Skip to main content
SpringerLink
Log in

Unique mode of cell death in freshly isolated adult rat ventricular cardiomyocytes exposed to hydrogen peroxide

  • Original Paper
  • Published:
Medical Molecular Morphology Aims and scope Submit manuscript

Abstract

To address whether adult rat ventricular cardiomyocytes (ARVCs) exposed to oxidant stress die via apoptosis (secondarily by necrosis) or primarily by necrosis, we exposed ARVCs to hydrogen peroxide (H2O2; 0.1–100 μM) for up to 24 h and then compared them with isoproterenol-induced apoptotic and Triton X-induced necrotic controls. Cellular shrinkage preceded plasma membrane disruption, reflected by trypan blue uptake in ARVCs exposed to lower concentrations of H2O2 (<1 μM; an apoptotic pattern), but the order was reversed in cells exposed to higher concentrations of H2O2 (>1 μM; a necrotic pattern). DNA fragmentation, caspase-3 activation, mitochondrial membrane potential preservation, and ATP preservation were all apparent in ARVCs treated with low H2O2 (0.5 μM), but not in those treated with high H2O2 (10 μM). In addition, electron microscopy revealed unique morphology in H2O2-treated ARVCs; i.e., the nuclei had a homogeneous ground glass-like appearance that was never accompanied by chromatin condensation. Apparently, high concentrations of H2O2 caused primary necrosis in ARVCs, whereas low concentrations induced biochemically comparable apoptosis, although the latter did not satisfy the morphological criteria of apoptosis. These findings caution against the use of oxidant stress, H2O2 in particular, as an inducer of apoptosis in ARVCs.

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.

Institutional subscriptions

Similar content being viewed by others

References

  1. Tsutsui H (2004) Novel pathophysiological insight and treatment strategies for heart failure: lessons from mice and patients. Circ J 68:1095–1103

    Article  PubMed  CAS  Google Scholar 

  2. Giordano FJ (2005) Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest 115:500–508

    PubMed  CAS  Google Scholar 

  3. von Harsdorf R, Li PF, Dietz R (1999) Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation 99:2934–2941

    Google Scholar 

  4. Kwon SH, Pimentel DR, Remondino A, Sawyer DB, Colucci WS (2003) H2O2 regulates cardiac myocyte phenotype via concentration-dependent activation of distinct kinase pathways. J Mol Cell Cardiol 35:615–621

    Article  PubMed  CAS  Google Scholar 

  5. Taimor G, Lorenz H, Hofstaetter B, Schlüter KD, Piper HM (1999) Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat. Cardiovasc Res 41:147–156

    Article  PubMed  CAS  Google Scholar 

  6. Suzuki K, Kostin S, Person V, Elsasser A, Schaper J (2001) Time course of the apoptotic cascade and effects of caspase inhibitors in adult rat ventricular cardiomyocytes. J Mol Cell Cardiol 33: 983–994

    Article  PubMed  CAS  Google Scholar 

  7. Bird SD, Doevendans PA, van Rooijen MA, Brutel de la Riviere A, Hassink RJ, Passier R, Mummery CL (2003) The human adult cardiomyocyte phenotype. Cardiovasc Res 58:423–434

    Article  PubMed  CAS  Google Scholar 

  8. Schaper J, Froede R, Hein S, Buck A, Hashizume H, Speiser B, Friedl A, Bleese N (1991) Impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy. Circulation 83:504–514

    PubMed  CAS  Google Scholar 

  9. Wyllie AH, Kerr JF, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306

    Article  PubMed  CAS  Google Scholar 

  10. Taatjes DJ, Sobel BE, Budd RC (2008) Morphological and cytochemical determination of cell death by apoptosis. Histochem Cell Biol 129:33–43

    Article  PubMed  CAS  Google Scholar 

  11. Piper HM, Probst I, Schwartz P, Hutter FJ, Spieckermann PG (1982) Culturing of calcium stable adult cardiac myocytes. J Mol Cell Cardiol 14:397–412

    Article  PubMed  CAS  Google Scholar 

  12. Maruyama R, Takemura G, Tohse N, Ohkusa T, Ikeda Y, Tsuchiya K, Minatoguchi S, Matsuzaki M, Fujiwara T, Fujiwara H (2006) Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes. Am J Physiol Heart Circ Physiol 290:H1493–H1502

    Article  PubMed  CAS  Google Scholar 

  13. Zaugg M, Xu W, Lucchinetti E, Shafiq SA, Jamali NZ, Siddiqui MA (2000) Beta-adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes. Circulation 102: 344–350

    PubMed  CAS  Google Scholar 

  14. Khalid MA, Ashraf M (1993) Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation. Is the hydroxyl radical really the most damaging radical species? Circ Res 72:725–736

    PubMed  CAS  Google Scholar 

  15. Kato S, Takemura G, Maruyama R, Aoyama T, Hayakawa K, Koda M, Kawase Y, Li Y, Minatoguchi S, Fujiwara T, Fujiwara H (2001) Apoptosis, rather than oncosis, is the predominant mode of spontaneous death of isolated adult rat cardiac myocytes in culture. Jpn Circ J 65:743–748

    Article  PubMed  CAS  Google Scholar 

  16. Richter C, Schweizer M, Cossarizza A, Franceshi C (1996) Control of apoptosis by the cellular ATP level. FEBS Lett 378:107–110

    Article  PubMed  CAS  Google Scholar 

  17. Majno G, Joris I (1995) Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol 146:3–15

    PubMed  CAS  Google Scholar 

  18. Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA (1995) Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-d-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci U S A 92:7162–7166

    Article  PubMed  CAS  Google Scholar 

  19. Lennon SV, Martin SJ, Cotter TG (1991) Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli. Cell Prolif 24:203–214

    Article  PubMed  CAS  Google Scholar 

  20. Reimer KA, Jennings RB, Hill ML (1981) Total ischemia in dog hearts, in vitro. 2. High energy phosphate depletion and associated defects in energy metabolism, cell volume regulation, and sarcolemmal integrity. Circ Res 49:901–911

    PubMed  CAS  Google Scholar 

  21. Bersohn MM, Philipson KD, Fukushima JY (1982) Sodiumcalcium exchange and sarcolemmal enzymes in ischemic rabbit hearts. Am J Physiol 242:C288–C295

    PubMed  CAS  Google Scholar 

  22. Halestrap AP, McStay GP, Clarke SJ (2002) The permeability transition pore complex: another view. Biochimie (Paris) 84:153–166

    CAS  Google Scholar 

  23. Crompton M (2003) On the involvement of mitochondrial intermembrane junctional complexes in apoptosis. Curr Med Chem 10:1473–1484

    Article  PubMed  CAS  Google Scholar 

  24. Broekemeier KM, Dempsey ME, Pfeiffer DR (1989) Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria. J Biol Chem 264:7826–7830

    PubMed  CAS  Google Scholar 

  25. Zamzami N, Kroemer G (2001) The mitochondrion in apoptosis: how Pandora’s box opens. Nat Rev Mol Cell Biol 2:67–71

    Article  PubMed  CAS  Google Scholar 

  26. Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H, Inohara H, Kubo T, Tsujimoto Y (2005) Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature (Lond) 434:652–658

    Article  CAS  Google Scholar 

  27. Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, Robbins J, Molkentin JD (2005) Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature (Lond) 434:658–662

    Article  CAS  Google Scholar 

  28. Tsujimoto Y, Shimizu S (2007) Role of the mitochondrial membrane permeability transition in cell death. Apoptosis 12:835–840

    Article  PubMed  CAS  Google Scholar 

  29. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S (1998) A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature (Lond) 391: 43–50

    Article  CAS  Google Scholar 

  30. Sahara S, Aoto M, Eguchi Y, Imamoto N, Yoneda Y, Tsujimoto Y (1999) Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation. Nature (Lond) 401:168–173

    Article  CAS  Google Scholar 

  31. Maruyama R, Takemura G, Aoyama T, Hayakawa K, Koda M, Kawase Y, Qiu X, Ohno Y, Minatoguchi S, Miyata K, Fujiwara T, Fujiwara H (2001) Dynamic process of apoptosis in adult rat cardiomyocytes analyzed using 48-hour videomicroscopy and electron microscopy: beating and rate are associated with the apoptotic process. Am J Pathol 159:683–691

    PubMed  CAS  Google Scholar 

  32. Kerr JF, Winterford CM, Harmon BV (1994) Apoptosis. Its significance in cancer and cancer therapy. Cancer (Phila) 73: 2013–3026

    Article  CAS  Google Scholar 

  33. Galluzzi L, Maiuri MC, Vitale I, Zischka H, Castedo M, Zitvogel L, Kroemer G (2007) Cell death modalities: classification and pathophysiological implications. Cell Death Differ 14: 1237–1243

    Article  PubMed  CAS  Google Scholar 

  34. Tanaka M, Ito H, Adachi S, Akimoto H, Nishikawa T, Kasajima T, Marumo F, Hiroe M (1994) Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res 75:426–433

    PubMed  CAS  Google Scholar 

  35. Matsuoka R, Ogawa K, Yaoita H, Naganuma W, Maehara K, Maruyama Y (2002) Characteristics of death of neonatal rat cardiomyocytes following hypoxia or hypoxia-reoxygenation: the association of apoptosis and cell membrane disintegrity. Heart Vessels 16:241–248

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Cardiology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu, 501-1194, Japan

    Kazuko Goto, Genzou Takemura, Rumi Maruyama, Munehiro Nakagawa, Akiko Tsujimoto, Hiromitsu Kanamori, Longhu Li, Itta Kawamura, Tomonori Kawaguchi, Toshiaki Takeyama, Hisayoshi Fujiwara & Shinya Minatoguchi

Authors
  1. Kazuko Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Genzou Takemura
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rumi Maruyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Munehiro Nakagawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Akiko Tsujimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hiromitsu Kanamori
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Longhu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Itta Kawamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Tomonori Kawaguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Toshiaki Takeyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Hisayoshi Fujiwara
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Shinya Minatoguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Genzou Takemura.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Goto, K., Takemura, G., Maruyama, R. et al. Unique mode of cell death in freshly isolated adult rat ventricular cardiomyocytes exposed to hydrogen peroxide. Med Mol Morphol 42, 92–101 (2009). https://doi.org/10.1007/s00795-009-0439-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00795-009-0439-x

Key words

Search

Navigation