BOOK REVIEWS

<div><p>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 <i>in vitro</i> 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. </p> </div

Reconstitution with phosphorylation resistant CcO Vb subunit attenuates hypoxia induced CcO dysfunction and ROS production.

<p>siRNA resistant (SiR) - wild type (WT) or S40A mutant CcO Vb were expressed in RAW 264.7 macrophages with CcO Vb knockdown (VbKD). A) Immunoblot of cell lysates showing CcO Vb levels in control, CcO Vb knockdown (VbKD) and si-RNA resistant CcO Vb expressing VbKD cells. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. Blots were stained with CcO Vb and SDHA antibodies. B) Blue Native PAGE performed with mitochondrial proteins from all cell types. 150µg of mitochondria from each sample was solubilized with Lauryl maltoside as described in Materials and Methods. Electrophoresis was carried out in a 6-13% gradient gel. Gel was destained to remove excess Coomassie stain and the bands were imaged in a scanner. C&D) Control, VbKD and SiR-wild type and S40A mutant cells were maintained under either normoxia or hypoxia. Mitochondria were isolated and proteins (25µg each) were used to measure CcO activity (C) and ROS production by Amplex red oxidation (D). n=4. **, p<0.005; ***, p<0.001.</p

Effects of antioxidants on mitochondrial PKAα subunit level and activity.

<p>RAW 264.7 macrophages were subjected to hypoxia for 12h with or without addition of 1µM Mito-CP or 10mM N-Acetyl Cysteine. At the end of hypoxia, part of the cells was used for measuring ROS production by DCFDA oxidation and the remaining for mitochondria isolation. Protein was estimated by Lowry’s method. A) PKA activity and ROS production (n=3). After hypoxia cells were plated in 96 well plate in phosphate buffered saline and incubated with DCFDA (1µM) for 15 minutes. Fluorescence was measured at Excitation 525nm and Emission 575nm. Corresponding PKA activity was measured in 10µg of mitochondrial protein, B) PKAα protein level. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. PKAα and CcO IVi1 antibodies were used for immunoblotting. Relative band intensities are given in parantheses. The blot is representative of two separate experiments. C) Effect of Mito-CP on CcO activity under hypoxia. CcO activity was measured with 10µg of mitochondria (n=4). **, p<0.005.</p

Role of hypoxia induced PKA on mitochondrial respiratory capacity.

<p>Macrophages grown under normoxic or hypoxic conditions in the presence or absence of H89 or KH7 were used for measuring Oxygen consumption in a Seahorse XF extracellular flux analyzer. Cells were maintained under either hypoxia or normoxia in presence of inhibitors for 12h. Before measurement of respiration, the cells were washed with fresh medium without inhibitors and plated on XF24 plates and incubated for additional 3h. A) Respiration measurement in presence of H89 (1µM) and KH7 (10µM) is shown. DNP=2,3 dinitrophenol. Rot/AA=Rotenone and Antimycin. This is a representative profile of three replicates B) The maximum OCR of cells treated with H89 or KH7 under hypoxia. Maximum OCR was calculated by subtracting residual OCR after adding Rotenone and Antimycin from OCR measured after uncoupling with 100µM DNP. The values were normalized to 100µg of total cellular protein. C) Maximum OCR in cells treated with different concentration of H89. Maximum OCR calculation and normalization were as described for B). Protection against loss of respiratory capacity by H89 was significant at 300nM and 500nM as indicated, compared to untreated hypoxic control. The values were derived from three separate experiments.*, p<0.05;**, p<0.005.</p

Hypoxia increases the intra mitochondrial pool of PKAα.

<p>A) Immunoblots of the PKAα subunit in mitochondria from normoxic and hypoxic macrophages. For trypsin treatment, mitochondria (100µg) were incubated with 15µg of trypsin at 4°C for 20 min. Tom20 and SDHA were used as outer and inner mitochondrial membrane markers, respectively. B) Mitochondria (100 µg) from normoxic and hypoxic cells were incubated in increasing amounts of digitonin (0-1% digitonin), in the presence of 15 µg Trypsin, as described in Materials and Methods. Mitochondrial pellets were separated by SDS PAGE and immunoblotted for PKAα, CcO IVi1 (inner membrane marker) and TOM20 (outer membrane marker). Upper panel: Immunoblots for normoxic cell mitochondria; lower panel: immunoblot for hypoxic cell mitochondria. The immunoblots are representative of three independent experiments. C) Immunofluorescence images of COS-7 cells transfected with cDNA for FLAG-tagged PKAα and grown under normoxic or hypoxic conditions. Cells were stained for FLAG (green) and CcO I (red) as described in Material and Methods.</p

Hypoxia induces mitochondrial PKA activity in RAW 264.7 macrophages.

<p>A) PKA activity in isolated mitochondrial and cytosol of RAW 264.7 macrophages subjected to hypoxia for 0-12h. The activity of the corresponding normoxic fraction was taken as 100% activity. The inset shows immunoblot of representative mitochondrial and cytosolic fractions for cross contamination. Blot was probed with antibodies to Actin and TOM20 as markers of cytosol and mitochondria. 30µg of mitochondrial and cytosolic proteins were loaded in each well. B) PKA activity in mitochondria isolated from normoxic and hypoxic cells. H89 (1µM) and MPI (1µM) were added at the start of hypoxia (n=3) C) Effect of KH7 on hypoxia induced mitochondrial PKA activity. RAW 264.7 cells were treated with indicated concentrations of KH7 at the start of hypoxia (n=3) D) Immunoblots showing level of PKAα in mitochondria isolated from normoxic and hypoxic cells with or without KH7 (10µM) treatment. Values in parantheses underneath each blot are relative intensities of the bands. Blot is representative of two separate experiments. E) cAMP levels in 10µg of whole cell lysates and isolated mitochondria from normoxic and hypoxic cells. The difference in cAMP level between normoxic and hypoxic total lysates was not significant. *, p<0.01; **, p<0.005; ***,p<0.001.</p

PKA inhibitor and mitochondrial antioxidant attenuate ischemia-reperfusion injury in perfused mouse heart.

<p>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-<i>Q</i>. <i>Spectrum</i> 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.</p

Effect of Mito-CP on mitochondrial membrane potential, ATP levels, Hydrogen peroxide levels and subcellular mitochondrial localisation.

<p><b><i>(A)</i></b> Mitochondrial membrane potential in Daudi cells and PBMCs with and without Mito-CP (1μM) and Dec-TPP⁺ (1μM) under hypoxia (5%O₂) and normoxia were measured using JC-1 dye. Data were obtained from three separate experiments and are expressed as mean ± SEM. * and **, significantly different when compared to control p<0.05 and p<0.01 respectively. (<b><i>B</i></b>) Cellular ATP levels in Daudi cells and PBMC were determined using a bioluminescence based assay kit with or without Mito-CP (1μM) and Dec-TPP⁺ (1μM) treatment under hypoxia (5%O₂) and normoxia. ATP concentration were determined using the standard ATP provided with the manufacturer’s kit (Molecular Probes, Eugene, OR, USA). Bar graph represented was normalised to protein concentration. Data were obtained from three separate experiments and are expressed as mean ± SEM. * and **, significantly different when compared to control p<0.05 and p<0.01 respectively. (<b><i>C</i></b>) Hydrogen peroxide levels in Daudi cells and PBMCs with and without Mito-CP (1μM) and Dec-TPP⁺ (1μM) under hypoxia (5%O₂) and normoxia were measured using Amplex red assay. Concentration of hydrogen peroxide were obtained using the standard hydrogen peroxide provided with the amplex red reagent manufacturer’s kit (Molecular Probes, Eugene, OR, USA). Bar graph represented was normalised to protein concentration. Data were obtained from three separate experiments and are expressed as mean ± SEM. *, significantly different when compared to control p<0.05. (<b><i>D</i></b>) Shows EPR monitoring of mitochondrial localization of Mito-CP in Daudi cells and PBMC under hypoxia (5%O₂) and normoxia. <b><i>(i)</i></b> EPR spectra were obtained from mitochondrial fraction of Daudi cells and PBMCs treated with and without Mito-CP. <b><i>(ii)</i></b> As was done for (i), Daudi cells and PBMCs were treated with Mito-CP (1μm). <b><i>(iii)</i></b> As was done for (i), Daudi cells and PBMCs were treated with Mito-CP under hypoxia. The parameters used in EPR spectra follow: G_xx = 2.0089, G_yy = 2.0058, G_zz = 2.0021, A_xx = 5.6, A_yy = 5.3, A_zz = 34 G, β = 60°, R_xx = 8.9×10⁷, r_yy = 8.9x10⁷, r_zz = 1.0x10⁷s^-i, Ψ = 60°, C₂₀ = 2.00. (<b><i>E</i></b>) Shows quantified data of total EPR signal intensity of Mito-CP in Daudi cells and PBMC under normoxia and hypoxia. Concentration of cell lysate used for analysis was determined by Bradford method and equal amount of concentration in each sample was analysed in EPR spectrometer. EPR signal intensity was normalized to the peak intensity obtained by Mito-CP alone before treatment. Data were obtained from three separate experiments and are expressed as mean ± SEM. * and ** denotes significantly different when compared to control p<0.05 and p<0.01 respectively.</p

Effect of Mito-CP on BAX mRNA expression levels and HIF-1α protein expression levels.

<p><b><i>(A)</i></b> Real time polymerase chain reaction were performed to quantify BAX mRNA levels in Daudi and PBMC with and without Mito-CP (1μM) treatment under hypoxia (5%O₂) and normoxia. Amplified BAX mRNA was analysed by melting curve analysis and fold change in expression in each experimental group were calculated by 2^-ΔΔCT. Data were obtained from three separate experiments and are expressed as mean ± SEM. * and ** denotes significantly different when compared to control p<0.05 and p<0.01 respectively. (<b><i>B</i></b>) Daudi cells and PBMCs were treated with and without Mito-CP (1μM) under hypoxia (5%O₂) and normoxia. HIF-1α levels were measured by western blot and densitometric analysis are shown as bar graphs. Data were obtained from three separate experiments and were expressed as by mean ± SEM. * denotes significantly different when compared to control p<0.05.</p

Effect of Mito-CP induced apoptosis by Annexin V-FITC staining.

<p>Characteristic phenomenon of Phosphotidyl serine externalisation and disruption in cell membrane in apoptotic cells were observed by staining cells with Annexin V FITC and Propidium Iodide. Daudi cells and PBMCs were treated with and without Mito-CP (1μM) under hypoxia (5% O₂) and normoxia for a period of 6 h. Cells were visualised under confocal laser scanning microscope and photographed. Images obtained were analysed by Image J software and the data is expressed as bar graph. Data were obtained from five different experiments and are expressed as mean ±SEM. * and **, significantly different compared to control p<0.05 and p<0.01 respectively.</p