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Oxidative stress induced mitochondrial protein kinase A mediates cytochrome c oxidase dysfunction.

Srinivasan S, Spear J, Chandran K, Joseph J, Kalyanaraman B, Avadhani NG - PLoS ONE (2013)

Bottom Line: Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS).Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production.The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

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PKA inhibitor and mitochondrial antioxidant attenuate ischemia-reperfusion injury in perfused mouse heart.A) Schematic drawing showing the protocol of ischemia-reperfusion used for each group. B) Myocardial ventricular volumes (cm3) of mouse hearts after 30 min ischemia and 120 min reperfusion. Insets show representative photographs of midventricular myocardium after staining with 1% triphenyltetrazolium chloride solution to delineate the necrotic zone. For treatments, H89 (1µM) (n=6) or Mito-CP (1µM) (n=3) were maintained in the perfusion medium throughout the duration of the experiment. C) cAMP levels in 10µg of total homogenate and isolated mitochondria from control and ischemia-reperfused mouse heart tissues. D-F) Mitochondria were isolated from non-ischemic, ischemia-reperfused and H89 or Mito-Q preconditioned heart and used for measuring Complex I (D), III (E), IV (F) and Aconitase (H) activities (n=3). Protein used was as follows: Complex I and III, 25µg, Complex IV, 1µg and Aconitase, 30µg G) Low temperature EPR spectra of non-ischemic hearts and hearts subjected to ischemia reperfusion with or without preconditioning with H89 or Mito-Q. Spectrum from MPTP treated brain slice is shown as positive control for [3Fe-4S]+ aconitase. Conditions of spectroscopy: microwave power and frequency, 5 mW and 9.635 GHz; modulation amplitude and frequency, 10 G and 100 kHz; time constant, 0.01 s; temperature range, 4-50 K. *, p<0.01; **, p<0.005.
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pone-0077129-g006: PKA inhibitor and mitochondrial antioxidant attenuate ischemia-reperfusion injury in perfused mouse heart.A) Schematic drawing showing the protocol of ischemia-reperfusion used for each group. B) Myocardial ventricular volumes (cm3) of mouse hearts after 30 min ischemia and 120 min reperfusion. Insets show representative photographs of midventricular myocardium after staining with 1% triphenyltetrazolium chloride solution to delineate the necrotic zone. For treatments, H89 (1µM) (n=6) or Mito-CP (1µM) (n=3) were maintained in the perfusion medium throughout the duration of the experiment. C) cAMP levels in 10µg of total homogenate and isolated mitochondria from control and ischemia-reperfused mouse heart tissues. D-F) Mitochondria were isolated from non-ischemic, ischemia-reperfused and H89 or Mito-Q preconditioned heart and used for measuring Complex I (D), III (E), IV (F) and Aconitase (H) activities (n=3). Protein used was as follows: Complex I and III, 25µg, Complex IV, 1µg and Aconitase, 30µg G) Low temperature EPR spectra of non-ischemic hearts and hearts subjected to ischemia reperfusion with or without preconditioning with H89 or Mito-Q. Spectrum from MPTP treated brain slice is shown as positive control for [3Fe-4S]+ aconitase. Conditions of spectroscopy: microwave power and frequency, 5 mW and 9.635 GHz; modulation amplitude and frequency, 10 G and 100 kHz; time constant, 0.01 s; temperature range, 4-50 K. *, p<0.01; **, p<0.005.

Mentions: In order to extend our findings on the role of PKA in hypoxia in cell culture model to hypoxic conditions found in pathologies, we tested the effect of PKA modulators on myocardial ischemia-reperfusion injury. The infarct size resulting from temporary global ischemia and 2 hours of reperfusion was measured in mouse hearts perfused with PKA modulators. In untreated ischemia-reperfused hearts, the infarct size amounted to 61.7 % of the total area at risk (Figure 6B). The hearts that were perfused with H89 showed nearly 50% decrease in infarct size, suggesting protective effect of PKA inhibition during ischemia. Pre-perfusion with Mito-CP, a mitochondria targeted antioxidant resulted in nearly 60% reduction in necrotic tissue compared to untreated ischemic heart. The level of protection was comparable with that of H89 treatment. Consistent with our results with hypoxic cell mitochondria, the mitochondrial cAMP levels in ischemic hearts was not increased. The total tissue content of cAMP was increased marginally (Figure 6C).


Oxidative stress induced mitochondrial protein kinase A mediates cytochrome c oxidase dysfunction.

Srinivasan S, Spear J, Chandran K, Joseph J, Kalyanaraman B, Avadhani NG - PLoS ONE (2013)

PKA inhibitor and mitochondrial antioxidant attenuate ischemia-reperfusion injury in perfused mouse heart.A) Schematic drawing showing the protocol of ischemia-reperfusion used for each group. B) Myocardial ventricular volumes (cm3) of mouse hearts after 30 min ischemia and 120 min reperfusion. Insets show representative photographs of midventricular myocardium after staining with 1% triphenyltetrazolium chloride solution to delineate the necrotic zone. For treatments, H89 (1µM) (n=6) or Mito-CP (1µM) (n=3) were maintained in the perfusion medium throughout the duration of the experiment. C) cAMP levels in 10µg of total homogenate and isolated mitochondria from control and ischemia-reperfused mouse heart tissues. D-F) Mitochondria were isolated from non-ischemic, ischemia-reperfused and H89 or Mito-Q preconditioned heart and used for measuring Complex I (D), III (E), IV (F) and Aconitase (H) activities (n=3). Protein used was as follows: Complex I and III, 25µg, Complex IV, 1µg and Aconitase, 30µg G) Low temperature EPR spectra of non-ischemic hearts and hearts subjected to ischemia reperfusion with or without preconditioning with H89 or Mito-Q. Spectrum from MPTP treated brain slice is shown as positive control for [3Fe-4S]+ aconitase. Conditions of spectroscopy: microwave power and frequency, 5 mW and 9.635 GHz; modulation amplitude and frequency, 10 G and 100 kHz; time constant, 0.01 s; temperature range, 4-50 K. *, p<0.01; **, p<0.005.
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pone-0077129-g006: PKA inhibitor and mitochondrial antioxidant attenuate ischemia-reperfusion injury in perfused mouse heart.A) Schematic drawing showing the protocol of ischemia-reperfusion used for each group. B) Myocardial ventricular volumes (cm3) of mouse hearts after 30 min ischemia and 120 min reperfusion. Insets show representative photographs of midventricular myocardium after staining with 1% triphenyltetrazolium chloride solution to delineate the necrotic zone. For treatments, H89 (1µM) (n=6) or Mito-CP (1µM) (n=3) were maintained in the perfusion medium throughout the duration of the experiment. C) cAMP levels in 10µg of total homogenate and isolated mitochondria from control and ischemia-reperfused mouse heart tissues. D-F) Mitochondria were isolated from non-ischemic, ischemia-reperfused and H89 or Mito-Q preconditioned heart and used for measuring Complex I (D), III (E), IV (F) and Aconitase (H) activities (n=3). Protein used was as follows: Complex I and III, 25µg, Complex IV, 1µg and Aconitase, 30µg G) Low temperature EPR spectra of non-ischemic hearts and hearts subjected to ischemia reperfusion with or without preconditioning with H89 or Mito-Q. Spectrum from MPTP treated brain slice is shown as positive control for [3Fe-4S]+ aconitase. Conditions of spectroscopy: microwave power and frequency, 5 mW and 9.635 GHz; modulation amplitude and frequency, 10 G and 100 kHz; time constant, 0.01 s; temperature range, 4-50 K. *, p<0.01; **, p<0.005.
Mentions: In order to extend our findings on the role of PKA in hypoxia in cell culture model to hypoxic conditions found in pathologies, we tested the effect of PKA modulators on myocardial ischemia-reperfusion injury. The infarct size resulting from temporary global ischemia and 2 hours of reperfusion was measured in mouse hearts perfused with PKA modulators. In untreated ischemia-reperfused hearts, the infarct size amounted to 61.7 % of the total area at risk (Figure 6B). The hearts that were perfused with H89 showed nearly 50% decrease in infarct size, suggesting protective effect of PKA inhibition during ischemia. Pre-perfusion with Mito-CP, a mitochondria targeted antioxidant resulted in nearly 60% reduction in necrotic tissue compared to untreated ischemic heart. The level of protection was comparable with that of H89 treatment. Consistent with our results with hypoxic cell mitochondria, the mitochondrial cAMP levels in ischemic hearts was not increased. The total tissue content of cAMP was increased marginally (Figure 6C).

Bottom Line: Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS).Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production.The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

Show MeSH
Related in: MedlinePlus