title: MitoNEET Protects HL-1 Cardiomyocytes from Oxidative Stress Mediated Apoptosis in an In Vitro Model of Hypoxia and Reoxygenation.
abstract: The iron-sulfur cluster containing protein mitoNEET is known to modulate the oxidative capacity of cardiac mitochondria but its function during myocardial reperfusion injury after transient ischemia is unknown. The purpose of this study was to analyze the impact of mitoNEET on oxidative stress induced cell death and its relation to the glutathione-redox system in cardiomyocytes in an in vitro model of hypoxia and reoxygenation (H/R). Our results show that siRNA knockdown (KD) of mitoNEET caused an 1.9-fold increase in H/R induced apoptosis compared to H/R control while overexpression of mitoNEET caused a 53% decrease in apoptosis. Necrosis was not affected. Apoptosis of both, mitoNEET-KD and control cells was diminished to comparable levels by using the antioxidants Tiron and glutathione compound glutathione reduced ethyl ester (GSH-MEE), indicating that mitoNEET-dependent apoptosis is mediated by oxidative stress. The interplay between mitoNEET and glutathione redox system was assessed by treating cardiomyocytes with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthio-carbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), known to effectively inhibit glutathione reductase (GSR) and to decrease the GSH/GSSG ratio. Surprisingly, inhibition of GSR-activity to 20% by 2-AAPA decreased apoptosis of control and mitoNEET-KD cells to 23% and 25% respectively, while at the same time mitoNEET-protein was increased 4-fold. This effect on mitoNEET-protein was not accessible by mitoNEET-KD but was reversed by GSH-MEE. In conclusion we show that mitoNEET protects cardiomyocytes from oxidative stress-induced apoptosis during H/R. Inhibition of GSH-recycling, GSR-activity by 2-AAPA increased mitoNEET-protein, accompanied by reduced apoptosis. Addition of GSH reversed these effects suggesting that mitoNEET can in part compensate for imbalances in the antioxidative glutathione-system and therefore could serve as a potential therapeutic approach for the oxidatively stressed myocardium.
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany.|Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany.
authors.forename: Anika
authors.initials: A
authors.lastname: Habener
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany.|Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Arpita
authors.initials: A
authors.lastname: Chowdhury
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Echtermeyer
authors.affiliation: Institute for Clinical Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Lichtinghagen
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany.|Department of Health Services Sciences, Faculty of Medicine and Health Sciences, University of Oldenburg, Oldenburg, Germany.
authors.forename: Gregor
authors.initials: G
authors.lastname: Theilmeier
authors.affiliation: Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Herzog
mesh_terms: D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics*; D002460:Cell Line; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism; D033862:Iron-Binding Proteins / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D015424:Reperfusion; D013859:Thiocarbamates / Q000494:pharmacology
chemical_list: C539612:2-acetylamino-3-(4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl)propionic acid; D000975:Antioxidants; D033862:Iron-Binding Proteins; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013859:Thiocarbamates; C519501:mitoNEET protein, mouse; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; C042431:S-ethyl glutathione; D005980:Glutathione Reductase; D005978:Glutathione; D000111:Acetylcysteine