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Long Noncoding RNA Dysregulation in Ischemic Heart Failure

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Long Noncoding RNA Dysregulation in Ischemic Heart Failure Greco et al. J Transl Med (2016) 14:183 DOI 10.1186/s12967-016-0926-5 Journal of Translational Medicine RESEARCH Open Access Long noncoding RNA dysregulation in ischemic heart failure Simona Greco1, Germana Zaccagnini1, Alessandra Perfetti1, Paola Fuschi1, Rea Valaperta1, Christine Voellenkle1, Serenella Castelvecchio1, Carlo Gaetano2, Nicoletta Finato3, Antonio Paolo Beltrami3, Lorenzo Menicanti1 and Fabio Martelli1* Abstract Background: Long noncoding RNAs (lncRNAs) are non-protein coding transcripts regulating a variety of physiologi- cal and pathological functions. However, their implication in heart failure is still largely unknown. The aim of this study is to identify and characterize lncRNAs deregulated in patients affected by ischemic heart failure. Methods: LncRNAs were profiled and validated in left ventricle biopsies of 18 patients affected by non end-stage dilated ischemic cardiomyopathy and 17 matched controls. Further validations were performed in left ventricle samples derived from explanted hearts of end-stage heart failure patients and in a mouse model of cardiac hypertro- phy, obtained by transverse aortic constriction. Peripheral blood mononuclear cells of heart failure patients were also analyzed. LncRNA distribution in the heart was assessed by in situ hybridization. Function of the deregulated lncRNA was explored analyzing the expression of the neighbor mRNAs and by gene ontology analysis of the correlating cod- ing transcripts. Results: Fourteen lncRNAs were significantly modulated in non end-stage heart failure patients, identifying a heart failure lncRNA signature. Nine of these lncRNAs (CDKN2B-AS1/ANRIL, EGOT, H19, HOTAIR, LOC285194/TUSC7, RMRP, RNY5, SOX2-OT and SRA1) were also confirmed in end-stage failing hearts. Intriguingly, among the conserved lncR- NAs, h19, rmrp and hotair were also induced in a mouse model of heart hypertrophy. CDKN2B-AS1/ANRIL, HOTAIR and LOC285194/TUSC7 showed similar modulation in peripheral blood mononuclear cells and heart tissue, suggesting a potential role as disease biomarkers. Interestingly, RMRP displayed a ubiquitous nuclear distribution, while H19 RNA was more abundant in blood vessels and was both cytoplasmic and nuclear. Gene ontology analysis of the mRNAs displaying a significant correlation in expression with heart failure lncRNAs identified numerous pathways and func- tions involved in heart failure progression. Conclusions: These data strongly suggest lncRNA implication in the molecular mechanisms underpinning HF. Background deregulated proteins and is characterized in the adult It is estimated that heart failure (HF) is the cardiovas- heart by the reactivation of fetal cardiac gene expres- cular disease with the worse rate of morbidity, mortal- sion [2]. This scenario of transcriptional control is also ity, accounting for one of the highest health care costs complicated by the addition of epigenetic mechanisms. in the western world. Indeed, the 1-year-survival rate of The encyclopedia of DNA elements (ENCODE) project patients with end-stage HF is about 50 % [1]. indicates that at least 80 % of the genome is functional The adverse left ventricle (LV) remodeling process, and is transcribed both into protein coding RNAs (about leading to the clinical syndrome of HF, involves several 1.1–1.5 %) and in a much larger quantity of non-coding, regulatory RNAs, arbitrarily divided into long (lncR- *Correspondence: [email protected] NAs, >200 nt), and short (<200 nt) ncRNAs [3, 4]. 1 IRCCS Policlinico San Donato, Via Morandi, 30, 20097 San Donato While dysregulation and role of short ncRNAs, in par- Milanese, Milan, Italy ticular of miRNAs, has been extensively explored [5–7], Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Greco et al. J Transl Med (2016) 14:183 Page 2 of 14 the involvement of lncRNAs in specific physiological and Table 1 Clinical characteristics of the study population pathological processes [5, 8], as well as in cardiovascular Characteristics HF patients diseases [9–16], is still in its early stages of study. Age (years) (median SE) 65.0 0.6 In this study, in order to investigate the molecular ± ± mechanisms underpinning HF during its progression, Sex (male/female) 17/1 we profiled the expression of 83 lncRNAs, known to be Diabetics (%) 28 BSA (m2) (median SE) 1.8 0.02 implicated in human diseases, in LV biopsies of non end- ± ± GFR (ml/min) (median SE) 64.2 1.6 stage HF patients. ± ± Glucose (mg/dl) (median SE) 105 1.5 ± ± Time from MI (months) (median SE) 5.5 10.6 Methods ± ± NYHA class (%) 1 5; 2 55; 3 39; 4 1 Patient selection and tissue collection = = = = DD (mm) (median SE) 62.5 0.5 The investigation was conducted in conformity with the ± ± SD (mm) (median SE) 52.0 0.5 principles outlined in the Helsinki Declaration and with ± ± LVEF (%) (median SE) 26.5 0.4 the Italian laws and guidelines, and was authorized by ± ± EDV (ml) (median SE) 216.5 2.7 local Ethics Committee (protocol #2438, 27/01/2009). ± ± ESV (ml) (median SE) 151.0 2.2 All specimens were taken after informed consent disclos- ± ± E/A (median SE) 1.25 0.1 ing future use for research. Left ventricle (LV) cardiac ± ± biopsies from patients affected by non end-stage dilated Medications (%) hypokinetic ischemic cardiomyopathy where collected Oral antidiabetic agents 15 during surgical ventricular restoration procedure per- Insulin 20 formed as described previously [17, 18]. For each patient, Statins 75 two biopsies were collected from the non-ischemic and ACEIs 70 dysfunctional remote myocardium: one was immediately β-Blockers 80 immersed in RNAlater (Qiagen GmbH) and stored at 4 °C Aspirin 95 for <24 h before RNA extraction, and the other one was Loop diuretics 85 fixed in 10 % neutral buffered formalin (NBF) and paraf- Nitrates 35 fin embedded for RNA in situ hybridization and immu- ACEI angiotensin converting enzymes inhibitor, BSA body surface area, nohistochemistry assays. HF patients’ characteristics are DD diastolic diameter, SD systolic diameter, E/A diastolic function ratio, EDV end- diastolic volume, ESV end-systolic volume, GFR glomerular filtration rate, described in Table 1. Peripheral blood mononuclear cells LVEF left ventricular ejection fraction, MI myocardial infarction, NYHA New York (PBMC) were isolated from the peripheral blood of HF Heart Association Functional Classification patients the day before surgery or from healthy controls by Histopaque Ficoll (Sigma Diagnostics, St. Louis, MO, USA) gradient centrifugation as described previously The control group was composed by age- and sex- [19]. matched subjects, who had died for causes different We have also analyzed 11 post-ischemic end-stage from stroke, ischemia, or cachexia for chronic diseases heart failure patients [10 males and 1 female, aged (n = 17, females/males = 6/10; 58.3 ± 3.4 years old). Left 59 ± 2.4 years (median ± SE)]. Among these, 8 LV ventricle samples were excised and processed with less samples were collected by an expert pathologist from than 30 min cold ischemic time and snap frozen (Xpress- explanted hearts of patients that underwent cardiac BANK, Asterand Biosciences). transplantation; the remaining 3 end-stage samples were collected during the implantation of LV assist device Mouse transverse aortic constriction (TAC) (LVAD). Care was taken to avoid necrotic or fibrotic 3 All experimental procedures complied with the Guide- areas. Samples of ~2 mm were snap frozen in liquid lines of the Italian National Institutes of Health and with nitrogen and kept at −80 °C until they were analyzed. the Guide for the Care and Use of Laboratory Animals The end-stage HF patients were classified as NYHA class (Institute of Laboratory Animal Resources, National 3 (82 %) and class 4 (18 %), showed a LV transverse diam- Academy of Sciences, Bethesda, MD, USA) and were eter of 130 ± 5.3 mm (median ± SE), measured by the approved by the institutional Animal Care and Use Com- pathologist after explantation, and ejection fraction of mittee (IACUC n. 709). TAC was performed in 2 months 22 ± 2.9 % (median ± SE). Sample harvesting was con- old C57BL/6 J male mice with a 27-gauge needle [20]. ducted after approval of the Ethics Committee of Udine Before all surgical procedures, mice were anesthetized (2 August 2011, reference number 47,831), in accordance with an intraperitoneal injection of 10 mg/kg Xilazine with the Declaration of Helsinki, once written informed (Intervet Farmaceutici, Italy) and 100 mg/kg Ketamine consent was obtained from each enrolled patient. (Ketavet 100; Intervet Farmaceutici, Italy). After the Greco et al. J Transl Med (2016) 14:183 Page 3 of 14 surgery, mice were allowed to recover at 37 °C. Sham RNA in situ hybridization assay and immunohistochemistry operated mice received the same procedure except for In situ hybridization was performed using the Quanti- the ligation of the aorta. Only mice showing a pressure Gene ViewRNA system (Affymetrix) according to
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