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.
Highlights
Oxidative stress is a critical factor for the enlargement of myocardial damage during reperfusion injury after transient myocardial ischemia [1,2,3] by inducing cardiomyocyte death through apoptosis and necrosis [4, 5]
MitoNEET plays a role for oxidative stress induced apoptosis in cardiac HL-1 cells In HL-1 cells knockdown of mitoNEET expression by small interfering RNA (siRNA) reduced both mitoNEET-mRNA and -protein to 25% quantified by real-time room temperature (RT)-PCR and Western Blot, respectively (S2 Fig)
We observed an increase of 1.9 fold in caspase-3 activity in the mitoNEET-KD cells after hypoxia and reoxygenation (H/R) compared to hypoxic control cells (Fig 1A, 1E and 1F)
Summary
Oxidative stress is a critical factor for the enlargement of myocardial damage during reperfusion injury after transient myocardial ischemia [1,2,3] by inducing cardiomyocyte death through apoptosis and necrosis [4, 5]. MitoNEET is a ubiquitously expressed iron-sulfur (Fe-S) protein with putative antioxidative capacity and with the highest level of mRNA seen in the heart [6]. Fe-S cluster containing proteins exhibit multiple functions depending on cluster ligands, their orientations and the local hydrogen-bonding arrangement [9]. They often take part in dynamic redox-sensitive activities; act as electron transport mediators; regulatory agents in gene expression and enzyme activity; act as a depot for sulfur as well as iron; and sensors for cellular oxygen [10,11,12]. Its role in cardiovascular disease states that are associated with oxidative stress induced damage has not been characterized yet
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
