Korean Circ J.  2012 Jan;42(1):23-32. 10.4070/kcj.2012.42.1.23.

Protective Effects of Peroxiredoxin on Hydrogen Peroxide Induced Oxidative Stress and Apoptosis in Cardiomyocytes

• Park KJ
• Kim YJ
• Kim J
• Kim SM
• Lee SY
• Bae JW
• Hwang KK
• Kim DW
• Cho MC

• KMID: 2297904
• DOI: http://doi.org/10.4070/kcj.2012.42.1.23

Abstract

BACKGROUND AND OBJECTIVES
The redox system is an important anti-oxidative system composed of thioredoxin, thioredoxin reductase, and peroxiredoxin (PRx). The fine details of PRx expression and its protective effects in various cells in cardiovascular tissue under oxidative stress created by hydrogen peroxide have not been fully elucidated.
SUBJECTS AND METHODS
Oxidative stress was induced by adding hydrogen peroxide at 0.25 mM for 2 hours to rat neonatal cardiomyocytes (rCMCs), rat vascular smooth muscle cells (rVSMCs), and human umbilical vein endothelial cells (HUVECs). Apoptosis was quantified by flow cytometry and the expression patterns of the six PRx isoforms were evaluated by western blotting in the three cell lines after hydrogen peroxide stimulation. Apoptosis and the cell survival signal pathway were evaluated by PRx1 gene delivery using lentiviral vector in hydrogen peroxide stimulated rCMCs versus green fluorescence protein gene delivery.
RESULTS
Hydrogen peroxide induced 25% apoptosis in rCMCs. Furthermore, the PRx1 and 5 isoforms were found to be overexpressed in hydrogen peroxide treated rCMCs, and PRx1 overexpression by gene delivery was found to reduce hydrogen peroxide induced rCMCs apoptosis significantly. In addition, this effect was found to originate from cell survival pathway modification.
CONCLUSION
Hydrogen peroxide induced significant oxidative stress in rCMCs, rVSMCs, and HUVECs, and PRx1 overexpression using a lentiviral vector system significantly reduced hydrogen peroxide induced rCMCs apoptosis by upregulation of cell survival signals and downregulation of apoptotic signals. These findings suggest that PRx1 could be used as a treatment strategy for myocardial salvage in conditions of oxidative stress.

Keyword

Peroxiredoxins; Myocytes, cardiac; Oxidative stress; Apoptosis

MeSH Terms

Animals
Apoptosis
Blotting, Western
Cell Line
Cell Survival
Down-Regulation
Flow Cytometry
Fluorescence
Human Umbilical Vein Endothelial Cells
Hydrogen
Hydrogen Peroxide
Muscle, Smooth, Vascular
Myocytes, Cardiac
Oxidation-Reduction
Oxidative Stress
Peroxiredoxins
Protein Isoforms
Rats
Signal Transduction
Thioredoxin-Disulfide Reductase
Thioredoxins
Up-Regulation
Hydrogen
Hydrogen Peroxide
Peroxiredoxins
Protein Isoforms
Thioredoxin-Disulfide Reductase
Thioredoxins

Figure

• 

  Fig. 1 FACS and the detection of rCMC, rVSMC, and HUVEC apoptosis under hydrogen peroxide-induced oxidative stress. Flow cytometric analysis showed significant increases in apoptosis after treatment with 0.1 mM hydrogen peroxide for 2 hours versus non-treated control rCMCs, rVSMCs, or HUVECs. *Compared to control, p<0.05. FACS: flow cytometry analysis, rCMCs: neonatal rat cardiomyocytes, rVSMCs: rat vascular smooth muscle cells, HUVECs: human umbilical vein endothelial cells, Con: control, H2O2: hydrogen peroxide.

• 

  Fig. 2 Temporal expression patterns of the 6 PRx isoforms in rCMCs, rVSMCs, and HUVECs exposed to hydrogen peroxide-induced oxidative stress. PRx1 and 5 were overexpressed at 30 and 120 minutes, whereas PRx6 was overexpressed at 120 minutes in rCMCs after hydrogen peroxide stimulation. All isoforms were overexpressed in rVSMCs at both time points. No significant change in the expression of PRx isoforms was observed in HUVECs. *Compared to control, p<0.05. PRx: peroxiredoxin, rCMCs: neonatal rat cardiomyocytes, rVSMCs: rat vascular smooth muscle cells, HUVECs: human umbilical vein endothelial cells, con: control, min: minutes.

• 

  Fig. 3 Confirmation of gene expression after the transfection of LeV-GFP into rCMCs. GFP expression in rCMCs/LeV-GFP. rCMCs (3×105 cell) were transfected with LeV-GFP for 16 hours at a multiplicity of infection of 1.5×107 IU. A: rCMCs appeared healthy under optical microscopy (×200). B: GFP expression in rCMCs transfected with LeV-GFP was observed in over 80% of cells by fluorescence microscopy (×200). LeV-GFP: GFP gene containing lentivirus vector, rCMCs: neonatal rat cardiomyocytes, GFP: green fluorescence protein.

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  Fig. 4 FACS and the quantitation of apoptosis in rCMCs transfected with LeV-PRx1 or LeV-GFP and exposed to hydrogen peroxide. The transfection of LeV-PRx1 significantly reduced apoptosis induced by 0.25 mM hydrogen peroxide for 2 hours as compared to LeV-GFP transfection (72.55±10.19% vs. 187.5±25.49%, p<0.05). *Compared to control, p<0.05, †Compared to LeV-GFP+H2O2, p<0.05. FACS: flow cytometric analysis, rCMCs: neonatal rat cardiomyocytes, LeV-PRx1: PRx1 gene containing lentivirus vector, LeV-GFP: green fluorescence protein gene containing lentivirus vector, Con: control, H2O2: hydrogen peroxide, rVSMCs: rat vascular smooth muscle cells.

• 

  Fig. 5 Expression patterns of PRx1 in rCMCs transfected with LeV-PRx1 in hydrogen peroxide induced oxidative stress. PRx1 expression patterns under various conditions of hydrogen peroxide induced oxidative stress and PRx1 gene delivery. Cells were exposed to 0.25 mM hydrogen peroxide for 2 hours. LeV-PRx1 transfection significantly upregulated PRx1 expression versus LeV-GFP transfection in hydrogen peroxide treated rCMCs. *Compared to control, p<0.05, †Compared to LeV-GFP+H2O2, p<0.05. rCMCs: rat cardiomyocytes, LeV-PRx1: PRx1 gene containing lentivirus vector, con: control, H2O2: hydrogen peroxide, LeV-GFP: green fluorescence protein gene containing lentivirus vector.

• 

  Fig. 6 Immunohistochemistry in rCMCs transfected with LeV-PRx1. PRx1 expression was upregulated in rCMCs/LeV-PRx1 as compared to rCMCs/LeV-GFP treated with 0.25 mM hydrogen peroxide for 2 hours. Cytolsolic isoforms, PRx1 was found to be highly expressed in the rCMCs/LeV-PRx1 (stained red). rCMCs: neonatal rat cardiomyocytes, LeV-PRx1: PRx1 gene containing lentivirus vector, LeV-GFP: green fluorescence protein gene containing lentivirus vector.

• 

  Fig. 7 Protein expression of caspase 3 and survivin in rCMCs transfected with LeV-Prx1. LeV-PRx transfection effectively reduced caspase 3 expression and enhanced survivin expression in rCMCs treated with 0.25 mM hydrogen peroxide for 2 hours. *Compared with control, p<0.05, †Compared with LeV-GFP+H2O2, p<0.05. rCMCs: rat cardiomyocytes, LeV-PRx1: PRx1 gene containing lentivirus vector, Con: control, H2O2: hydrogen peroxide, LeV-GFP: GFP gene containing lentivirus vector.

• 

  Fig. 8 Bax/Bcl-2 ratios in LeV-PRx1 versus LeV-GFP transfected rCMCs treated with hydrogen peroxide. Bax/Bcl-2 ratio was significantly lower in LeV-PRx1 than in LeV-GFP transfected rCMCs treated with 0.25 mM hydrogen peroxide for 2 hours. *Compared to control, p<0.05, †Compared to LeV-GFP+H2O2, p<0.05. LeV-PRx1: PRx1 gene containing lentivirus vector, LeV-GFP: GFP gene containing lentivirus vector, rCMCs: rat cardiomyocytes, Con: control, H2O2: hydrogen peroxide, neonatal rat cardiomyocytes.

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