Skip to main content
SpringerLink
Log in

Nuclear Factor E2-Related Factor 2-Dependent Myocardiac Cytoprotection Against Oxidative and Electrophilic Stress

  • Published:
Cardiovascular Toxicology Aims and scope Submit manuscript

Abstract

Nuclear factor E2-related factor 2 (Nrf2) is a critical regulator of cytoprotective gene expression. However, the role of this transcription factor in myocardiac cytoprotection against oxidative and electrophilic stress remains unknown. This study was undertaken to investigate if Nrf2 signaling could control the constitutive and inducible expression of antioxidants and phase 2 enzymes in primary cardiomyocytes as well as the susceptibility of these cells to oxidative and electrophilic injury. The basal expression of a series of antioxidants and phase 2 enzymes was significantly lower in cardiomyocytes from Nrf2−/− mice than those from wild-type littermates. Incubation of wild-type cardiomyocytes with 3H-1,2-dithiole-3-thione (D3T) led to significant induction of various antioxidants and phase 2 enzymes, including catalase, glutathione, glutathione peroxidase (GPx), glutathione reductase, glutathione S-transferase, NAD(P)H:quinone oxidoreductase 1, and heme oxygenase-1. The inducibility of the above cellular defenses except GPx by D3T was abolished in Nrf2−/− cardiomyocytes. As compared to wild-type cells, Nrf2−/− cardiomyocytes were much more susceptible to cell injury induced by H2O2, peroxynitrite, and 4-hydroxy-2-nonenal. Treatment of wild-type cardiomyocytes with D3T, which upregulated the cellular defenses, resulted in increased resistance to the above oxidant- and electrophile-induced cell injury, whereas D3T treatment of Nrf2−/− cardiomyocytes provided no cytoprotection. This study demonstrates that Nrf2 is an important factor in controlling both constitutive and inducible expression of a wide spectrum of antioxidants and phase 2 enzymes in cardiomyocytes and is responsible for protecting these cells against oxidative and electrophilic stress. These findings also implicate Nrf2 as an important signaling molecule for myocardiac cytoprotection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Abbreviations

ARE:

Antioxidant response element

CDNB:

1-chloro-2,4-dinitrobenzene

D3T:

3H-1,2-dithiole-3-thione

DCIP:

2,6-dichloroindophenol

FBS:

Fetal bovine serum

γGCL:

γ-Glutamylcysteine ligase

GCLC:

γ-Glutamylcysteine ligase catalytic subunit

GPx:

Glutathione peroxidase

GR:

Glutathione reductase

GSH:

Reduced glutathione

GSSG:

Oxidized form of glutathione

GST:

Glutathione S-transferase

HNE:

4-hydroxy-2-nonenal

HO-1:

Heme oxygenasse-1

MTT:

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

NQO1:

NAD(P)H:quinone oxidoreductase 1

Nrf2:

Nuclear factor E2-related factor 2

PBS:

Phosphate buffered saline

SIN-1:

3-morpholinosydnonimine

RT-PCR:

Reverse transcriptase-polymerase chain reaction

SOD:

Superoxide dismutase

References

  1. McCord, J. M. (1985). Oxygen-derived free radicals in postischemic tissue injury. The New England Journal of Medicine, 312, 159–163.

    PubMed  CAS  Google Scholar 

  2. Villamena, F. A., & Zweier, J. L. (2004). Detection of reactive oxygen, nitrogen species by EPR spin trapping. Antioxidants & Redox Signaling, 6, 619–629. doi:10.1089/152308604773934387.

    Article  CAS  Google Scholar 

  3. Kutala, V. K., Khan, M., Angelos, M. G., & Kuppusamy, P. (2007). Role of oxygen in postischemic myocardial injury. Antioxidants & Redox Signaling, 9, 1193–206. doi:10.1089/ars.2007.1636.

    Article  CAS  Google Scholar 

  4. Kang, Y. J. (2007). Antioxidant defense against anthracycline cardiotoxicity by metallothionein. Cardiovascular Toxicology, 7, 95–100 doi:10.1007/s12012-007-0007-3.

    Article  PubMed  CAS  Google Scholar 

  5. Salvemini, D., Wang, Z. Q., Zweier, J. L., Samouilov, A., Macarthur, H., Misko, T. P., Currie, M. G., Cuzzocrea, S., Sikorski, J. A., & Riley, D. P. (1999). A nonpeptidyl mimic of superoxide dismutase with therapeutic activity in rats. Science, 286, 304–306. doi:10.1126/science.286.5438.304.

    Article  PubMed  CAS  Google Scholar 

  6. Lefer, D. J., & Granger, N. (2000). Oxidative stress and cardiac disease. The American Journal of Medicine, 109, 315–323. doi:10.1016/S0002-9343(00)00467-8.

    Article  PubMed  CAS  Google Scholar 

  7. Steinberg, D. (2002). Atherogenesis in perspective: Hypercholesterolemia and inflammation as partners in crime. Nature Medicine, 8, 1211–1217. doi:10.1038/nm1102–1211.

    Article  PubMed  CAS  Google Scholar 

  8. Bolli, R., Becker, L., Gross, G., Mentzer, R. Jr., Balshaw, D., & Lathrop, D. A. (2004). NHLBI working group on the translation of therapies for protecting the heart from ischemia. Myocardial protection at a crossroads: The need for translation into clinical therapy. Circulation Research, 95, 125–134. doi:10.1161/01.RES.0000137171.97172.d7.

    Article  PubMed  CAS  Google Scholar 

  9. Lonn, E., Bosch, J., Yusuf, S., Sheridan, P., Pogue, J., Arnold, J. M., Ross, C., Arnold, A., Sleight, P., Probstfield, J., & Dagenais, G. R. (2005). HOPE and HOPE-TOO trial investigators. Effects of long-term vitamin E supplementation on cardiovascular events and cancer: A randomized controlled trial. Journal of the American Medical Association, 293, 1338–1347. doi:10.1001/jama.293.11.1338.

    Article  PubMed  Google Scholar 

  10. Bailey, D. M., Raman, S., McEneny, J., Young, I. S., Parham, K. L., Hullin, D. A., Davies, B., McKeeman, G., McCord, J. M., & Lewis, M. H. (2006). Vitamin C prophylaxis promotes oxidative lipid damage during surgical ischemia-reperfusion. Free Radical Biology & Medicine, 40, 591–600. doi:10.1016/j.freeradbiomed.2005.09.024.

    Article  CAS  Google Scholar 

  11. Itoh, K., Chiba, T., Takahashi, S., Ishii, T., Igarashi, K., Katoh, Y., Oyake, T., Hayashi, N., Satoh, K., Hatayama, I., Yamamoto, M., & Nabeshima, Y. (1997). An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochemical and Biophysical Research Communications, 236, 313–322. doi:10.1006/bbrc.1997.6943.

    Article  PubMed  CAS  Google Scholar 

  12. Motohashi, H., & Yamamoto, M. (2004). Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends in Molecular Medicine, 10, 549–557. doi:10.1016/j.molmed.2004.09.003.

    Article  PubMed  CAS  Google Scholar 

  13. Zhu, H., Zhang, L., Itoh, K., Yamamoto, M., Ross, D., Trush, M. A., Zweier, J. L., & Li, Y. (2006). Nrf2 controls bone marrow stromal cell susceptibility to oxidative and electrophilic stress. Free Radical Biology & Medicine, 41, 132–143. doi:10.1016/j.freeradbiomed.2006.03.020.

    Article  CAS  Google Scholar 

  14. Spitz, D. R., & Oberley L. W. (1989). An assay for superoxide dismutase activity in mammalian tissue homogenates. Analytical Biochemistry, 179, 8–18. doi:10.1016/0003-2697(89)90192-9.

    Article  PubMed  CAS  Google Scholar 

  15. Aebi, H. (1984). Catalase in vitro. Methods in Enzymology, 105, 121–127. doi:10.1016/S0076-6879(84)05016-3.

    Article  PubMed  CAS  Google Scholar 

  16. Flohé L, & Günzler W. A. (1984). Assays of glutathione peroxidase. Methods in Enzymology, 105, 114–121. doi:10.1016/S0076–6879(84)05015-1.

    Article  PubMed  Google Scholar 

  17. Habig, W. H., Pabst, M. J., & Jakoby, W. B. (1974). Glutathione S-transferases: The first enzymatic step in mercapturic acid formation. The Journal of Biological Chemistry, 249, 7130–7139.

    PubMed  CAS  Google Scholar 

  18. Naughton, P., Foresti, R., Bains, S. K., Hoque, M., Green, C. J., & Motterlini, R. (2002). Induction of heme oxygenase 1 by nitrosative stress. A role for nitroxyl anion. The Journal of Biological Chemistry, 277, 40666–40674. doi:10.1074/jbc.M203863200.

    Article  PubMed  CAS  Google Scholar 

  19. Zhu, H., Zhang, L., Trush, M. A., & Li, Y. (2007). Upregulation of endogenous glutathione system by 3H-1,2-dithiole-3-thione in pancreatic RINm5F beta-cells as a novel strategy for protecting against oxidative beta-cell injury. Free Radical Research, 41, 242–250. doi:10.1080/10715760601009586.

    Article  PubMed  CAS  Google Scholar 

  20. Jia, Z., Zhu, H., Misra, H. P., & Li, Y. (2008). Potent induction of total cellular GSH and NQO1 as well as mitochondrial GSH by 3H-1,2-dithiole-3-thione in SH-SY5Y neuroblastoma cells and primary human neurons: Protection against neurocytotoxicity elicited by dopamine, 6-hydroxydopamine, 4-hydroxy-2-nonenal, or hydrogen peroxide. Brain Research, 1197, 159–169. doi:10.1016/j.brainres.2007.12.044.

    Article  PubMed  CAS  Google Scholar 

  21. Halford, W. P., Falco, V. C., Gebhardt, B. M., & Carr, D. J. (1999). The inherent quantitative capacity of the reverse transcription-polymerase chain reaction. Analytical Biochemistry, 266, 181–191. doi:10.1006/abio.1998.2913.

    Article  PubMed  CAS  Google Scholar 

  22. Hayes, J. D., Flanagan, J. U., & Jowsey, I. R. (2005). Glutathione transferases. Annual Review of Pharmacology and Toxicology, 45, 51–88. doi:10.1146/annurev.pharmtox.45.120403.095857.

    Article  PubMed  CAS  Google Scholar 

  23. Abraham, N. G., & Kappas, A. (2005). Heme oxygenase and the cardiovascular-renal system. Free Radical Biology & Medicine, 39, 1–25. doi:10.1016/j.freeradbiomed.2005.03.010.

    Article  CAS  Google Scholar 

  24. Ryter, S. W., Alam, J., & Choi, A. M. (2006). Heme oxygenase-1/carbon monoxide: From basic science to therapeutic applications. Physiological Research, 86, 583–650. doi:10.1152/physrev.00011.2005.

    Article  CAS  Google Scholar 

  25. Szabó C, Ischiropoulos, H., & Radi, R. (2007) Peroxynitrite: Biochemistry, pathophysiology and development of therapeutics. Nature Reviews. Drug Discovery, 6, 662–680. doi:10.1038/nrd2222.

    Article  PubMed  CAS  Google Scholar 

  26. Lucas, D. T., & Szweda, L. I. (1998). Cardiac reperfusion injury: Aging, lipid peroxidation, and mitochondrial dysfunction. Proceedings of the National Academy of Sciences of the United States of America, 95, 510–514. doi:10.1073/pnas.95.2.510.

    Article  PubMed  CAS  Google Scholar 

  27. Shinmura, K., Bolli, R., Liu, S. Q., Tang, X. L., Kodani, E., Xuan, Y. T., Srivastava, S., & Bhatnagar A (2002). Aldose reductase is an obligatory mediator of the late phase of ischemic preconditioning. Circulation Research, 91, 240–246. doi:10.1161/01.RES.0000029970.97247.57.

    Article  PubMed  CAS  Google Scholar 

  28. Cho, H. Y., Reddy, S. P., Debiase, A., Yamamoto, M., & Kleeberger, S. R. (2005). Gene expression profiling of Nrf2-mediated protection against oxidative injury. Free Radical Biology & Medicine, 38, 325–343. doi:10.1016/j.freeradbiomed.2004.10.013.

    Article  CAS  Google Scholar 

  29. Kwak, M. K., Wakabayashi, N., Itoh, K., Motohashi, H., Yamamoto, M., & Kensler, T. W. (2003). Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway: Identification of novel gene clusters for cell survival. The Journal of Biological Chemistry, 278, 8135–8145. doi:10.1074/jbc.M211898200.

    Article  PubMed  CAS  Google Scholar 

  30. Zhu, H., Itoh, K., Yamamoto, M., Zweier, J. L., & Li, Y. (2005). Role of Nrf2 signaling in regulation of antioxidants, phase 2 enzymes in cardiac fibroblasts: Protection against reactive oxygen and nitrogen species-induced cell injury. FEBS Letters, 579, 3029–3036. doi:10.1016/j.febslet.2005.04.058.

    Article  PubMed  CAS  Google Scholar 

  31. Anderson, M. E. (1998). Glutathione: An overview of biosynthesis and modulation. Chemico-Biological Interactions, 111–112, 1–14. doi:10.1016/S0009-2797(97)00146-4.

    Article  PubMed  Google Scholar 

  32. Banning, A., Deubel, S., Kluth, D., Zhou, Z., & Brigelius-Flohe, R. (2005). The GI-GPx gene is a target for Nrf2. Molecular and Cellular Biology, 25, 4914–4923. doi:10.1128/MCB.25.12.4914-4923.2005.

    Article  PubMed  CAS  Google Scholar 

  33. Li, Y., Lafuente, A., & Trush, M. A. (1994). Characterization of quinone reductase, glutathione and glutathione S-transferase in human myeloid cell lines: Induction by 1,2-dithiole-3-thione and effects on hydroquinone-induced cytotoxicity. Life Sciences, 54, 901–916. doi:10.1016/0024-3205(94)00626-1.

    Article  PubMed  CAS  Google Scholar 

  34. Ross, D. (2004). Quinone reductases multitasking in the metabolic world. Drug Metabolism Reviews, 36, 639–654. doi:10.1081/DMR-200033465.

    Article  PubMed  CAS  Google Scholar 

  35. Siegel, D., Gustafson D. L, Dehn, D. L., Han, J. Y., Boonchoong, P., Berliner, L. J., & Ross, D. (2004). NAD(P)H: Quinone oxidoreductase 1: Role as a superoxide scavenger. Molecular Pharmacology, 65, 1238–1247. doi:10.1124/mol.65.5.1238.

    Article  PubMed  CAS  Google Scholar 

  36. Zhu, H., Jia, Z., Mahaney, J. E., Ross, D., Misra, H. P., Trush, M. A., & Li, Y. (2007). The highly expressed and inducible endogenous NAD(P)H: Quinone oxidoreductase 1 in cardiovascular cells acts as a potential superoxide scavenger. Cardiovascular Toxicology, 7, 202–211. doi:10.1007/s12012-007-9001-z.

    Article  PubMed  CAS  Google Scholar 

  37. Asher, G., Lotem, J., Kama, R., Sachs, L., & Shaul, Y. (2002). NQO1 stabilizes p53 through a distinct pathway. Proceedings of the National Academy of Sciences of the United States of America, 99, 3099–3104. doi:10.1073/pnas.052706799.

    Article  PubMed  CAS  Google Scholar 

  38. Juan, S. H., Lee, T. S., Tseng, K. W., Liou, J. Y., Shyue, S. K., Wu, K. K., & Chau, L. Y. (2001). Adenovirus-mediated heme oxygenase-1 gene transfer inhibits the development of atherosclerosis in apolipoprotein E-deficient mice. Circulation, 104, 1519–1525. doi:10.1161/hc3801.095663.

    Article  PubMed  CAS  Google Scholar 

  39. Liu, X., Simpson, J. A., Brunt, K. R., Ward, C. A., Hall, S. R., Kinobe, R. T., Barrette, V., Tse, M. Y., Pang, S. C., Pachori, A. S., Dzau, V. J., Ogunyankin, K. O., & Melo, L. G. (2007). Preemptive heme oxygenase-1 gene delivery reveals reduced mortality and preservation of left ventricular function 1 yr after acute myocardial infarction. The American Journal of Physiology, 293, H48–H59.

    CAS  Google Scholar 

  40. Sies, H., Sharov, V. S., Klotz, L. O., & Briviba, K. (1997). Glutathione peroxidase protects against peroxynitrite-mediated oxidations: A new function for selenoproteins as peroxynitrite reductase. The Journal of Biological Chemistry, 272, 27812–27817. doi:10.1074/jbc.272.44.27812.

    Article  PubMed  CAS  Google Scholar 

  41. Xie, C., Lovell, M. A., Xiong, S., Kindy, M. S., Guo J.-T., Xie, J., Amaranth, V., Montine, T. J., & Markesbery, W. R. (2001). Expression of glutathione-S-transferase isozyme in the SY5Y neuroblastoma cell line increases resistance to oxidative stress. Free Radical Biology & Medicine, 31, 73–81. doi:10.1016/S0891-5849(01)00553-6.

    Article  CAS  Google Scholar 

  42. Yang, Y., Cheng J.-Z., Singhal, S. S., Saini, M., Pandya, U., Awasthi, S., & Awasthi, Y. C. (2001). Role of glutathione S-transferases in protection against lipid peroxidation. The Journal of Biological Chemistry, 276, 19220–19230. doi:10.1074/jbc.M100551200.

    Article  PubMed  CAS  Google Scholar 

  43. Sayre, L. M., Lin, D., Yuan, Q., Zhu, X., & Tang, X. (2006). Protein adducts generated from products of lipid oxidation: Focus on HNE and one. Drug Metabolism Reviews, 38, 651–675. doi:10.1080/03602530600959508.

    Article  PubMed  CAS  Google Scholar 

  44. Cao, Z., Hardej, D., Trombetta, L. D., & Li, Y. (2003). The role of chemically induced glutathione and glutathione S-transferase in protecting against 4-hydroxy-2-nonenal-mediated cytotoxicity in vascular smooth muscle cells. Cardiovascular Toxicology, 3, 165–177. doi:10.1385/CT:3,2:165.

    Article  PubMed  CAS  Google Scholar 

  45. Li, Y., Cao, Z., Zhu, H., & Trush, M. A. (2005). Differential roles of 3H-1,2-dithiole-3-thione-induced glutathione, glutathione S-transferase and aldose reductase in protecting against 4-hydroxy-2-nonenal toxicity in cultured cardiomyocytes. Archives of Biochemistry and Biophysics, 439, 80–90. doi:10.1016/j.abb.2005.05.008.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grant R01HL71190 from the National Institutes of Health and a grant from Harvey Peters Research Foundation (YL).

Author information

Authors and Affiliations

  1. Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA, 24060, USA

    Hong Zhu, Zhenquan Jia, Bhaba R. Misra, Hara P. Misra & Yunbo Li

  2. Davis Heart and Lung Research Institute, The Ohio State University College of Medicine, Columbus, OH, 43210, USA

    Li Zhang

  3. Cardiology Division, Brigham and Women’s Hospital, Harvard Medical School, 20 Shattuck St., Thorn 1130, Boston, MA, 02115, USA

    Zhuoxiao Cao

  4. Center for TARA and Institute for Basic Medical Sciences, University of Tsukuba, Tsukuba, 305-8577, Japan

    Masayuki Yamamoto

  5. Division of Toxicology, Department of Environmental Health Sciences, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 21205, USA

    Michael A. Trush

  6. Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24061, USA

    Hara P. Misra & Yunbo Li

Authors
  1. Hong Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhenquan Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bhaba R. Misra
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Li Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhuoxiao Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Masayuki Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael A. Trush
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hara P. Misra
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yunbo Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Hara P. Misra or Yunbo Li.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhu, H., Jia, Z., Misra, B.R. et al. Nuclear Factor E2-Related Factor 2-Dependent Myocardiac Cytoprotection Against Oxidative and Electrophilic Stress. Cardiovasc Toxicol 8, 71–85 (2008). https://doi.org/10.1007/s12012-008-9016-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12012-008-9016-0

Keywords

Search

Navigation