title: MicroRNA-25-dependent up-regulation of NADPH oxidase 4 (NOX4) mediates hypercholesterolemia-induced oxidative/nitrative stress and subsequent dysfunction in the heart.
abstract: 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 12weeks. 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 myocardial microRNA expression, and by down-regulating microRNA-25 increases NOX4 expression and consequently 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.
authors.affiliation: Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Szeged, Hungary.
authors.forename: Zoltán V
authors.initials: ZV
authors.lastname: Varga
authors.forename: Krisztina
authors.initials: K
authors.lastname: Kupai
authors.forename: Gergő
authors.initials: G
authors.lastname: Szűcs
authors.forename: Renáta
authors.initials: R
authors.lastname: Gáspár
authors.forename: János
authors.initials: J
authors.lastname: Pálóczi
authors.forename: Nóra
authors.initials: N
authors.lastname: Faragó
authors.forename: Agnes
authors.initials: A
authors.lastname: Zvara
authors.forename: László G
authors.initials: LG
authors.lastname: Puskás
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Rázga
authors.forename: László
authors.initials: L
authors.lastname: Tiszlavicz
authors.forename: Péter
authors.initials: P
authors.lastname: Bencsik
authors.forename: Anikó
authors.initials: A
authors.lastname: Görbe
authors.forename: Csaba
authors.initials: C
authors.lastname: Csonka
authors.forename: Péter
authors.initials: P
authors.lastname: Ferdinandy
authors.forename: Tamás
authors.initials: T
authors.lastname: Csont
mesh_terms: D000818:Animals; D006321:Heart; D006331:Heart Diseases / Q000209:etiology* / Q000235:genetics* / Q000378:metabolism; D006937:Hypercholesterolemia / Q000150:complications* / Q000235:genetics*; D007150:Immunohistochemistry; D008297:Male; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000235:genetics / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D035683:MicroRNAs; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat
keywords: Cholesterol; Hyperlipidemia; Metabolic syndrome; Nitrosative stress; Peroxynitrite; Superoxide