TIP 30 counteracts cardiac hypertrophy and failure by inhibiting translational elongation
2019; Springer Nature; Volume: 11; Issue: 10 Linguagem: Inglês
10.15252/emmm.201810018
ISSN1757-4684
AutoresAndrea Grund, Malgorzata Szaroszyk, Mortimer Korf‐Klingebiel, Mona Malek Mohammadi, Felix A. Trogisch, Ulrike Schrameck, Anna Gigina, Christopher Tiedje, Matthias Gaestel, Theresia Kraft, Jan Hegermann, Sándor Bátkai, Thomas Thum, Andreas Perrot, Cristobal G. dos Remedios, Eva Riechert, Mirko Völkers, Shirin Doroudgar, Andreas Jungmann, Ralf Bauer, Xiaoke Yin, Manuel Mayr, Kai C. Wollert, Andreas Pich, Hua Xiao, Hugo A. Katus, Johann Bauersachs, Oliver J. Müller, Joerg Heineke,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoArticle30 August 2019Open Access Source DataTransparent process TIP30 counteracts cardiac hypertrophy and failure by inhibiting translational elongation Andrea Grund Andrea Grund Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Department of Cardiovascular Research, European Center for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Search for more papers by this author Malgorzata Szaroszyk Malgorzata Szaroszyk Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Mortimer Korf-Klingebiel Mortimer Korf-Klingebiel Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Mona Malek Mohammadi Mona Malek Mohammadi Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Department of Cardiovascular Research, European Center for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Search for more papers by this author Felix A Trogisch Felix A Trogisch Department of Cardiovascular Research, European Center for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Search for more papers by this author Ulrike Schrameck Ulrike Schrameck Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Anna Gigina Anna Gigina Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Christopher Tiedje Christopher Tiedje orcid.org/0000-0002-3013-6967 Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Matthias Gaestel Matthias Gaestel orcid.org/0000-0002-4944-4652 Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Theresia Kraft Theresia Kraft Institute for Molecular and Cellphysiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Jan Hegermann Jan Hegermann Research Core Unit Electron Microscopy, Hannover Medical School, Hannover, Germany Search for more papers by this author Sandor Batkai Sandor Batkai Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany Search for more papers by this author Thomas Thum Thomas Thum Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany Cluster of Excellence Rebirth, Hannover Medical School, Hannover, Germany Search for more papers by this author Andreas Perrot Andreas Perrot Experimental and Clinical Research Center, A Joint Cooperation of Max-Delbrück Center for Molecular Medicine and Charité-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Cris dos Remedios Cris dos Remedios Sydney Heart Bank, University of Sydney, Sydney, NSW, Australia Search for more papers by this author Eva Riechert Eva Riechert Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Mirko Völkers Mirko Völkers orcid.org/0000-0003-2344-1856 Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Search for more papers by this author Shirin Doroudgar Shirin Doroudgar Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Search for more papers by this author Andreas Jungmann Andreas Jungmann Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Ralf Bauer Ralf Bauer Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Xiaoke Yin Xiaoke Yin King's British Heart Foundation Centre, King's College London, London, UK Search for more papers by this author Manuel Mayr Manuel Mayr King's British Heart Foundation Centre, King's College London, London, UK Search for more papers by this author Kai C Wollert Kai C Wollert Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Cluster of Excellence Rebirth, Hannover Medical School, Hannover, Germany Search for more papers by this author Andreas Pich Andreas Pich Core Unit Proteomics, Hannover Medical School, Hannover, Germany Search for more papers by this author Hua Xiao Hua Xiao Department of Physiology, Michigan State University, East Lansing, MI, USA Search for more papers by this author Hugo A Katus Hugo A Katus Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Search for more papers by this author Johann Bauersachs Johann Bauersachs Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Cluster of Excellence Rebirth, Hannover Medical School, Hannover, Germany Search for more papers by this author Oliver J Müller Oliver J Müller Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Department of Internal Medicine III, Cardiology, Angiology and Intensive Care Medicine, Universitätsklinikum Schleswig-Holstein, Kiel, Germany Search for more papers by this author Joerg Heineke Corresponding Author Joerg Heineke [email protected] orcid.org/0000-0002-1541-3030 Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Department of Cardiovascular Research, European Center for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Cluster of Excellence Rebirth, Hannover Medical School, Hannover, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Search for more papers by this author Andrea Grund Andrea Grund Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Department of Cardiovascular Research, European Center for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Search for more papers by this author Malgorzata Szaroszyk Malgorzata Szaroszyk Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Mortimer Korf-Klingebiel Mortimer Korf-Klingebiel Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Mona Malek Mohammadi Mona Malek Mohammadi Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Department of Cardiovascular Research, European Center for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Search for more papers by this author Felix A Trogisch Felix A Trogisch Department of Cardiovascular Research, European Center for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Search for more papers by this author Ulrike Schrameck Ulrike Schrameck Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Anna Gigina Anna Gigina Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Christopher Tiedje Christopher Tiedje orcid.org/0000-0002-3013-6967 Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Matthias Gaestel Matthias Gaestel orcid.org/0000-0002-4944-4652 Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany Search for more papers by this author Theresia Kraft Theresia Kraft Institute for Molecular and Cellphysiology, Hannover Medical School, Hannover, Germany Search for more papers by this author Jan Hegermann Jan Hegermann Research Core Unit Electron Microscopy, Hannover Medical School, Hannover, Germany Search for more papers by this author Sandor Batkai Sandor Batkai Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany Search for more papers by this author Thomas Thum Thomas Thum Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany Cluster of Excellence Rebirth, Hannover Medical School, Hannover, Germany Search for more papers by this author Andreas Perrot Andreas Perrot Experimental and Clinical Research Center, A Joint Cooperation of Max-Delbrück Center for Molecular Medicine and Charité-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Cris dos Remedios Cris dos Remedios Sydney Heart Bank, University of Sydney, Sydney, NSW, Australia Search for more papers by this author Eva Riechert Eva Riechert Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Mirko Völkers Mirko Völkers orcid.org/0000-0003-2344-1856 Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Search for more papers by this author Shirin Doroudgar Shirin Doroudgar Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Search for more papers by this author Andreas Jungmann Andreas Jungmann Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Ralf Bauer Ralf Bauer Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Xiaoke Yin Xiaoke Yin King's British Heart Foundation Centre, King's College London, London, UK Search for more papers by this author Manuel Mayr Manuel Mayr King's British Heart Foundation Centre, King's College London, London, UK Search for more papers by this author Kai C Wollert Kai C Wollert Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Cluster of Excellence Rebirth, Hannover Medical School, Hannover, Germany Search for more papers by this author Andreas Pich Andreas Pich Core Unit Proteomics, Hannover Medical School, Hannover, Germany Search for more papers by this author Hua Xiao Hua Xiao Department of Physiology, Michigan State University, East Lansing, MI, USA Search for more papers by this author Hugo A Katus Hugo A Katus Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Search for more papers by this author Johann Bauersachs Johann Bauersachs Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Cluster of Excellence Rebirth, Hannover Medical School, Hannover, Germany Search for more papers by this author Oliver J Müller Oliver J Müller Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Department of Internal Medicine III, Cardiology, Angiology and Intensive Care Medicine, Universitätsklinikum Schleswig-Holstein, Kiel, Germany Search for more papers by this author Joerg Heineke Corresponding Author Joerg Heineke [email protected] orcid.org/0000-0002-1541-3030 Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany Department of Cardiovascular Research, European Center for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Cluster of Excellence Rebirth, Hannover Medical School, Hannover, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany Search for more papers by this author Author Information Andrea Grund1,2, Malgorzata Szaroszyk1, Mortimer Korf-Klingebiel1, Mona Malek Mohammadi1,2, Felix A Trogisch2, Ulrike Schrameck1, Anna Gigina1, Christopher Tiedje3, Matthias Gaestel3, Theresia Kraft4, Jan Hegermann5, Sandor Batkai6, Thomas Thum6,7, Andreas Perrot8, Cris dos Remedios9, Eva Riechert10, Mirko Völkers10,11, Shirin Doroudgar10,11, Andreas Jungmann10, Ralf Bauer10, Xiaoke Yin12, Manuel Mayr12, Kai C Wollert1,7, Andreas Pich13, Hua Xiao14, Hugo A Katus10,11, Johann Bauersachs1,7, Oliver J Müller10,11,15,‡ and Joerg Heineke *,1,2,7,11,‡ 1Department for Cardiology and Angiology, Hannover Medical School, Hannover, Germany 2Department of Cardiovascular Research, European Center for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany 3Institute of Cell Biochemistry, Hannover Medical School, Hannover, Germany 4Institute for Molecular and Cellphysiology, Hannover Medical School, Hannover, Germany 5Research Core Unit Electron Microscopy, Hannover Medical School, Hannover, Germany 6Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany 7Cluster of Excellence Rebirth, Hannover Medical School, Hannover, Germany 8Experimental and Clinical Research Center, A Joint Cooperation of Max-Delbrück Center for Molecular Medicine and Charité-Universitätsmedizin Berlin, Berlin, Germany 9Sydney Heart Bank, University of Sydney, Sydney, NSW, Australia 10Department of Cardiology, Angiology and Pneumology, Medical Faculty of Heidelberg, University of Heidelberg, Heidelberg, Germany 11DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany 12King's British Heart Foundation Centre, King's College London, London, UK 13Core Unit Proteomics, Hannover Medical School, Hannover, Germany 14Department of Physiology, Michigan State University, East Lansing, MI, USA 15Department of Internal Medicine III, Cardiology, Angiology and Intensive Care Medicine, Universitätsklinikum Schleswig-Holstein, Kiel, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49-621-383-71855; Fax: +49-621-383-71851; E-mail: [email protected] EMBO Mol Med (2019)11:e10018https://doi.org/10.15252/emmm.201810018 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pathological cardiac overload induces myocardial protein synthesis and hypertrophy, which predisposes to heart failure. To inhibit hypertrophy therapeutically, the identification of negative regulators of cardiomyocyte protein synthesis is needed. Here, we identified the tumor suppressor protein TIP30 as novel inhibitor of cardiac hypertrophy and dysfunction. Reduced TIP30 levels in mice entailed exaggerated cardiac growth during experimental pressure overload, which was associated with cardiomyocyte cellular hypertrophy, increased myocardial protein synthesis, reduced capillary density, and left ventricular dysfunction. Pharmacological inhibition of protein synthesis improved these defects. Our results are relevant for human disease, since we found diminished cardiac TIP30 levels in samples from patients suffering from end-stage heart failure or hypertrophic cardiomyopathy. Importantly, therapeutic overexpression of TIP30 in mouse hearts inhibited cardiac hypertrophy and improved left ventricular function during pressure overload and in cardiomyopathic mdx mice. Mechanistically, we identified a previously unknown anti-hypertrophic mechanism, whereby TIP30 binds the eukaryotic elongation factor 1A (eEF1A) to prevent the interaction with its essential co-factor eEF1B2 and translational elongation. Therefore, TIP30 could be a therapeutic target to counteract cardiac hypertrophy. Synopsis How overshooting protein synthesis and growth are prevented in the heart during pathological overload or in cardiomyopathy is not known. The tumor suppressor protein TIP30 is shown here to act as an endogenous brake on cardiac hypertrophy by inhibiting translational elongation. TIP30 binds the eukaryotic elongation factor (eEF1A), thereby interfering with binding of the guanine exchange factor eEF1B and inhibiting reconstitution of inactive into active, GTP-bound eEF1A. Endogenous TIP30 levels are downregulated in the myocardium of patients suffering from hypertrophic cardiomyopathy or end-stage heart failure, while the levels of eEF1A remain unchanged. Reduced myocardial TIP30 with unchanged eEF1A levels in mice lead to exaggerated cardiac protein synthesis and growth as well as cardiac dysfunction during pathological overload. Exaggerated cardiac growth in mice with reduced TIP30 levels is normalized by pharmacological inhibition of eEF1A as well as by cardiomyocyte specific expression of TIP30. Overexpression of TIP30 effectively inhibits pathological cardiac hypertrophy and dysfunction during pathological overload or in cardiomyopathy. Introduction Pathological cardiac hypertrophy, which predisposes to the development of heart failure, frequently develops as consequence of ventricular pressure overload, myocardial infarction or due to inherited cardiomyopathy (Heineke & Molkentin, 2006; Hill & Olson, 2008). It is associated with decreased cardiac function, increased cardiomyocyte size, interstitial fibrosis, and capillary rarefaction (Hein et al, 2003). Many signaling proteins were identified that act in concert to trigger transcription of a pro-hypertrophic gene program (Heineke & Molkentin, 2006; Hill & Olson, 2008). This gene program entails mainly qualitative changes in gene expression, but does not account for the quantitative changes during cardiac growth, which are characterized by strong accumulation of newly synthesized proteins that can lead to enlargement of the heart by more than 50% (Nagatomo et al, 1999; McDermott et al, 2012). The strong increase in cardiac protein content mainly results from enhanced protein synthesis within the first 1–5 days of pressure overload with or without a significant decrease in protein degradation (Nagatomo et al, 1999; McDermott et al, 2012). After 10–14 days, hypertrophy reaches its maximum and a new steady state is attained, in which protein synthesis equals protein degradation and cardiac mass remains stable (Nagatomo et al, 1999). mTOR as catalytic subunit of the mTOR containing multiprotein complex 1 (mTORC1) promotes cardiac protein synthesis and hypertrophy mainly by fostering translational initiation (Laplante & Sabatini, 2012). mTORC1 inhibition by rapamycin or its partial deletion in zebrafish improves cardiac function, although its complete genetic abrogation in cardiomyocytes induces cardiomyopathy (Shioi et al, 2003; McMullen et al, 2004; Ma & Blenis, 2009; Ding et al, 2011; Zhang et al, 2011b). As overgrowth of the myocardium is associated with poor prognosis during disease (Levy et al, 1990), the identification of currently largely undefined endogenous negative regulators of hypertrophy at the level of cardiomyocyte protein synthesis might reveal interesting future therapeutic targets, especially when their abundance is dysregulated in failing hearts. Protein synthesis is a tightly regulated process that is initiated at the start codon by the 80S ribosome and continues into elongation wherein the peptide chain increases its length cyclically one amino acid at a time (Sasikumar et al, 2012). Translational elongation is catalyzed by the eukaryotic translation elongation factor 1A (eEF1A), which in its active GTP-bound form binds and delivers amino acid loaded tRNAs to the A-site of the ribosome. By formation of the correct codon–anti-codon pair between tRNA and mRNA, a conformational change in the ribosome leads to GTP hydrolysis and release of then inactive, GDP-bound eEF1A. GDP needs to be actively exchanged for GTP by the guanine nucleotide exchange factor (GEF) eEF1B2, in order to enable eEF1A to participate in another round of elongation. Here, we characterized the 30 kDa protein TIP30 (also termed Htatip2) as inhibitor of mRNA translation and cardiac hypertrophy and revealed that it protects against heart failure during pathological stimulation. TIP30 is ubiquitously expressed and is acting as tumor suppressor, since reduced TIP30 levels were found in human cancers and were related to enhanced tumor growth and metastasis formation (Shtivelman, 1997; Ito et al, 2003; Zhao et al, 2007; Li et al, 2009). Moreover, homozygous (Tip30−/−, KO) and heterozygous (Tip30+/−, Het) Tip30 knock-out mice develop malignant tumors starting at 18–20 months of age (Ito et al, 2003; Li et al, 2013; Chen et al, 2014). The role of TIP30 in the heart, however, had so far not been analyzed. TIP30 is well conserved across species, and crystallographic analyses suggest binding of NADPH, but found enzymatic activity of TIP30 to be very unlikely (El Omari et al, 2005). Instead, it was suggested that TIP30 might play a regulatory role by mediating protein interactions (El Omari et al, 2005; Nakahara et al, 2009). Accordingly, we demonstrate here that TIP30 interacts with eEF1A to prevent association with its co-factor eEF1B2, thereby blocking translational elongation and cardiomyocyte hypertrophy. Results TIP30 deficiency facilitates cardiac hypertrophy and failure To assess the functional role of TIP30 during cardiac overload, we subjected heterozygous (Het, with a 50–60% reduction of cardiac TIP30) and homozygous Tip30 knock-out (KO, completely deficient of TIP30) as well as wild-type (WT) mice to sham or transverse aortic constriction (TAC) surgery (Fig 1A). While no phenotypic differences were noted after sham operation, Het and KO mice developed more cardiac hypertrophy (i.e., increased heart weight/tibia length ratio, HW/TL; Fig 1B) 6 weeks after TAC surgery. Het, but not KO or WT mice exerted enhanced pulmonary congestion (increased lung weight/TL; Fig 1C) as sign of cardiac dysfunction after TAC. Accordingly, echocardiography revealed decreased cardiac systolic function (fractional area change) in Het mice and increased cardiac dilation (LVEDA) in Het and KO mice versus WT mice 6 weeks after TAC (Fig 1D and E). Increased dilation and wall thickness of the left ventricle (indicative of enhanced hypertrophy), as well as cardiac dysfunction, were already observed in Het (but not KO) versus WT mice 2 weeks after TAC in echocardiography (Fig EV1A–D). Because Het mice therefore showed a more prominent phenotype than KO mice, we carried out most of the following experiments in Het in comparison with WT mice. To rule out principal differences in the degree of pressure overload after TAC between both genotypes, we conducted Doppler measurements of right versus left carotid artery blood flow. The results indicated that a similar degree of left ventricular pressure overload was reached in Het and WT mice 2 days after TAC versus sham surgery (Fig EV1E). Figure 1. TIP30 deficiency results in enhanced cardiac hypertrophy during pathological overload A. Schematic representation of the study design and Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild-type (WT), heterozygous (Het), and homozygous knock-out (KO) mice under basal conditions. B–I. Quantification of heart weight (HW)/tibia length (TL) ratio (B), lung weight (LuW/TL) ratio (C), echocardiographic fractional area change (D) and left ventricular end-diastolic area (LVEDA; E), dP/dt max and dP/dt min (Millar catheter; F, G), and α-MHC and β-MHC transcript abundance (H, I). N = 4–18 mice/group, all 6 weeks after TAC or sham surgery. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. One-way ANOVA with Sidak's multiple comparisons test. J, K. Representative images of Sirius red-stained heart sections (scale bar: 1 mm) (J) and fibrosis quantification (K) of indicated mice 6 weeks after TAC or sham surgery. N = 3-5 mice/group. L. Quantification of cardiomyocyte area of isolated adult cardiac myocytes (N = 4–11 mice/group) of indicated mice 6 weeks after TAC or sham surgery. *P < 0.05. One-way ANOVA with Sidak's multiple comparisons test. M, N. Microscopy images of heart sections of indicated mice 6 weeks after TAC surgery stained for isolectin B4 (green) and WGA (red, M) and quantification of capillaries per myocyte (N). (N = 5–7 mice/group, scale bar: 50 μm). **P < 0.01. One-way ANOVA with Sidak's multiple comparisons test. O. Schematic representation of AAV-TopT-TIP30 study design. P. Western blot analysis for TIP30 and GAPDH in hearts from TIP30 wild-type (WT) and heterozygous (Het) after AAV-TropT-TIP30 or AAV-control (AAV-Con) injection followed by 6 weeks of TAC surgery. Q, R. Quantification of HW/TL ratio (Q) and LuW/TL (R) ratio in AAV-Con or AAV-TropT-TIP30 treated Tip30 heterozygous (Het) or WT mice 6 weeks after TAC or sham surgery (N = 5–11 mice/group). *P < 0.05, ***P < 0.001. One-way ANOVA with Sidak's multiple comparisons test. Data information: Data are shown as mean ± SEM. Source data are available online for this figure. Source Data for Figure 1 [emmm201810018-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Additional characterization of TIP30-deficient mice during transverse aortic constriction (TAC) A–D. Fractional area change (A), left ventricular end-diastolic area (LVEDA; B), average diastolic wall thickness (C), and heart rate (D) as determined by echocardiography 2 weeks after sham or TAC surgery in Tip30 Het, KO, or WT mice (N = 6–17 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with Sidak's multiple comparisons test. E. Doppler measurements of right (RCA) to left (LCA) carotid artery blood flow in TIP30 Het, or WT mice 2 days after TAC surgery (N = 5–7 mice/group). ***P < 0.001, ****P < 0.0001. One-way ANOVA with Sidak's multiple comparisons test. F. Quantification of maximal systolic pressure in indicated mice 6 weeks after TAC or sham surgery (N = 4–18 mice/group). *P < 0.05, ***P < 0.001. One-way ANOVA with Sidak's multiple comparisons test. G. Ventricular cardiomyocyte sarcomere shortening at 1, 2, and 4 Hz of isolated adult cardiomyocytes from TIP30 Het, or WT mice 2 weeks after TAC surgery (N = 5 mice/group). H, I. Microscopy images of heart sections of indicated mice 6 weeks after TAC surgery stained for PDGFRα (green), WGA (red), and DAPI (blue) (scale bar: 50 μm) and quantification of PDGFRα-positive cells per myocyte (N = 2–5 mice/group, I). J. Quantification of cleaved caspase 3-positive cardiomyocytes in hearts of Tip30 Het and WT mice 6 weeks after TAC (N = 3–5 mice/group). K. Electron microscopy images of heart sections of indicated mice 6 weeks after TAC surgery (scale bar = 5 μm). L. Representative confocal microscopy images of heart sections of TIP30 Het mice subjected to 6 weeks of TAC surgery and AAV-TropT-TIP30 or AAV-control treatment. Red: TIP30, green: WGA, and blue: DAPI (scale bar: 20 μm). M. Western blot analysis for TIP30 and GAPDH from isolated adult cardiomyocytes 2 weeks after injection of AAV-control or AAV-TropT-TIP30. Data information: Data are shown as mean ± SEM. Source data are available online for this figure. Download figure Download PowerPoint Direct analysis of left ventricular pressure development by catheterization revealed decreased left ventricular contractility (dP/dt max), relaxation (dP/dt min), and systolic pressure in Het versus WT mice during pressure overload (Figs 1F and G, and EV1F). Single cardiomyocyte contractility, however, was not different between WT and Het cardiomyocytes after TAC at three different pacing rates (Fig EV1G). Six weeks after TAC surgery, we found a similarly reduced expression of α-myosin heavy chain (α-MHC), but significantly more increased β-MHC expression in the myocardium of Het mice (Fig 1H and I). Cardiac fibrosis was not different between the experimental groups (Fig 1J and K), and accordingly, the number of PDGFRα-positive cardiac fibroblasts was also not changed between them (Fig EV1H and I). In line with the increased HW/TL ratio, enlarged cardiomyocytes were found in Het versus WT mice after TAC (Fig 1L). This augmented cardiomyocyte growth was not accompanied by growth of the cardiac micro-vasculature, since we detected a prominent reduction of the capillary/cardiomyocyte ratio selectively in Het mice after TAC (Fig 1M and N). As capillary rarefaction during pressure overload is known to be maladaptive, it might at least partial
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