Protection of peroxiredoxin II on oxidative stress-induced cardiomyocyte death and apoptosis

Abstract

Peroxiredoxin II, a cytosolic isoform of the antioxidant enzyme family, has been implicated in cancer-associated cell death and apoptosis, but its functional role in the heart remains to be elucidated. Interestingly, the expression levels of peroxiredoxin II were decreased in mouse hearts upon ischemia-reperfusion, while they were elevated in two genetically modified hyperdynamic hearts with phospholamban ablation or protein phosphatase 1 inhibitor 1 overexpression. To delineate the functional significance of altered peroxiredoxin II expression, adenoviruses encoding sense or antisense peroxiredoxin II were generated; cardiomyocytes were infected, and then subjected to H(2)O(2) treatment to mimic oxidative stress-induced cell death and apoptosis. H(2)O(2) stimulation resulted in a significant decrease of endogenous peroxiredoxin II expression, along with reduced cell viability in control cells. However, overexpression of peroxiredoxin II significantly protected from H(2)O(2)-induced apoptosis and necrosis, while downregulation of this enzyme promoted the detrimental effects of oxidative stress in cardiomyocytes. The beneficial effects of peroxiredoxin II were associated with increased Bcl-2 expression, decreased expression of Bax and attenuated activity of caspases 3, 9 and 12. Furthermore, there were no significant alterations in the expression levels of the other five isoforms of peroxiredoxin, as well as active catalase or glutathione peroxidase-1 after ischemia-reperfusion or H(2)O(2) treatment. These findings suggest that peroxiredoxin II may be a unique antioxidant in the cardiac system and may represent a potential target for cardiac protection from oxidative stress-induced injury.

Fig. 1

Alterations of peroxiredoxin II expression in the hearts and isolated cardiomyocytes as well as cell viability upon H₂O₂ treatment. Hearts from phospholamban deficient (a: PLN KO) and protein phosphatase 1 inhibitor-1 overexpression (b: I-1 OE) mice were homogenized and processed for quantitative immunoblotting for the expression of peroxiredoxin II. c Wild type hearts were subjected to ex vivo Langendorff perfusion, consisting of 40 min ischemia (pre I/R) followed by 60 min reperfusion (post I/R) and the levels of peroxiredoxin II were determined; n = 6 hearts for each group. Values are mean ± SE, *P < 0.05, compared to pre I/R or wild type values. d Quantitative immunoblotting and relative expressions of peroxiredoxin II (prxII) in cultured cardiomyocytes (24 h) in response to treatment with various H₂O₂ doses for 2 h. Calsequestrin was used as a loading control (n = 7 hearts for each group). e Cardiomyocyte viability was analyzed by MTT assay after H₂O₂ (50 μM) treatment for 2 h; n = 6 hearts for each group. Values are mean ± SE, *P < 0.05, compared to control

Fig. 2

Effects of peroxiredoxin II (prxII) overexpression and downregulation on cardiomyocyte viability and nuclear fragmentation upon H₂O₂ treatment. a Cardiomyocytes infected with adenoviruses: Ad.GFP, Ad.prxII (sense) or Ad.prxII-AS (antisense), under light (×40, left panel) and fluorescence (×40, right panel) microscope. b Quantitative immunobloting of peroxiredoxin II expression levels in infected cardiomyocytes. Calsequestrin was used as a loading control; n = 8 hearts for each group. Values are mean ± SE, *P < 0.05, Vs. Ad.GFP group. Cardiomyocytes were treated with H₂O₂ (50 μM) for 2 h after 24 h viral infection, then (c) MTT assay was used to analyze the cell viability, and (d) cell nuclear fragmentation was determined by Hoechst staining. n = 4 hearts for each group. Values are mean ± SE, *P < 0.05, Vs. control (control without H₂O₂ treatment). ^P < 0.05, Vs. Ad.GFP

Fig. 3

Effects of peroxiredoxin II (prxII) overexpression and downregulation on intracellular lipid peroxidation (TBARS) and lactate dehydrogenase (LDH) release upon H₂O₂ treatment. Cardiomyocytes were isolated, infected with Ad.GFP, Ad.prxII and Ad.prxII-AS for 24 h, and then treated with 50 lM H₂O₂ for 2 h. Cells were harvested, intracellular lipid peroxidation, TBARS (a) and LDH release (b) were determined; n = 4 hearts for each group. Values are mean ± SE, *P < 0.05, Vs. control (control without H₂O₂ treatment), ^P < 0.05, Vs. Ad.GFP

Fig. 4

Effects of peroxiredoxin II (prxII) overexpression and downregulation on apoptosis-related proteins in cardiomyocytes after H₂O₂ treatment. Cardiomyocytes were isolated, infected with Ad. GFP, Ad.prxII and Ad.prxII-AS for 24 h, and then treated with 50 lM H₂O₂ for 2 h. Cells were harvested, lysed and processed for quantitative immunobloting to determine the expression levels of Bcl-2, Bax, active caspase 3, active caspase 9 and active caspase 12. a Representative immunoblots for Bcl-2, Bax, active caspases 3, 9 and 12. b Quantitation of these proteins; n = 4–6 hearts for each group. Values are mean ± SE, *P < 0.05, Vs. control (control without H₂O₂ treatment), ^P < 0.05, Vs. Ad.GFP

Fig. 5

Effect of cardiac ischemia-reperfusion injury and H₂O₂ stimulation on the expression of peroxiredoxin family members, catalase and glutathione peroxidase (GHPx-1). a and b WT hearts were subjected to ex vivo Langendorff perfusion, as described in Fig. 1. The levels of the other five peroxiredoxin family members, including peroxiredoxin I, III, IV, V and VI, as well as two representative cytosolic house keeping proteins: catalase and GHPx-1 were determined. c and d Cardiomyocytes were isolated and cultured for 24 h, then treated with 50 μM H₂O₂ for 2 h. Cells were harvested, lysed and processed for western blot to examine the expression levels of the same proteins as those in (a) and (b); n = 4 hearts for each group, Values are mean ± SE, *P ≤ 0.05, Vs. per I/R or control cells

Fig. 6

Effect of overexpression or downregulation of peroxiredoxin II on the expression levels of catalase, GHPx-1 and peroxiredoxin I. Cardiomyocytes were isolated, infected with Ad.GFP, Ad.prxII and Ad.prxII-AS for 24 h, and then some of the cells were treated with 50 μM H₂O₂ for 2 h. Cells were then harvested, lysed and processed for western blot to examine the expression levels of catalase, GHPx-1 and peroxiredoxin I. a Representative immunoblots and b quantitation of the expressions of catalase, GHPx-1 and peroxiredoxin I; n = 4 hearts for each group. Values are mean ± SE

Fig. 7

Proposed scheme for the effects of peroxiredoxin II (prx II) in H₂O₂ or oxidative stress-induced myocyte apoptosis and necrosis