Abstract
In this study, we have characterized the cellular source and mechanism for the enhanced generation of reactive oxygen species (ROS) in the myocardium during Trypanosoma cruzi infection. Cardiac mitochondria of infected mice, as compared to normal controls, exhibited 63.3% and 30.8% increase in ROS-specific fluorescence of dihydroethidium (detects O2 •−) and amplex red (detects H2O2), respectively. This increase in ROS level in cardiac mitochondria of infected mice was associated with a 59% and 114% increase in the rate of glutamate/malate- (complex I substrates) and succinate- (complex II substrate) supported ROS release, respectively, and up to a 74.9% increase in the rate of electron leakage from the respiratory chain when compared to normal controls. Inhibition studies with normal cardiac mitochondria showed that rotenone induced ROS generation at the QNf-ubisemiquinone site in complex I. In complex III, myxothiazol induced ROS generation from a site located at the Qo center that was different from the Qi center of O2 •− generation by antimycin. In cardiac mitochondria of infected mice, the rate of electron leakage at complex I during forward (complex I-to-complex III) and reverse (complex II-to-complex I) electron flow was not enhanced, and complex I was not the main site of increased ROS production in infected myocardium. Instead, defects of complex III proximal to the Qo site resulted in enhanced electron leakage and ROS formation in cardiac mitochondria of infected mice. Treatment of infected mice with phenyl-α-tert-butyl-nitrone (PBN) improved the respiratory chain function, and, subsequently, decreased the extent of electron leakage and ROS release. In conclusion, we show that impairment of the Qo site of complex III resulted in increased electron leakage and O2 •− formation in infected myocardium, and was controlled by PBN.
Article PDF
Similar content being viewed by others
References
Aleardi AM, Benard G et al (2005) “Gradual alteration of mitochondrial structure and function by beta-amyloids: importance of membrane viscosity changes, energy deprivation, reactive oxygen species production, and cytochrome c release”. J Bioenerg Biomembr 37(4):207–25
Boveris A, Oshino N et al (1972) “The cellular production of hydrogen peroxide”. Biochem J 128(3):617–30
Bradford MM (1976) “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding”. Anal Biochem 72:248–54
Braun HP, Schmitz UK (1995) “Are the ‘core’ proteins of the mitochondrial bc1 complex evolutionary relics of a processing protease?”. Trends Biochem Sci 20(5):171–5
Carrasco Guerra HA, Palacios-Pru E et al (1987) “Clinical, histochemical, and ultrastructural correlation in septal endomyocardial biopsies from chronic chagasic patients: detection of early myocardial damage”. Am Heart J 113(3):716–24
Crofts AR (2004) “The cytochrome bc1 complex: function in the context of structure”. Annu Rev Physiol 66:689–733
Degli Esposti M, Ghelli A et al (1993) “Complex I and complex III of mitochondria have common inhibitors acting as ubiquinone antagonists”. Biochem Biophys Res Commun 190(3):1090–6
Floyd RA, Hensley K et al (2002) “Nitrones as neuroprotectants and antiaging drugs”. Ann N Y Acad Sci 959:321–9
Garg N, Popov VL et al (2003) “Profiling gene transcription reveals a deficiency of mitochondrial oxidative phosphorylation in Trypanosoma cruzi-infected murine hearts: implications in chagasic myocarditis development”. Biochim Biophys Acta 1638(2):106–20
Gellerich FN, Trumbeckaite S et al (1999) “Impaired energy metabolism in hearts of septic baboons: diminished activities of Complex I and Complex II of the mitochondrial respiratory chain”. Shock 11(5):336–41
Genova ML, Pich MM et al (2004) “The mitochondrial production of reactive oxygen species in relation to aging and pathology”. Ann N Y Acad Sci 1011:86–100
Genova ML, Ventura B et al (2001) “The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2”. FEBS Lett 505(3):364–8
Gyulkhandanyan AV, Pennefather PS (2004) “Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress”. J Neurochem 90(2):405–21
Harper A (1963) “Glucose-6-Phosphatase”. Methods of enzymatic analysis:788–792
Iwata S, Lee JW et al (1998) “Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex”. Science 281(5373):64–71
Jarreta D, Orus J et al (2000) “Mitochondrial function in heart muscle from patients with idiopathic dilated cardiomyopathy”. Cardiovasc Res 45(4):860–5
Krishnamoorthy G, Hinkle PC (1988) “Studies on the electron transfer pathway, topography of iron-sulfur centers, and site of coupling in NADH-Q oxidoreductase”. J Biol Chem 263(33):17566–75
Ksenzenko M, Konstantinov AA et al (1983) “Effect of electron transfer inhibitors on superoxide generation in the cytochrome bc1 site of the mitochondrial respiratory chain”. FEBS Lett 155(1):19–24
Kudin AP, Bimpong-Buta NY et al (2004) “Characterization of superoxide-producing sites in isolated brain mitochondria”. J Biol Chem 279(6):4127–35
Kushnareva Y, Murphy AN et al (2002) “Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state”. Biochem J 368(Pt 2):545–53
Lemasters JJ (1984) “The ATP-to-oxygen stoichiometries of oxidative phosphorylation by rat liver mitochondria. An analysis of ADP-induced oxygen jumps by linear nonequilibrium thermodynamics”. J Biol Chem 259(21):13123–30
Lui NS, Roels OA et al (1968) “Subcellular distribution of enzymes in Ochromonas malhamensis”. J Protozool 15(3):536–42
Ohnishi ST, Ohnishi T et al (2005) “A possible site of superoxide generation in the complex I segment of rat heart mitochondria”. J Bioenerg Biomembr 37(1):1–15
Palacios-Pru E, Carrasco H et al (1989) “Ultrastructural characteristics of different stages of human chagasic myocarditis”. Am J Trop Med Hyg 41(1):29–40
Parker WD Jr, Boyson SJ et al (1990) “Evidence for a defect in NADH: ubiquinone oxidoreductase (complex I) in Huntington’s disease”. Neurology 40(8):1231–4
Robertson DE, Ding H et al (1993) “Hydroubiquinone-cytochrome c2 oxidoreductase from Rhodobacter capsulatus: definition of a minimal, functional isolated preparation”. Biochemistry 32(5):1310–7
Sanz A, Caro P et al (2005) “Dietary restriction at old age lowers mitochondrial oxygen radical production and leak at complex I and oxidative DNA damage in rat brain”. J Bioenerg Biomembr 37(2):83–90
Schapira AH (1999) “Mitochondrial involvement in Parkinson’s disease, Huntington’s disease, hereditary spastic paraplegia and Friedreich’s ataxia”. Biochim Biophys Acta 1410(2):159–70
Starkov AA, Fiskum G (2001) “Myxothiazol induces H(2)O(2) production from mitochondrial respiratory chain”. Biochem Biophys Res Commun 281(3):645–50
Toth PP, Ferguson-Miller SM et al (1986) “Isolation of highly coupled heart mitochondria in high yield using a bacterial collagenase”. Methods Enzymol 125:16–27
Towbin JA, Bowles KR et al (1999) “Etiologies of cardiomyopathy and heart failure”. Nat Med 5(3):266–7
Turrens JF, Alexandre A et al (1985) “Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria”. Arch Biochem Biophys 237(2):408–14
Vyatkina G, Bhatia V et al (2004) “Impaired mitochondrial respiratory chain and bioenergetics during chagasic cardiomyopathy development”. Biochim Biophys Acta 1689:162–173
Wen J-J, Bhatia V et al (2006a) “Phenyl-alpha-tert-butyl nitrone reverses mitochondrial decay in acute Chagas disease”. Am J Pathol 169(6):1953–64
Wen J-J, Garg N (2004) “Oxidative modifications of mitochondrial respiratory complexes in response to the stress of Trypanosoma cruzi infection”. Free Radic Biol Med 37(12):2072–81
Wen J-J, Vyatkina G et al (2004) “Oxidative damage during chagasic cardiomyopathy development: Role of mitochondrial oxidant release and inefficient antioxidant defense”. Free Radic Biol Med 37(11):1821–33
Wen J-J, Yachelini PC et al (2006b) “Increased oxidative stress is correlated with mitochondrial dysfunction in chagasic patients”. Free Rad Biol Med 41:270–276
World Health Organization (2002) Control of Chagas disease: Second report of the WHO expert committee. WHO Technical Report Series 905. UNDP/World Bank/WHO, Geneva, Switzerland
Young TA, Cunningham CC et al (2002) “Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol”. Arch Biochem Biophys 405(1):65–72
Zacks MA, Wen J-J et al (2005) “An overview of chagasic cardiomyopathy: pathogenic importance of oxidative stress”. Ann Acad Bras Cienc 77:695–715
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wen, JJ., Garg, N.J. Mitochondrial generation of reactive oxygen species is enhanced at the Qo site of the complex III in the myocardium of Trypanosoma cruzi-infected mice: beneficial effects of an antioxidant. J Bioenerg Biomembr 40, 587–598 (2008). https://doi.org/10.1007/s10863-008-9184-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10863-008-9184-4