A gene therapeutic approach to inhibit calcium and integrin binding protein 1 ameliorates maladaptive remodelling in pressure overload (pdf)

Article PDF cannot be displayed. You can download it here:

https://academic.oup.com/cardiovascres/article-pdf/115/1/71/29816739/cvy154.pdf

A gene therapeutic approach to inhibit calcium and integrin binding protein 1 ameliorates maladaptive remodelling in pressure overload

Cardiovascular Research (2019) 115, 71–82 doi:10.1093/cvr/cvy154 A gene therapeutic approach to inhibit calcium and integrin binding protein 1 ameliorates maladaptive remodelling in pressure overload 1 Klinik für Kardiologie und Angiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany; 2Abteilung für Herz- und Kreislaufforschung, European Center for Angioscience (ECAS), Medizinische Fakultät Mannheim, Universität Heidelberg, Ludolf-Krehl-Straße 7-11, 68167, Mannheim, Germany; 3Klinik für Hämatologie, Hämostaseologie, Onkologie und Stammzelltransplantation; 4Leibniz Forschungslaboratorien für Biotechnologie und künstliche Organe, Klinik für Herz-, Thorax-, Transplantations- und Gefäßchirurgie; 5Cluster of Excellence-Rebirth, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, 30625, Hannover, Germany; 6Institut für Pharmakologie und Toxikologie and Rudolf Virchow Zentrum für Experimentelle Biomedizin, Universität Würzburg, Versbacher Straße 9, 97078, Würzburg, Germany; 7Klinik für Kardiologie, Angiologie und Pneumologie, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany; 8DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg, Mannheim, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany; and 9Klinik für Innere Medizin III, Universitätsklinikum Schleswig-Holstein, Arnold-Heller-Straße 3, 24105, Kiel, Germany Received 6 September 2017; revised 22 March 2018; editorial decision 9 June 2018; accepted 17 June 2018; online publish-ahead-of-print 20 June 2018 Time for primary review: 36 days Aims Chronic heart failure is becoming increasingly prevalent and is still associated with a high mortality rate. Myocardial hypertrophy and fibrosis drive cardiac remodelling and heart failure, but they are not sufficiently inhibited by current treatment strategies. Furthermore, despite increasing knowledge on cardiomyocyte intracellular signalling proteins inducing pathological hypertrophy, therapeutic approaches to target these molecules are currently unavailable. In this study, we aimed to establish and test a therapeutic tool to counteract the 22 kDa calcium and integrin binding protein (CIB) 1, which we have previously identified as nodal regulator of pathological cardiac hypertrophy and as activator of the maladaptive calcineurin/NFAT axis. .................................................................................................................................................................................................... Methods Among three different sequences, we selected a shRNA construct (shCIB1) to specifically down-regulate CIB1 by 50% upon adenoviral overexpression in neonatal rat cardiomyocytes (NRCM), and upon overexpression by an and results adeno-associated-virus (AAV) 9 vector in mouse hearts. Overexpression of shCIB1 in NRCM markedly reduced cellular growth, improved contractility of bioartificial cardiac tissue and reduced calcineurin/NFAT activation in response to hypertrophic stimulation. In mice, administration of AAV-shCIB1 strongly ameliorated eccentric cardiac hypertrophy and cardiac dysfunction during 2 weeks of pressure overload by transverse aortic constriction (TAC). Ultrastructural and molecular analyses revealed markedly reduced myocardial fibrosis, inhibition of hypertrophy associated gene expression and calcineurin/NFAT as well as ERK MAP kinase activation after TAC in AAV-shCIB1 vs. AAV-shControl treated mice. During long-term exposure to pressure overload for 10 weeks, AAV-shCIB1 treatment maintained its anti-hypertrophic and anti-fibrotic effects, but cardiac function was no longer improved vs. AAVshControl treatment, most likely resulting from a reduction in myocardial angiogenesis upon downregulation of CIB1. .................................................................................................................................................................................................... Conclusions Inhibition of CIB1 by a shRNA-mediated gene therapy potently inhibits pathological cardiac hypertrophy and fibrosis during pressure overload. While cardiac function is initially improved by shCIB1, this cannot be kept up during persisting overload. 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 hypertrophy • CIB1 • Gene therapy • Heart failure • shRNA * Corresponding author. Tel: þþ49 621 383 71855; fax: þþ49 621 383 71851, E-mail: † Present address. Klinik für Thorax-, Herz- und Gefäßmedizin, Universitätsmedizin Göttingen, 37075, Göttingen, Germany. C The Author(s) 2018. For permissions, please email: . Published on behalf of the European Society of Cardiology. All rights reserved. V Andrea Grund1, Malgorzata Szaroszyk1, Janina K. Döppner1, Mona Malek Mohammadi1,2, Badder Kattih1,2, Mortimer Korf-Klingebiel1, Anna Gigina1, Michaela Scherr3, George Kensah4,5†, Monica Jara-Avaca4,5, Ina Gruh4,5, Ulrich Martin4,5, Kai C. Wollert1,5, Antje Gohla6, Hugo A. Katus7,8, Oliver J. Müller7,8,9, Johann Bauersachs1,5, and Joerg Heineke1,2,5,8* 72 Introduction .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . Methods An extended version of this section is contained in the Supplementary material online, Supplementary Methods. Animal experiments All procedures involving the use and care of animals were performed according to the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Approval was granted by the local state authorities (Reference no. 33.14-42502-04-14/1574). AAV9 vectors were injected subcutaneously into 6 week old mice (5 1011 viral genomes/ml in PBS). TAC surgery was performed in 8 weeks old mice by subjecting the aorta to a defined 26 gauge constriction as described previously.19,20 Non-invasive echocardiography with a linear 30 MHz transducer (Vevo 770, Visualsonics) was used to record or calculate LV end-diastolic area (LVEDA), end-diastolic average wall thickness, (Wth) and end-diastolic volume (EDV) as well as end-systolic area (LVESA) and end-systolic volume (ESV). Ejection fraction was calculated as [(EDV - ESV)/EDV] 100. Relative wall thickness (RWT) was calculated as Wth/LVEDA. Downregulation of CIB1 in vivo AAV vectors were produced, purified, and titrated using standard procedures.21,22 Isolation and cul (...truncated)