Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria.

Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 min global ischemia or following 40 min of perfusion alone as controls. H(2)O(2) release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H(2)O(2) was increased by 178 +/- 14% and 179 +/- 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, H(2)O(2) increased by 272 +/- 22% and 171 +/- 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H(2)O(2) production by 179 +/- 24% and 155 +/- 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of H(2)O(2) from complex III. Thus ischemic damage to the ETC increased both the capacity and the net production of H(2)O(2) from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury.

[1]  M. Ikeda-Saito,et al.  Aging decreases electron transport complex III activity in heart interfibrillar mitochondria by alteration of the cytochrome c binding site. , 2001, Journal of molecular and cellular cardiology.

[2]  C. Hoppel,et al.  Preservation of cardiolipin content during aging in rat heart interfibrillar mitochondria. , 2002, The journals of gerontology. Series A, Biological sciences and medical sciences.

[3]  T. Fiore,et al.  Mitochondrial dysfunction associated with cardiac ischemia/reperfusion can be attenuated by oxygen tension control. Role of oxygen-free radicals and cardiolipin. , 2005, Biochimica et biophysica acta.

[4]  T. Slabe,et al.  Myocardial ischemia decreases oxidative phosphorylation through cytochrome oxidase in subsarcolemmal mitochondria. , 1997, The American journal of physiology.

[5]  G. Paradies,et al.  Interaction of peroxidized cardiolipin with rat‐heart mitochondrial membranes: Induction of permeability transition and cytochrome c release , 2006, FEBS letters.

[6]  G. Paradies,et al.  Decrease in Mitochondrial Complex I Activity in Ischemic/Reperfused Rat Heart: Involvement of Reactive Oxygen Species and Cardiolipin , 2004, Circulation research.

[7]  Peipei Ping,et al.  Role of the mitochondrial permeability transition in myocardial disease. , 2003, Circulation research.

[8]  C. Hoppel,et al.  Blockade of Electron Transport before Cardiac Ischemia with the Reversible Inhibitor Amobarbital Protects Rat Heart Mitochondria , 2006, Journal of Pharmacology and Experimental Therapeutics.

[9]  C. Hoppel,et al.  Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. , 1977, The Journal of biological chemistry.

[10]  E. Cadenas,et al.  Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis. , 2007, The Biochemical journal.

[11]  G. Gross,et al.  Blocking Na(+)/H(+) exchange reduces [Na(+)](i) and [Ca(2+)](i) load after ischemia and improves function in intact hearts. , 2001, American journal of physiology. Heart and circulatory physiology.

[12]  J. Williams A METHOD FOR THE SIMULTANEOUS QUANTITATIVE ESTIMATION OF CYTOCHROMES A, B, C1, AND C IN MITOCHONDRIA. , 1964, Archives of biochemistry and biophysics.

[13]  D. Yellon,et al.  Realizing the clinical potential of ischemic preconditioning and postconditioning , 2005, Nature Clinical Practice Cardiovascular Medicine.

[14]  G. Finet,et al.  Postconditioning the Human Heart , 2005, Circulation.

[15]  L. Hue,et al.  Global ischaemia induces a biphasic response of the mitochondrial respiratory chain. Anoxic pre-perfusion protects against ischaemic damage. , 1992, The Biochemical journal.

[16]  L. Partridge,et al.  Superoxide and hydrogen peroxide production by Drosophila mitochondria. , 2003, Free radical biology & medicine.

[17]  T. Slabe,et al.  Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin. , 2004, American journal of physiology. Heart and circulatory physiology.

[18]  P. Bernardi,et al.  Opening of the Mitochondrial Permeability Transition Pore Causes Depletion of Mitochondrial and Cytosolic NAD+and Is a Causative Event in the Death of Myocytes in Postischemic Reperfusion of the Heart* , 2001, The Journal of Biological Chemistry.

[19]  C. Hoppel,et al.  Mitochondrial dysfunction in cardiac disease: ischemia--reperfusion, aging, and heart failure. , 2001, Journal of molecular and cellular cardiology.

[20]  J. Zweier,et al.  Evidence that mitochondrial respiration is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow. , 1993, The Journal of biological chemistry.

[21]  L. Becker New concepts in reactive oxygen species and cardiovascular reperfusion physiology. , 2004, Cardiovascular research.

[22]  P. Bernardi,et al.  Mitochondria and reperfusion injury. The role of permeability transition. , 2003, Basic research in cardiology.

[23]  L. Argaud,et al.  Mitochondrial permeability transition pore and postconditioning. , 2006, Cardiovascular research.

[24]  T. Vanden Hoek,et al.  Generation of superoxide in cardiomyocytes during ischemia before reperfusion. , 1999, American journal of physiology. Heart and circulatory physiology.

[25]  B. Trumpower,et al.  Superoxide anion generation by the cytochrome bc1 complex. , 2003, Archives of biochemistry and biophysics.

[26]  C. Hoppel,et al.  Reversible Blockade of Electron Transport during Ischemia Protects Mitochondria and Decreases Myocardial Injury following Reperfusion , 2006, Journal of Pharmacology and Experimental Therapeutics.

[27]  Ken-ichi Yoshida,et al.  A Possible Site of Superoxide Generation in the Complex I Segment of Rat Heart Mitochondria , 2005, Journal of bioenergetics and biomembranes.

[28]  L. Kevin,et al.  Ischemic preconditioning alters real-time measure of O2 radicals in intact hearts with ischemia and reperfusion. , 2003, American journal of physiology. Heart and circulatory physiology.

[29]  B. Robinson,et al.  Superoxides from mitochondrial complex III: the role of manganese superoxide dismutase. , 2000, Free radical biology & medicine.

[30]  C. Hoppel,et al.  Blockade of Electron Transport during Ischemia Protects Cardiac Mitochondria* , 2004, Journal of Biological Chemistry.

[31]  E. Cadenas,et al.  Voltage-dependent Anion Channels Control the Release of the Superoxide Anion from Mitochondria to Cytosol* , 2003, The Journal of Biological Chemistry.

[32]  D. Kerr,et al.  Deficiency of the reduced nicotinamide adenine dinucleotide dehydrogenase component of complex I of mitochondrial electron transport. Fatal infantile lactic acidosis and hypermetabolism with skeletal-cardiac myopathy and encephalopathy. , 1987, The Journal of clinical investigation.

[33]  C. Elger,et al.  Characterization of Superoxide-producing Sites in Isolated Brain Mitochondria* , 2004, Journal of Biological Chemistry.

[34]  G. R. Williams,et al.  Inhibition of electron and energy transfer in mitochondria. I. Effects of Amytal, thiopental, rotenone, progesterone, and methylene glycol. , 1963, The Journal of biological chemistry.

[35]  C. Ragan,et al.  Proteins, polypeptides, prosthetic groups, and enzymic properties of complexes I, II, III, IV, and V of the mitochondrial oxidative phosphorylation system. , 1979, Methods in enzymology.

[36]  G. Barja Mitochondrial Free Radical Production and Aging in Mammals and Birds a , 1998, Annals of the New York Academy of Sciences.

[37]  M. Brand,et al.  Topology of Superoxide Production from Different Sites in the Mitochondrial Electron Transport Chain* , 2002, The Journal of Biological Chemistry.

[38]  J. Flaherty,et al.  Effects of the superoxide radical scavenger superoxide dismutase, and of the hydroxyl radical scavenger mannitol, on reperfusion injury in isolated rabbit hearts , 1992, Cardiovascular Drugs and Therapy.

[39]  I. A. Rose,et al.  Mechanism of aconitase action. I. The hydrogen transfer reaction. , 1967, The Journal of biological chemistry.

[40]  G. Cecchini,et al.  MnSOD in mouse heart: acute responses to ischemic preconditioning and ischemia-reperfusion injury. , 2005, American journal of physiology. Heart and circulatory physiology.

[41]  L. Gille,et al.  The ubiquinol/bc1 redox couple regulates mitochondrial oxygen radical formation. , 2001, Archives of biochemistry and biophysics.

[42]  J. Okun,et al.  Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase)* , 1999, The Journal of Biological Chemistry.

[43]  H. Westerhoff,et al.  A wave of reactive oxygen species (ROS)-induced ROS release in a sea of excitable mitochondria. , 2006, Antioxidants & redox signaling.

[44]  C. Hoppel,et al.  Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion. , 2007, American journal of physiology. Cell physiology.

[45]  Qun Chen,et al.  Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain. , 2006, Free radical biology & medicine.

[46]  M. Ikeda-Saito,et al.  Ischemic injury to mitochondrial electron transport in the aging heart: damage to the iron-sulfur protein subunit of electron transport complex III. , 2001, Archives of biochemistry and biophysics.

[47]  T. Yagi,et al.  The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I , 2006, FEBS letters.

[48]  M. L. Genova,et al.  The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron–sulfur cluster N2 , 2001, FEBS letters.

[49]  J. Turrens,et al.  Mitochondrial formation of reactive oxygen species , 2003, The Journal of physiology.

[50]  G. Freeman,et al.  Role of 4-hydroxynonenal in modification of cytochrome c oxidase in ischemia/reperfused rat heart. , 2001, Journal of molecular and cellular cardiology.

[51]  A. Murphy,et al.  Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state. , 2002, The Biochemical journal.

[52]  S. Cortassa,et al.  Mitochondrial ion channels: gatekeepers of life and death. , 2005, Physiology.

[53]  C. Hoppel,et al.  Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria. , 1991, The Journal of biological chemistry.

[54]  A. Camara,et al.  Inhibited mitochondrial respiration by amobarbital during cardiac ischaemia improves redox state and reduces matrix Ca2+ overload and ROS release. , 2007, Cardiovascular research.

[55]  H. Nohl Generation of superoxide radicals as byproduct of cellular respiration. , 1994, Annales de biologie clinique.

[56]  J. Turrens,et al.  Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. , 1980, The Biochemical journal.

[57]  William Rouslin Mitochondrial complexes I, II, III, IV, and V in myocardial ischemia and autolysis. , 1983, The American journal of physiology.

[58]  B. Trumpower,et al.  Regulatory Interactions between Ubiquinol Oxidation and Ubiquinone Reduction Sites in the Dimeric Cytochrome bc1 Complex* , 2006, Journal of Biological Chemistry.

[59]  Y. Chen,et al.  Sensitivity of protein sulfhydryl repair enzymes to oxidative stress. , 1997, Free radical biology & medicine.

[60]  Brian O'Rourke,et al.  Synchronized Whole Cell Oscillations in Mitochondrial Metabolism Triggered by a Local Release of Reactive Oxygen Species in Cardiac Myocytes* , 2003, Journal of Biological Chemistry.