- Email: [email protected]
Reactive oxygen species production by flavin dehydrogenases of the mitochondrial respiratory chain
S100
Abstracts
and 2′,3′-cNADP [1], and the neuronal protein p42IP4 [2]. We compared the mPTP opening parameters in non-synaptic brain mitochondria from young and old rats. In mitochondria from old rats (N18 months), mPTP opening occurred at a lower threshold calcium concentration than in mitochondria from young rats (b3 months). mPTP opening in mitochondria from old rats was accelerated by 2′,3′-cAMP, which also lowered the threshold calcium concentration. In non-synaptic mitochondria from old rats, the CNP level was decreased by 87%. This was accompanied with decreased levels of voltage-dependent anion channel (VDAC) and of p42IP4. Thus, reduced mitochondrial level of CNP and activity could lead to a rise in the concentration of the mPTP promoter 2′,3′-cAMP. We propose that in aging diminished levels of these proteins lead to mitochondrial dysfunction, in particular, to decreased threshold calcium concentration for mPTP induction. The level of CNP and p42IP4 and, probably VDAC, might be essential for myelination and electrical activity of axons. That might be the steps of age-related mitochondrial dysfunction, resulting in myelin and axonal pathology. We demonstrate that the levels of three mitochondrial proteins (CNP, p42IP4, and VDAC) decreased in mitochondria with aging. These three proteins are related to the calcium-transporting system in mitochondria and the regulation of mPTP function. A diminished level of these proteins leads to mitochondrial dysfunction, in particular, increased calcium uptake and decrease of threshold calcium concentration. That causes mPTP induction and might be the initial step of mitochondrial dysfunction, resulting in myelin and axonal pathology. References [1] T. Azarashvili, O. Krestinina, A. Galvita, D. Grachev, Y. Baburina, R. Stricker, Y. Evtodienko, G. Reiser, Am J Physiol Cell Physiol 296 (2009) C1428–1439. [2] A. Galvita, D. Grachev, T. Azarashvili, Y. Baburina, O. Krestinina, R. Stricker, G. Reiser, J Neurochem 109 (2009) 1701–1713. doi:10.1016/j.bbabio.2012.06.269
13P9 Mitochondrial dysfunction in MnSOD knockout mice A.P. Kudin, E. Taheizadeh-Fard, W.S. Kunz Division of Neurochemistry, Department of Epileptology and Life & Brain Center, University of Bonn, Sigmund-Freud-Str. 25, D-53105, Bonn, Germany E-mail: [email protected] Oxidative stress is implicated to be the cause of several pathological conditions in the cell. Therefore mechanisms reducing ROS levels in the cell are carefully investigated. One of the antioxidant enzyme MnSOD, which is located in the mitochondrial matrix, attracts special attention, because mitochondria are suggested to be a major source of ROS production. Introduced in the 90th of the last century, MnSOD knockout mouse is a useful model to investigate the consequences of reduction of antioxidant reactions within mitochondria. This knockout mouse model was generated via inactivation of the MnSOD gene by homologous recombination [1]. Despite of the available data in characterizing the pathological phenotype of MnSOD knockout mouse, conclusive results concerning homozygous mice are still limited, because homozygous mutant mice die during the first days after birth. Therefore, we analyzed the impact of MnSOD knockout on mitochondria from different tissues of newborn mice. Mitochondrial function was investigated in the brain, liver, heart and muscle. The enzyme activities of aconitase, citrate synthase and mitochondrial complexes I and IV were measured in tissue homogenates from wild type, heterozygous and homozygous mice. In accordance with the literature aconitase activity was reduced in all tissues under investigation. A significant reduction of complex I activity was observed in
liver homogenates of homozygous mice. Additionally, we analyzed oxygen consumption in tissue homogenates. Interestingly, while complex I activity in brain homogenates was only slightly reduced, respiration rates with complex I supported substrates were significantly decreased in homozygous mouse brain homogenates. Taken together, our findings demonstrate a tissue specific distribution of mitochondrial dysfunction in MnSOD knockout mice. Reference [1] Y. Li, T. Huang, E.J. Carlson, S. Melov, P.C. Ursell, J.L. Olson, L.J. Noble, M.P. Yoshimura, C. Berger, P.H. Chan, D.C. Wallace, C.J. Epstein, Nat. Genet. 11 (1995) 376–381. doi:10.1016/j.bbabio.2012.06.270
13P10 Oxidation of cardiolipin in liposomes: A new insight into the primary steps of mitochondria-triggered apoptosis A.V. Lokhmatikov, N. Voskoboynikova, D.A. Cherepanov, H.-J. Steinhoff, V.P. Skulachev, A.Y. Mulkidjanian School of Physics, University of Osnabrück, D-49069 Osnabrück, Germany A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119992, Russia A.N. Frumkin Institute of Electrochemistry of the RAS, Leninsky Prospect 31, Moscow, 119991, Russia E-mail: [email protected] Cardiolipin is an unusual lipid that is present almost exclusively in the coupling membranes. Cardiolipin is abundant in the inner mitochondrial membrane, being tightly bound within the proteins of respiratory complexes and apparently required for their proper functioning. In addition, cardiolipin represents the major target for reactive oxygen species and its oxidation is regarded as a primary signal of mitochondria-induced apoptosis [1,2]. The present study introduces an approach where the impact of diverse antioxidants on the peroxidation of cardiolipin could be investigated with a liposome model based on the cardiolipin from bovine heart. The data obtained suggest how the changes in the condition of the respiratory chain enzymes can trigger the oxidation of cardiolipin molecules. References [1] V.E. Kagan, V.A. Tyurin, J. Jiang, Y.Y. Tyurina, V.B. Ritov, A.A. Amoscato, A.N. Osipov, N.A. Belikova, A.A. Kapralov, V. Kini, I.I. Vlasova, Q. Zhao, M. Zou, P. Di, D.A. Svistunenko, I.V. Kurnikov, G.G. Borisenko, Nat. Chem. Biol. 1 (2005) 223–232. [2] V.P. Skulachev, Y.N. Antonenko, D.A. Cherepanov, B.V. Chernyak, D.S. Izyumov, L.S. Khailova, S.S. Klishin, G.A. Korshunova, K.G. Lyamzaev, O.Y. Pletjushkina, V.A. Roginsky, T.I. Rokitskaya, F.F. Severin, I.I. Severina, R.A. Simonyan, M.V. Skulachev, N.V. Sumbatyan, E.I. Sukhanova, V.N. Tashlitsky, T.A. Trendeleva, M.Y. Vyssokikh, R.A. Zvyagilskaya, Biochim. Biophys. Acta 1797 (2010) 878–889. doi:10.1016/j.bbabio.2012.06.271
13P11 Reactive oxygen species production by flavin dehydrogenases of the mitochondrial respiratory chain T. Mráček, E. Holzerová, N. Kovářová, P. Ješina, Z. Drahota, J. Houštěk Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic E-mail: [email protected]
Abstracts
Overproduction of reactive oxygen species (ROS) may cause cell and tissue damage and is implicated in a range of different pathologies. Enzymes of the mitochondrial respiratory chain, namely complexes I and III, belong to the important cellular sources of ROS. However, ROS production is also associated with flavin dehydrogenases — succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (mGPDH) [1]. In our previous studies we demonstrated that both SDH and mGPDH can support ROS production. In the case of SDH, most ROS are produced at the level of complex III, while with mGPDH, the prominent ROS source is dehydrogenase itself and coenzyme Q (CoQ) pool [2]. We therefore decided to characterise the mechanism of ROS production by these dehydrogenases in more detail. We used mild detergents to solubilise mitochondrial membranes into individual complexes and supercomplexes as a tool for elucidating their role in ROS production. These experiments uncovered important differences between SDH and mGPDH associated ROS production both in terms of intensity and mechanism. Solubilisation of SDH led to the loss of enzyme activity, caused by the depletion of CoQ. Exogenous CoQ had pro-oxidant activity, which implicated CoQ and not SDH as the place of electron leak to superoxide. In contrast, basal mGPDH ROS production was several times higher and was further increased by enzyme solubilisation. Exogenous CoQ prevented leak of electrons towards oxygen and had an anti-oxidant effect. Further we used native electrophoretic systems to isolate the enzyme and proved that mGPDH itself is the place of electron leak and ROS production. We also demonstrated mGPDH association into homooligomeric forms as well as in high molecular weight supercomplex. All these forms were capable of ROS production. This project has been funded by GACR P303/10/P227. References [1] T. Mracek, P. Jesina, P. Krivakova, R. Bolehovska, Z. Cervinkova, Z. Drahota, et al., Biochim. Biophys. Acta 1726 (2005) 217–223. [2] M. Vrbacky, Z. Drahota, T. Mracek, A. Vojtiskova, P. Jesina, P. Stopka, et al., Biochim. Biophys. Acta 1767 (2007) 989–997. doi:10.1016/j.bbabio.2012.06.272
S101
13P12 Real-time tracking of mitochondrial hydrogen peroxide in single rat cardiomyocytes during simulated ischaemia/reperfusion Andreas M. Rossbach, Tatyana Andrienko, Andrew P. Halestrap Department of Biochemistry, School of Medical Sciences, University Walk, Clifton, Bristol, BS8 1TD, UK E-mail: [email protected] Reperfusion injury is mediated by reactive oxygen species (ROS) produced during ischaemia–reperfusion (IR) which leads to the opening of the mitochondrial permeability transition pore (MPTP). ROS production has previously been monitored in isolated cardiomyocytes by fluorescence microscopy with varying methods, cell types and results. We have developed a real-time ischaemia–reperfusion protocol for fluorescent microscopy that exposes isolated rat cardiomyocytes to conditions closely matching global ischaemia–reperfusion in the perfused heart. Temperature and [O2] are tightly controlled and continuously monitored. We have previously confirmed that neither DHE nor DCF are suitable dyes to monitor ROS production in situ. Instead, we have employed the mitochondria-targeted H2O2-sensitive fluorescent dye MitoPY1 for live monitoring of ROS production. We have also targeted Ca2 +, ΔΨm and the cells' oxidative state with additional co-stains and markers. In addition, we are developing a protocol for real-time multiwavelength surface fluorescence and reflectance measurement to monitor autofluorescence and ROS production in the more pathophysiologically relevant Langendorff-perfused rat heart. Our results to date show that fluorescence of MitoPY1 in cells remains stable in single myocytes during the normoxic equilibration period but increases briefly during the initial period of simulated ischaemia. A large rise in fluorescence is observed during reperfusion prior to hypercontraction and death of the cells. We aim to establish an experimental method using single myocytes that complements the reflectance/fluorescence studies in the perfused hearts to elucidate further the cellular events during IR.
doi:10.1016/j.bbabio.2012.06.273
Abstracts
and 2′,3′-cNADP [1], and the neuronal protein p42IP4 [2]. We compared the mPTP opening parameters in non-synaptic brain mitochondria from young and old rats. In mitochondria from old rats (N18 months), mPTP opening occurred at a lower threshold calcium concentration than in mitochondria from young rats (b3 months). mPTP opening in mitochondria from old rats was accelerated by 2′,3′-cAMP, which also lowered the threshold calcium concentration. In non-synaptic mitochondria from old rats, the CNP level was decreased by 87%. This was accompanied with decreased levels of voltage-dependent anion channel (VDAC) and of p42IP4. Thus, reduced mitochondrial level of CNP and activity could lead to a rise in the concentration of the mPTP promoter 2′,3′-cAMP. We propose that in aging diminished levels of these proteins lead to mitochondrial dysfunction, in particular, to decreased threshold calcium concentration for mPTP induction. The level of CNP and p42IP4 and, probably VDAC, might be essential for myelination and electrical activity of axons. That might be the steps of age-related mitochondrial dysfunction, resulting in myelin and axonal pathology. We demonstrate that the levels of three mitochondrial proteins (CNP, p42IP4, and VDAC) decreased in mitochondria with aging. These three proteins are related to the calcium-transporting system in mitochondria and the regulation of mPTP function. A diminished level of these proteins leads to mitochondrial dysfunction, in particular, increased calcium uptake and decrease of threshold calcium concentration. That causes mPTP induction and might be the initial step of mitochondrial dysfunction, resulting in myelin and axonal pathology. References [1] T. Azarashvili, O. Krestinina, A. Galvita, D. Grachev, Y. Baburina, R. Stricker, Y. Evtodienko, G. Reiser, Am J Physiol Cell Physiol 296 (2009) C1428–1439. [2] A. Galvita, D. Grachev, T. Azarashvili, Y. Baburina, O. Krestinina, R. Stricker, G. Reiser, J Neurochem 109 (2009) 1701–1713. doi:10.1016/j.bbabio.2012.06.269
13P9 Mitochondrial dysfunction in MnSOD knockout mice A.P. Kudin, E. Taheizadeh-Fard, W.S. Kunz Division of Neurochemistry, Department of Epileptology and Life & Brain Center, University of Bonn, Sigmund-Freud-Str. 25, D-53105, Bonn, Germany E-mail: [email protected] Oxidative stress is implicated to be the cause of several pathological conditions in the cell. Therefore mechanisms reducing ROS levels in the cell are carefully investigated. One of the antioxidant enzyme MnSOD, which is located in the mitochondrial matrix, attracts special attention, because mitochondria are suggested to be a major source of ROS production. Introduced in the 90th of the last century, MnSOD knockout mouse is a useful model to investigate the consequences of reduction of antioxidant reactions within mitochondria. This knockout mouse model was generated via inactivation of the MnSOD gene by homologous recombination [1]. Despite of the available data in characterizing the pathological phenotype of MnSOD knockout mouse, conclusive results concerning homozygous mice are still limited, because homozygous mutant mice die during the first days after birth. Therefore, we analyzed the impact of MnSOD knockout on mitochondria from different tissues of newborn mice. Mitochondrial function was investigated in the brain, liver, heart and muscle. The enzyme activities of aconitase, citrate synthase and mitochondrial complexes I and IV were measured in tissue homogenates from wild type, heterozygous and homozygous mice. In accordance with the literature aconitase activity was reduced in all tissues under investigation. A significant reduction of complex I activity was observed in
liver homogenates of homozygous mice. Additionally, we analyzed oxygen consumption in tissue homogenates. Interestingly, while complex I activity in brain homogenates was only slightly reduced, respiration rates with complex I supported substrates were significantly decreased in homozygous mouse brain homogenates. Taken together, our findings demonstrate a tissue specific distribution of mitochondrial dysfunction in MnSOD knockout mice. Reference [1] Y. Li, T. Huang, E.J. Carlson, S. Melov, P.C. Ursell, J.L. Olson, L.J. Noble, M.P. Yoshimura, C. Berger, P.H. Chan, D.C. Wallace, C.J. Epstein, Nat. Genet. 11 (1995) 376–381. doi:10.1016/j.bbabio.2012.06.270
13P10 Oxidation of cardiolipin in liposomes: A new insight into the primary steps of mitochondria-triggered apoptosis A.V. Lokhmatikov, N. Voskoboynikova, D.A. Cherepanov, H.-J. Steinhoff, V.P. Skulachev, A.Y. Mulkidjanian School of Physics, University of Osnabrück, D-49069 Osnabrück, Germany A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119992, Russia A.N. Frumkin Institute of Electrochemistry of the RAS, Leninsky Prospect 31, Moscow, 119991, Russia E-mail: [email protected] Cardiolipin is an unusual lipid that is present almost exclusively in the coupling membranes. Cardiolipin is abundant in the inner mitochondrial membrane, being tightly bound within the proteins of respiratory complexes and apparently required for their proper functioning. In addition, cardiolipin represents the major target for reactive oxygen species and its oxidation is regarded as a primary signal of mitochondria-induced apoptosis [1,2]. The present study introduces an approach where the impact of diverse antioxidants on the peroxidation of cardiolipin could be investigated with a liposome model based on the cardiolipin from bovine heart. The data obtained suggest how the changes in the condition of the respiratory chain enzymes can trigger the oxidation of cardiolipin molecules. References [1] V.E. Kagan, V.A. Tyurin, J. Jiang, Y.Y. Tyurina, V.B. Ritov, A.A. Amoscato, A.N. Osipov, N.A. Belikova, A.A. Kapralov, V. Kini, I.I. Vlasova, Q. Zhao, M. Zou, P. Di, D.A. Svistunenko, I.V. Kurnikov, G.G. Borisenko, Nat. Chem. Biol. 1 (2005) 223–232. [2] V.P. Skulachev, Y.N. Antonenko, D.A. Cherepanov, B.V. Chernyak, D.S. Izyumov, L.S. Khailova, S.S. Klishin, G.A. Korshunova, K.G. Lyamzaev, O.Y. Pletjushkina, V.A. Roginsky, T.I. Rokitskaya, F.F. Severin, I.I. Severina, R.A. Simonyan, M.V. Skulachev, N.V. Sumbatyan, E.I. Sukhanova, V.N. Tashlitsky, T.A. Trendeleva, M.Y. Vyssokikh, R.A. Zvyagilskaya, Biochim. Biophys. Acta 1797 (2010) 878–889. doi:10.1016/j.bbabio.2012.06.271
13P11 Reactive oxygen species production by flavin dehydrogenases of the mitochondrial respiratory chain T. Mráček, E. Holzerová, N. Kovářová, P. Ješina, Z. Drahota, J. Houštěk Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic E-mail: [email protected]
Abstracts
Overproduction of reactive oxygen species (ROS) may cause cell and tissue damage and is implicated in a range of different pathologies. Enzymes of the mitochondrial respiratory chain, namely complexes I and III, belong to the important cellular sources of ROS. However, ROS production is also associated with flavin dehydrogenases — succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (mGPDH) [1]. In our previous studies we demonstrated that both SDH and mGPDH can support ROS production. In the case of SDH, most ROS are produced at the level of complex III, while with mGPDH, the prominent ROS source is dehydrogenase itself and coenzyme Q (CoQ) pool [2]. We therefore decided to characterise the mechanism of ROS production by these dehydrogenases in more detail. We used mild detergents to solubilise mitochondrial membranes into individual complexes and supercomplexes as a tool for elucidating their role in ROS production. These experiments uncovered important differences between SDH and mGPDH associated ROS production both in terms of intensity and mechanism. Solubilisation of SDH led to the loss of enzyme activity, caused by the depletion of CoQ. Exogenous CoQ had pro-oxidant activity, which implicated CoQ and not SDH as the place of electron leak to superoxide. In contrast, basal mGPDH ROS production was several times higher and was further increased by enzyme solubilisation. Exogenous CoQ prevented leak of electrons towards oxygen and had an anti-oxidant effect. Further we used native electrophoretic systems to isolate the enzyme and proved that mGPDH itself is the place of electron leak and ROS production. We also demonstrated mGPDH association into homooligomeric forms as well as in high molecular weight supercomplex. All these forms were capable of ROS production. This project has been funded by GACR P303/10/P227. References [1] T. Mracek, P. Jesina, P. Krivakova, R. Bolehovska, Z. Cervinkova, Z. Drahota, et al., Biochim. Biophys. Acta 1726 (2005) 217–223. [2] M. Vrbacky, Z. Drahota, T. Mracek, A. Vojtiskova, P. Jesina, P. Stopka, et al., Biochim. Biophys. Acta 1767 (2007) 989–997. doi:10.1016/j.bbabio.2012.06.272
S101
13P12 Real-time tracking of mitochondrial hydrogen peroxide in single rat cardiomyocytes during simulated ischaemia/reperfusion Andreas M. Rossbach, Tatyana Andrienko, Andrew P. Halestrap Department of Biochemistry, School of Medical Sciences, University Walk, Clifton, Bristol, BS8 1TD, UK E-mail: [email protected] Reperfusion injury is mediated by reactive oxygen species (ROS) produced during ischaemia–reperfusion (IR) which leads to the opening of the mitochondrial permeability transition pore (MPTP). ROS production has previously been monitored in isolated cardiomyocytes by fluorescence microscopy with varying methods, cell types and results. We have developed a real-time ischaemia–reperfusion protocol for fluorescent microscopy that exposes isolated rat cardiomyocytes to conditions closely matching global ischaemia–reperfusion in the perfused heart. Temperature and [O2] are tightly controlled and continuously monitored. We have previously confirmed that neither DHE nor DCF are suitable dyes to monitor ROS production in situ. Instead, we have employed the mitochondria-targeted H2O2-sensitive fluorescent dye MitoPY1 for live monitoring of ROS production. We have also targeted Ca2 +, ΔΨm and the cells' oxidative state with additional co-stains and markers. In addition, we are developing a protocol for real-time multiwavelength surface fluorescence and reflectance measurement to monitor autofluorescence and ROS production in the more pathophysiologically relevant Langendorff-perfused rat heart. Our results to date show that fluorescence of MitoPY1 in cells remains stable in single myocytes during the normoxic equilibration period but increases briefly during the initial period of simulated ischaemia. A large rise in fluorescence is observed during reperfusion prior to hypercontraction and death of the cells. We aim to establish an experimental method using single myocytes that complements the reflectance/fluorescence studies in the perfused hearts to elucidate further the cellular events during IR.
doi:10.1016/j.bbabio.2012.06.273