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Nfat and miR-25 cooperate to reactivate the transcription factor Hand2 in heart failure

Nature Cell Biology volume 15, pages 1282–1293 (2013)Cite this article

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Abstract

Although aberrant reactivation of embryonic gene programs is intricately linked to pathological heart disease, the transcription factors driving these gene programs remain ill-defined. Here we report that increased calcineurin/Nfat signalling and decreased miR-25 expression integrate to re-express the basic helix-loop-helix (bHLH) transcription factor dHAND (also known as Hand2) in the diseased human and mouse myocardium. In line, mutant mice overexpressing Hand2 in otherwise healthy heart muscle cells developed a phenotype of pathological hypertrophy. Conversely, conditional gene-targeted Hand2 mice demonstrated a marked resistance to pressure-overload-induced hypertrophy, fibrosis, ventricular dysfunction and induction of a fetal gene program. Furthermore, in vivo inhibition of miR-25 by a specific antagomir evoked spontaneous cardiac dysfunction and sensitized the murine myocardium to heart failure in a Hand2-dependent manner. Our results reveal that signalling cascades integrate with microRNAs to induce the expression of the bHLH transcription factor Hand2 in the postnatal mammalian myocardium with impact on embryonic gene programs in heart failure.

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Figure 1: Hand2 is reactivated in heart failure and sufficient to drive cardiac remodelling.
Figure 2: Conditional Hand2 gene targeting.
Figure 3: Gene profiling of Hand2 target genes.
Figure 4: Transcriptional regulation of cardiac Hand2.
Figure 5: Post-transcriptional regulation of Hand2 expression.
Figure 6: miR-25 silencing exacerbates cardiac remodelling.
Figure 7: miR-25 silencing exacerbates cardiac remodelling.
Figure 8: Hand2 regulation in the postnatal myocardium by a combined calcineurin/Nfat transcriptional and miR-25 post-transcriptional axis.

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Acknowledgements

We gratefully acknowledge G. Summer for bioinformatics help and G. van Hout for technical support. T.T. was supported by the German Research Foundation (TH903/11-1) and the REBIRTH Exellence Cluster. T.E. was supported by the German Research Foundation (ES88/12-1) and the DZHK, the German Centre for Cardiovascular Research funded by the German Ministry of Research and Education (BMBF). P.A.d.C.M. was supported by a Leducq Career Development Award and the Dutch Heart Foundation grant NHS2010B261. L.J.D.W. acknowledges support from the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development (ZonMW) and the Royal Netherlands Academy of Sciences. L.J.D.W. was further supported by a VIDI award 917-863-72 from the ZonMW; the Dutch Heart Foundation program grant NHS2007B167; the Fondation Leducq Transatlantic Network of Excellence program 08-CVD-03 and grant 311549 from the European Research Council (ERC).

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Author notes

  1. Ellen Dirkx and Monika M. Gladka: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Cardiology, CARIM School for Cardiovascular Diseases, Maastricht University, 6229 ER Maastricht, The Netherlands

    Ellen Dirkx, Monika M. Gladka, Leonne E. Philippen, Virginie Kinet, Stefanos Leptidis, Hamid el Azzouzi, Kanita Salic, Servé Olieslagers, Nicole Bitsch, Natasja Kisters, Sandrine Seyen, Stephane Heymans, Paul G. A. Volders, Paula A. da Costa Martins & Leon J. De Windt

  2. Centre d’Etude de la Sensori-motricité, UMR 8194 CNRS, Université Paris Descartes, 75006 Paris, France

    Anne-Sophie Armand & Christophe Chanoine

  3. Interuniversity Cardiology Institute Netherlands, Royal Netherlands Academy of Sciences, 3511 GC Utrecht, The Netherlands

    Stefanos Leptidis, Hamid el Azzouzi, Kanita Salic & Meriem Bourajjaj

  4. Department of Circulation and Medical Imaging, Norwegian University of Science and Technology (NTNU), NO-7491 Trondheim, Norway

    Gustavo J. J. da Silva

  5. Department of Medical Physiology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands

    Roel van der Nagel

  6. Department of Pathology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands

    Roel de Weger

  7. Texas Heart Institute, Houston, Texas 77030, USA

    Yuka Morikawa

  8. Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School, D-30625 Hannover, Germany

    Thomas Thum

  9. National Heart and Lung Institute, Imperial College London, London SW7 2AZ, UK

    Thomas Thum

  10. Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, Goethe University, D-60590 Frankfurt am Main, Germany

    Stefanie Dimmeler

  11. Department of Pathology and Cell Biology, Columbia University, New York 10032, USA

    Peter Cserjesi

  12. Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany

    Thomas Eschenhagen

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  1. Ellen Dirkx
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Contributions

E.D., P.A.d.C.M, M.M.G., L.E.P., K.S., M.B. and N.K. performed northern blots and quantitative real-time PCR experiments. M.M.G., S.O. and S.S. performed western blots. E.D. and M.M.G. performed luciferase assays. S.L. performed bioinformatics analyses. E.D., V.K. and S.O. performed chromatin immunoprecipitation assays. A-S.A. and C.C. performed mouse embryo studies. Y.M., P.C., S.O. and L.J.D.W. created genetically modified mouse models. H.e.A. and N.B. performed surgical procedures in mouse models. E.D., P.A.d.C.M., M.M.G., R.N., K.S., N.B. and G.J.J.d.S. performed echocardiography and histology in mouse models. E.D., P.A.d.C.M., M.M.G., A-S.A., K.S., S.L. and L.J.D.W. analysed data. C.C., S.H., P.G.A.V., T.T., S.D., P.C. and T.E. provided reagents, models or data. E.D., P.A.d.C.M. and L.J.D.W. designed the study. E.D., P.A.d.C.M. and L.J.D.W. wrote the manuscript. P.A.d.C.M. and L.J.D.W. acquired funding for the study. E.D. and M.M.G. contributed equally as joint first authors. P.A.d.C.M. and L.J.D.W. contributed equally as joint last authors.

Corresponding authors

Correspondence to Paula A. da Costa Martins or Leon J. De Windt.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Additive cardiomyocyte hypertrophy by Hand2 overexpression in combination with overexpression with MEF2, NFAT or GATA4.

(a) Confocal microscopy images of neonatal rat cardiomyocytes infected with AdGFP, AdMEF2A, AdNFAT or AdGATA4 alone (top row). Confocal microscopy images of neonatal rat cardiomyocyte pre-infected with AdHand2 and infected with AdGFP, AdMEF2A, AdNFAT or AdGATA4 (bottom row). Nuclei were visualized with DAPI and cells were stained with an antibody against α-actinin (red). Scale bar, 50 μm. (b) Quantification of cell surface area in conditions in (a), n refers to number of microscopic fields. *P<0.05 vs control group; #P<0.05 vs experimental group (error bars are s.e.m.). Source data are shown in Supplementary Table 9.

Supplementary Figure 2 Effect of tamoxifen treatment on cardiac function.

(a) Real-time PCR analysis of Hand2, Nppa and Nppb transcript abundance in hearts from MHC-MerCreMer transgenic (MHC-MCM) mice treated with vehicle or tamoxifen at a dose of 40 mg/kg/day for 5 consecutive days dissolved in sunflower oil as described previously or tamoxifen at a dose of 45 mg/kg/day for 5 consecutive days dissolved in peanut oil (this study), n refers to number of hearts. (b) Quantification of the left ventricular mass, (c) fractional shortening (FS), (d) left ventricular internal diameter in systole (LVIDs) and (e) E/A ratio from pulsed-wave Doppler imaging of the ratio of blood flow through the mitral valve during early (E) versus late (A) diastole in MHC-MCM mice, injected with vehicle (PBS) or 45 mg/kg/day tamoxifen dissolved in peanut oil, n refers to number of animals. (f) Real-time PCR analysis of Rcan1.4 expression in hearts from mice with indicated genotype, pre-treated with vehicle or tamoxifen and subjected to sham or TAC surgery, n refers to number of hearts. *P<0.05 vs control group; #P<0.05 vs experimental group (error bars are s.e.m.). Source data are shown in Supplementary Table 9.

Supplementary Figure 3 Real-time PCR validation of microarray results in pressure overloaded Hand2F/F and MCM-Hand2F/F mice.

(a) Validation of the gene profiling microarray results from Supplementary Tables 1 and 2 by quantitative real-time PCR, n refers to number of hearts. Relative mRNA expression levels were determined for transcripts that were increased in expression in pressure overloaded Hand2-deficient hearts (Abcc8, Ephb3, Smad6) or (b) downregulated in pressure overloaded Hand2-deficient hearts (Enpp1, Cacna1c, Lama2, Actb, Robo2 and Id1), n refers to number of hearts. *P<0.05 vs control group (error bars are s.e.m.). Source data are shown in Supplementary Table 9.

Supplementary Figure 4 miR-25, miR-92a, miR-92b and miR-1 expression levels in heart disease.

(a) Location of potential miR-92a, miR-92b and miR-1 seed regions in human and mouse Hand2 3′UTR. (b) Northern blot analysis of miR-92a expression in hearts from nontransgenic littermates (nTg), calcineurin transgenic mice (MHC-CnA) and mice subjected to transverse aortic constriction (TAC). Quantification of Rnu6-2 corrected Northern blot signals for miR-92a (right panel), n refers to number of hearts. (c) Northern blot analysis of miR-92b expression in hearts from nontransgenic littermates (nTg), calcineurin transgenic mice (MHC-CnA) and mice subjected to transverse aortic constriction (TAC). Quantification of Rnu6-2 corrected northern blot miR-92b signals (right panel), n refers to number of hearts. (d) Northern blot analysis of miR-1 expression levels in hearts from nTg mice, MHC-CnA mice and mice subjected to TAC. Quantification of Rnu6-2 corrected Northern blot miR-1 signals (right panel), n refers to number of hearts. (e) Real-time PCR analysis of miR-92a expression in hearts from mice treated with scrambled antagomir (ctrl antagomir) or antagomir specific for miR-92a (antagomir-92a), n refers to number of hearts. (f) Western blot analysis of endogenous Hand2 or GAPDH protein expression in hearts from mice treated with ctrl antagomir or antagomir-92a. (g) Quantification of GAPDH corrected Hand2 Western blot signals from (f), n refers to number of hearts. (h) Real-time PCR analysis of Hand2 transcript abundance in hearts from mice treated with ctrl antagomir or antagomir-92a, n refers to number of hearts. (i) Schematic representation of the precursor sequence of hsa-miR-25 and conservation level of the mature miR-25-3p strand. (j) Endogenous miR-25 expression in human fetal myocardium and human adult myocardium indicating a higher expression level of miR-25 in the adult human heart, n refers to number of hearts. (k) Northern blot analysis of miR-25 expression in multiple mouse organs. (l) Activity assay of luciferase reporter construct harboring an intact or mutated Hand2 3′UTR after transfection with premiR- 25, pre-miR-92a, pre-miR-92b or pre-miR-1 in neonatal rat cardiomyocytes. A scrambled precursor miR (scr.pre-miR) was used as a control, n refers to number of transfection experiments. (m) Confocal microscopy images of neonatal rat cardiomyocytes transfected with scrambled anti-miR (scr.anti-miR) or anti-miR for miR-25 (anti-miR-25) and treated with PE for 24 h. Quantification of cell surface areas from these conditions (lower panel), n refers to number of microscopic fields. *P<0.05 vs control group (error bars are s.e.m.). Source data are shown in Supplementary Table 9.

Supplementary Figure 5 Downregulation of the miR-106b∼25 cluster in the adult heart involves NFAT-dependent transcriptional repression.

(a) Confocal microscopy images of neonatal rat cardiomyocytes transfected with scrambled precursor (scr.pre-miR), synthetic precursor scrambled anti-miR (scr.anti-miR) or anti-miR for miR-92a (pre-miR-92b, anti-miR-92b), miR-92b (pre-miR-92b, anti-miR-92b) and treated with or without PE for 24 h. Scale bar, 50 μm. (b) Quantification of cell surface areas from conditions in (a), n refers to number of microscopic fields. (c) Confocal microscopy images of neonatal rat cardiomyocytes infected with AdLacz (control adenovirus), AdVIVIT (adenovirus overexpressing the VIVIT optimized NFAT inhibitory peptide), AdVIVIT + pre-miR-25 and AdHand2 + pre-miR-25. In all conditions cells were treated with PE or AdCnA for 24h. Scale bar, 50 μm. (d) Quantification of cell surface areas from conditions in (c), n refers to number of microscopic fields. (e) Pri-miR-25, pri-miR-106b and pri-miR-93 are down-regulated in two animal models of heart failure: TAC hearts and MHC-CnA Tg hearts, n refers to number of hearts. (f) Pre-miR-25, pre-miR-106b and pre-miR-93 are downregulated in two animal models of heart failure: TAC hearts and MHC-CnA Tg hearts, n refers to number of hearts. (g) Real-time PCR analysis of the MCM7-001 and MCM7-010 transcripts in wild-type (wt) versus MHC-CnA Tg hearts, n refers to number of hearts. (h) Real-time PCR analysis of miR-25 expression of neonatal rat cardiomyocytes infected with AdLacz, AdCnA, AdVIVIT, and AdCnA with AdVIVIT, n refers to number of dishes. (i) Real-time PCR analysis of MCM7-001 and MCM7-010 expression of neonatal rat cardiomyocytes infected with AdLacz, AdCnA, AdVIVIT, and AdCnA with AdVIVIT, n refers to number of dishes. *P<0.05 vs control group; #P<0.05 vs experimental group (error bars are s.e.m.). Source data are shown in Supplementary Table 9.

Supplementary Figure 6 Specific miR-25 silencing with antagomir-25.

(a) Real-time PCR analysis of miR-93 expression in hearts from mice receiving control (ctrl) antagomir or antagomir against miR-25 (antagomir-25) and subjected to sham or transverse aortic constriction (TAC) surgery, n refers to number of hearts. (b) Real-time PCR analysis of miR-92b expression in hearts from mice receiving control (ctrl) antagomir or antagomir against miR-25 and subjected to sham or transverse aortic constriction (TAC) surgery, n refers to number of hearts. (c) Real-time PCR analysis of miR-148a expression in hearts from mice receiving control (ctrl) antagomir or antagomir against miR-25 and subjected to sham or transverse aortic constriction (TAC) surgery, n refers to number of hearts. (d) Real-time PCR analysis of relative transcript abundance for the fetal marker genes Nppa, Nppb, Acta1 and Myh7 in hearts from mice treated with ctrl antagomir or antagomir-25 following sham or TAC surgery, n refers to number of hearts. *P<0.05 vs control group (error bars are s.e.m.). Source data are shown in Supplementary Table 9.

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Dirkx, E., Gladka, M., Philippen, L. et al. Nfat and miR-25 cooperate to reactivate the transcription factor Hand2 in heart failure. Nat Cell Biol 15, 1282–1293 (2013). https://doi.org/10.1038/ncb2866

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