Uncoupling of mitochondrial oxidative phosphorylation alters lipid peroxidation-derived free radical production but not recovery of postischemic rat hearts and post-hypoxic endothelial cells

  • I. E. Blasig
  • B. F. Dickens
  • W. B. Weglicki
  • J. H. Kramer
Part of the Developments in Molecular and Cellular Biochemistry book series (DMCB, volume 18)


The contribution of mitochondrial free radical production towards the initiation of lipid peroxidation (LPO) and functional injury in the post-ischemic heart is unclear. Using the isolated rat heart model, the effects of the uncoupler of mitochondrial oxidative phosphorylation dinitrophenol (DNP, 50 µM final) on post-ischemic lipid peroxidation-derived free radical production and functional recovery were assessed. Hearts were subjected to 30 min total global ischemia followed by 15 min of reperfusion in the presence of DNP. As expected, DNP enhanced oxygen consumption before (11.3 ± 0.9 jimol/min, p < 0.001) and during reperfusion (at 10 min: 7.9 ± 0.7 µumol/min), compared to the heart with control treatment (8.2 ± 0.5 and 6.7 ± 0.3, respectively). This effect was only associated with a higher incidence of ventricular tachycardia during reperfusion (80 vs. 50% for control treatment, p < 0.05). Electron spin resonance spectroscopy (ESR) and spin trapping with α-phenyl-tert-butylnitrone (PBN, 3 mM final) were used to monitor free radical generation during reperfusion. The vascular concentration of PBN-radical adducts (untreated: 6.4 ± 1.0 nM, at 10 min) decreased in the presence of DNP (1.7 ± 0.4 nM, p < 0.01). The radical concentration inversely correlated with myocardial oxygen consumption. Total liberation of free radical adducts during the initial 10 min of reperfusion was reduced by DNP (0.59 ± 0.09 nmol, p < 0.01) compared to the respective control treatment (1.26 ± 0.16 nmol). Similar effects, prevention of PBN adduct formation and unchanged viability in the presence of DNP, were obtained with endothelial cells during post-hypoxic reoxygenation. Since inhibition of mitochondrial phosphorylation can inhibit the formation of LPO-derived free radicals after an ischemic/hypoxic interval, mitochondria may represent an important source of free radicals capable of initiating lipid peroxidative injury during reperfusion/reoxygenation. (Mol Cell Biochem 160/161:167–177, 1996)

Key words

ESR spin trapping lipid radicals mitochondrial uncoupling oxygen consumption myocardial reperfusion endothelial cell reoxygenation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Blasig IE, Shuter S, Garlick P, Slater TF: Relative time-profiles for free radical trapping, coronary flow, enzyme leakage, arrhythmias and function during myocardial reperfusion. Free Rad Biol Med 16: 35–41, 1994PubMedCrossRefGoogle Scholar
  2. 2.
    Damerau W, Ibel J, Thürich T, Assadnazari A, Zimmer G: Generation of free radicals in Langendorff and working hearts during normoxia, hypoxia, and reoxygenation. Basic Res Cardiol 88: 141–119, 1993PubMedGoogle Scholar
  3. 3.
    Kumar C, Okuda M, Ikai I, Chance B: Luminol enhanced chemiluminescence of the perfused rat heart during ischemia and reperfusion. FEBS Lett 272: 121–124, 1990PubMedCrossRefGoogle Scholar
  4. 4.
    Mak IT, Kramer JH, Weglicki WB: Antioxidant properties of active and inactive isomers of nicardipin in cardiac membranes, endothelial cells, and perfused rat hearts. Coronary Artery Disease 3: 1095–1103, 1992CrossRefGoogle Scholar
  5. 5.
    Nohl H, Stolze K, Napetschnig S, Ishikawa T: Is oxidative stress primarily involved in reperfusion injury of the ischemic heart? Free Rad Biol Med 11:581–588, 1991PubMedCrossRefGoogle Scholar
  6. 6.
    Pietri S, Culcasi M, Stella L, Cozzone PJ: Ascorbyl free radical as a reliable indicator of free radical-mediated myocardial ischemic and postischemic injury. A real-time continuous flow ESR study. Eur J Biochem 193:845–854, 1990PubMedCrossRefGoogle Scholar
  7. 7.
    Powell RS, Hall D: Use of salicylate as a probe for -OH formation in isolated ischemic rat hearts. Free Rad Biol Med 9: 133–141, 1990PubMedCrossRefGoogle Scholar
  8. 8.
    Bolli R, Jeroudi MO, Patel BS, Aruoma Oi, Halliwell B, Lai EK, McCay PB: Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion: Evidence that myocardial ‘stunning’ is manifestation of reperfusion injury. Circ Res 65: 607–622, 1989PubMedGoogle Scholar
  9. 9.
    Paraidathanthiu T, DeGroot H, Kehrer JP: Production of reactive oxygen by mitochondria from normoxic and hypoxic rat heart tissue. Free Rad Biol Med 13: 289–297, 1992CrossRefGoogle Scholar
  10. 10.
    Rashba YE, Vartanyan LS, Bayder LM, Krinitskaya LA: Quantitative estimation of the rate of superoxide radical generation in mitochondrial membranes by ESR-technique. Biofizika USSR 34: 57–62, 1989Google Scholar
  11. 11.
    Baker JE, Kalyanaraman B: Ischemia-induced changes in myocardial paramagnetic metabolites: Implications for intracellular oxy-radical generation. FEBS Lett 244: 311–314, 1989PubMedCrossRefGoogle Scholar
  12. 12.
    Ambrosio G, Zweier JL, Duilio C, Santoro G, Elia PP, Tritto I, Chiariello M, Flaherty JT: Inhibition of mitochondrial respiration and lipid peroxidation in reperfused hearts. Circulation 80(4): 11–31 (Abstract), 1989Google Scholar
  13. 13.
    Prasad MR, Engelman RM, Jones RM, Das DK: Effects of oxyradicals on oxymyoglobin. Deoxygenation, haem removal and iron release. Biochem J 263: 731–736, 1989PubMedGoogle Scholar
  14. 14.
    Zweier JL, Kuppusami P, Williams R, Rayburn BK, Smith D, Weisfeldt ML, Flaherty JT: Measurement and characterization of postischemic free radical generation in the isolated perfused heart. J Biol Chem 264: 18890–18895, 1989PubMedGoogle Scholar
  15. 15.
    Ueta H, Ogura R, Sugiyama M, Kagiyama A, Shin G: O2-spin trapping on cardiac submitochondrial particles isolated from ischemic and non-ischemic myocardium. J Mol Cell Cardiol 22: 893–899, 1990PubMedCrossRefGoogle Scholar
  16. 16.
    Otani H, Tanaka H, Inoue T, Umemoto M, Omoto K, Tanaka K, Sato T, Osako T, Masuda A, Nonoyama A, Kagawa T: In vitro study on contribution of oxidative metabolism of isolated rabbit heart mitochondria to myocardial reperfusion injury. Circulation Res 55: 168–175, 1984PubMedGoogle Scholar
  17. 17.
    Davies MJ, Garlick PB, Slater TF, Hearse DJ: Detection of free radical production during myocardial reperfusion injury using electron spin resonance spin trapping. In: C. Rice-Evans and T. Dormandy (eds). Free radicals: Chemistry, pathology and medicine. Richelieu Press, London, 1988, pp 303–319Google Scholar
  18. 18.
    Atiq Khalid M, Samara ZO, Ashraf M: Association of endogenously produced OH radicals with anoxia reoxygenation injury in isolated rat myocytes. FASEB J 5(5): A1282 (Abstract), 1991Google Scholar
  19. 19.
    Henry TD, Archer SL, Nelson D, Weir EK, From AHL: Postischemic oxygen radical production varies with duration of ischemia. Am J Physiol 264: H1478–H1484, 1993PubMedGoogle Scholar
  20. 20.
    Kramer JH, Misik V, Weglicki WB: Lipid peroxidation-derived free radical production and postischemic myocardial reperfusion injury. Ann N Y Acad Sci 723: 180–196, 1994PubMedCrossRefGoogle Scholar
  21. 21.
    Freedman AM, Kramer JH, Mak IT, Cassidy MM, Weglicki WB: Propranolol preserves ultrastructure in adult cardiocytes exposed to an-oxia/reoxygenation: A morphometric analysis. Free Rad Biol Med 11: 197–206, 1991PubMedCrossRefGoogle Scholar
  22. 22.
    Kramer JH, Mak IT, Freedman AM, Weglicki WB: Propranolol reduces anoxia/reoxygenation-mediated injury of adult myocytes through an anti-radical mechanism. J Mol Cell Cardiol 23: 1231–1244, 1991PubMedCrossRefGoogle Scholar
  23. 23.
    Dickens BF, Weglicki WB, Li Y-S, Mak IT: Magnesium deficiency in vitro enhances free radical-induced intracellular oxidation and cytotoxicity in endothelial cells. FEBS Lett 311: 187–191, 1992PubMedCrossRefGoogle Scholar
  24. 24.
    Kramer JH, Dickens BF, Misik V, Weglicki WB: Phospholipid hydroperoxides are precursors of lipid alkoxyl radicals produced from anoxia/reoxygenated endothelial cells. J Mol Cell Cardiol 27: 371–381, 1995PubMedCrossRefGoogle Scholar
  25. 25.
    Blasig IE, Schönheit K: Improvement of heart function by the spin trap alpha-phenyl-t-butyl nitrone possibly caused by its nitroxyl radical adduct. In. K.J.A. Davies (ed.) Oxidative damage and repair: Chemical, biological and medical aspects. Pergamon Press, New York, 1992, pp 421–425Google Scholar
  26. 26.
    Li X-Y, Sun J-Z, Bradamante S, Piccinini F, Bolli R: Effects of the spin trap alpha-phenyl N-tert-butyl nitrone on myocardial function and flow: a dose-response study in the open chest dog and in the isolated rat heart. Free Rad Biol Med 14: 277–285, 1993PubMedCrossRefGoogle Scholar
  27. 27.
    Konorev EA, Baker JE, Joseph J, Kalyanaraman B: Vasodilatory and toxic effects of spin traps on aerobic cardiac function. Free Rad Biol Med 14: 127–137, 1993PubMedCrossRefGoogle Scholar
  28. 28.
    Pissarek M, Jänichen F, Blasig IB, Haseloff RF, Keller T, Tapp E, Krause E-G: Cardioprotective potency of the radical scavenger S-2-(3 aminopropionylamino) ethylphosphorothioic acid in the post-ischemic rat heart. Mol Cell Biochem 145: 121–129, 1995PubMedCrossRefGoogle Scholar
  29. 29.
    Hearse DJ, Tosaki A: Free radicals and reperfusion-induced arrhythmias: protection by spin trap agent PBN in the rat heart. Circ Res 60:375–383, 1987PubMedGoogle Scholar
  30. 30.
    Tosaki A, Haseloff RF, Hellegouarch A, Schonheit K, Martin VV, Das DK, Blasig IE: Does the antiarrhythmic effect of DMPO originate from its oxygen radical trapping property or the structure of the molecule itself? Basic Res Cardiol 87: 536–547, 1992PubMedCrossRefGoogle Scholar
  31. 31.
    Biasig IE, Volodarski LB, Tosaki: A. Nitrone spin trap compounds. Mode of cardioprotective action. Pharm Pharmacol Lett 3: 135–138, 1993Google Scholar
  32. 32.
    Bradamante S, Monti E, Paracchini L, Lazzarini E, Piccini F: Protective activity of the spin trap tert-butyl-alpha-phenyl nitrone (PBN) in reperfused rat heart. J Mol Cell Cardiol 24: 375–386,1992PubMedCrossRefGoogle Scholar
  33. 33.
    Cova D, De Angelis L, Monti E, Piccinini F: Subcellular distribution of two spin trapping agents in rat heart: Possible explanation for their different protective effects against doxorubicin-induced cardiotoxicity Free Rad Res Commun 15: 353–360, 1991CrossRefGoogle Scholar
  34. 34.
    Loschen G, Azzi A, Flohe L: Mitochondrial H2O2 formation and energy conservation. In. L. Flohe (ed.) Glutathione. Thieme-Verlag, Stuttgart, 1974, pp 228–236Google Scholar
  35. 35.
    Blasig IE, Ebert B, Hennig C, Pali T, Tosaki A: Inverse relationship between ESR spin trapping of oxyradicals and degree of functional recovery during myocardial reperfusion in isolated working rat heart. Cardiovasc Res 24: 263–270, 1990PubMedCrossRefGoogle Scholar
  36. 36.
    Kloner RA, Przyklenk K, Whittaker P: Deleterious effects of oxygen radicals in ischemia/reperfusion. Resolved and unresolved issues. Circulation 80: 1115–1127, 1989PubMedCrossRefGoogle Scholar
  37. 37.
    Zweier JL, Kuppusamy P Lutty GA: Measurement of endothelial cell free radical generation: Evidence for a central mechanism of free radical injury in postischemic tissues. Proc Natl Acad Sci USA 85: 4046–4050, 1988PubMedCrossRefGoogle Scholar
  38. 38.
    Dickens BF, Weglicki WB, Li Y-S, Kramer JH: Rapid alkoxyl radical production during endothelial cell hypoxia/reoxygenation. FASEB J 5(5): A1283 (Abstract), 1991Google Scholar
  39. 39.
    Blasig IE, Steinschneider AY, Lakomkin VL, Ledenev AN, Korchazkina OV, Ruuge EK: ESR spin trapping and NMR spectroscopy of the same heart shows correlation between energy depression and radical formation during post-ischemic reperfusion. FEBS Lett 267:29–32, 1990PubMedCrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • I. E. Blasig
    • 1
  • B. F. Dickens
    • 2
  • W. B. Weglicki
    • 2
  • J. H. Kramer
    • 2
  1. 1.Forschungsgruppe Radikal- und Zellbiochemie, Forschungsinstitut für Molekulare PharmakologieForschungsverbund Berlin e.V.BerlinGermany
  2. 2.Departments of Medicine and Physiology, Division of Experimental MedicineThe George Washington University Medical CenterUSA

Personalised recommendations