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Am J Physiol Heart Circ Physiol 294: H2637-H2645, 2008. First published April 11, 2008; doi:10.1152/ajpheart.91476.2007
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Differential loss of cytochrome-c oxidase subunits in ischemia-reperfusion injury: exacerbation of COI subunit loss by PKC-{varepsilon} inhibition

Qilin Yu,1,2 Tiffany Nguyen,1,2 Mourad Ogbi,1,2 Robert W. Caldwell,1 and John A. Johnson1,2

1Department of Pharmacology and Toxicology, School of Medicine, and 2The Program in Regenerative Medicine, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia

Submitted 14 December 2007 ; accepted in final form 4 April 2008


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
We have previously described a PKC-{varepsilon} interaction with cytochrome oxidase subunit IV (COIV) that correlates with enhanced CO activity and cardiac ischemic preconditioning (PC). We therefore investigated the effects of PC and ischemia-reperfusion (I/R) injury on CO subunit levels in an anesthetized rat coronary ligation model. Homogenates prepared from the left ventricular regions at risk (RAR) and not at risk (RNAR) for I/R injury were fractionated into cell-soluble (S), 600 g low-speed centrifugation (L), gradient-purified mitochondrial (M), and 100,000 g particulate (P) fractions. In RAR tissue, PC (2 cycles of 5-min ischemia and 5-min reperfusion) decreased the COI in the P fraction (~29% of total cellular COI), suggesting changes in interfibrillar mitochondria. After 30 min of ischemia and 120 min of reperfusion, total COI levels decreased in the RAR by 72%. Subunit Va was also downregulated by 42% following prolonged I/R in the RAR. PC administered before I/R reduced the loss of COI in the M and P fractions ~30% and prevented COVa losses completely. We observed no losses in subunits Vb and VIIa following I/R alone; however, significant losses occurred when PC was administered before prolonged I/R. Delivery of a cell-permeable PKC-{varepsilon} translocation inhibitor ({varepsilon}V1-2) to isolated rat hearts before prolonged I/R dramatically increased COI loss, suggesting that PKC-{varepsilon} protects COI levels. We propose that additional measures to protect CO subunits when coadministered with PC may improve its cardioprotection against I/R injury.

preconditioning; protein kinase C; cytochrome oxidase subunit; coronary ligation; mitochondria

CARDIAC ISCHEMIA-REPERFUSION (I/R) injury is a major cause of death and morbidity in the Western world, and considerable research has targeted interventional therapies for this condition. Cardiac ischemic preconditioning (PC), a series of brief ischemia and reperfusion exposures administered before a prolonged I/R insult, is experimentally the most potent form of cardiac protection against I/R injury (44). More than 20 years of intense study have revealed numerous mechanisms by which PC protects the myocardium, including modest elevation of reactive oxygen species (ROS) (23, 39, 49, 58), nitric oxide (23, 58), mitochondrial ATP-sensitive potassium (mKATP) channels (18, 31, 32, 39, 60), heat shock proteins (18, 34), and activation of protein kinase cascades (17). Protein kinase C (PKC) isozyme-induced alterations in mitochondrial functions also have been proposed to play important roles in cardiac PC (45). For example, the PKC-{varepsilon} isozyme has been reported to form signaling clusters inside cardiac mitochondria with MAP kinase (4) and tyrosine kinases (56), inhibit the opening of the mitochondrial permeability transition pore (3), preserve cytochrome oxidase (CO) activity (16, 47, 48), and act in a cascade involving PKG-mediated opening of the mKATP channel (13). The role of the PKC-{delta} isozyme is less clear, since it has been reported to translocate to mitochondria to contribute to either PC (47, 61) or I/R injury (11, 42, 43).

CO has 13 subunits and catalyzes the final enzymatic reactions in the electron transport chain (ETC) (59). It contributes significantly to maintenance of the mitochondrial electrochemical/proton gradient and the production of ATP (24) and is inhibited following prolonged I/R (55). Previously, Prabu et al. (51) reported impaired CO function following I/R injury in isolated rabbit hearts involving PKA-mediated phosphorylation and degradation of CO I, IVi, and Vb subunits. However, there have been few cardiac studies exploring the regulation of CO following PC and even fewer exploring its regulation by individual PKC isozymes (16, 45, 47, 48). Our laboratory recently demonstrated that PKC-{varepsilon} coimmunoprecipitates with the number IV subunit of CO (COIV), which correlates with elevated CO activity during cardiac PC (16, 48). We hypothesize that there are multiple sites of PKC interaction with CO, and therefore, we determined which CO subunit levels were altered following PC, I/R, and PC + I/R treatments.

Losses in CO subunits have been reported previously in models of cardiac failure. Sayen et al. (53) demonstrated ~25% decreases in the COI and COIV subunits in transgenic mice expressing constitutively active calcineurin, a model of pathological hypertrophy and heart failure (53). Kuo et al. (27) demonstrated loss of the COVb subunit in ventricular tissue isolated from rats subjected to prolonged aortic banding. The COII subunit also has been reported to decline following short aortic cross-clamping procedures (12) and experimental heart failure in canine cardiac myocytes (54). In contrast, McLeod et al. (38) reported that delayed PC enhanced mRNA and protein levels of COIV in a rat coronary ligation model. Javadov et al. (20) reported that inhibition of the Na+/H+ exchanger attenuated downregulation of mRNA for the COI and COIV subunits following chronic coronary artery ligation. In transgenic mice deficient in the COVIa-H subunit, defects in diastolic function were observed (52). Finally, overexpression of the metallothionein protein in transgenic mice reduced doxorubicin-induced cardiomyopathy in conjunction with elevated COVa subunit levels (40). However, none of these studies focused on subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, and generally, few CO subunits were evaluated, which provided only partial information on which aspects of CO functions would be modified following cardiac damage. Our results suggest differential sensitivities of CO subunits to PC and prolonged I/R exposures and varying degrees of CO subunit protection when PC precedes the index I/R insult. We therefore propose that failure to protect CO subunits could limit the recovery of aerobic ATP production and the extent of long-term survivability of heart cells following PC protocols.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Myocardial PC and I/R injury. Our in situ anesthetized rat coronary ligation model of PC and I/R injury has been described previously in detail (16). For PC, two cycles of a 5-min occlusion followed by a 5-min reperfusion were induced. For I/R injury, the occlusion was elicited for 30 min followed by a 120-min reperfusion period. Coronary artery occlusion was confirmed by epicardial cyanosis and a decrease in blood pressure. All experimental protocols involving the use of animals were approved by The Medical College of Georgia Institutional Animal Care and Use Committee and conformed with the Helsinki Agreement for the humane care and use of laboratory animals.

Determination of the myocardial region at risk. The left ventricular region at risk (RAR) was determined as described previously (16, 36, 37). After the final reperfusion period, the coronary artery was again occluded. The RAR was determined by a lack of staining with Evans blue dye (2%), which was injected into the left ventricle. The entire heart was excised, and left ventricular RAR and region not at risk (RNAR) tissue were used in Western blot analyses as described below.

Isolated heart preparations. Adult Sprague-Dawley rats (250 g) were heparinized and anesthetized with ketamine-xylazine as previously described (16). Hearts were rapidly excised, rinsed in oxygenated (95% O2-5% CO2), chilled Krebs buffer (115 mM NaCl, 4 mM KCl, 1.1 mM MgSO4·7H2O, 1.3 mM KH2PO4, 24 mM NaHCO3, 1.0 mM CaCl2, and 5.5 mM glucose, pH 7.4), and attached by the aorta to a Langendorff perfusion apparatus. Hearts were retrograde perfused with the 37°C Krebs buffer at a constant flow rate of 11 ml/min (average perfusion pressure 70 mmHg) for 5 min to wash away residual blood. A water-filled balloon-tipped Millar catheter was inserted into the left ventricle through the left atrium. Left ventricular end-diastolic pressure was adjusted to 5 mmHg during the initial equilibration, and the volume of the balloon was not subsequently altered. Coronary perfusion pressure, left ventricular developed pressure, and heart rate (HR) were recorded on a Grass polygraph via a pressure transducer. Hearts were then paced at 280 beats/min using a Grass SD stimulator and equilibrated for 20 min. One group of hearts was perfused with 1 µM concentrations of the human immunodeficiency virus (HIV)-Tat carrier-carrier control peptide (NH-YGRKKRRQRRR-YGRKKRRQRRR-COOH) for 10 min and then subjected to 30 min of no-flow global ischemia and 90 min of normoxic reperfusion. We observed no effects of the carrier-carrier peptide on COI levels compared with hearts receiving no peptide (not shown). A second group of hearts was administered the PKC-{varepsilon} translocation inhibitor {varepsilon}V1-2 coupled to the HIV-Tat protein transducing domain (NH-YGRKKRRQRRR-EAVSLKPT-COOH) for 10 min before 30 min of global ischemia and 90 min of reperfusion. Effluents from hearts following basal and prolonged I/R exposures were collected and assayed for cardiac troponin I (cTnI).

Subcellular fractionation and Percoll/Optiprep gradients. The isolation of soluble (S), low-speed centrifugation (L), density gradient-purified mitochondrial (M), and 100,000 g particulate (P) cell fractions and the preparation and use of Percoll/Optiprep density gradients to isolate mitochondria have been described previously in detail (16). We also previously determined the average percentages of total homogenate protein found in the S, L, M, and P fractions to be 18.8, 66.8, 9.4, and 5.4%, respectively (16). Therefore, to truly quantitate the percentages of each CO subunit in the S, L, M, and P fractions ideally, we would not load equal concentrations of protein on gels from each fraction as we did. However, we could not consistently obtain accurate densitometry values for each CO subunit when less protein was used for some of the fractions. To account for this in our studies with anesthetized control (normoxic) rats, we present raw data first, using equivalent amounts of protein for each fraction, and then report these data after normalization to account for the different percentages of total homogenate protein found in each (S, L, M, and P) fraction.

Western blot analyses. Samples were subjected to SDS-PAGE on 18.75% acrylamide-6 M urea gels (26) and transferred onto nitrocellulose paper. The resulting blots were probed for CO subunits using 125I-labeled protein A detection (21, 22) or enhanced chemiluminescence (GE Healthcare). CO subunit antisera were obtained from GE Healthcare and Mitosciences (Eugene, OR) and used at the following dilutions: COI, 1:500; COIII, 1:250; COIV, 1:1,000; COVa, 1:250; COVb, 1:250; COVIb, 1:500; and COVIIa, 1:100.

Statistical analyses. Values are means ± SE from a minimum of four animals, as indicated. Differences between two groups were assessed using unpaired Student's t-test, and comparisons among multiple groups were made using one-way ANOVA with Bonferroni's post hoc test. A P value ≤0.05 was considered significant.


   RESULTS AND DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
Baseline hemodynamic parameters. Control, ischemic PC, 30-min ischemia-120-min reperfusion (I/R), and PC + I/R exposures were administered to adult Sprague-Dawley rats using an open-chest coronary ligation model (16). The means ± SE of heart weight-to-body weight ratio (in grams) for control (1.0 ± 0.1/286 ± 36), PC (0.9 ± 0.1/321 ± 13), I/R (0.9 ± 0.1/305 ± 19), and PC + I/R animals (1.0 ± 0.1 /291 ± 27) were not significantly different. Furthermore, following PC cycles, 30 min of ischemia, or 30 min of ischemia followed by up to 120 min of reperfusion, we observed no significant differences in HR or mean arterial blood pressure (MAP) (Table 1). It is important to note that we did not measure coronary blood flow directly to the nonischemic (RNAR) myocardial regions. However, since we observed no significant change in HR or MAP (measured via a carotid catheter) following PC, I/R, or PC + I/R exposures, we predict no significant difference in RNAR blood flow for any of these groups.


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  		Table 1. Hemodynamic parameters of Sprague-Dawley rats exposed to control, cardiac ischemic PC, and I/R protocols

 
Ischemic PC reduces I/R injury-induced serum cTnI release in Sprague-Dawley rats. Previously, we used tetrazolium-based staining of ventricular tissue sections to confirm that PC induces ~50% reduction in infarct size (and release of cTnI into rat serum) in this model (16). In the present study, we monitored cTnI release into rat serum and then used hearts from the same animals for CO subunit Western blots (see Figs. 1, Go3, 4, and Go6). We observed no significant cTnI release in the sham surgery control and PC alone treatment groups. In contrast, animals subjected to prolonged I/R demonstrated considerable cTnI release (11.2 ± 0.7 ng/ml, n = 9), which was reduced by more than one-half when animals were given PC before I/R (4.9 ± 0.2 ng/ml, n = 9). This reduction was significant at the P ≤ 0.001 level.


Figure 1
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  		Fig. 1. Imunnodetection of the I, III, IV, Va, Vb, VIb, and VIIa subunits of cytochrome-c oxidase (CO) in subcellular fractions isolated from control adult rat ventricular myocardium. Sprague-Dawley rats were subjected to sham anesthesia and mock surgery (no coronary ligation) as previously described (16). Left ventricular tissue was homogenized, separated into soluble (S), low-speed centrifugation (L), density gradient-purified mitochondrial (M), and 100,000 g particulate (P) cell fractions, and each fraction was subjected to SDS-PAGE and Western blot analysis using commercially available CO subunit antisera. Typical CO subunit autoradiographs are shown at the top of each histogram. Histograms represent densitometric data (means ± SE) from 9 (COI), 5 (COIII), 9 (COIV), 5 (COVa), 5 (COVb), 5 (COVIa), and 5 animals (COVIIa).

 

Figure 2
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  		Fig. 2. Percoll/Optiprep density gradients are required for optimal detection of COI in subsarcolemmal (SSM) and interfibrillar mitochondria (IFM). SSM and IFM were isolated as previously described (50), and each mitochondria population was then divided into 2 equal groups. The first group from SSM and IFM populations was isolated using the procedures of Hoppel (50) and used directly for experiments (SSM and IFM columns). The second group was first isolated using the procedure of Hoppel to obtain SSM and IFM, and then SSM and IFM were further purified using a Percoll/Optiprep gradient (16) (SSM gradient and IFM gradient). Next, 50 µg of non-gradient- or Percoll/Optiprep gradient-purified SSM and IFM were subjected to Western blot analyses using anti-COI (open bars) and anti-COIV antisera (hatched bars). Typical autoradiographs are shown at top of each histogram. Densitometry values (means ± SE), expressed as the percentage of maximal CO subunit levels, from 4 experiments, are depicted in histograms. *P < 0.5 vs. non-gradient-purified mitochondria.

 

Figure 3
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  		Fig. 3. Ischemic preconditioning (PC) of adult rat myocardium diminishes COI. Rats were anesthetized and subjected to brief left anterior descending coronary artery ligations and reperfusions as outlined in MATERIALS AND METHODS to invoke PC. The left ventricular region at risk (RISK) was determined, and tissue was taken from the RISK and region not at risk (NR). All other details are as described in Fig. 1. The number of animals monitored were 9 (COI), 7 (COIII), 7 (COIV), 5 (COVa), 6 (COVb), 6 (COVIa), and 6 (COVIIa). *P ≤ 0.05, RISK vs. NR groups.

 

Figure 4
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  		Fig. 4. Prolonged ischemia-reperfusion (I/R) exposures downregulate COI and COVa. All experimental conditions and analyses are as described in Fig. 3, except prolonged I/R alone (see MATERIALS AND METHODS) was the treatment instead of PC. The number of animals monitored were 9 (COI), 5 (COIII), 8 (COIV), 6 (COVa), 4 (COVb), 5 (COVIa), and 5 (COVIIa). *P ≤ 0.05, RISK vs. NR groups.

 

Figure 5
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  		Fig. 5. COI and COIV in isolated SSM and IFM are not degraded by exogenously added trypsin. SSM and IFM were isolated according to the procedures of Hoppel (50) and then further purified over a Percoll/Optiprep gradient (16). SSM and IFM were then incubated on ice for 10 min with increasing concentrations of trypsin. Western blots were then conducted using antisera to the COI and COIV subunits. A representative autoradiograph is shown at top of each histogram. Histograms represent densitometry values (means ± SE), expressed as a percentage of maximal CO subunit levels, taken from 4 independent experiments.

 

Figure 6
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  		Fig. 6. Ischemic PC only partially prevents CO subunit losses following prolonged I/R injury. All details are as described in Fig. 3, except PC + I/R (see MATERIALS AND METHODS) was the treatment. The number of animals monitored were 9 (COI), 7 (COIII), 7 (COIV), 6 (COVa), 6 (COVb), 7 (COVIa), and 6 (COVIIa). *P ≤ 0.05, RISK vs. NR groups.

 
Baseline distributions of CO subunits in Sprague-Dawley rat myocardium. Rats were anesthetized and subjected to a mock surgery without coronary ligation. Left ventricular tissue was obtained from comparable anatomic regions to those used for our PC and I/R experiments (e.g., Figs. 3, 4–6), and S, L, M, and P fractions were then subjected to Western blot analysis. We observed CO subunit expression in the L, M, and P fractions (Fig. 1). This confirmed their expression in both SSM (M fraction) and IFM (L and P fractions) and demonstrated no substantial CO leakage out of mitochondria, since we did not observe CO subunit immunoreactivity in the S fraction (Fig. 1). The percentages of each CO subunit in S, L, M, and P fractions under baseline conditions are summarized in Table 2. When normalized for protein (Table 2), most CO subunit immunoreactivity was found in the L and M fractions with considerably less existing in the P fraction. However, ~58% of COI existed in the P fraction (Table 2). These studies demonstrate the importance of monitoring all cellular mitochondria when determining the levels of cardiac CO subunits.


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  		Table 2. CO subunit expression in S, L, M, and P fractions isolated from the left ventricles of control rats

 
Gradient purification of cardiac SSM yields greater COI but not COIV immunoreactivity. It was possible that the Percoll/Optiprep gradients used to isolated our M fraction (SSM) might have selected for more viable mitochondria than the crude centrifugation methods used to isolate our L and P (IFM) fractions. To test this possibility, we purified SSM and IFM from rat heart ventricles using the procedures of Hoppel's group (50). Next, SSM and IFM were split into two equal groups. One group each from SSM and IFM was further purified over our Percoll/Optiprep gradient (16), and the second group was used directly as purified with the Hoppel protocol (50). We next subjected SSM and IFM protein to Western blot analyses with COI and COIV antisera. We observed no significant differences in COIV immunoreactivity among any of the groups tested (Fig. 2). This suggested that our Percoll/Optiprep gradients alone did not alter the number of SSM or IFM isolated. However, we did observe a striking 2.4 ± 0.2-fold improvement in COI immunoreactivity when SSM were purified using the Percoll/Optiprep gradient vs. the non-gradient-purified SSM. In contrast, this enhanced COI recovery was not observed when IFM were purified over the Percoll/Optiprep gradient. The reason for the better recovery of COI by the gradient in SSM is unknown. However, if we had not used the gradient to purify our M fraction, it is likely that we would have underestimated COI levels in the SSM. Furthermore, since the gradient purification method does not alter COI levels in the IFM, we do not believe that any changes in the levels of COI (as will be shown for PC and I/R groups later in this study) can be explained by our use of the gradient to purify the M fraction and the lack of gradient purification of the L and P fractions.

COI decreases following cardiac ischemic PC in the P but not the L and M fractions. Our previous work indicated that there is no significant infarct in hearts isolated from rats receiving PC involving two cycles of 5-min ischemia and 5-min reperfusion (16). In the present study, we also found no significant elevation of cTnI following PC alone, consistent with PC not causing infarction. We therefore hypothesized that there would be no loss of CO subunits following PC. Adult Sprague-Dawley rats were exposed to PC, and the myocardial RAR and RNAR for I/R injury were determined (Fig. 3). Compared with RNAR, COI immunoreactivity in the RAR declined by 51 + 14% (P ≤ 0.01) in the P fraction (Fig. 3). Since on average the P fraction contains 57.6% of the total COI (Table 2), PC caused an ~29% reduction in total COI levels. This suggests that COI in IFM (P fraction) is partially downregulated following PC, whereas COI in the SSM (M fraction) is unaffected. In contrast, we did not observe significant losses in any of the other CO subunits tested following PC (Fig. 3). In fact, PC increased COVIb levels by 87 ± 19% (P ≤ 0.01) in the L fraction. Collectively, these results indicate that the PC-induced loss of COI is not a general cytotoxic response.

CO has been hypothesized to be a rate-limiting step in the ETC (59). However, experiments using isolated mitochondria indicate that CO exists in excess (14, 15, 30), but the presence of unlimited amounts of respiratory substrates and the absence of crucial cytosolic/cell factors complicate the interpretation of these results. In addition, CO reserve could be very limited if it is heavily damaged during cardiac I/R injury and low-oxygen states persist. We currently do not know whether the decrease in COI following PC is substantial enough to compromise mitochondrial respiration or elevate mitochondrial ROS. ROS are necessary for the induction of many PC responses, and the loss of COI could lead to elevated mitochondrial ROS production. However, mitochondria possess substantial antioxidant defense mechanisms (1) that could neutralize elevations in ROS that might arise from the partial loss of COI.

Alternatively, there have been reports suggesting that inhibition of ETC function preceding prolonged I/R injury is protective. For example, Lesnefsky et al. (29) found that blockade of complex I with rotenone before prolonged ischemia preserved cardiolipin, cytochrome c content, and mitochondrial respiration through complex IV (29). In addition, Chen et al. (10) demonstrated that reversible inhibition of complex I with amobarbital, before prolonged I/R in isolated rat hearts, protected ETC function and lowered diastolic pressures. This same group, however, reported that inhibition of the ETC protects myocardium via a mechanism distinct from that of PC (57). Adding to the complexity of this issue, Ludwig et al. (35) reported that isoflurane-induced cardioprotection and ROS production were attenuated by the ETC complex III inhibitor myxothiazole in rabbits. From these studies, it appears that, at least in some cases, inhibition of ETC function can be cardioprotective. In the instances mentioned above, however, the extent of ETC inhibition was much greater than would be predicted for the ~30% loss of COI we observed following PC.

Further support for a role of altered ETC function in PC comes from the effects of nitric oxide (NO), a PC mimetic. Treatment with NO donors at concentrations below those required to induce vasodilation preconditions the heart (23, 58). When administered at physiological oxygen concentrations, NO binds to CO and inhibits the enzyme by competing with oxygen for its binding site on the COI subunit (41). In heart cells there is also a modest burst of ROS following NO donor (23, 58) and mKATP channel activator treatments (39, 49). Modest elevation of mitochondrial ROS is therefore necessary for PC to occur (39, 49), and extensive deficits in CO would be expected to facilitate the backup of electrons in the iron-sulfur clusters and cytochromes of complexes I and III. Unpaired electrons leaking from these sites can combine with oxygen to form superoxide, which in turn can lead to elevation of other mitochondrial ROS such as H2O2 and peroxynitrite (1). It also has been reported that NO-induced PC involves nitration of PKC-{varepsilon} and facilitation of its interaction with mitochondrial receptors for activated C-kinase (RACKS) (5). Furthermore, Xu et al. (61) reported diminished oxygen demand following PC, which correlated with reduced oxidation of cytochromes a and a3 (an indirect estimate of electron transfer through COI). This effect was lost when hearts were exposed to the nitric oxide synthase inhibitor N-nitro-L-arginine (61). Another interesting phenomenon that has been proposed is that drugs which activate the mKATP channel also may inhibit complex II of the ETC (2, 8, 33). Under normoxic conditions (when ATP is abundant), mKATP is thought to be inhibited, but during PC it opens, allowing K+ entry into mitochondria. This modestly diminishes (uncouples) the electrochemical gradient across the mitochondrial inner membrane, which in turn stimulates respiration (8, 33) and ROS production (39, 49). Therefore, inhibition of ETC function may provide one mechanism of cardioprotection, but further study is needed to clarify whether this applies to the modest loss of COI we observed in the P fraction of our experiments.

Prolonged I/R exposures cause differential loss of COI and COVa. A 30-min ischemia-120-min reperfusion exposure causes ~50% infarction in the RAR of our model, which correlates with elevated levels of serum cTnI (16). Our Western blots indicate that despite substantial I/R-induced cell death (based on tetrazolium chloride staining and cTnI release) in the RAR of these hearts, the immunoreactivity of COIII, COIV, COVIb, and COVIIa subunits was highly preserved (Fig. 4). The most sensitive CO subunit was COI, which was downregulated in the RAR by 72 ± 9% (P ≤ 0.003) following IR. We found very little COI in the L fractions from the RAR and RNAR; however, COI downregulation occurred in IFM (P) and SSM (M) fractions (Fig. 4). In contrast, the COIII subunit actually showed a 3.9-fold increase (P ≤ 0.04) in immunoreactivity in the L fraction with no change in the M and P fractions in the RAR following I/R (Fig. 4). Whether this COIII elevation is involved in the mechanisms of I/R damage or is merely an attempt by the SSM in the L fraction to upregulate or preserve a crucial portion of the CO catalytic core is currently unknown. COVa showed 37 ± 12, 45 ± 15, and 55 ± 10% losses in the L, M, and P fractions, respectively, when taken from RAR tissue following prolonged I/R, and COVb showed no significant losses in any fraction. In addition, our results indicate that because of its extreme stability, COIV immunoreactivity could serve as a reliable loading control for total mitochondrial protein in gels (see Fig. 3–6) even after prolonged I/R.

Exposure of isolated SSM and IFM to trypsin does not alter COI or COIV levels. Since trypsin was used to digest our cardiac ventricular tissue, we determined whether the loss of CO subunits in PC (Fig. 3) and I/R (Fig. 4) reflected enhanced CO subunit sensitivity to trypsin in our homogenization steps. Trypsin is too large to passively enter intact mitochondria, so proteins in the inner mitochondrial membrane should not be proteolyzed when trypsin is added to intact mitochondria. We also reasoned that the most likely condition to cause increased sensitivity to trypsin in our study would be our prolonged I/R exposures. We therefore exposed Langendorff heart preparations to 30 min of no-flow global ischemia followed by 90 min of normoxic reperfusion. We next isolated SSM and IFM using the Hoppel procedure (50) and then further purified both SSM and IFM using our standard Percoll/Optiprep density gradient (16). In our standard S, L, M, and P fractionation protocol, ventricular tissue containing IFM is exposed to 375 units of trypsin per milliliter on ice for 10 min. However, since IFM are attached to myofibrils, it is unlikely that the IFM would directly encounter this full level of trypsin activity. For this reason we exposed isolated SSM and IFM for 10 min on ice to a range of trypsin concentrations and then monitored COI and COIV in Western blots. We observed no significant loss of COI or COIV even when SSM or IFM were incubated with 10 times the concentration of trypsin used in our S, L, M, and P fractionation (Fig. 5). These results indicate that COI losses in response to PC or I/R are not likely to be the result of enhanced sensitivity of either SSM or IFM to trypsin in homogenization and mitochondrial isolation steps.

Ischemic PC only partially protects CO subunits against I/R injury. When rats were administered PC before I/R, the COVa subunit was protected by nearly 100% in L, M, and P fractions (Fig. 6). These results suggest that COVa can be fully protected following PC + I/R despite the fact that tetrazolium chloride staining and cTnI release indicate an ~25% cell mortality (16). In contrast, we observed no protection of COI levels in the M fraction. However, COI immunoreactivity was substantially greater in the P fraction following PC + I/R, suggesting that PC before I/R may better protect COI in IFM than in SSM (Fig. 6). This may have important consequences, since the SSM have been proposed to supply energy for sarcolemmal functions such as ion channels, glucose uptake, the Na+/K+-ATPase, and others. Our results are consistent with diminished energy production contributing to deficits in these enzymatic activities following I/R. In contrast, IFM are thought to contribute energy for the actomyosin ATPase, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and contractile processes. Our results suggest a major loss in IFM COI following I/R and a greater effect of PC to preserve COI in IFM than was observed for the SSM (Fig. 6). The major protection of COI following PC + I/R occurred in the P fraction in which COI was restored to 51 ± 11% (P ≤ 0.02) of the RNAR COI levels. Also of major interest, PC + I/R treatments actually caused greater losses of the COVb subunit [47 ± 13% (P < 0.02) losses in the M and 57 ± 12% (P < 0.01) losses in the P fraction] than were observed for I/R alone. Similarly, we observed no losses in COVIIa following I/R (Fig. 4). However, we observed 42 ± 18% (P ≤ 0.05) losses of COVIIa in the P fraction following PC + I/R (Fig. 6). Finally, there were no significant losses of the COIII, COIV, or COVIb subunits following PC + I/R (Fig. 6).

A PKC-{varepsilon} translocation inhibitor accelerates the loss of COI following prolonged I/R injury. It is well established that the PKC-{varepsilon} isozyme is cardioprotective in models of cardiac PC (19). We evaluated the effects of a cell-permeable PKC-{varepsilon}-selective translocation inhibitor {varepsilon}V1-2 (9, 19) on the loss of COI in I/R injury in isolated rat hearts (Fig. 7). After 30 min of global ischemia and 90 min of reperfusion, cTnI release was elevated in the carrier-carrier control hearts (3.5 ng/ml) and the {varepsilon}V1-2 hearts (6.7 ng/ml). This corresponded to a 1.9-fold greater release of cTnI from the {varepsilon}V1-2 hearts than from the carrier-carrier control hearts exposed to I/R. These results confirm significant infarction in the carrier-carrier control group, which was amplified in hearts pretreated with {varepsilon}V1-2 before the I/R insult. Our findings are therefore consistent with the removal of PKC-{varepsilon} cardioprotective mechanisms in the {varepsilon}V1-2 hearts. In these experiments, we observed very little COI in the L fraction and were unable to determine the effects of the PKC-{varepsilon} inhibitor on COI in this fraction. However, with the PKC-{varepsilon} inhibitor present, the I/R-induced losses in the M and P fractions were found to be 52 ± 10 (P ≤ 0.04) and 70 ± 7.4% (P ≤ 0.02) greater than in the carrier-carrier groups subjected to I/R (Fig. 7A). This indicated a greater than twofold COI loss in these fractions, suggesting that PKC-{varepsilon} protects COI levels in both SSM and IFM. In contrast, the {varepsilon}V1-2 inhibitor had no effect on the COIV subunit (Fig. 7B). Our results confirm a major loss of COI following I/R, which is dramatically increased when PKC-{varepsilon} function is blocked by {varepsilon}V1-2.


Figure 7
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  		Fig. 7. Introduction of a PKC-{varepsilon} translocation inhibitor into isolated rat hearts exacerbates I/R-induced loss of COI. Langendorff heart preparations from Sprague-Dawley rats were equilibrated at 37°C and then perfused with the human immunodeficiency virus (HIV)-Tat carrier-carrier control peptide (carrier; A) or the HIV-Tat-coupled PKC-{varepsilon} translocation inhibitor ({varepsilon}V1-2) for 10 min. Next, hearts were subjected to 30 min of global ischemia followed by 90 min of reperfusion (I/R). Ventricles were then harvested, fractionated into S, L, M, and P fractions, and subjected to Western blot analyses for COI and COIV as described for Fig. 3. A single representative autoradiograph is show at top of each histogram. Histograms represents data from 3 independent hearts in each treatment group. All data are means ± SE, expressed as a percentage of maximal densitometry values. *P ≤ 0.05, carrier control peptide and {varepsilon}V1-2 groups.

 
Significance of PC- and I/R-induced changes in CO subunits. Mammalian CO in vivo requires interactions between each of its subunits for optimal functioning. The mitochondrial coded subunits I–III form the active sites for the acceptance and transfer of electrons and protons to oxygen and for the pumping of protons out of the mitochondrial matrix (6, 24). Our data indicate a major loss of COI content following I/R that is only partially reversed by PC (Figs. 4 and 6). In addition, we observed this protection in the P fraction, which contains IFM. Had we monitored only SSM, we would have missed this protective effect. Another important point is that most PC studies monitor the effects of cardiac protection immediately after an I/R insult. However, the loss of COI would have detrimental consequences for long-term myocardial survival, since COI contains the aa3 and CuB redox centers, which are required for the four-electron reduction of diatomic oxygen to water (6, 24). The COII subunit contains the two-copper binding site for ferrocytochrome c and along with COI and COIII comprises an important component of the enzyme's catalytic core. It also has been reported that COII is substantially downregulated following I/R and cardiac failure (12, 54). However, despite exhaustive efforts, we were unable to monitor COII in Western blots using commercially available antisera. Subunit I is positioned between subunits II and III, and the function of each of these subunits is dependent on one another. Some studies have indicated that the primary electron acceptor site for electrons transferred from ferrocytochrome c is cytochrome a on COI, which in turn transfers the electrons to the CuA redox center of COII. Loss of CO subunits comprising the mitochondrially encoded catalytic core (e.g., COI, COII, and COIII) would be predicted to elevate mitochondrial ROS, which would likely damage the intramitochondrial genes and or ribosomes attempting to replenish lost CO subunits, particularly because mitochondria lack most of the DNA repair enzymes found in the cell nucleus (28).

CO subunits other than COI, COII, and COIII are coded for in the cell nucleus, and their precise functions are only partially understood. The COIV subunit reportedly binds ATP when mitochondrial ATP levels are high and only if the COI subunit has been phosphorylated by PKA (24). ATP binding to COIV is thought to result in conformational changes and inhibition of CO activity. COIV also contains three predicted phosphorylation sites for PKC, and we recently showed that interaction of PKC-{varepsilon} with COIV correlates with higher CO activities following PMA treatment (47) and hypoxic PC (48) or ischemic PC (16). We were surprised to determine that neither prolonged I/R nor PC + I/R caused substantial loss of COIV (Figs. 4 and 6). It is possible that COIV is an important localizing protein for PKC-{varepsilon} in cardiac mitochondria, hence positioning it to mediate a cardioprotective role such as preservation of COI levels.

We also found that I/R induced an ~50% decrease in the COVa subunit (Fig. 5) in both SSM and IFM. Previous studies employing cross-linking reagents have indicated that COVa is a "near neighbor" to COI and COII (7). It also has been reported to contain a binding site for 3,5-diiodothyronine, which when bound can abolish the allosteric inhibition of CO by ATP binding to COIV (25). The net effect of this is a rapid increase in respiration via complex IV. In our studies, the COVa subunit was protected 100% when PC was administered before I/R, which suggests preservation of an important CO regulation. The COVb subunit contains a zinc binding site, but its function is unknown (6). COVb levels declined by 41 + 9% following PC + I/R, which represented a substantially greater loss than was observed following I/R alone (Fig. 6 vs. Fig. 4). These results further support the hypothesis that PC does not optimally protect all CO subunits.

In vivo, the COVIb subunit is thought to bind to a second COVIb subunit, allowing the formation of CO holoenzyme dimers (6). In the dimeric conformation, binding of one molecule of cytochrome c to one COII subunit inhibits binding of cytochrome c to the second COII subunit (6). This effect apparently allows electrons to flow to fewer CO molecules under low-oxygen conditions, which makes the reduction of molecular oxygen to water more efficient (6, 24). COVIb levels were increased in the L fraction following PC alone (Fig. 3). Whether this increase plays a role in PC via improved CO efficiency is currently unknown. We also observed no loss in COVIb following I/R, suggesting its functions may be at least partially intact following I/R. Finally, the functions of the COVIIa subunit have not been defined, and we also observed no losses of COVIIa following prolonged I/R and only minor losses following PC + I/R. Our major findings indicate that the mitochondrially encoded COI subunit is not completely protected by PC. In addition, the COVb and COVIIa subunits show greater losses following PC + I/R than with I/R alone (Fig. 4 vs. Fig. 6). We hypothesize that additional strategies to better protect these subunits should improve the recovery of aerobic respiration and prolong survival of the myocardium following I/R injury.


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This research was funded by National Heart, Lung, and Blood Institute Grant R01 HL076805 (to J. A. Johnson).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Johnson, Dept. of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300 (e-mail: jjohnson{at}mail.mcg.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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