Skip to main content

Advertisement

SpringerLink
Log in

Morphological alterations induced by doxorubicin on H9c2 myoblasts: nuclear, mitochondrial, and cytoskeletal targets

  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

Abstract

Doxorubicin (Dox) is a very potent antineoplastic agent used against several types of cancer, despite a cumulative cardiomyopathy that reduces the therapeutic index for treatment. H9c2 myoblast cells have been used as an in vitro model to study biochemical alterations induced by Dox treatment on cardiomyocyte cells. Despite the extensive work already published, few data are available regarding morphological alterations of H9c2 cells during Dox treatment. The purpose of the present work was to evaluate Dox-induced morphological alterations in H9c2 myoblasts, focusing especially on the nuclei, mitochondria, and structural fibrous proteins. Treatment of H9c2 cell with low concentrations of Dox causes alterations in fibrous structural proteins including the nuclear lamina and sarcomeric cardiac myosin, as well as mitochondrial depolarization and fragmentation, membrane blebbing with cell shape changes, and phosphatidylserine externalization. For higher Dox concentrations, more profound alterations are evident, including nuclear swelling with disruption of nuclear membrane structure, mitochondrial swelling, and extensive cytoplasm vacuolization. The results obtained indicate that Dox causes morphological alterations in mitochondrial, nuclear, and fibrous protein structures in H9c2 cells, which are dependent on the drug concentration. Data obtained with the present study allow for a better characterization of the effects of Dox on H9c2 myoblasts, used as a model to study Dox-induced cardiotoxicity. The results obtained also provide new and previously unknown targets that can contribute to understand the mechanisms involved in the cardiotoxicity of Dox.

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

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

DIC:

differential interference contrast

Dox:

doxorubicin

LDH:

lactate dehydrogenase

PS:

phosphatidylserine

ROS:

reactive oxygen species

TMRM:

tetramethylrhodamine methyl ester

References

  • Ben Yaou R, Gueneau L, Demay L, et al. Heart involvement in lamin A/C related diseases. Arch Mal Coeur Vaiss. 2006;99(9):848–55.

    PubMed  CAS  Google Scholar 

  • Bergmeyer HU, Bernt E. Lactate-dehydrogenase, UV-assay with pyruvate and NADH. In: Bergmeyer HU, editor. Methods of enzymatic analysis (vol. 2). New York: Academic; 1974.

    Google Scholar 

  • Berthiaume JM, Wallace KB. Adriamycin-induced oxidative mitochondrial cardiotoxicity. Cell Biol Toxicol. 2007;23(1):15–25.

    Article  PubMed  CAS  Google Scholar 

  • Box VG. The intercalation of DNA double helices with doxorubicin and nagalomycin. J Mol Graph Model. 2007;26(1):14–9.

    Article  PubMed  CAS  Google Scholar 

  • Bridger JM, Foeger N, Kill IR, et al. The nuclear lamina. Both a structural framework and a platform for genome organization. FEBS J. 2007;274(6):1354–61.

    Article  PubMed  CAS  Google Scholar 

  • Brostrom MA, Reilly BA, Wilson FJ, et al. Vasopressin-induced hypertrophy in H9c2 heart-derived myocytes. Int J Biochem Cell Biol. 2000;32(9):993–1006.

    Article  PubMed  CAS  Google Scholar 

  • Chua CC, Liu X, Gao J, et al. Multiple actions of pifithrin-alpha on doxorubicin-induced apoptosis in rat myoblastic H9c2 cells. Am J Physiol Heart Circ Physiol. 2006;290(6):H2606–13.

    Article  PubMed  CAS  Google Scholar 

  • Croft DR, Coleman ML, Li S, et al. Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration. J Cell Biol. 2005;168(2):245–55.

    Article  PubMed  CAS  Google Scholar 

  • Cutts SM, Nudelman A, Rephaeli A, et al. The power and potential of doxorubicin-DNA adducts. IUBMB Life. 2005;57(2):73–81.

    Article  PubMed  CAS  Google Scholar 

  • Cutts SM, Parsons PG, Sturm RA, et al. Adriamycin-induced DNA adducts inhibit the DNA interactions of transcription factors and RNA polymerase. J Biol Chem. 1996;271(10):5422–9.

    Article  PubMed  CAS  Google Scholar 

  • Davies KJ, Doroshow JH. Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase. J Biol Chem. 1986;261(7):3060–7.

    PubMed  CAS  Google Scholar 

  • Dechat T, Shimi T, Adam SA, et al. Alterations in mitosis and cell cycle progression caused by a mutant lamin A known to accelerate human aging. Proc Natl Acad Sci U S A. 2007;104(12):4955–60.

    Article  PubMed  CAS  Google Scholar 

  • Dudnakova TV, Lakomkin VL, Tsyplenkova VG, et al. Alterations in myocardial cytoskeletal and regulatory protein expression following a single doxorubicin injection. J Cardiovasc Pharmacol. 2003;41(5):788–94.

    Article  PubMed  CAS  Google Scholar 

  • Fadeel B, Kagan VE. Apoptosis and macrophage clearance of neutrophils: regulation by reactive oxygen species. Redox Rep. 2003;8(3):143–50.

    Article  PubMed  CAS  Google Scholar 

  • Frost BM, Eksborg S, Bjork O, et al. Pharmacokinetics of doxorubicin in children with acute lymphoblastic leukemia: multi-institutional collaborative study. Med Pediatr Oncol. 2002;38(5):329–37.

    Article  PubMed  Google Scholar 

  • Gabrielson K, Bedja D, Pin S, et al. Heat shock protein 90 and ErbB2 in the cardiac response to doxorubicin injury. Cancer Res. 2007;67(4):1436–41.

    Article  PubMed  CAS  Google Scholar 

  • Garrido C, Galluzzi L, Brunet M, et al. Mechanisms of cytochrome c release from mitochondria. Cell Death Differ. 2006;13(9):1423–33.

    Article  PubMed  CAS  Google Scholar 

  • Grimmond HE, Beerman T. Alteration of chromatin structure induced by the binding of adriamycin, daunorubicin and ethidium bromide. Biochem Pharmacol. 1982;31(21):3379–86.

    Article  PubMed  CAS  Google Scholar 

  • Hescheler J, Meyer R, Plant S, et al. Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ Res. 1991;69(6):1476–86.

    PubMed  CAS  Google Scholar 

  • Iwasaki T, Suzuki T. Ultrastructural alterations of the myocardium induced by doxorubicin. A scanning electron microscopic study. Virchows Arch B Cell Pathol Incl Mol Pathol 1991;60(1):35–9.

    Article  PubMed  CAS  Google Scholar 

  • Jang YM, Kendaiah S, Drew B, et al. Doxorubicin treatment in vivo activates caspase-12 mediated cardiac apoptosis in both male and female rats. FEBS Lett. 2004;577(3):483–90.

    Article  PubMed  CAS  Google Scholar 

  • Jones SM, Kirby MS, Harding SE, et al. Adriamycin cardiomyopathy in the rabbit: alterations in contractile proteins and myocyte function. Cardiovasc Res. 1990;24(10):834–42.

    Article  PubMed  CAS  Google Scholar 

  • Joyeux M, Godin-Ribuot D, Faure P, et al. Heat stress protects against electrophysiological damages induced by acute doxorubicin exposure in isolated rat hearts. Cardiovasc Drugs Ther. 2001;15(3):219–24.

    Article  PubMed  CAS  Google Scholar 

  • Kalyanaraman B, Joseph J, Kalivendi S, et al. Doxorubicin-induced apoptosis: implications in cardiotoxicity. Mol Cell Biochem 2002;234–5(1–2):119–24.

    Article  Google Scholar 

  • Kim DS, Kim HR, Woo ER, et al. Inhibitory effects of rosmarinic acid on adriamycin-induced apoptosis in H9c2 cardiac muscle cells by inhibiting reactive oxygen species and the activations of c-Jun N-terminal kinase and extracellular signal-regulated kinase. Biochem Pharmacol. 2005;70(7):1066–78.

    Article  PubMed  CAS  Google Scholar 

  • Kim DS, Woo ER, Chae SW, et al. Plantainoside D protects adriamycin-induced apoptosis in H9c2 cardiac muscle cells via the inhibition of ROS generation and NF-kappaB activation. Life Sci. 2007;80(4):314–23.

    Article  PubMed  CAS  Google Scholar 

  • Kim JE, Han BS, Choi WS, et al. Temporospatial sequence of cellular events associated with etoposide-induced neuronal cell death: role of antiapoptotic protein Bcl-X(L). J Neurosci Res. 2001;66(6):1074–82.

    Article  PubMed  CAS  Google Scholar 

  • Kimes BW, Brandt BL. Properties of a clonal muscle cell line from rat heart. Exp Cell Res. 1976;98(2):367–81.

    Article  PubMed  CAS  Google Scholar 

  • L’Ecuyer T, Horenstein MS, Thomas R, et al. Anthracycline-induced cardiac injury using a cardiac cell line: potential for gene therapy studies. Mol Genet Metab. 2001;74(3):370–9.

    Article  PubMed  CAS  Google Scholar 

  • L’Ecuyer T, Sanjeev S, Thomas R, et al. DNA damage is an early event in doxorubicin-induced cardiac myocyte death. Am J Physiol Heart Circ Physiol. 2006;291(3):H1273–80.

    Article  PubMed  CAS  Google Scholar 

  • Li K, Sung RY, Huang WZ, et al. Thrombopoietin protects against in vitro and in vivo cardiotoxicity induced by doxorubicin. Circulation. 2006;113(18):2211–20.

    Article  PubMed  CAS  Google Scholar 

  • Lieberman J, Fan Z. Nuclear war: the granzyme A-bomb. Curr Opin Immunol 2003;15(5):553–9.

    Article  PubMed  CAS  Google Scholar 

  • Lushnikova EL, Klinnikova MG, Molodykh OP, et al. Morphological manifestations of heart remodeling in anthracycline-induced dilated cardiomyopathy. Bull Exp Biol Med. 2004;138(6):607–12.

    Article  PubMed  CAS  Google Scholar 

  • Menna P, Salvatorelli E, Minotti G. Doxorubicin degradation in cardiomyocytes. J Pharmacol Exp Ther. 2007;322(1):408–19.

    Article  PubMed  CAS  Google Scholar 

  • Merten KE, Jiang Y, Feng W, et al. Calcineurin activation is not necessary for doxorubicin-induced hypertrophy in H9c2 embryonic rat cardiac cells: involvement of the phosphoinositide 3-kinase-Akt pathway. J Pharmacol Exp Ther. 2006;319(2):934–40.

    Article  PubMed  CAS  Google Scholar 

  • Muntoni F. Cardiomyopathy in muscular dystrophies. Curr Opin Neurol. 2003;16(5):577–83.

    Article  PubMed  Google Scholar 

  • Oliveira PJ, Wallace KB. Depletion of adenine nucleotide translocator protein in heart mitochondria from doxorubicin-treated rats–relevance for mitochondrial dysfunction. Toxicology 2006;220(2–3):160–8.

    Article  PubMed  CAS  Google Scholar 

  • Palle J, Frost BM, Peterson C, et al. Doxorubicin pharmacokinetics is correlated to the effect of induction therapy in children with acute myeloid leukemia. Anticancer Drugs. 2006;17(4):385–92.

    Article  PubMed  CAS  Google Scholar 

  • Park C, So HS, Shin CH, et al. Quercetin protects the hydrogen peroxide-induced apoptosis via inhibition of mitochondrial dysfunction in H9c2 cardiomyoblast cells. Biochem Pharmacol. 2003;66(7):1287–95.

    Article  PubMed  CAS  Google Scholar 

  • Perrot A, Sigusch HH, Nagele H, et al. Genetic and phenotypic analysis of dilated cardiomyopathy with conduction system disease: demand for strategies in the management of presymptomatic lamin A/C mutant carriers. Eur J Heart Fail. 2006;8(5):484–93.

    Article  PubMed  CAS  Google Scholar 

  • Potemski P, Polakowski P, Wiktorowska-Owczarek AK, et al. Amifostine improves hemodynamic parameters in doxorubicin-pretreated rabbits. Pharmacol Rep. 2006;58(6):966–72.

    PubMed  CAS  Google Scholar 

  • Prasad S, Soldatenkov VA, Srinivasarao G, et al. Intermediate filament proteins during carcinogenesis and apoptosis (Review). Int J Oncol. 1999;14(3):563–70.

    PubMed  CAS  Google Scholar 

  • Rasola A, Bernardi P. The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis. Apoptosis. 2007;12(5):815–33.

    Article  PubMed  CAS  Google Scholar 

  • Santos DL, Moreno AJ, Leino RL, et al. Carvedilol protects against doxorubicin-induced mitochondrial cardiomyopathy. Toxicol Appl Pharmacol. 2002;185(3):218–27.

    Article  PubMed  CAS  Google Scholar 

  • Schafer ZT, Kornbluth S. The apoptosome: physiological, developmental, and pathological modes of regulation. Dev Cell. 2006;10(5):549–61.

    Article  PubMed  CAS  Google Scholar 

  • Shimasaki H, Ueta N, Mowri HO, et al. Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes. Biochim Biophys Acta. 1984;792(2):123–9.

    PubMed  CAS  Google Scholar 

  • Solem LE, Heller LJ, Wallace KB. Dose-dependent increase in sensitivity to calcium-induced mitochondrial dysfunction and cardiomyocyte cell injury by doxorubicin. J Mol Cell Cardiol. 1996;28(5):1023–32.

    Article  PubMed  CAS  Google Scholar 

  • Spallarossa P, Garibaldi S, Altieri P, et al. Carvedilol prevents doxorubicin-induced free radical release and apoptosis in cardiomyocytes in vitro. J Mol Cell Cardiol. 2004;37(4):837–46.

    Article  PubMed  CAS  Google Scholar 

  • Takemura G, Fujiwara H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog Cardiovasc Dis. 2007;49(5):330–52.

    Article  PubMed  CAS  Google Scholar 

  • Taylor AL, Bulkley BH. Acute adriamycin cardiotoxicity: morphologic alterations in isolated perfused rabbit heart. Lab Invest. 1982;47(5):459–64.

    PubMed  CAS  Google Scholar 

  • Terman A, Brunk UT. Autophagy in cardiac myocyte homeostasis, aging, and pathology. Cardiovasc Res. 2005;68(3):355–65.

    Article  PubMed  CAS  Google Scholar 

  • Ueno M, Kakinuma Y, Yuhki K, et al. Doxorubicin induces apoptosis by activation of caspase-3 in cultured cardiomyocytes in vitro and rat cardiac ventricles in vivo. J Pharmacol Sci. 2006;101(2):151–8.

    Article  PubMed  CAS  Google Scholar 

  • Unverferth DV, Magorien RD, Unverferth BP, et al. Human myocardial morphologic and functional changes in the first 24 hours after doxorubicin administration. Cancer Treat Rep. 1981;65(11–12):1093–7.

    PubMed  CAS  Google Scholar 

  • Villani F, Galimberti M, Monti E, et al. Effect of ICRF-187 pretreatment against doxorubicin-induced delayed cardiotoxicity in the rat. Toxicol Appl Pharmacol. 1990;102(2):292–9.

    Article  PubMed  CAS  Google Scholar 

  • von Harsdorf R, Li PF, Dietz R. Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation. 1999;99(22):2934–41.

    Google Scholar 

  • Wallace KB. Doxorubicin-induced cardiac mitochondrionopathy. Pharmacol Toxicol. 2003;93(3):105–15.

    Article  PubMed  CAS  Google Scholar 

  • Wattanapitayakul SK, Chularojmontri L, Herunsalee A, et al. Screening of antioxidants from medicinal plants for cardioprotective effect against doxorubicin toxicity. Basic Clin Pharmacol Toxicol. 2005;96(1):80–7.

    Article  PubMed  CAS  Google Scholar 

  • Wayman N, McDonald MC, Thompson AS, et al. 5-Aminoisoquinolinone, a potent inhibitor of poly (adenosine 5'-diphosphate ribose) polymerase, reduces myocardial infarct size. Eur J Pharmacol. 2001;430(1):93–100.

    Article  PubMed  CAS  Google Scholar 

  • Xu MF, Tang PL, Qian ZM, et al. Effects by doxorubicin on the myocardium are mediated by oxygen free radicals. Life Sci. 2001;68(8):889–901.

    Article  PubMed  CAS  Google Scholar 

  • Yan L, Vatner DE, Kim SJ, et al. Autophagy in chronically ischemic myocardium. Proc Natl Acad Sci U S A. 2005;102(39):13807–12.

    Article  PubMed  CAS  Google Scholar 

  • Yue TL, Wang C, Romanic AM, et al. Staurosporine-induced apoptosis in cardiomyocytes: a potential role of caspase-3. J Mol Cell Cardiol. 1998;30(3):495–507.

    Article  PubMed  CAS  Google Scholar 

  • Zhou S, Palmeira CM, Wallace KB. Doxorubicin-induced persistent oxidative stress to cardiac myocytes. Toxicol Lett. 2001;121(3):151–7.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

This work was supported by NIH grant HL 58016 to K.W., by FCT grant PTDC-SAU-OSM-64084-2006 to P.J.O. and by fellowships SFRH/BD/10251/2002 and SFRH/BPD/8359/2002 from the Portuguese Foundation for Science and Technology to V.S. and P.J.O., respectively.

Author information

Authors and Affiliations

  1. Center for Neurosciences and Cellular Biology, Department of Zoology, University of Coimbra, 3004-517, Coimbra, Portugal

    Vilma A. Sardão & Paulo J. Oliveira

  2. Department of Anatomy, Microbiology and Pathology, University of Minnesota Medical School, Duluth, USA

    Jon Holy

  3. Center for Neurosciences and Cellular Biology, Department of Biochemistry, Medical School, University of Coimbra, Coimbra, Portugal

    Catarina R. Oliveira

  4. Department of Biochemistry and Molecular Biology, University of Minnesota Medical School, Duluth, USA

    Kendall B. Wallace

Authors
  1. Vilma A. Sardão
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paulo J. Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jon Holy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Catarina R. Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kendall B. Wallace
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vilma A. Sardão.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sardão, V.A., Oliveira, P.J., Holy, J. et al. Morphological alterations induced by doxorubicin on H9c2 myoblasts: nuclear, mitochondrial, and cytoskeletal targets. Cell Biol Toxicol 25, 227–243 (2009). https://doi.org/10.1007/s10565-008-9070-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10565-008-9070-1

Keywords

Search

Navigation