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.

Fig 1. MitoNEET plays a role for oxidative stress induced apoptosis in cardiac HL-1 cells.

HL-1 cells were transfected with silencing RNA (siRNA) directed against mitoNEET and non-specific-siRNA as control and subjected to 3h hypoxia followed by 1h of reoxygenation (H/R). (A) Densitometric analysis of Western Blots revealed aggravated activation of caspase-3 in mitoNEET-knockdown (mitoN-KD) cells after H/R compared to hypoxic controls (n = 11). (B) MitoNEET-KD showed no effect on lactate dehydrogenase (LDH) release after H/R measured in culture supernatants (n = 10). LDH was quantified as U/L by a routine clinical analyzer and is expressed as % of H/R control. (C,D) Overexpression of mitoNEET in HL-1 cells caused a significant decrease in apoptosis in mitoNEET overexpressing cells compared to H/R control cells. Expression of mitoNEET (n = 5) and cleaved caspase (n = 7) is shown in representative Western Blots and was densitometrically measured as % of H/R control. (E-G) H/R-induced apoptosis in control- and mitoNEET-KD cells was reduced by two different antioxidants, superoxide scavenger Tiron (10 mM, n = 5) and esterified glutathione compound GSH-MEE (glutathione reduced ethyl ester, 2 mM, n = 6) as demonstrated by representative Western Blots (F-G). Data were analyzed densitometrically, normalized to housekeeping gene expression and are expressed as % of H/R control.

Fig 2. Intracellular ROS-production in HL-1 cells is not directly affected by mitoNEET.

HL-1 cells were transfected with silencing RNA (siRNA) directed against mitoNEET and non-specific-siRNA as control and subjected to 3h hypoxia followed by 1h of reoxygenation (H/R). Using the fluorescent probe DCF-DA intracellular ROS-production was analyzed in a microplate reader. After 3h hypoxia intracellular ROS is 2.6 fold and after 60 min of reoxygenation even 5.4 fold increased compared to normoxic control cells (n = 12). Addition of antioxidative superoxide scavenger Tiron (10 mM; n = 6) and GSH-MEE (2 mM; n = 9) reduced the amount of ROS significantly. MitoNEET-KD doesn’t show any effect on the amount of intracellular ROS measured within the different conditions.

Fig 3. Chemical inhibition of glutathione reductase (GSR) reduces oxidative stress-induced apoptosis.

(A) Chemical GSR-inhibitor 2-AAPA (10 μM) reduced GSR-activity to 20% (n = 5). GSR-activity was determined by a colorimetric glutathione reductase assay. Thereby GSR reduces GSSG to GSH that reacts with 5, 5′-Dithiobis (2-nitrobenzoic acid) (DTNB) to generate yellow TNB^2-, which was measured at 405 nm in a microplate reader. GSR-activity was calculated, normalized to protein amount (mU/mg protein) and is expressed as % of H/R control. (B) Blockade of GSR-activity by 10 μM 2-AAPA decreased levels of activated caspase-3 in HL-1 cells after H/R (n = 5). (C) Representative Western Blot image is shown. Data were densitometrically analyzed and are expressed as % of H/R control. (D) TUNEL staining was utilized to determine apoptosis in HL-1 cells by the use of MEBSTAIN Apoptosis Kit II according to manufacturer`s protocol (MBL, Woburn, MA, USA). Data are presented as TUNEL-positive nuclei per total nuclei (n = 5).

Fig 4. MitoNEET is increased by 2-AAPA.

(A) Addition of GSR-inhibitor 2-AAPA (10 μM) led to a mild increase of mitoNEET-mRNA (n = 5) (B) while protein was increased 4-fold (n = 6) after H/R. (C) Representative Western Blots is presented. Data are expressed as % of H/R control. Real-time RT-PCR signals were normalized to hypoxanthine phosphoribosyl-transferase (HPRT) gene expression and data are expressed as 2^-ΔΔCT.

Fig 5. 2-AAPA decreases H/R induced apoptosis in mitoNEET-KD cells and increases mitoNEET-protein despite KD.

(A) Western Blot analysis of activated caspase-3 revealed reduced apoptosis in 2-AAPA treated mitoNEET-KD cells to the same level seen in 2-AAPA treated HL-1 cells after H/R (n = 6). (B) siRNA transfection decreased mitoNEET-mRNA efficiently in the presence and absence of chemical GSR-inhibitor 2-AAPA (10 μM, n = 5) determined by real-time RT-PCR. (C) MitoNEET-protein of mitoNEET-KD-cells was significantly higher after addition of 2-AAPA compared to mitoNEET-KD-cells without 2-AAPA treatment (n = 5) measured after H/R. Representative Western Blots are shown (A, C) and data are expressed as % of H/R control. Real-time RT-PCR data are expressed as 2^-ΔΔCT.

Fig 6. 2-AAPA effect on mitoNEET is abolished by addition of glutathione compound GSH-MEE.

MitoNEET-protein amount of HL-1 cells was clearly increased after addition of GSR-inhibitor 2-AAPA (10 μM) determined after H/R by Western Blot. This effect was completely reversed by additon of glutathione compound GSH-MEE (2 mM, n = 4–5) as demonstrated by a representative Western Blot. Data are expressed as % of H/R control.

Fig 7. Schematic view.

(i) MitoNEET-knockdown increases oxidative stress induced apoptosis. (ii) 2-AAPA inhibits GSR activity and diminishes the GSH/GSSG ratio resulting in decreasesd H/R induced apoptosis and an increase in mitoNEET-protein.