Molecular and Cellular Biochemistry

, Volume 337, Issue 1–2, pp 25–38

Opening of the mitoKATP channel and decoupling of mitochondrial complex II and III contribute to the suppression of myocardial reperfusion hyperoxygenation

  • Bin Liu
  • Xuehai Zhu
  • Chwen-Lih Chen
  • Keli Hu
  • Harold M. Swartz
  • Yeong-Renn Chen
  • Guanglong He


Diazoxide, a mitochondrial ATP-sensitive potassium (mitoKATP) channel opener, protects the heart from ischemia–reperfusion injury. Diazoxide also inhibits mitochondrial complex II-dependent respiration in addition to its preconditioning effect. However, there are no prior studies of the role of diazoxide on post-ischemic myocardial oxygenation. In the current study, we determined the effect of diazoxide on the suppression of post-ischemic myocardial tissue hyperoxygenation in vivo, superoxide (O2−•) generation in isolated mitochondria, and impairment of the interaction between complex II and complex III in purified mitochondrial proteins. It was observed that diazoxide totally suppressed the post-ischemic myocardial hyperoxygenation. With succinate but not glutamate/malate as the substrate, diazoxide significantly increased ubisemiquinone-dependent O2−• generation, which was not blocked by 5-HD and glibenclamide. Using a model system, the super complex of succinate-cytochrome c reductase (SCR) hosting complex II and complex III, we also observed that diazoxide impaired complex II and its interaction with complex III with no effect on complex III. UV–visible spectral analysis revealed that diazoxide decreased succinate-mediated ferricytochrome b reduction in SCR. In conclusion, our results demonstrated that diazoxide suppressed the in vivo post-ischemic myocardial hyperoxygenation through opening the mitoKATP channel and ubisemiquinone-dependent O2−• generation via inhibiting mitochondrial complex II-dependent respiration.


Mitochondria Diazoxide Superoxide Ischemia reperfusion Oxygenation 





5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide


5,5-Dimethyl-1-pyrroline N-oxide


Diethylenetriaminepentaacetic acid


Electron paramagnetic resonance


Carbonyl cyanide-p-tri-fluromethoxyphenyl-hydrazone






Ischemic preconditioning


Mitochondrial ATP-sensitive potassium channel


3-Nitropropionic acid




Hydroxyl radical

\( P_{{{\text{O}}_{ 2} }} \)

Tissue oxygen tension


Pharmacological preconditioning








Ubiquinol cytochrome c reductase or complex III




Ubisemiquinone at the QO site


Reactive oxygen species


Succinate-cytochrome c reductase or super complex containing complex II and complex III


Sub-mitochondrial particles


Superoxide dismutase


Succinate ubiquinone reductase or complex II


Tetraethyl ammonium


  1. 1.
    Murry CE, Jennings RB, Reimer KA (1986) Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74:1124–1136PubMedGoogle Scholar
  2. 2.
    Yellon DM, Downey JM (2003) Preconditioning the myocardium: from cellular physiology to clinical cardiology. Physiol Rev 83:1113–1151PubMedGoogle Scholar
  3. 3.
    Zhu X, Liu B, Zhou S et al (2007) Ischemic preconditioning prevents in vivo hyperoxygenation in postischemic myocardium with preservation of mitochondrial oxygen consumption. Am J Physiol Heart Circ Physiol 293:H1442–H1450CrossRefPubMedGoogle Scholar
  4. 4.
    Carroll R, Gant VA, Yellon DM (2001) Mitochondrial K(ATP) channel opening protects a human atrial-derived cell line by a mechanism involving free radical generation. Cardiovasc Res 51:691–700CrossRefPubMedGoogle Scholar
  5. 5.
    Ozcan C, Holmuhamedov EL, Jahangir A et al (2001) Diazoxide protects mitochondria from anoxic injury: implications for myopreservation. J Thorac Cardiovasc Surg 121:298–306CrossRefPubMedGoogle Scholar
  6. 6.
    Przyklenk K, Maynard M, Whittaker P (2006) First molecular evidence that inositol trisphosphate signaling contributes to infarct size reduction with preconditioning. Am J Physiol Heart Circ Physiol 291:H2008–H2012CrossRefPubMedGoogle Scholar
  7. 7.
    Schafer G, Wegener C, Portenhauser R et al (1969) Diazoxide, an inhibitor of succinate oxidation. Biochem Pharmacol 18:2678–2681CrossRefPubMedGoogle Scholar
  8. 8.
    Ovide-Bordeaux S, Ventura-Clapier R, Veksler V (2000) Do modulators of the mitochondrial K(ATP) channel change the function of mitochondria in situ? J Biol Chem 275:37291–37295CrossRefPubMedGoogle Scholar
  9. 9.
    Hanley PJ, Mickel M, Loffler M et al (2002) K(ATP) channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart. J Physiol 542:735–741CrossRefPubMedGoogle Scholar
  10. 10.
    Lim KH, Javadov SA, Das M et al (2002) The effects of ischaemic preconditioning, diazoxide and 5-hydroxydecanoate on rat heart mitochondrial volume and respiration. J Physiol 545:961–974CrossRefPubMedGoogle Scholar
  11. 11.
    Drose S, Brandt U, Hanley PJ (2006) K+-independent actions of diazoxide question the role of inner membrane KATP channels in mitochondrial cytoprotective signaling. J Biol Chem 281:23733–23739CrossRefPubMedGoogle Scholar
  12. 12.
    Minners J, Lacerda L, Yellon DM et al (2007) Diazoxide-induced respiratory inhibition—a putative mitochondrial K(ATP) channel independent mechanism of pharmacological preconditioning. Mol Cell Biochem 294:11–18CrossRefPubMedGoogle Scholar
  13. 13.
    Bacsi A, Woodberry M, Widger W et al (2006) Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells. Mitochondrion 6:235–244CrossRefPubMedGoogle Scholar
  14. 14.
    Gomez-Lazaro M, Galindo MF, Melero-Fernandez de Mera RM et al (2007) Reactive oxygen species and p38 mitogen-activated protein kinase activate Bax to induce mitochondrial cytochrome c release and apoptosis in response to malonate. Mol Pharmacol 71:736–743CrossRefPubMedGoogle Scholar
  15. 15.
    Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol 552:335–344CrossRefPubMedGoogle Scholar
  16. 16.
    Swartz HM, Bacic G, Friedman B et al (1994) Measurements of pO2 in vivo, including human subjects, by electron paramagnetic resonance. Adv Exp Med Biol 361:119–128PubMedGoogle Scholar
  17. 17.
    Friedman BJ, Grinberg OY, Grinberg SA et al (1997) Myocardial oxygen tension in isolated erythrocyte-perfused rat hearts and comparison with crystalloid media. J Mol Cell Cardiol 29:2855–2858CrossRefPubMedGoogle Scholar
  18. 18.
    Zhao X, He G, Chen YR et al (2005) Endothelium-derived nitric oxide regulates postischemic myocardial oxygenation and oxygen consumption by modulation of mitochondrial electron transport. Circulation 111:2966–2972CrossRefPubMedGoogle Scholar
  19. 19.
    Khan M, Iyyapu KM, Kutala V et al (2008) Sulfaphenazole protects heart against ischemia–reperfusion injury and cardiac dysfunction by overexpression of iNOS leading to enhancement of nitric-oxide bioavailability and tissue oxygenation. Antioxid Redox Signal 11:725–738Google Scholar
  20. 20.
    Villamena FA, Zweier JL (2004) Detection of reactive oxygen and nitrogen species by EPR spin trapping. Antioxid Redox Signal 6:619–629CrossRefPubMedGoogle Scholar
  21. 21.
    Quast U, Cook NS (1989) In vitro and in vivo comparison of two K+ channel openers, diazoxide and cromakalim, and their inhibition by glibenclamide. J Pharmacol Exp Ther 250:261–271PubMedGoogle Scholar
  22. 22.
    Xu Y, Liu B, Zweier JL et al (2008) Formation of hydrogen peroxide and reduction of peroxynitrite via dismutation of superoxide at reperfusion enhances myocardial blood flow and oxygen consumption in postischemic mouse heart. J Pharmacol Exp Ther 327:402–410CrossRefPubMedGoogle Scholar
  23. 23.
    Costa AD, Quinlan CL, Andrukhiv A et al (2006) The direct physiological effects of mitoK(ATP) opening on heart mitochondria. Am J Physiol Heart Circ Physiol 290:H406–H415CrossRefPubMedGoogle Scholar
  24. 24.
    Dzeja PP, Bast P, Ozcan C et al (2003) Targeting nucleotide-requiring enzymes: implications for diazoxide-induced cardioprotection. Am J Physiol Heart Circ Physiol 284:H1048–H1056PubMedGoogle Scholar
  25. 25.
    Ferranti R, da Silva MM, Kowaltowski AJ (2003) Mitochondrial ATP-sensitive K+ channel opening decreases reactive oxygen species generation. FEBS Lett 536:51–55CrossRefPubMedGoogle Scholar
  26. 26.
    Han D, Antunes F, Canali R et al (2003) Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol. J Biol Chem 278:5557–5563CrossRefPubMedGoogle Scholar
  27. 27.
    Rigobello MP, Folda A, Scutari G et al (2005) The modulation of thiol redox state affects the production and metabolism of hydrogen peroxide by heart mitochondria. Arch Biochem Biophys 441:112–122CrossRefPubMedGoogle Scholar
  28. 28.
    Chen YR, Chen CL, Pfeiffer DR et al (2007) Mitochondrial complex II in the post-ischemic heart: oxidative injury and the role of protein S-glutathionylation. J Biol Chem 282:32640–32654CrossRefPubMedGoogle Scholar
  29. 29.
    Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  30. 30.
    Boveris A, Cadenas E (1975) Mitochondrial production of superoxide anions and its relationship to the antimycin insensitive respiration. FEBS Lett 54:311–314CrossRefPubMedGoogle Scholar
  31. 31.
    Salvemini D, Wang ZQ, Zweier JL et al (1999) A nonpeptidyl mimic of superoxide dismutase with therapeutic activity in rats. Science 286:304–306CrossRefPubMedGoogle Scholar
  32. 32.
    Iqbal J, Whitney P (1991) Use of cyanide and diethyldithiocarbamate in the assay of superoxide dismutases. Free Radic Biol Med 10:69–77CrossRefPubMedGoogle Scholar
  33. 33.
    Chen YR, Chen CL, Yeh A et al (2006) Direct and indirect roles of cytochrome b in the mediation of superoxide generation and NO catabolism by mitochondrial succinate-cytochrome c reductase. J Biol Chem 281:13159–13168CrossRefPubMedGoogle Scholar
  34. 34.
    Sato T, O’Rourke B, Marban E (1998) Modulation of mitochondrial ATP-dependent K+ channels by protein kinase C. Circ Res 83:110–114PubMedGoogle Scholar
  35. 35.
    Hu H, Sato T, Seharaseyon J et al (1999) Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels. Mol Pharmacol 55:1000–1005PubMedGoogle Scholar
  36. 36.
    Han D, Williams E, Cadenas E (2001) Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space. Biochem J 353:411–416CrossRefPubMedGoogle Scholar
  37. 37.
    Frejaville C, Karoui H, Tuccio B et al (1995) 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals. J Med Chem 38:258–265CrossRefPubMedGoogle Scholar
  38. 38.
    Holmuhamedov EL, Jahangir A, Oberlin A et al (2004) Potassium channel openers are uncoupling protonophores: implication in cardioprotection. FEBS Lett 568:167–170CrossRefPubMedGoogle Scholar
  39. 39.
    Kopustinskiene DM, Jovaisiene J, Liobikas J et al (2002) Diazoxide and pinacidil uncouple pyruvate-malate-induced mitochondrial respiration. J Bioenerg Biomembr 34:49–53CrossRefPubMedGoogle Scholar
  40. 40.
    Andrukhiv A, Costa AD, West IC et al (2006) Opening mitoKATP increases superoxide generation from complex I of the electron transport chain. Am J Physiol Heart Circ Physiol 291:H2067–H2074CrossRefPubMedGoogle Scholar
  41. 41.
    Velayutham M, Muthukumaran RB, Sostaric JZ et al (2007) Interactions of the major metabolite of the cancer chemopreventive drug oltipraz with cytochrome c: a novel pathway for cancer chemoprevention. Free Radic Biol Med 43:1076–1085CrossRefPubMedGoogle Scholar
  42. 42.
    Halliwell B, Whiteman M (2004) Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 142:231–255CrossRefPubMedGoogle Scholar
  43. 43.
    Dikalov S, Griendling KK, Harrison DG (2007) Measurement of reactive oxygen species in cardiovascular studies. Hypertension 49:717–727CrossRefPubMedGoogle Scholar
  44. 44.
    Fernandes MA, Santos MS, Moreno AJ et al (2004) Glibenclamide interferes with mitochondrial bioenergetics by inducing changes on membrane ion permeability. J Biochem Mol Toxicol 18:162–169CrossRefPubMedGoogle Scholar
  45. 45.
    Engbersen R, Masereeuw R, van Gestel MA et al (2005) Glibenclamide depletes ATP in renal proximal tubular cells by interfering with mitochondrial metabolism. Br J Pharmacol 145:1069–1075CrossRefPubMedGoogle Scholar
  46. 46.
    Cadenas E, Boveris A (1980) Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria. Biochem J 188:31–37PubMedGoogle Scholar
  47. 47.
    Brookes PS (2005) Mitochondrial H(+) leak and ROS generation: an odd couple. Free Radic Biol Med 38:12–23CrossRefPubMedGoogle Scholar
  48. 48.
    Lesnefsky EJ, Gudz TI, Migita CT et al (2001) Ischemic injury to mitochondrial electron transport in the aging heart: damage to the iron-sulfur protein subunit of electron transport complex III. Arch Biochem Biophys 385:117–128CrossRefPubMedGoogle Scholar
  49. 49.
    Petrosillo G, Ruggiero FM, Di Venosa N et al (2003) Decreased complex III activity in mitochondria isolated from rat heart subjected to ischemia and reperfusion: role of reactive oxygen species and cardiolipin. Faseb J 17:714–716CrossRefPubMedGoogle Scholar
  50. 50.
    Becker LB, vanden Hoek TL, Shao ZH et al (1999) Generation of superoxide in cardiomyocytes during ischemia before reperfusion. Am J Physiol 277:H2240–H2246PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2009

Authors and Affiliations

  • Bin Liu
    • 1
    • 2
  • Xuehai Zhu
    • 3
  • Chwen-Lih Chen
    • 4
  • Keli Hu
    • 5
  • Harold M. Swartz
    • 6
  • Yeong-Renn Chen
    • 4
  • Guanglong He
    • 7
    • 8
  1. 1.The Center for Biomedical EPR Spectroscopy and ImagingDavis Heart and Lung Research InstituteColumbusUSA
  2. 2.Shantou University Medical CollegeShantouChina
  3. 3.Key Laboratory of Organ TransplantationMinistry of Education Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and TechnologyWuhanChina
  4. 4.Davis Heart and Lung Research InstituteColumbusUSA
  5. 5.Division of Pharmacology, College of PharmacyThe Ohio State UniversityColumbusUSA
  6. 6.The EPR Center for the Study of Viable SystemsDartmouth Medical SchoolHanoverUSA
  7. 7.The Center for Biomedical EPR Spectroscopy and ImagingDavis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal MedicineColumbusUSA
  8. 8.Davis Heart and Lung Research Institute, Division of Cardiovascular MedicineDepartment of Internal MedicineColumbusUSA

Personalised recommendations