart failure.

a–c, Western blots of human heart samples. Ctrl: non-failing non-transplantable, n = 6 (a–c). HF: non-ischaemic idiopathic cardiomyopathy in end-stage heart failure, n = 6 (a, b). iHF: ischaemic heart in end-stage heart failure, n = 6 (c). Quantification presented in Fig. 1.

Extended Data Figure 2 Mouse model of systolic heart failure.

a–d, Systolic diameter (a), diastolic diameter (b), systolic volume (c), and diastolic volume (d); n values indicated in Fig. 1a; ANOVA, Tukey’s pairwise post-hoc test. e, f, Haematoxylin and eosin oedema liquid (pink transudate fluid) in lung tissue 3 weeks after myocardial infarction, sham (n = 3) (e) and myocardial infarction (n = 5) (f); scale bar, 50 μm. g, h, Prussian blue haemosiderin (blue) in lung tissue 3 weeks after myocardial infarction, sham (n = 3) (g) and myocardial infarction (n = 5) (h); scale bar, 50 μm. i, Natriuretic peptide B (BNP) in blood serum 3 weeks after myocardial infarction, Mann–Whitney U-test (i). j, Weight gain 3 weeks after myocardial infarction (j), t-test. k, l, Longitudinal echocardiography beginning 3 weeks after myocardial infarction; data are a subset of Fig. 2c. Control sham and control myocardial infarction samples were split by Cre genotype or by injection type, indicated in parenthesis (k). No significant effect of Cre or tamoxifen (Tam) was observed; ANOVA, Tukey’s pairwise post-hoc test (k). Data are mean ± s.e.m., P > 0.05 non-significant (NS), *P < 0.05, **P < 0.01, ***P < 0.001.

Extended Data Figure 3 Histological analysis at 3 weeks after myocardial infarction.

Masson’s trichrome of serial sagittal sections 3 weeks after myocardial infarction, no tamoxifen was delivered; genotype is indicated, n = 3 per group, scar boundaries (open arrows), cardiomyocytes in the ischaemic region (solid arrows); scale bar, 2 mm.

Extended Data Figure 4 Histological analysis at 4 weeks after myocardial infarction.

Masson’s trichrome of serial sagittal sections 4 weeks after myocardial infarction, 1 week after tamoxifen; control (αMHC-mcm; ROSAmT/mG) and SalvCKO (αMHC-mcm; ROSAmT/mG; Salvfl/fl), n = 3 per group, scar boundaries (open arrows), cardiomyocytes in the ischaemic region (solid arrows); scale bar, 2 mm.

Extended Data Figure 5 Histological analysis at 6 weeks after myocardial infarction.

Masson’s trichrome of serial sagittal sections 6 weeks after myocardial infarction, 3 weeks after tamoxifen; control (αMHC-mcm; ROSAmT/mG) and SalvCKO (αMHC-mcm; ROSAmT/mG; Salvfl/fl), n = 3 per group, scar boundaries (open arrows), cardiomyocytes in the ischaemic region (solid arrows); scale bar, 2 mm.

Extended Data Figure 6 Vessel growth in the border zone of Hippo-deficient mouse hearts.

a, qPCR of known markers of heart failure at 6 weeks after myocardial infarction; myosin heavy chain 6 (Myh6), myosin heavy chain 7 (Myh7), natriuretic peptide A (Nppa), natriuretic peptide B (Nppb), n = 3 per group; ANOVA, Bonferroni’s post-hoc test. b, Masson’s trichrome (scale bar, 100 μm) and immunofluorescence staining for isolectin B4 (scale bar, 25 μm) and CD31 (scale bar, 25 μm), control (n = 3) SalvCKO (n = 5). c–e, Quantification 9 weeks after myocardial infarction in the border zone for capillary density (c), isolectin+ vessels (d), and CD31+ cells (e), control (n = 3) SalvCKO (n = 5), Mann–Whitney U-test. f, qPCR of angiogenic growth factors, cardiomyocyte-specific TRAP RNA, 6 weeks after myocardial infarction, angiopoietin 1 (Angpt1) and 2 (Angpt2), fibroblast growth factor 14 (Fgf14) and 18 (Fgf18), vascular endothelial growth factor B (Vegfb) and C (Vegfc), n = 3 per group; ANOVA, Bonferroni’s post-hoc test. Data are mean ± s.e.m., P > 0.05 non-significant (NS), *P < 0.05, **P < 0.01, ***P < 0.001.

Extended Data Figure 7 TRAP RNA sequencing reproducibility.

a, Reproducibility correlation matrices of the RNA-seq read count, linear regression, n = 3 per group. b, c, Plot of the per-gene s.d. across samples, against the rank (mean) and read count, variance stabilizing transformation, total RNA-seq (b), and TRAP RNA-seq (c). d, log2(fold change) between TRAP and total RNA-seq for control myocardial infarction and SalvCKO myocardial infarction was highly correlated.

Extended Data Figure 8 A reparative molecular response to heart failure in Hippo-deficient hearts.

a, Gene lists of the top 10 genes with the highest fold change in each GO category for total RNA: SalvCKO myocardial infarction versus control myocardial infarction. b, Boxplot of the normalized read count for Tnnt2, Cdh5, and Malat1. The bars indicate minimum and maximum values. c, Volcano plot TRAP-seq: control myocardial infarction versus control sham. d, e, GO upregulated (d) and downregulated (e) genes. f, g, Gene lists of the top ten genes with the highest fold change in each GO category for TRAP RNA: control myocardial infarction versus control sham (f); TRAP RNA: SalvCKO myocardial infarction versus control myocardial infarction (g).

Extended Data Figure 9 Requirement of Park2 in the regenerating mouse heart.

a–c, Human heart western blots; quantification presented in Fig. 4, tubulin# blot is repeated from Extended Data Fig. 1. d, Scar size 21 days after myocardial infarction in P1 Park2 wild-type (+/+) and null (−/−) mice; Mann–Whitney U-test. e, f, Echocardiography: ejection fraction (EF) (e), and fractional shortening (FS) (f); ANOVA, Bonferroni’s post-hoc test. g, Cardiomyocyte cell size measured by cross-sectional area, mean (red dashed line), t-test. h, Masson’s trichrome, 21 days after myocardial infarction in P1 Park2 wild-type (+/+) (n = 8) and null (−/−) (n = 4) mice; scale bar, 2 mm. i, Summary of results indicating Park2 is necessary for cardiac regeneration. Data are mean ± s.e.m., P > 0.05 non-significant (NS), *P < 0.05, **P < 0.01, ***P < 0.001.

Extended Data Figure 10 Requirement of Park2 in the P8 Hippo-deficient regenerating mouse heart.

a, Mitochondrial DNA content 4 days after myocardial infarction in P8 control and SalvCKO (n = 3 per group). b, Park2 protein levels in border zone (BZ) and distal zone (DZ) myocardium at 4 days after myocardial infarction in P8 control and SalvCKO (n = 3 per group). c, Scar size 21 days after myocardial infarction in P8 Park2 wild-type (+/+) and mutant (mut; −/− or +/−) mice, in combination with SalvCKO; ANOVA, Bonferroni’s post-hoc test. d, e, Echocardiography: ejection fraction (EF) (d) and fractional shortening (FS) (e); ANOVA, Bonferroni’s post-hoc test. f, Masson’s trichrome, 21 days after myocardial infarction in P8 Park2 mutant (n = 20) and SalvCKO; Park2 double-mutant mice (n = 15); scale bar, 2 mm. g, Summary of results indicating Park2 is necessary for regeneration. Data are mean ± s.e.m., P > 0.05 non-significant (NS), *P < 0.05, **P < 0.01, ***P < 0.001.

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This file contains Supplementary Figure 1 and Supplementary Table 1. Supplementary Figure 1 shows the uncropped scans with size marker indications and Supplementary Table 1 shows a list of qPCR primers. (PDF 4977 kb)

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Leach, J., Heallen, T., Zhang, M. et al. Hippo pathway deficiency reverses systolic heart failure after infarction. Nature 550, 260–264 (2017). https://doi.org/10.1038/nature24045

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