Inducible Nitric Oxide Synthase in Cardiac Adaptation to Ischemia

  • Ján Slezák
  • Jan Styk
  • Olga Slezakova
  • Gerd Wallukat
  • Peter Karczewski
  • Wolfgang Schulze
  • Igor B. Buchwalow
Part of the Progress in Experimental Cardiology book series (PREC, volume 6)


Cardiac ischaemia/reperfusion is well known as a disease of the myocytes, it is now clear that reactive oxygen species and their intermediates in conjunction with increased nitric oxide (NO) production during and after ischemia determine the second window of protection. However, regulatory mechanisms of NO modulation during ischaemia/reperfusion are poorly understood. Assuming that increased expression of NO synthases (NOS) could be involved in mediation of cardioprotective effect of preconditioning, we preconditioned cultured neonatal rat cardiocytes with hypoxia, 10εM norepinephrine, or 1εM H2O2. Immunohistochemical assay of an inducible NOS isoform (NOS2) revealed that norepinephrine, hypoxia and H2O2 enhanced the expression of NOS2 in cardiomyocytes, both along contractile fibers and in a cytoplasmic granular component, apparently in mitochondria. Inhibition of NOS2 expression or NOS2 activity abolished the protective preconditioning effect of hypoxia treatment. Hence, NO is implicated as a trigger in this model of preconditioning via activation of inducible NOS isoform. These data provide evidence that an increased NO generation due to induction of inducible NOS, following ischaemia/reperfusion, might be associated with increased myocardial tolerance to infarction.

Key words

nitric oxide synthase myocardium ischemia reperfusion preconditioning cardiac adaptation 


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  1. 1.
    Slezák J, Tribulová N, Pristačová J, Uhrík B, Thomas T, Khaper N, Kaul N, Singal PK. 1995. Hydrogen peroxide changes in ischemic and reperfused heart. Cytochemical and x-ray microanalysis. Amer J Pathol 147:772–781.Google Scholar
  2. 2.
    Pecháň I, Holomáň M, Záhorec R, Rendeková V, Gabauer I. 1996. Antioxidant vitamin and phosphocreatine as protective agents in cardiac surgery. Cor Europeum 5:69–73.Google Scholar
  3. 3.
    Tritto I, D’Andrea D, Eramo N, Scognamiglio A, De Simone C, Violante A, Esposito A, Chiariello M, Ambrosio G. 1997. Oxygen radicals can induce preconditioningin Rabbit Hearts. Circ Res 80:743–748.PubMedCrossRefGoogle Scholar
  4. 4.
    Sun JZ, Tang XL, Park SW, Qiu Y, Turrens JF, Bolli R. 1996. Evidence for an esssential role of reactive oxygen species in the genesis of late preconditioning against myocardial stunning in concious pigs. J Clin Invest 97:562–576.PubMedCrossRefGoogle Scholar
  5. 5.
    Yasmin W, Strynadka KD, Schulz R. 1997. Generation of peroxynitrite contributes to ischemia-reperfusion injury in isolated rat hearts. Cardiovasc Res 97:422–432.CrossRefGoogle Scholar
  6. 6.
    Buchwalow IB, Schulze W, Karczewski P, Kostic MM, Wallukat G, Morwinski R, Krause EG, Muller J, Paul M, Slezak J, Luft FC, Haller H. 2001. Inducible nitric oxide synthase in the myocard. Mol Cell Biochem 217:73–82.PubMedCrossRefGoogle Scholar
  7. 7.
    Buchwalow IB, Podzuweit T, Böcker W, Samoilova VE, Thomas S, Wellner M, Baba HA, Robenek H, Schnekenburger J, Lerch MM. 2002. Vascular smooth muscle and nitric oxide synthase. FASEB J Vol. 16, In Press.Google Scholar
  8. 8.
    Punkt K, Zaitsev S, Park JK, Wellner M, Buchwalow IB. 2001. Nitric oxide synthase isoforms I, III and protein kinase-Cq in skeletal muscle fibres of normal and Streptozotocin-induced diabetic rats with and without Ginkgo Biloba extract treatment. Histochem J 33:213–219.PubMedCrossRefGoogle Scholar
  9. 9.
    Punkt K, Zaitsev S, Wellner M, Schreiter T, Fitzl G, Buchwalow IB. 2002. Myopathy-dependent changes in activity of ATPase, SDH and GPDH and NOS expression in the different fibre types of hamster muscles. Acta histochem, In press.Google Scholar
  10. 10.
    Liu P, Hock CE, Nagele R, Wong PY. 1997. Formation of nitric oxide, superoxide, and peroxynitrite in myocardial ischemia-reperfusion injury in rats. Am J Physiol 97:H2327–H2336.Google Scholar
  11. 11.
    Bolli R. 2001. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol 33:1897–1918.PubMedCrossRefGoogle Scholar
  12. 12.
    Bolli R, Bhatti ZA, Tang XL, Qiu Y, Zhang Q, Guo Y, Jadoon AK. 1997. Evidence that late preconditioning against myocardial stunning in conscious rabbits is trigger by the generation of nitric oxide. Circ Res 97:42–52.CrossRefGoogle Scholar
  13. 13.
    Yan YS, Davani S, Chocron S, Kantelip B, Muret P, Kantelip JP. 2001. Effects of L-rginine administration before cardioplegic arrest on ischemia-reperfusion injury. Annals of Thoracic Surgery 72:1985–1990.PubMedCrossRefGoogle Scholar
  14. 14.
    Song W, Furman BL, Parratt JR. 1996. Delayed protection against ischaemia-induced ventricular arrhythmias and infarct size Hmitation by the prior administration of Escherichia coli endotoxin. Br J Pharmacol 96:2157–2163.CrossRefGoogle Scholar
  15. 15.
    Rajani V, Hussain Y, Bolla BS, De Guzman FQ, Montiague RR, Igic R, Rabito SF. 1997. Attenuation of epinephrine-induced dysrhythmias by bradykinin: role of nitric oxide and prostaglandins. Am J Cardiol 97:153A–157A.CrossRefGoogle Scholar
  16. 16.
    Parratt JR, Vegh A, Papp JG. 1995. Bradykinin as an endogenous myocardial protective substance with particular reference to ischemic preconditioning-a brief review of the evidence. Can J Physiol Pharmacol 95:837–842.CrossRefGoogle Scholar
  17. 17.
    Meng X, Ao L, Brown JM, Fullerton DA, Banerjee A, Harken AH. 1997. Nitric oxide synthase is not involved in cardiac contractile dysfunction in a rat model of endotoxemia without shock. Shock 97:111–118.CrossRefGoogle Scholar
  18. 18.
    Zhao L, Weber PA, Smith JR, Comerford ML, Elliott GT. 1997. Role of inducible nitric oxide synthase in pharmacological “preconditioning” with monophosphoryl lipid. AJ Mil Cell Cardiol 97:1567–1576.CrossRefGoogle Scholar
  19. 19.
    Imagawa JI, Baxter GF, Yellon DM. 1997. Genistein, a tyrosine kinase inhibitor, blocks the “second window of protection” 48 h after ischemic preconditioning in the rabbit. J Mol Cell Cardiol 97:1885–1893.CrossRefGoogle Scholar
  20. 20.
    Mei DA, Elliott GT, Gross GJ. 1996. KATP channels mediate late preconditioning against infarction produced by monophosphoryl lipid A. Am J Physiol 96:H2723–H2729.Google Scholar
  21. 21.
    Ostadal B, Ostadalova I, Kolar F, Parratt JR. 1996. Tolerance to ischemia and ischemic preconditioning in neonatal rat hearts. J Mol Cell Cardiol 28:A33 (Abstract).CrossRefGoogle Scholar
  22. 22.
    Kinugawa KI, Kohmoto O, Yao A, Serizawa T, Takahashi T. 1997. Cardiac inducible nitric oxide synthase negatively modulates myocardial function in cultured rat myocytes. Am J Physiol 97:H35–H47.Google Scholar
  23. 23.
    Banerjee A, Gamboni Robertson F, Mitchell MB, Rehring TF, Butler K, Cleveland J, Meldrum DR, Shapiro JI, Meng XZ. 1996. Stress-induced cardioadaptation reveals a code linking hormone receptors and spatial redistribution of PKC isoforms. Ann NY Acad Sei 96:226–239.CrossRefGoogle Scholar
  24. 24.
    Halle W, Wllenberger A. 1970. Differentiation and behaviour of isolated embryonic and neonatal heart cells in chemically defined medium. AmJ Cardiol 25:292–299.CrossRefGoogle Scholar
  25. 25.
    Wallukat G, Wollenberger A. 1993. Supersensitivity to -adrenoceptor stimulation evoked in cultured neonatal rat heart myocytes by L(+)-lactate and pyruvate. J Auton Pharmacol 13:1–14.PubMedCrossRefGoogle Scholar
  26. 26.
    Buchwalow IB, Schulze W, Kostic MM, Wallukat G, Morwinski R. 1997. Intracellular localization of inducible nitric oxide synthase in neonatal rat cardiomyocytes in culture. Acta Histochem 99:231–240.PubMedCrossRefGoogle Scholar
  27. 27.
    Ravingerová T, Barančík M, Pancza D, Styk J, Ziegelhoffer A, Schaper W, Slezak J. 1996. Contribution to the factors involved in the protective effect of ischemic preconditioning: the role of catecholamines and protein kinase C. Annals of the New York Academy of Sciences 793:43–54.PubMedCrossRefGoogle Scholar
  28. 28.
    Ravingerová T, Wu S, Pancza D, Džurba A, Ziegelhoffer A, Parratt J. 1997. Pretreatment with catecholamines can suppress severe ventricular arrhythmias in rats: relevance to ischaemic preconditioning. Exp Clin Cardiol 2:19–25.Google Scholar
  29. 29.
    Ravingerová T. 1995. Mimicking preconditioning with catecholamines. In: Myocardial Preconditioning. Ch. L. Wainwright, J.R. Parratt, eds. Austin: RG Landes Company, pp. 167–184.Google Scholar
  30. 30.
    Kobzik L, Stringer B, Balligand JL, Reid MB, Stamler S. 1995. Endothelial type nitric oxide synthase in skeletal muscle fibers: Mitochondrial relationships. Biochem Biophys Res Comm 221:375–381.CrossRefGoogle Scholar
  31. 31.
    Hare JM, Colucci WS. 1995. Role of nitric oxide in the regulation of myocardial function. Prog Cardiovasc Dis 38:155–156.PubMedCrossRefGoogle Scholar
  32. 32.
    Xie J, Wang Y, Summer WR, Greenberg SS. 1996. Ouabain enhances basal release of nitric oxide from carotid artery. Am J Med Sei 93:157–163.Google Scholar
  33. 33.
    Murphy ME, Brayden JE. 1995 Nitric oxide hyperpolarizes rabbit mesenteric arteries via ATP-sensitive channels. J Physiol (Lond) 486:47–58.Google Scholar
  34. 34.
    Moncada S, Higgs A. 1993. The L-arginine-nitric oxide pathway. N Engl J Med 329:2002–2012.PubMedCrossRefGoogle Scholar
  35. 35.
    Oyama J, Shimokawa H, Momii H, Cheng X, Fukuyama N, Arai Y, Egashira K, Nakazawa H, Takeshita A. 1998. Role of nitric oxide and peroxynitrite in the cytokine induced sustained myocardial dysfunction in dogs in vivo. J Clin Invest 101:2207–2214.PubMedCrossRefGoogle Scholar
  36. 36.
    Iwashina M, Shichiri M, Marumo F, Hirata Y. 1998. Transfection of inducible nitric oxide synthase geen causes apoptosis in vascular smooth muscle cells. Circulation 98:1212–1218.PubMedCrossRefGoogle Scholar
  37. 37.
    Pinsky D, Cai B, Yang X, Rodrigues C, Sciacca RR, Cannon PJ. 1995. The lethal effect of cytokine-induced nitric oxide on cardiac myocytes blocked by nitric oxide synthase antagonism or transforming growth factor b. J Clin Invest 95:677–685.PubMedCrossRefGoogle Scholar
  38. 38.
    Buchwalow IB, Schulze W, Slezak J, Luft FC, Haller H. 1998. Biochemical and physical factors involved in reducing nitric oxide bioactivity in hypertensive heart and kidney. Circulation 98:604.CrossRefGoogle Scholar
  39. 39.
    Napoli C, Ignarro LJ. 2001. Nitric oxide and atherosclerosis. Nitric Oxide-Biol Chem 5:88–97.CrossRefGoogle Scholar
  40. 40.
    Slezák J, Ravingerová T, Ziegelhoffer A, Barančk J, Styk J, Breier A, Tribulová N. 1996. Contribution to mechanisms involved in protective effect of ischemic preconditioning. J Mol Cell Cardiol 28:102.Google Scholar
  41. 41.
    Slezák J, Styk J, Buchwalow I, Tribulová N, Slezaková O, Singal PK, Schulze W. 1998. The role of H2O2 and nitric oxide synthase in adaptation of the heart to ischemia. Kutaisis Samedicino Jurnaly 1:52.Google Scholar
  42. 42.
    Xuan Y-T, Tang X-L, Qiu Y, Banerje S, Takano H, Han H, Bolli R. 1998. Direct evidence that inducible nitric oxide synthase mediates the late phase of ischemic preconditioning in conscious rabbits. Abstracts of 71st Scientific Sessions of American Heart Association Dallas Texas, November 8–11, p. 2194.Google Scholar
  43. 43.
    Igarashi J, Masashi N, Hoshida S, Yamashita N, Kosaka H, Hori M, Kuzuya T, Tada M. 1998. Inducible nitric oxide synthase augments injury elecited by oxidative stress in rat cardiac myocytes. Am J Physiol 274:C245–C252.PubMedGoogle Scholar
  44. 44.
    Opie LH. 1989. Reperfusion injury and ist pharmacological reduction. Circulation 80:1049–1062.PubMedCrossRefGoogle Scholar
  45. 45.
    Bolli R, Manchikalapudi S, Tang XL, Takano H, Qiu Y, Guo Y, Zhang Q, Jadoon AK. 1998. The protective effect of late preconditioning against myocardial stunning in conscious rabbits is mediated by nitric oxide synthase. Evidence that nitric oxide acts as a trigger and as a mediator of the late phase of ischemic preconditioning. Circ Res 81:1094–1107.CrossRefGoogle Scholar
  46. 46.
    Jones WK, Flaherty MP, Tang X-L, Qiu Y, Banerjee S, Bolli R. 1998. Ischemic preconditioning upregulates expression of iNOS mRNA in conscious rabbits via an NO-dependent mechanism. Abstracts of 71st Scientific Sessions of American Heart Association, Dallas, TX, November 8–11, p. 3257.Google Scholar
  47. 47.
    Guo Y, Jones WK, Tang X-L, Wu W-J, Qiu Y, Yang Z, Bao W, Flaherty M, Shirk G, Bolli R. 1998. Targeted disruption of iNOS gene abrogates the late phase of ischemic preconditioning. Abstracts of 71st Scientific Sessions of American Heart Association, Dallas, TX, November 8–11, p. 2191.Google Scholar
  48. 48.
    Slezák J, Buchwalow I, Styk J, Slezáková O, Schulze W, Wallukat G, Karczewski P. 1999. Inducible nitric oxide synthase mediates delays hypoxic resistance of preconditioned cultures of neonatal cardiomyocytes. J Mol Cell Cardiol 1:A70.Google Scholar
  49. 49.
    Slezák J, Buchwalow I, Styk J, Slezáková O, Schulze W, Wallukat G, Karczewski P. 2000. Inducible nitric oxide synthase mediates delays hypoxic resistance of preconditioned cultures of neonatal cardiomyocytes. International Symposium: Adaptation of the Heart, May 22–24.Google Scholar

Copyright information

© Springer Science+Business Media New York 2003

Authors and Affiliations

  • Ján Slezák
    • 1
  • Jan Styk
    • 1
  • Olga Slezakova
    • 1
  • Gerd Wallukat
    • 2
  • Peter Karczewski
    • 2
  • Wolfgang Schulze
    • 2
  • Igor B. Buchwalow
    • 2
    • 3
  1. 1.Institute for Heart Research, Slovak Academy of Sciences; Faculty of MedicineCommenius UniversityBratislavaSlovak Republic
  2. 2.Max-Delbruck-Center for Molecular MedicineBerlinGermany
  3. 3.Department of Medicine B and Central Ultrastructure Research Team, IZKFUniversity of MünsterMünsterGermany

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