Original article MicroRNA-25-dependent up-regulation of NADPH oxidase 4 (NOX4) mediates hypercholesterolemia-induced oxidative/nitrative stress and subsequent dysfunction in the heart Zoltán V. Varga a,b , Krisztina Kupai a , Gergő Szűcs a , Renáta Gáspár a , János Pálóczi a , Nóra Faragó c , Ágnes Zvara c , László G. Puskás c , Zsolt Rázga d , László Tiszlavicz d , Péter Bencsik a,e , Anikó Görbe a,e , Csaba Csonka a,e , Péter Ferdinandy a,b,e , Tamás Csont a,e, ⁎ a Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Szeged, Hungary b Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary c Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary d Department of Pathology, University of Szeged, Szeged, Hungary e Pharmahungary Group, Szeged, Hungary abstract article info Article history: Received 6 December 2012 Received in revised form 13 May 2013 Accepted 17 May 2013 Available online 27 May 2013 Keywords: Hyperlipidemia Peroxynitrite Superoxide Metabolic syndrome Cholesterol Nitrosative stress Diet-induced hypercholesterolemia leads to oxidative/nitrative stress and subsequent myocardial dysfunction. However, the regulatory role of microRNAs in this phenomenon is unknown. We aimed to investigate, whether hypercholesterolemia-induced myocardial microRNA alterations play a role in the development of oxidative/ nitrative stress and in subsequent cardiac dysfunction. Male Wistar rats were fed with 2% cholesterol/0.25% cholate-enriched or standard diet for 12 weeks. Serum and tissue cholesterol levels were significantly elevated by cholesterol-enriched diet. Left ventricular end-diastolic pressure was significantly increased in cholesterol- fed rats both in vivo and in isolated perfused hearts, indicating diastolic dysfunction. Myocardial expression of microRNAs was affected by cholesterol-enriched diet as assessed by microarray analysis. MicroRNA-25 showed a significant down-regulation as detected by microarray analysis and QRT-PCR. In silico target prediction revealed NADPH oxidase 4 (NOX4) as a putative target of microRNA-25. NOX4 protein showed significant up-regulation in the hearts of cholesterol-fed rats, while NOX1 and NOX2 remained unaffected. Cholesterol- feeding significantly increased myocardial oxidative/nitrative stress as assessed by dihydroethidium staining, protein oxidation assay, and nitro-tyrosine ELISA, respectively. Direct binding of microRNA-25 mimic to the 3′ UTR region of NOX4 was demonstrated using a luciferase reporter assay. Transfection of a microRNA-25 mimic into primary cardiomyocytes decreased superoxide production, while a microRNA-25 inhibitor resulted in an up-regulation of NOX4 protein and an increase in oxidative stress that was attenuated by the NADPH oxidase inhibitor diphenyleneiodonium. Here we demonstrated for the first time that hypercholesterolemia affects myo- cardial microRNA expression, and by down-regulating microRNA-25 increases NOX4 expression and conse- quently oxidative/nitrative stress in the heart. We conclude that hypercholesterolemia-induced microRNA alterations play an important role in the regulation of oxidative/nitrative stress and in consequent myocardial dysfunction. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Incidence of metabolic diseases (including obesity, type II diabe- tes, high blood pressure, and dyslipidemia) leading to severe cardio- vascular complications is constantly growing worldwide. The cost of this both in human lives and in dollars is overwhelming. According to the American Heart Association 2012 Heart Disease and Stroke Statistics, elevated cholesterol level is still the leading risk factor for heart diseases. Almost 34 million people in the US have elevated total cholesterol level [1]. While metabolic diseases and especially hy- percholesterolemia have been studied extensively for many years, the underlying genetic networks and molecular signaling pathways re- main to be identified. It is well known, that hypercholesterolemic patients have a much higher morbidity and mortality from cardiovascular diseases, consid- ering the central role of elevated cholesterol levels in the develop- ment of atherosclerosis. However, several studies have shown that hypercholesterolemia exerts direct myocardial effects independent of the development of atherosclerosis both in humans [2–4] and Journal of Molecular and Cellular Cardiology 62 (2013) 111–121 ⁎ Corresponding author at: Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Dóm tér 9, Szeged, H-6720, Hungary. Tel.: +36 62 545 096; fax: +36 62 545 097. E-mail address: csont.tamas@med.u-szeged.hu (T. Csont). 0022-2828/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.yjmcc.2013.05.009 Contents lists available at SciVerse ScienceDirect Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc