Deletion of delta-like 1 homologue accelerates fibroblast–myofibroblast differentiation and induces myocardial fibrosis (pdf)

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Deletion of delta-like 1 homologue accelerates fibroblast–myofibroblast differentiation and induces myocardial fibrosis

BASIC SCIENCE European Heart Journal (2019) 40, 967–978 doi:10.1093/eurheartj/ehy188 Myocardial disease Deletion of delta-like 1 homologue accelerates fibroblast–myofibroblast differentiation and induces myocardial fibrosis 1 Department of Medicine, Cardiovascular Research Institute; 2Department of Pathology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA; 3Centro de Biologı́a Molecular ‘Severo Ochoa’ (CSIC-UAM), Nicolás Cabrera 1, Campus UAM, 28049 Madrid, Spain; and 4Department of Inorganic and Organic Chemistry and Biochemistry, Pharmacy School/Biomedical Unit/CRIB, University of Castilla-La Mancha/CSIC, Dr. José Marı́a Sánchez Ibá~ nez Street, s/n 02008 Albacete, Spain Received 9 November 2017; revised 14 February 2018; editorial decision 21 March 2018; accepted 21 March 2018; online publish-ahead-of-print 13 April 2018 See page 979 for the editorial comment on this article (doi: 10.1093/eurheartj/ehy307) Aims Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. ................................................................................................................................................................................................... Methods Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardioand results fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is medi- ated by TGF-b1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-b1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-b1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR370 mediated Dlk1’s regulation of cardiac fibroblast–myofibroblast differentiation by directly targeting TGFb-R2/Smad3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. ................................................................................................................................................................................................... Conclusion These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differ- entiation by interfering with TGFb/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFb signalling leads to chronic fibrosis. 䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏䊏 Keywords Cardiac fibrosis • Fibroblast–myofibroblast transdifferentiation Translational perspective Myocardial fibrosis is a major determinant of clinical outcomes in patients with heart failure. In fact, accumulating experimental and clinical evidences suggest that emergence of myofibroblasts and early activation of pro-fibrotic signalling pathways occur before adverse .. .. .. .. .. .. .. .. .. • Dlk1 • miR-370 • TGF-b signalling ventricular remodelling occurs, and prior to heart failure progression. However, the molecular factors that control progression of the fibrotic response are still unclear. In this regard, delta-like homologue-1 (Dlk1) is demonstrated to negatively regulate cardiac fibroblast-to-myofibroblast differentiation and control myocardial fibrosis. These novel anti-fibrogenic properties of Dlk1 epitomize a * Corresponding author. Tel: 1-212-824-8905, Fax: 1-212-241-4080, Email: C The Author(s) 2018. For permissions, please email: . Published on behalf of the European Society of Cardiology. All rights reserved. V Patricia Rodriguez1, Yassine Sassi1, Luca Troncone1, Ludovic Benard1, Kiyotake Ishikawa1, Ronald E. Gordon2, Santiago Lamas3, Jorge Laborda4, Roger J. Hajjar1, and Djamel Lebeche1* 968 potential intervention strategy in cases where aberrant signalling leads to chronic cardiac fibrosis. Introduction Methods An extended methods section is provided in Supplementary material online. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Animals and echocardiography The Mount Sinai Institutional Animal Care and Use Committees approved handling of animals in accordance with the ‘Principles of Laboratory Animal Care by the National Society for Medical research and the Guide for the Care and Use of Laboratory Animals’ (NIH Publication No. 86-23, revised 1996). Homozygous male Dlk1/ mice (mix background SvJ129xC57BL/6)6 and wild types were anesthetized with intraperitoneal ketamine (100 lg/g) for echocardiographic analysis at 7, 13, and 19 weeks of age using GE Vivid 7. Isolation of mouse cardiac myocytes, cardiac fibroblasts, adenoviral infection, and microRNA mimics and anti-miRs transfection Cardiomyocytes and fibroblasts were enzymatically isolated from wildtype and Dlk1-/- mice hearts and processed as detailed in the extended methods section in Supplementary material online. Cell proliferation assays Cardiac fibroblasts were seeded in a 96 multi-well plate at a density of 2000 cells/well. Incorporation of BrdU was assessed at 16, 20, and 24 h following manufactures’ instructions (Roche). Cell proliferation was assayed in the presence of 10% FBS. Results Cardiac myocytes and fibroblasts express different delta-like homologue 1 isoforms We first evaluated the Dlk1 expression pattern in the heart. We found that isolated cardiac fibroblasts and cardiomyocytes express different Dlk1 isoforms with fibroblasts displaying higher ex (...truncated)