Adaptive changes in the expression of nuclear and mitochondrial encoded subunits of cytochrome c oxidase and the catalytic activity during hypoxia C. Vijayasarathy 1, * ,† , Shirish Damle 1, *, Subbuswamy K. Prabu 1, *, Cynthia M. Otto 2 and Narayan G. Avadhani 1 1 Department of Animal Biology and 2 Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA The effects of physiologically relevant hypoxia on the catalytic activity of cytochrome c oxidase (CytOX), mito- chondrial gene expression, and both nuclear and mito- chondrial encoded CytOX mRNA levels were investigated in murine monocyte macrophages, mouse C2C12 skeletal myocytes and rat adrenal pheochromocytoma PC12 cells. Our results suggest a coordinated down regulation of mito- chondrial genome-coded CytOX I and II and nuclear genome-coded CytOX IV and Vb mRNAs during hypoxia. Hypoxia also caused a severe decrease in mitochondrial transcription rates, and associated decrease in mitochondrial transcription factor A. The enzyme from hypoxia exposed cells exhibited altered subunit content as revealed by blue native gel electrophoresis. There was a generalized decline in mitochondrial function that led to a decrease in total cellular heme and ATP pools. We also observed a decrease in mitochondrial heme aa 3 content and decreased levels of CytOX subunit I, IV and Vb, though the catalytic efficiency of the enzyme (TN for cytochrome c oxidase) remained nearly the same. Increased glycolytic flux and alterations in the kinetic characteristics of the CytOX might be the two mechanisms by which hypoxic cells maintain adequate ATP levels to sustain life processes. Reoxygenation nearly com- pletely reversed hypoxia-mediated changes in CytOX mRNA contents, rate of mitochondrial transcription, and the catalytic activity of CytOX enzyme. Our results show adaptive changes in CytOX structure and activity during physiological hypoxia. Keywords: hypoxia; cytochrome c oxidase; subunit content; mitochondrial genome transcription. Cytochrome c oxidase (CytOX) is the terminal oxidase of the mitochondrial electron transport chain [1–5], which catalyzes the reduction of the dioxygen (O 2 ) to water and harnesses the free energy of the reaction to phosphorylate ADP to ATP. Heme and Cu, which transfer electrons from ferrocytochrome c to molecular oxygen, constitute the catalytic site of the enzyme complex. The three catalytic subunits, CytOX I, II and III are coded by the mitochon- drial DNA and are synthesized within mitochondria. Heme a, heme a 3 and Cu b are ligated to subunit I, while Cu a is ligated to subunit II which is also the binding site for cytochrome c [4,5]. The remaining 10 subunits of the mammalian enzyme, namely, IV, Vb, VIa, VIb, VIc, VIIa, VIIb and VIII are encoded by the nuclear genome, synthesized in the cytosol and imported into mitochondria [1–3]. Some of the nuclear-encoded subunits in the mam- mals are regulated developmentally and occur as tissue specific isoforms [6,7]. Although the nuclear encoded subunits, such as CytOX VIa and VIb, have been shown to enhance the catalytic efficiency of the enzyme [8,9], the precise role of many nuclear-encoded subunits in the mammalian enzyme complex remains unknown. Oxygen as a substrate and heme as a prosthetic group, are closely interlinked in the function of the enzyme complex. Studies in yeast have shown that both oxygen and heme act as physiological modulators and regulate the expression of the enzyme complex [10]. In the yeast CytOX complex, the nuclear encoded subunit V is expressed as two distinct isoforms, Va and Vb, that are regulated by heme and O 2 [11]. In the mammalian systems, however, the differential expres- sion of nuclear encoded subunits in response to different physiological factors has not been investigated in detail. In a previous study we demonstrated that administration of inhibitors of heme biosynthesis (succinyl acetone and cobalt chloride) to mice, resulted in a 50% reduction in mitochondrial genome encoded CytOX I and II mRNAs and nuclear genome encoded CytOX Vb mRNA in heme depleted tissues [12]. Heme depletion was also accom- panied by a 50–80% reduction in intramitochondrial Correspondence to N. G. Avadhani, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA, 19104, USA. Fax: + 215 573 6651, Tel.: + 215 898 8819, E-mail: narayan@vet.upenn.edu Abbreviations: CytOX, cytochrome c oxidase; mtTFA, mitochondrial transcription factor A; SMP, submitochondrial particles; TN, turn- over number; BN/PAGE, blue native gel electrophoresis. *Note: these authors contributed equally to this work. Present address: UAE University, Faculty of Medicine and Health Sciences, Department of Biochemistry, Al Ain, United Arab Emirates. (Received 8 November 2002, revised 18 December 2002, accepted 3 January 2003) Eur. J. Biochem. 270, 871–879 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03447.x transcription and translation rates. Surprisingly, the enzyme from heme-depleted tissues showed twofold to fourfold higher turnover rates for cytochrome c oxidation, suggesting alterations in the kinetic characteristics of the enzyme following heme reduction. These studies suggested that heme might regulate not only the mammalian CytOX gene expression but also the catalytic activity of the enzyme by affecting its stability or composition. Although succinylacetone and CoCl 2 are known inhibitors of heme biosynthesis, these agents also elicit nonspecific and toxic effects in animals. To ascertain that the effects of these agents, on the catalytic activity and subunit composition of CytOX were related to their hypoxia-specific effects, we have extended our investigation to physiological hypoxia in cultured cells. We focused our attention on the expression of mitochondrial genome encoded catalytic subunits I and II, and the nuclear genome encoded subunits, IV, Vb and VIIa. The mammalian CytOX subunit IV is a homolog of the yeast subunit V, with the latterexpressedinanO 2 and heme regulated manner. In this study therefore, we investigated how O 2 modulates the expression of mRNAs for CytOX subunits and also the catalytic activity of the enzyme complex. Our results show that the levels of some of the select mitochondrial and nuclear genome encoded CytOX mRNAs are uniformly down regulated during hypoxia. Results also show changes in the composition and activity of the enzyme complex, which is accompanied by alterations in cellular ATP and heme pools. Materials and methods Cell culture The following cell lines were used in this study: RAW 264.7 mouse monocyte macrophages, C2C12 mouse skeletal muscle cells and PC12 rat adrenal pheochromocytoma cells. Mouse macrophages were cultured in Dulbecco’s modified Eagles medium supplemented with 10% (v/v) heat inactivated fetal bovine serum (Gibco) and 100 lgÆmL )1 penicillin/streptomycin. C2C12 cells were cultured as mono- layers in Falcon tissue culture dishes in a medium containing 10% (v/v) fetal bovine serum (Gibco) and 90% (v/v) high glucose Dulbecco’s modified Eagles medium supplemented with 100 lgÆmL )1 penicillin/streptomycin. The myocytes were induced to differentiate into myoblasts by replacing the medium at confluence with a fresh medium containing 2% (v/v) fetal bovine serum. These myoblasts were further grown for a period of 3 days when 70–80% of the cells fuse to form multinucleated myotubes. Rat pheochromocytoma (PC12) cells were cultured in Dulbecco’s modified Eagle’s medium F-12 containing 15 m M Hepes buffer, L -glutamine, 10% fetal bovine serum and penicillin/streptomycin (100 lgÆmL )1 ). All the cells were grown to 80–90% confluence in a controlled humidified environment (21% O 2 ,5%CO 2 , remainder N 2 at 37 °C). Exposure of cells to hypoxia The normal range of tissue oxygen tension (in nonpul- monary tissues) measured under in vivo conditions ranges from 5 to 71 Torr, with most tissues maintaining a pO 2 of 40 Torr or less. Simulation of realistic in vivo hypoxia requires that O 2 tension is maintained at less than 5 Torr [13]. We have used modular incubator chambers (Billups- Rothernberg, CA, USA) for creation of a nonfluctuating hypoxic environment. The chambers were maintained at 37 °C in a humidified incubator. Cells grown in tissue culture dishes were introduced into the chambers that were directly connected to certified premixed compressed gas cylinders. The modular chambers were purged with a constant flow of premixed gas that was certified to contain 1 Torr (hypoxic) or 141 Torr of oxygen (normoxic), all with 5% CO 2 and balance nitrogen (BOC gases; Murray Hill, NJ, USA). Based on the barometric pressure and atmospheric humidity, these levels approximately corres- pond to 0.1 and 21% of oxygen, respectively. Normally the cells were exposed to either normoxic or hypoxic conditions for a period of 6–12 h. In each experiment, a group of cells that were exposed to hypoxic conditions for 6–12 h were re-exposed to normoxic conditions for an additional period of 6 h. Collection of cells and fractionation At the end of the culture period, the cells were rapidly chilled on ice and were rinsed twice with ice cold NaCl/P i to remove any residual, media, and dead cells. The cells were harvested by centrifugation at 1500 g for 5 min and subsequently used to prepare total cell lysates or subcellular fractions as needed. For the preparation of total cell extracts the cells were suspended in a lysis buffer (100 m M Hepes, pH 7.5, 10% Sucrose, 0.1 m M dithiothreitol, 0.1% Chaps and 150 m M NaCl and protease inhibitors [5 lgÆmL )1 pepstatin A, 5 lgÆmL )1 aprotinin, and 1 m M phenylmethanesulfonyl fluoride]) and lysed by three cycles of freezing and thawing in liquid nitrogen. The lysates were centrifuged at 4 °Cfor 30 min in an Eppendorf centrifuge tube to remove debris and unlysed cells. The supernatant was collected and stored at )80 °C till further use. Protein concentrations were determined using Lowry’s method [14]. Mitochondria were isolated form intact cells by differen- tial centrifugation as described earlier [17]. The cell pellets were suspended in H medium (70 m M sucrose, 220 m M mannitol, 2.5 m M Hepes, pH 7.4 and 2 m M EDTA) and ruptured by homogenization through a Dounce homo- genizer. Submitochondrial particles (SMP) were prepared according to the method of Pederson et al. [18], and washed three times with mitochondrial isolation buffer. All steps of subcellular fractionation and isolation of SMP were carried out at 4 °C. Northern blot analysis RNA was isolated from cells grown under hypoxic and normoxic conditions using the guanidine thiocyanate procedure previously described [15]. Total RNA (25 lg) was analyzed by Northern blot hybridization with 32 P-labeled mouse cDNA probes for CytOX subunits I, II, IV, Vb and VIIa under standard conditions (Schleicher & Schuell Laboratory Manual). Gel-purified double stranded DNA probes were labeled with 32 PdCTP (6000 CiÆmmol )1 , Dupont, NEN) by random primer extension using the Klenow polymerase. The same blots 872 C. Vijayasarathy et al.(Eur. J. Biochem. 270) Ó FEBS 2003 were stripped and rehybridized with a 32 P-labeled 18S DNA probe to evaluate loading levels [16]. The Northern blots were imaged and quantified using the Bio-Rad GS-525 Molecular Imager. Mitochondrial transcription The rate of transcription in isolated mitochondrial parti- cles was measured essentially as described previously [19]. Freshly prepared mitoplasts were suspended in RNA synthesis buffer at the final concentration of 5 mgÆmL )1 protein. The reaction mixture consisted of 10 m M Hepes, pH 7.4, 60 m M KCl, 10 m M MgCl 2 ,5m M 2-mercapto- ethanol, 10 m M KH 2 PO 4 (pH 7.4), 0.14 M sucrose, 2 m M ATP, 1 m M each of GTP and CTP, 5 m M pyruvate, 5 m M creatine phosphate, 0.2 mgÆmL )1 creatine phosphokinase and 100 l M each of 20 L -amino acids. The reaction was initiated by the addition of 200 lCiÆmL )1 of [ 32 P]UTP (400 CiÆmmol )1 ) and was allowed to proceed for 45 min at 33 °C. Aliquots were used to determine the level of 32 P incorporation in to RNA as described before [19]. At the end of incubation, the mitochondria were pelleted by centrifugation at 10 000 g for 10 min and 32 P-labeled mitochondrial RNA was isolated as previously described [15]. The rate of in vitro transcription was measured by dot blot analysis. For this purpose, the plasmid DNA carrying the mouse CytOX I and II encoding region of mitochon- drial DNA, was immobilized on a Nytran membrane (Schleicher & Schuell). The membrane was probed with 32 P-labeled mitochondrial RNA, subjected to autoradio- graphy and quantified in a Bio-Rad GS 525 Molecular Imager. Spectrophotometric analysis of CytOX activity and heme content CytOX was assayed in membrane fragments (SMP) by the method of Smith [20], wherein the rate of oxidation of ferrocytochrome cwas measured by following the decrease in absorbency of its a band at 550 nm. The reaction medium contained 50 m M PO 4 (pH 7.0), 1% sodium cholate, 80 l M ferrocytochrome c,1m M EDTA and 1–2 lg of protein in a total volume of 1 mL. Reaction rates were measured using Cary-1E spectrophotometer (Varian Instruments Walnut Creek, CA, USA). First order rate constants were calculated from mean values of four measurements. The heme aa 3 content was calculated from the difference spectra (dithio- nate/ascorbate reduced minus ferricyanide oxidized) of mitochondria or SMP solubilized in 2% lauryl maltoside using an absorption coefficient of 24 m M )1 Æcm )1 at 605– 630 nm [21]. Electrophoresis of proteins and immunoblot analysis Proteins were subjected to electrophoresis on 12–18% SDS/ polyacrylamide gels as described by Laemmli [22]. The conditions for immunoblot analysis of proteins were similar to that described earlier [23]. Polyclonal antibody against purified mouse mitochondrial transcription factor (mtTFA) was a gift from David Clayton (Howard Hughes Medical Institute, Chevy Chase, MD, USA). The immunoblot was developed using the Super Signal ULTRA chemilumines- cent substrate kit from Pierce Chemical Co. The blots were imaged and quantified in a Bio-Rad Fluor-S imaging system. Blue native gel electrophoresis of mitochondrial membrane complexes Blue native gel electrophoresis (BN/PAGE) was carried out following the method of Schagger and Von Jagow on 6–13% gradient acrylamide gels [24]. SMP (30–50 lg) were solubilized in ice-cold detergent buffer (1% digitonin, 0.1 m M EDTA, 50 m M NaCl, 10% glycerol, 20 m M Tris- Hcl, pH 7.4) and centrifuged at 100 000 g for 20 min to remove any insoluble material. The supernatant, 45 lLwas mixedwith5lL of loading dye (5% Serva Blue G, 500 m M amino-n-caproic acid, 100 m M Bis-Tris, pH 7.0) and analyzed by BN/PAGE. Marker proteins such as b-amy- lase, 200 kDa; apo-ferritin, 443 kDa and thyroglobulin, 669 kDa (Sigma Chemical Company) were included as standards. Electrophoresis was carried out initially at 100 V until the protein samples were within the stacking gel, and then at a constant current of 18 mA (500 V) for 5–6 h. The proteins were transblotted onto a poly(vinylidene difluoride) membrane and probed with subunit-specific monoclonal or polyclonal antibodies and appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. The immunoblot was developed using the Super Signal ULTRA chemiluminescent substrate kit (Pierce Chemical Co), imaged and quantitated in a Bio-Rad Fluor-S imaging sys- tem. Subunit-specific monoclonal antibodies for CytOX I, IV and Vb proteins were obtained from Molecular Probes (Eugene, OR, USA) and the specificity of each antibody was tested by immunoblot analysis of purified CytOX complex. Polyclonal antibody to rat liver F 1 ATPase was a kind gift from P. L. Pederson (Johns Hopkins University, Baltimore, MD, USA). Measurement of cellular ATP levels Cellular ATP levels were measured using a somatic cell ATP assay kit (Sigma Chemical Co, St Louis, MO, USA), which is based on the assay of ATP driven luciferin luciferase assay system. Cells were lysed with ATP releasing agent as per manufacturer’s protocol and ATP levels were measured in a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA), using appropriate controls and blanks. For measur- ing the respiration driven ATP synthesis, mitochondria were incubated for 3 min in a medium consisting of 150 m M KCl, 25 m M Tris/HCl pH 7.4, 2 m M EDTA, 10 m M KH 2 PO 4 , 0.25 M sucrose, 0.1% bovine serum albumin, 0.3 m M ADP, and 5 m M succinate. At the end of 3 min, mitochondria were solubilized and ATP levels were measured as described above. Assays for other enzyme activities Extracts of isolated mitochondria were assayed for NADH- ubiquinone oxidoreductase (Complex-1) [25], cytosolic fractions (105 000 g supernatant fractions) were used for assaying isocitrate dehydrogenase [26], hexokinase [27] and phosphofructokinase [28] enzyme activities by published methods. Ó FEBS 2003 Cytochrome c oxidase subunit expression in hypoxia (Eur. J. Biochem. 270) 873 Results As a measure of the hypoxic effect on CytOX gene expression, we measured the steady state CytOX mRNA levels. The Northern blot in Fig. 1 shows the effect of hypoxia on the levels of mitochondrial genome- and nuclear genome-coded mRNAs for CytOX subunits. There were no detectable changes in mRNA levels for CytOX subunits in macrophages (Fig. 1A), PC12 and C2C12 cells (results not shown) after 3 h of exposure to hypoxia. As CytOX has a low K m for oxygen, it is conceivable that an adaptive response to hypoxia might only be seen after a long period of exposure to low O 2 . Changes in mRNA levels became apparent only after 6 h of exposure to hypoxia. The mitochondrial genome coded CytOX I and II mRNAs were reduced by 60–70% in macrophages (Fig. 1A), PC12 cells (Fig. 1B) and differentiated C2C12 myotubes (Fig. 1C), after hypoxic exposures ranging from 6 to 10 h. The time point at which a 40–60% reduction in mRNA levels occurred varied between different cell types. Macrophages and C2C12 myotubes showed a 50% reduction in mRNA levels after 6 h of exposure to hypoxia, while PC12 cells showed a similar reduction after 10 h of exposure to hypoxia. In contrast, undifferentiated C2C12 myocytes did not show any change in mRNA levels even after 12-h exposure to hypoxia (Fig. 1D). Figure 1(A–C) shows the effects of hypoxia on the steady state levels of nuclear genome encoded CytOX IV and Vb mRNAs. There was no change in the levels of these mRNAs in macrophages at 6 h of hypoxia, the time point at which there was a 50% reduction in mitochondrial genome encoded subunit I and II mRNAs (Fig. 1A). However, changes in subunit IV and Vb mRNA levels became apparent after prolonged exposure to hypoxia. In both PC12 cells (Fig. 1B) and differentiated C2C12 myotube (Fig. 1C), CytOX IV and Vb mRNA levels were reduced by 30–60% at 10 h exposure to hypoxic conditions. These results suggest that changes in mitochondrial gene expres- sion precede changes in the expression of nuclear genome coded CytOX subunits during hypoxia. We also tested the level of mRNA for subunit VIIa, which is expressed as isolog H and L. The L isolog is ubiquitously expressed in all the tissues whereas the H isolog is detected in the heart and skeletal muscle tissues [3]. The Northern blot in Fig. 1E shows that in both macrophages and PC12 cells more than 50% reduction in CytOX VIIa (L) mRNA level was observed following 10 h of exposure to hypoxia. The mRNA levels reverted to near control levels in cells subjected to normoxia following exposure to hypoxia (Fig. 1E). These results together show that physiological hypoxia in cells causes (a) progressive decrease in the nuclear and mitochondrial genome encoded mRNAs for CytOX subunits and (b) the nuclear and mitochondrial genes coding for CytOX are coordinately down regulated during hypoxia. Although not shown, the reversibility of mRNA levels and also other biochemical parameters tested in this study were limited to hypoxic exposure up to a threshold limit. This threshold limit ranged from 10 to 16 h for different cells. Our results point to differential sensitivity of cell types to hypoxia, though the effect on CytOX gene expression was similar in all the cell types studied. Based on Fig. 1. Effects of hypoxia on the steady state levels of nuclear and mitochondrial coded CytOX mRNAs. Northern blot analysis was car- ried out with total RNA (25 lg each) from macrophages (A, E), PC12 cells (B,E), myotubes (C) and myocytes (D) exposed to normoxia (141 Torrr O 2 )orhypoxia(1TorrO 2 ) for 10 h. Hybridization with 32 P-labeled probes was carried out as described in the Materials and Methods section. The stripped blots were rehybridized with a [ 32 P]18S rDNA probe to determine the RNA loading. The blots were scanned in a Bio-Rad GS-525 Molecular Imager. The values were normalized to the 18S rRNA level in each lane. 874 C. Vijayasarathy et al.(Eur. J. Biochem. 270) Ó FEBS 2003 this observation we restricted our subsequent investigations to macrophages and PC12 cells. In order to understand the basis for reduced CytOX I and II mRNA levels, we studied the rate of transcrip- tion in mitochondria from cells cultured under hypoxic environment. Mitochondrial transcription rates were meas- ured by extent of incorporation of 32 P-labeled UTP into RNA. It is seen from Fig. 2A that mitochondria from PC12 cells and macrophages exposed to hypoxia for 8 h show 50% and about 75% reduced transcription, respectively. The effect on the transcription rates of CytOX I and II mRNAs was further ascertained by slot blot hybridization of nascent 32 P-labeled RNA to mitochondrial DNA frag- ment encoding the CytOX I and II mRNAs. The hybrid- ization patterns (Fig. 2B,C) show that the transcription of CytOX I and II mRNAs in both PC12 cells and macroph- ages were inhibited by over 60% when cells were exposed to hypoxia for 8 h. Because each strand of the mitochondrial genome is transcribed as a single unit originating from one or limited number of strand specific promoters [29], a reduction in subunit I and II transcript levels reflects an overall reduction in mitochondrial genome transcription rates. We also determined the levels of mitochondrial tran- scription factor mtTFA by immunoblot analysis. The 29 kDa MtTFA is a mitochondrial specific transcription factor coded by the nuclear genome, which is implicated in the regulation of mitochondrial genome transcription [29,30]. Both macrophages and PC12 cells had a 60–80% reduction in mtTFA levels following exposure to hypoxia as compared to the controls (Fig. 2D). However, mtTFA levels were restored to near normal levels following exposure of cells to normoxic conditions. It is likely that the reduced mtTFA level is a factor in reduced mitochondrial mRNA levels during hypoxia. Fig. 2. Hypoxia mediated inhibition of mitochondrial transcription. In organelle RNA synthesis with isolated mitochondrial particles from cells grown under control (141 Torrr O 2 ) and hypoxia (1 Torr O 2 ) for 10 h was carried out using [a- 32 P]UTP as described in Materials and methods. Rate of 32 P incorporation in the RNA fraction was determined as described before [21]. RNA isolated from in vitro incubated mitochondria (5 lgeach) was hybridized to CytOX subunit I and II encoding DNA from the mouse mitochondrial genome by slot blot hybridization. The blot was quantified in a Bio-Rad GS-525 Molecular Imager. Rates of 32 P incorporation by mitochondria from these cells (A), transcription rates as in PC12 cells (B) and macrophages (C) were shown. (D) shows the levels of mtTFA in PC12 cells and macrophages grown under normoxia and hypoxia by immunoblot analysis. Immunoblot analysis was carried out as described in Materials and methods using 30 lg mitochondrial protein in each case. Ó FEBS 2003 Cytochrome c oxidase subunit expression in hypoxia (Eur. J. Biochem. 270) 875 Oxygen is essential for the biosynthesis of heme and hence heme is considered, an oxygen sensor [31]. Addition- ally, our previous study [11] indicated that heme not only regulates the catalytic activity of the CytOX complex but may also affect its stability. Based on these observations, we determined heme aa 3 levels as well as the catalytic activity of the CytOX complex in cells exposed to physiologically relevant hypoxia. The heme aa 3 content of SMP directly represents the enzyme-associated heme. In both PC12 cells and macrophages a 38–55% decrease in heme aa 3 content was seen after 10 h of exposure to hypoxia (Table 1). At this time point, the CytOX activity (mol cytochrome c oxi- dizedÆmin )1 Æmg SMP )1 ) was reduced only marginally in PC12 cells but was decreased by 52% in macrophages (Table 1). With the accompanying reduction in heme aa 3 levels, the enzyme activity per mg SMP protein indicates a change in the catalytic efficiency (TN) of the enzyme complex in PC12 cells following exposure to hypoxia (Table 1). Hypoxia induced heme depletion in PC12 cells, probably caused a small, but significant increase in TN. However, both the CytOX activity and heme content were reduced by about 50% in macrophages exposed to hypoxia, thus indicating a major change in the CytOX content in these cells. These results suggest that the CytOX activity in the two cell types is differentially modulated in response to hypoxia. The heme a and a 3 moieties are associated with the mitochondrial genome encoded CytOX subunit I [4,5]. The effects of hypoxic inhibition of transcription on the subunit contents of the complex were assessed by BN/ PAGE, which allows the separation of large oligomeric complexes based predominantly on size. Equal amounts of SMP protein from control and hypoxia exposed macro- phages were resolved on BN/PAGE and transferred to PVDF membrane. The enzyme resolved as two major complexes, which comigrated with Apo-ferritin and b-amylase. Based on the rates of migration, the slow migrating complex may be a dimmer and the faster migrating complex migrating with an apparent molecular mass of 200 kDa may be the monomeric form. It is interesting that the levels of mitochondrial encoded CytOX I and nuclear encoded CytOX IV and Vb in both complexes were reduced, although we observed a more pronounced reduction in the putative dimeric form, which is thought to be the more active form (Fig. 3A). It is also seen that the level of ATPase complex as determined by immunoblotting with antibody to the F 1 ATPase did not change under these conditions (Fig. 3B). Quantification of the blots shows that the levels of CytOX subunits I, IV and Vb were reduced by 50–75% in the two complexes combined as compared to cells grown under normoxia (see Fig. 3C). A nearly 50% reduction in enzyme activity (Table 1) under hypoxia seems to accompany a change in the subunit content of CytOX subunits I, IV and Vb as seen from BN/PAGE analysis (Fig. 3A). Table 1. Effect of hypoxia on CytOX activity, mitochondrial heme aa 3 content and TN (s )1 ) in macrophages and PC12 cells. CytOX activity in submitochondrial particles (SMP) from standard (141 Torr O 2 ) and hypoxia (1 Torr O 2 ) exposed cells were assayed by measuring the rate of oxidation of ferrocytochrome c at 550 nm. Ferrocytochrome c concentrations were determined using an absorption coefficient (reduced-oxidized) at 550 nm of 21.1 m M )1 Æcm )1 andthevaluesexpressedasmolÆmin )1 Æmg )1 protein. For the measurement of heme aa3 levels, the SMP were solubilized in laurylmaltoside and heme aa3 levels were calculated from the difference spectra (dithionate/ascorbate reduced vs. air oxidized) using an absorption coefficient 24 m M )1 Æcm )1 at 605–630 nm. Values represent average of three separate experiments. TN, turnover number. Macrophages PC12 Cells Normoxia Hypoxia Normoxia Hypoxia CytOX activity (nmolÆmin )1 Æmg )1 SMP) 2429 1160 2633 2166 Heme aa3 content (nmolÆmg )1 SMP )1 ) 0.225 0.103 0.179 0.112 TN (nmolÆnmol heme aa À1 3 Æs )1 ) 180 187 245 314 Fig. 3. Hypoxia induced changes in CytOX complex. SMP (30 lg protein) from standard (141 Torr O 2 )orhypoxia(1TorrO 2 )exposed cells were solubilized by treatment with 1% digitonin and analyzed by BN/PAGE on 6–13% acrylamide gels as described in Materials and methods. Proteins were transblotted to poly(vinylidene difluoride) membrane and probed with subunit specific monoclonal antibodies and HRP-conjugated anti-(mouse IgG) Ig (A). Stripped blots were also probed with antibody to F 1 ATPaseandusedasaloadingcontrol (B). Blots were imaged and quantified using BIO-RAD GS-525 Molecular imager and the difference in band intensities were depicted as a bar chart (C). 876 C. Vijayasarathy et al.(Eur. J. Biochem. 270) Ó FEBS 2003 We also tested other mitochondrial functional param- eters including ATP synthesis and the activities of the Krebs cycle enzyme, isocitrate dehydrogenase, and the electron transport enzyme NADH:ubiqunone oxidoreduc- tase (Complex I). As shown in Table 2, there was a marked difference in the total cell ATP synthesis as well as mitochondrial respiration-coupled ATP synthesis in macrophages and PC12 cells. The rate of ATP synthesis in control PC12 cells was nearly twice that of macrophages suggesting vastly different energy needs of these cell types. The mitochondrial respiration-coupled ATP synthesis was inhibited in cells grown under hypoxic conditions but the degree of inhibition was dependent on cell type. In macrophages, there was a 22% reduction in ATP synthesis compared to a steep 56% reduction in PC12 cells. The activities of isocitrate dehydrogenase and complex I were inhibited in both cell types by 35–50% level of control cells. Most notably, the glycolytic pathway enzymes, hexokinase and PFK were increased nearly two fold in both cell types (Table 2). These results are consistent with the generalized down regulation of mitochondrial functions during hyp- oxia and a compensatory up-regulation of alternate energy generating systems. Discussion Studies in yeast have demonstrated that oxygen acts as a molecular switch and alters the expression of the two nuclear genome coded isoforms of CytOX V [10,32]. The regulation of genes CytOX5a and CytOX5b, coding for the two isofoms Va and Vb, parallels that of genes CYC1 and CYC7, which encode iso-1 and iso-2 of yeast cytochrome c, respectively. CytOX 5a and CYC1 are coexpressed under aerobic conditions (O 2 >0.5l M ), whereas CytOX 5b and CYC7 are co-expressed under hypoxic (O 2 <0.5l M )and heme deficient conditions [11]. The coexpression of specific subunit V and cytochrome c isoforms indicates that these isoform pairs act synergistically to regulate electron transfer rates in enzyme function. These variant subunit isoforms have been shown to affect the turnover rate (TN) of the holoenzyme markedly by altering the rates of intramole- cular electron transfer between heme a and the binuclear reaction center. Thus the yeast, CytOX V, functions as an oxygen/heme sensor [32]. This investigation was undertaken, to determine the effect of hypoxia on (a) CytOX gene expression and (b) CytOX activity. The objective was to determine if oxygen/heme dependent regulation of mammalian CytOX genes is similar to that observed in the yeast system. Our results show that the levels of mitochondrial and nuclear genome encoded CytOX mRNAs are uniformly reduced during hypoxia (Fig. 1). We show that the reduction in mitochondrial mRNAs may be due to reduced mitochondrial genome transcription (Fig. 2). Although, the precise reasons for hypoxia-induced reduction in the levels of nuclear genome coded CytOX Vb and IV mRNA remain unknown, reduced transcription is a likely possibility. Transcription factors NRF1 and NRF2 (the latter factor also called GABP) that have direct roles in the regulation of CytOX IV and Vb genes are known to be modulated by oxidative stress [3,33]. Although reasons for reduced mitochondrial transcription remain unclear, altered activity of mtTFA (Fig. 2D) and phosphorylation of mtRNA polymerase (results not shown) are the likely possibilities. Our results therefore show for the first time that in mammalian cells physiologically relevant hypoxia induces a coordinated down regulation of both the nuclear and mitochondrial genes coding for the CytOX enzyme complex. These results are also consistent with a coordinated up or down regulation of the nuclear and mitochondrial genes under various physiological and pathological conditions such as cardiac growth, develop- ment and cardiac hypertrophy [35,36]. The restoration of mRNA levels within 3–6 h following reoxygenation indicates that the decreased ATP levels, which follow reduced respiration (O 2 uptake) during hypoxia, might be one of the mechanisms for reduced transcription rates (Fig. 1 and Table 2). This is supported by the observations of Schumacker et al. who have noted an inhibition of cellular respiration and suppression of ATP utilization during hypoxia [37–40]. The mammalian mito- chondrial RNA polymerase requires a high concentration of ATP (0.5–1 m M ) for maximal activity. Narasimhan and Attardi [41] showed that a high concentration of 5¢-adenylylimidodiphosphate was able to stimulate the Table 2. Effect of hypoxia on some biochemical parameters related to mitochondrial function. ATP levels in total cell extracts were measured using the somatic cell ATP assay kit, which is based on the assay of ATP driven luciferin luciferase assay system. ATP levels were measured in a TD-20/20 luminometer. Respiration coupled ATP synthesis by isolated mitochondria was measured by incubating mitochondria in a medium supplemented with ADP and succinate as described in the Materials and methods section. Hexokinase and phosphofructokinase activities were measured in the cytosolic fractions. Isocitrate dehydrogenase and NADH:ubiqinone oxidoreductase (complex I) activities were measured in isolated mitochondria by standard methods as indicated in the Materials and methods section. Values are given as means ± SD calculated from four estimates. Macrophages PC12 Cells Normoxia Hypoxia Normoxia Hypoxia Total cellular ATP (nmolÆmg protein )1 ) 10.3 ± 1.132 7.04 ± 0.774 22 ± 1.986 11 ± 1.431 Respiration coupled ATP synthesis in isolated mitochondria (nmolÆmg protein )1 ) 36 ± 3.24 28 ± 5.7 84 ± 9.52 37 ± 5.12 Enzyme activity Isocitrate dehydrogenase (lmolÆmin )1 Æmg protein )1 ) 0.022 ± 0.0024 0.014 ± 0.0013 0.010 ± 0.009 0.006 ± 0.0048 Complex I (lmolÆmin )1 Æmg protein )1 ) 0.157 ± 0.0200 0.088 ± 0.0079 0.148 ± 0.0237 0.082 ± 0.0111 Hexokinase (lmolÆmin )1 Æmg protein )1 ) 0.016 ± 0.0021 0.028 ± 0.0034 0.048 ± 0.0067 0.068 ± 0.0986 Phosphofructokinase (lmolÆmin )1 Æmg protein )1 ) 0.026 ± 0.0030 0.056 ± 0.0050 0.051 ± 0.0068 0.077 ± 0.0073 Ó FEBS 2003 Cytochrome c oxidase subunit expression in hypoxia (Eur. J. Biochem. 270) 877 transcription in vitro in the presence of a low concentration of ATP. These studies suggest that while at low concentra- tions ATP is a substrate for mitochondrial RNA poly- merase, at high concentrations it has a regulatory function. Reduction in cellular ATP levels (Table 2) might also be one of the factors for the down regulation of nuclear genes coding for the mitochondrial proteins. It is likely that the decreased mitochondrial enzyme activities that were observed during the exposure of lung macrophages and rat L8 myocytes to 96 h of mild hypoxia (15–20 Torr) might be related to such a coordinated down regulation of mitochondrial and nuclear genes [42]. Similar to that reported for chemical hypoxia with CoCl 2 and succinyl acetone [12], physiologically relevant hypoxia in cultured cells also leads to rapid depletion of heme aa 3 pools (Table 1). The observed heme depletion is closely associated with lowered CytOX subunits I, IV and Vb content and altered enzyme activity. Notably the reduced subunit level is more apparent in the slow migrating putative dimeric form of the enzyme (Fig. 3), suggesting that reduced heme and altered subunit levels may interfere with the formation of the more active dimeric complex. As heme is involved in reactions that transfer electrons from cytochrome c to molecular oxygen, its depletion reflects alterations in the catalytic efficiency of the enzyme complex as assessed by the TN for cytochrome c oxidation or oxygen utilization (Table 1). Even under hypoxia induced heme depletion and reduced enzyme content the TN of the CytOX complex for cytochrome c oxidation essentially remained unaltered in macrophages, while the TN was slightly enhanced in PC12 cells (Table 1). This is in sharp contrast to twofold to fourfold higher TN rates for cytochrome c oxidation, which we reported for the enzyme from heme-depleted tissues in CoCl 2 treated animals [12]. Although the mechanism of action of CoCl 2 is not clearly known, it is generally believed that its action mimics hypoxia by interfering with the incorporation of iron into heme and thus limiting the utilization of molecular O 2 . While heme depletion is common to both physiological and chemical hypoxia as shown by our studies, physiological hypoxia is also characterized by the deficiency of molecular O 2 . Employing a variety of experimental systems that included hepatocytes or isolated mitochondria, Schumacker et al. demonstrated the inhibition of CytOX during hypoxia [37–40]. They proposed that inhibition of CytOX is an adaptive response to inhibition of respiration during hypoxia. In a polarographic assay that employed TMPD as a substrate, they observed that the decrease in TN occurred more rapidly at 0 l M oxygen compared with 25 or 50 l M (approximately 13–27 Torr) O 2 concentrations. However, the data on TN of the enzyme was obtained using isolated bovine enzyme exposed to varying levels of hypoxic conditions. Our results suggest that such a decrease in TN of the enzyme might be a late event in intact cells exposed to hypoxia. It is more likely that these differences in TN represent the different stages of adaptive response to hypoxia. Based on the results of our study and those of Schumacker, it is reasonable to conclude that the effects of hypoxia on CytOX gene expression and its activity are secondary to suppression of respiration during hypoxia. In the absence of differential regulation of a specific nuclear gene coding for the subunits of CytOX, changes in the microenvironment of the cell may induce alterations in the catalytic efficiency of mammalian enzyme. Selective loss of CytOX subunits I/II and IV might be important factors in altered catalytic activity [12]. 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Adaptive changes in the expression of nuclear and mitochondrial encoded subunits of cytochrome c oxidase and the catalytic activity during hypoxia C. . nuclear gene coding for the subunits of CytOX, changes in the microenvironment of the cell may induce alterations in the catalytic efficiency of mammalian