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TOXICOLOGY
Departments of Pharmacology and Toxicology (K.E.M., W.P., J.C., Y.J.K.) and Medicine (W.F., L.Z., J.B.K., Y.J.K.), University of Louisville School of Medicine, Louisville, Kentucky
Received May 17, 2005; accepted August 31, 2005.
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Abstract |
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-oxidation of fatty acids and glycolysis, and proteins involved in regulation of cardiac muscle contraction. However, the most dominant modification by MT is the cytochrome c oxidase subunit Va (CCO-Va). In response to DOX treatment, a specific isoform of CCO-Va was enhanced in the MT-TG but not in the WT mouse hearts. Because CCO-Va is a critical component in the mitochondrial electron transport chain, the results suggest that the cardioprotective effect of MT may be related to an increased expression or a differential modification of CCO-Va.
). Previous studies using a cardiac-specific metallothionein (MT)-overexpressing transgenic (MT-TG) mouse model have demonstrated that MT protects from DOX-induced oxidative cardiac injury (Kang et al., 1997, 2000; Sun et al., 2001). MT overexpression significantly inhibits DOX-induced apoptosis in the transgenic myocardium both in vivo (Kang et al., 2000) and in vitro (Wang et al., 2001a). Furthermore, MT prevents DOX-induced myocardial apoptosis via inhibition of DOX-activated p38 mitogen-activated protein kinase (Kang et al., 2000) and mitochondrial cytochrome c release and caspase-3 activation (Wang et al., 2001a). Studies using primary cultures of neonatal mouse cardiomyocytes have shown that DOX-generated reactive oxygen species (ROS) accumulate predominantly in the mitochondria (Wang et al., 2001a). MT eliminated the accumulation of ROS in the mitochondria (Wang et al., 2001a), although MT is not localized in the mitochondria (Zhou and Kang, 2000).
The accumulation of ROS in the mitochondria seems to be an important cellular event triggering apoptosis involved in the DOX-induced cardiac injury. Therefore, it is important to define the molecular mechanisms by which MT inhibits the accumulation of ROS in the mitochondria. Recent developments in proteomics have permitted robust investigations into the molecular mechanisms underlying cardiovascular disease and have allowed for identification of protein modifications (Arrell et al., 2001). Proteomics techniques also offer the ability to simultaneously monitor differential changes in protein expression in response to various stimuli and under different physiological and pathological conditions (Macri and Rapundalo, 2001). In previous studies, proteomics approaches have been used to identify DOX-induced protein changes in MCF-7 human breast cancer cells (Chen et al., 2002). Proteomics techniques would also offer a powerful approach to understanding molecular mechanisms of MT protection against DOX cardiotoxicity.
In the present study, we used the unique cardiac-specific MT-TG mouse model in concert with expression proteomic approaches to identify differences in global proteomic profiles between the WT and MT-TG mice in their responses to DOX cardiotoxicity, focusing on the effect of MT on mitochondrial protection and thereby inhibition of myocardial oxidative injury. Our data indicate that cytochrome c oxidase subunit Va (CCO-Va) is among several proteins that are differentially expressed or modified in response to DOX treatment in the MT-TG mouse hearts relative to controls. Because CCO-Va is a critical component of the terminal enzyme of the mitochondrial electron transport chain, the results obtained suggest that the cardioprotective effect of MT may be related to an increased expression or a differential modification of CCO-Va.
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Materials and Methods |
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Animals. Male Friend Virus B-type WT mice and cardiac-specific MT-TG mice (8–10 weeks) were maintained at the University of Louisville Research Resources Center at 22°C with a 12-h light/dark cycle and were allowed free access to rodent chow and tap water. The cardiac-specific MT-TG mice were produced and characterized as previously reported (Kang et al., 1997). The transgenic founder mice were bred with WT mice of the same strain. Transgenic litter mates were identified by a pigment transgene marker (dark eye and fur) that was coinjected with the MT transgene into the early embryo when the transgenic mice were produced. Transgenic and WT mice were randomly assigned to control or treatment groups and were treated i.p. with either 0.9% NaCl or with DOX at a single dose of 20 mg/kg body weight. All animal procedures were approved by the Institutional Animal Care and Use Committee, which is certified by the American Association for Accreditation of Laboratory Animal Care.
Sample Preparation. Three days after DOX treatment, mice were sacrificed and the hearts were removed and homogenized in solubilization buffer containing 8 M urea, 2 M thiourea, 2% CHAPS, 65 mM dithiothreitol (DTT), 1% n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent; Calbiochem, San Diego, CA), and 1% protease inhibitor cocktail. Homogenates were allowed to incubate at room temperature for 30 min and were then centrifuged twice at 14,000g for 15 min. The supernatant was then collected, and the protein concentration of each sample was determined using the Bradford assay (Bio-Rad). Initial studies indicated considerable variations in protein levels among individual animals, thereby complicating data analysis. To eliminate the variations, three hearts from each treatment group were pooled to run the gel electrophoresis and imaging analysis, and four gel electrophoresis and analyses were conducted for each treatment group. The same experiment was repeated three times (a total of nine mice and 12 gels for each treatment group).
Two-Dimensional Gel Electrophoresis. First dimension isoelectric focusing (IEF) was performed using 18-cm immobilized pH gradient (IPG) strips with a pH range of 3 to 10 using a passive in-gel rehydration method. Briefly, 400-µg protein samples from each experimental group (Con-WT, Con-TG, DOX-WT, and DOX-TG) were diluted in a rehydration buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 65 mM DTT, 1% Zwittergent, 0.80% ampholytes, pH 3 to 10, and 0.01% bromphenol blue to a volume of 400 µl and allowed to incubate at room temperature for 30 min to allow the samples to be fully solubilized. After incubation, protein samples in rehydration buffer were added to IPG strips and the strips were allowed to rehydrate overnight at room temperature. IEF was performed at 5000 V for 24 h for a total of 100,000 V/h using a Genomic Solutions pHaser Isoelectric Focusing System. After IEF was completed, IPG strips were equilibrated for 15 min in equilibration buffer containing 45 mM Tris-HCl, pH 7.0, 6 M urea, 130 mM DTT, 30% glycerol, 1.6% SDS, and 0.002% bromphenol blue followed by a second 15-min incubation in the same buffer. The IPG strips were then equilibrated for an additional 15 min in the same buffer containing 4.5% iodoacetamide in place of DTT. SDS-polyacrylamide gel electrophoresis (PAGE) was performed on 18-cm IPG strips using 12.5% Duracryl Tricine Chemistry gels at 4°C for 5 h. Gels were then stained with Sypro Ruby, and gel images were captured using the ProXPRESS 2D Proteomic Imaging System (PerkinElmer Life and Analytical Sciences, Boston, MA) with excitation and emission wavelengths of 480 and 620 nm, respectively.
Two-Dimensional Gel Image Analysis. Gels were matched and analyzed for protein expression using Investigator HT Analyzer software (Genomic Solutions). Prior to image analysis, Sypro Ruby-stained gels were inverted so that the density of the fluorescent-stained spots could be detected as dark protein spots on a light background. For each treatment group, an analysis set containing eight gels (of 12 gels) was created, protein spots on each gel were detected, and the imaging was digitalized and statistically analyzed. A representative gel was presented as a reference gel for each treatment group. Protein spots in each gel were then automatically matched to the corresponding protein spots in the reference gel, and any incorrectly matched protein spots were corrected manually. Individual protein spot numbers were then synchronized to the corresponding protein spot number in the reference gel. An average mode of background subtraction was used to normalize the intensity volume of each spot for compatibility of intensity among gels. To accurately compare the measurements of protein spots across different gels and to correct for any protein loading differences, a normalization volume was calculated for each protein spot in a given gel by dividing the volume of the individual spot by the total volume of all of the valid spots in the gel. Normalization volumes were then transferred to Microsoft Excel, and an average normalization volume was determined for each protein spot in each treatment group. For each protein spot, three separate fold changes were then calculated (Con-TG/Con-WT, DOX-WT/Con-WT, and DOX-TG/Con-TG).
Peptide Mass Fingerprinting. Protein samples of interest were excised from gels and taken for identification using peptide mass fingerprinting. Samples were excised from gels and digested with trypsin as described previously (Jensen et al., 1999). Initially, protein spots were minced into small gel pieces and incubated for 15 min in 50 mM NH4HCO3. Acetonitrile was then added to the gel suspension, and the mixture was incubated for 15 min at room temperature. The wash buffer was then removed, and the gel was dried via vacuum centrifugation. Subsequently, the gel pieces were rehydrated in 20 mM DTT in 100 mM NH4HCO3 and incubated at 56°C for 45 min. The gel pieces were then incubated in 55 mM iodoacetamide in 100 mM NH4HCO3 at room temperature for 30 min in the dark. After alkylation of the sulfhydryl groups, gel pieces were washed a second time in 50 mM NH4HCO3 and 50% acetonitrile for 15 min and dried via vacuum centrifugation. Finally, protein gel pieces were incubated in 25 ng/µl modified trypsin at 37°C overnight. Tryptic digests then underwent matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide analysis.
Tryptic digests were initially mixed with an equal volume of 
-cyano-4-hydroxy-trans-cinnamic acid matrix. The mixture was then deposited on an -cyano-4-hydroxy-trans-cinnamic acid matrix surface and allowed to dry. The sample was subsequently washed with 5% formic acid to remove any water-soluble compounds. MALDI-TOF peptide fingerprint mass spectra were acquired with a TofSpec 2E MALDI-TOF mass spectrometer (Waters, Milford, MA) with a nitrogen laser. Peptide masses acquired by MALDI-TOF peptide analysis were searched against a subset of mouse proteins in the National Center for Biotechnology Information (NCBI) protein database using the Mascot search engine (Matrix Science, London, UK) (www.matrixscience.com). The search was based on three assumptions: first, that the polypeptides are monoisotopic; second, that the peptides may be oxidized at methionine residues; and third, that the peptides are carbamidomethylated at cysteine residues. Also, one missed trypsin cleavage and a mass tolerance of 100 ppm were allowed for matching the peptide mass values.
Two-Dimensional Gel Electrophoresis-Linked Immunoblot Analysis. First-dimension IEF was performed using 7-cm IPG strips with a pH range of 4 to 7 as above. Five micrograms of protein sample from DOX-TG was diluted in rehydration buffer containing IPG buffer, pH 4 to 7, in place of ampholytes, pH 3 to 10, to a volume of 125 µl. As before, rehydrated sample was added to IPG strips, and the strips were allowed to rehydrate overnight at room temperature. IEF was performed at 5000 V for 2 h 40 min for a total of 8000 V/h. Strips were equilibrated as before, and SDS-PAGE was performed using 17% Tris-glycine gels. Protein was then transferred to polyvinylidene difluoride membrane for immunoblot analysis. Nonspecific binding was blocked via incubation of the membrane in blocking buffer (5% nonfat dry milk in Tris-buffered saline) for 2 h followed by incubation at 4°C overnight in primary antibody (monoclonal anticytochrome c oxidase subunit Va, 1:2000) diluted in blocking buffer. The membrane was washed in TBST (Tris-buffered saline, 1% Tween 20) and incubated for 2 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000). The blot was then processed and visualized by chemiluminescence (GE Healthcare).
Statistical Analysis. All of the data are expressed as mean ± S.E. (n = 8). The data were analyzed according to a 2 x 2 factorial design. After a significant interaction was detected by the multiway analysis of variance, the significance of the main effects was further determined. The level of significance was considered when p < 0.05 as well as when a 2-fold difference, either increase or decrease, between the treated and control groups was detected.
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Results |
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; Sun et al., 2001).
Conventional two-dimensional gel electrophoresis (IEF/SDS-PAGE) was performed using total cardiac lysate to identify differences in proteomic profiles between the WT and MT-TG mouse hearts and their responses to DOX toxicity. The results presented in Fig. 1 show the effects of MT on DOX-induced changes in cardiac proteomic profiles. The gels were stained with Sypro Ruby to visualize proteins. Image analysis using Investigator HT Analyzer software revealed that 90 individual protein spots were differentially expressed (p < 0.05 and a 2-fold or more difference, either increase or decrease, between the groups) in either WT or MT-TG mouse hearts in response to DOX treatment. -Fold changes were calculated by dividing the average normalization volume of the spots from the eight DOX-treated gels by the average normalization volume of the spots from the eight control gels. Of these spots, 21 protein spots were increased and 18 were decreased in the WT mouse hearts (DOX-WT/Con-WT) but not changed in the MT-TG mouse hearts (DOX -TG/Con-TG). In the MT-TG mouse hearts, 11 protein spots were increased and 10 were decreased but not changed in the WT mouse hearts. Image analysis also revealed that three protein spots were increased in the WT mouse hearts while being decreased in the MT-TG mouse hearts after DOX treatment and that another five protein spots were decreased in the WT mouse hearts while being increased in the MT-TG mouse hearts after DOX treatment. Another 13 proteins were increased, and nine proteins were decreased in both WT and MT-TG mouse hearts after DOX treatment. All of the 90 protein spots were analyzed by MALDI-TOF mass spectrometry, and 18 were identified successfully (Fig. 2). These proteins included those involved in cellular antioxidant defense, nuclear-encoded subunits of the mitochondrial electron transport chain, enzymes involved in -oxidation of fatty acids and glycolysis, and proteins involved in regulation of cardiac muscle contraction (Table 1).
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The individual protein spot that showed the greatest change in staining intensity after DOX treatment was spot 3 (Fig. 3). After DOX treatment, spot 3 was found to be dramatically increased in the MT-TG mouse hearts in comparison with the MT-TG controls, whereas in the DOX-treated WT mouse hearts, spot 3 was found to be decreased slightly in comparison with the WT control (Table 1). Peptide masses acquired from the mass spectrum of spot 3 matched seven theoretical peptide mass values of tryptic digests of CCO-Va (Fig. 4A) and demonstrated a 48% coverage of the sequence (Fig. 4B). Interestingly, immunoblot analysis of the total proteins isolated from the hearts did not reveal significant difference in CCO-Va among the four treatment groups (data not shown). Subsequent MALDI-TOF mass spectrometry analysis of protein spots adjacent to spot 3 in DOX-treated transgenic heart 2D gels identified that spot 2 was also CCO-Va (Fig. 5A). Two-dimensional gel electrophoresis-linked immunoblot analysis of CCO-Va in the DOX-treated MT-TG mouse hearts revealed two specific spots for CCO-Va (Fig. 5B). MALDI-TOF mass spectrometry analysis also identified that both of these spots were CCO-Va. The quantitative changes between the two spots were significantly different among the treatment groups; however, the combined total amounts of the two spots were not significantly different. In particular, CCO-Va in spot 3 was significantly increased and in spot 2 was significantly decreased in response to DOX treatment in the MT-TG mouse hearts.
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Discussion |
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; Goormaghtigh et al., 1980; Cova et al., 1992) and recent studies using more advanced techniques (Kang et al., 1996, 1997; Sun et al., 2001; Wang et al., 2001a) have shown that ROS accumulation in mitochondria of cardiomyocytes is highly responsible for the oxidative damage to the heart. In particular, ROS accumulation in mitochondria triggers myocyte apoptosis (Kang et al., 2000; Wang et al., 2001a,b). MT inhibits DOX-induced myocardial apoptosis (Kang et al., 2000; Wang et al., 2001a) as well as ROS accumulation in mitochondria (Wang et al., 2001a). However, MT is not localized in mitochondria in the heart (Zhou and Kang, 2000; Ye et al., 2001). An important question that needs to be addressed is how MT affects DOX-induced ROS accumulation in mitochondria.
DOX-induced mitochondrial ROS accumulation is related to the mitochondrial oxidative phosphorylation process. DOX has been shown to act as a redox-cycling alternate electron acceptor and thereby leads to the uncoupling of mitochondrial oxidative phosphorylation (Wallace and Starkov, 2000). DOX is converted to reduced semiquinone free radical after accepting electrons from complex I of the mitochondrial respiratory chain (Davies and Doroshow, 1986). This semiquinone free radical intermediate is highly unstable and rapidly reacts with molecular oxygen to generate superoxide free radical with regeneration of intact DOX (Jung and Reszka, 2001). Furthermore, redox cycling of DOX in the mitochondria was shown to generate not only superoxide but also H2O2 and more importantly hydroxyl radical (Doroshow and Davies, 1986). The production and accumulation of ROS in the mitochondria is therefore thought to be a major mechanism leading to DOX-induced cardiotoxicity.
In mammals, mitochondrial oxidative phosphorylation is catalyzed by five membrane-bound protein complexes, NADH-ubiquinol oxidoreductase (complex I), succinateubiquinol oxidoreductase (complex II), ubiquinol-cytochrome c oxidoreductase (complex III), CCO (complex IV), and ATP synthase (complex V). These protein complexes are composed of multiple subunits encoded by both mitochondrial DNA and nuclear DNA. The mitochondrion contributes 13 of these subunits, whereas the remaining subunits (approximately 70) are encoded by nuclear DNA. Because MT is not localized in myocardial mitochondria (Zhou and Kang, 2000; Ye et al., 2001), it is likely that MT modifies mitochondrial proteins that are encoded by nuclear DNA. CCO-Va would be such a protein (Grossman and Lomax, 1997).
CCO (complex IV) is the terminal enzyme of the mitochondrial respiratory chain and as such catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. CCO is the primary determinant of molecular oxygen consumption in the cell and is thought to play an important role in the regulation of energy production (Capaldi, 1990; Poyton and McEwen, 1996). In mammals, CCO is composed of 13 subunits (three mitochondrial-encoded subunits and 10 nuclear encoded subunits), some of which occur as tissue-specific isoforms (Grossman and Lomax, 1997). The three mitochondrial-encoded subunits compose the catalytic center of the enzyme and are essential and sufficient to carry out the catalytic function of CCO (Grossman and Lomax, 1997). The exact function of the nuclear-encoded subunits of CCO has long been a topic of debate. However, experimental studies suggest that these nuclear-encoded subunits are involved in the regulation of CCO activity, including the rate of respiration as well as the efficiency of proton translocation (Kadenbach, 2003). The nuclear-encoded subunits of CCO, including CCO-Va, are therefore thought to be of importance in the regulation of oxidative phosphorylation.
A specific function for CCO-Va has been shown in reconstituted CCO from bovine heart. The thyroid hormone 3,5-diiodothyronine was found to specifically bind to CCO-Va using the radioactive hormone [3-125I]5-diiodothyronine (Arnold et al., 1998). This binding abolishes the allosteric inhibition of CCO activity by ATP and leads to stimulation of respiration (Arnold et al., 1998). Further function of CCO-Va in mitochondrial oxidative phosphorylation has yet to be determined, and it is unknown whether CCO-Va is subjected to post-translational modification in the heart. However, in the present study, the expression proteomic analysis indeed identified two different CCO-Va protein spots in the 2D gels. Interestingly, MT overexpression in the heart did not change the total concentrations of CCO-Va under the treatment with DOX relative to saline-treated controls; however, MT caused a shift of the dominant isoforms, enhancing one isoform and reducing the other. We postulate that this alteration would be due to a post-translational modification of CCO-Va and that such modification could enhance the oxidative phosphorylation reaction, thereby increasing the utilization of molecular oxygen and reducing the opportunity for the generation and accumulation of ROS in the mitochondria.
Besides the CCO-Va, which showed the most dominant change in the MT-TG mouse heart in response to DOX treatment, some other proteins also showed differential changes. The significance of these changes is subjected to further analysis. Further work is also necessary to elucidate the mechanism by which CCO-Va is modified by MT and the functional significance of this modification in protection from DOX cardiotoxicity. The present study demonstrates that modification of CCO-Va in the heart is likely involved in MT protection from DOX cardiotoxicity, directing our future effort on the mechanistic understanding of MT modification of this protein and functional significance of the modification.
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Acknowledgements |
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Footnotes |
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K.E.M. and W.F. contributed equally to this work.
ABBREVIATIONS: DOX, doxorubicin; MT, metallothionein; MT-TG, metallothionein-overexpressing transgenic; ROS, reactive oxygen species; WT, nontransgenic wild type; CCO-Va, cytochrome c oxidase subunit Va; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; NCBI, National Center for Biotechnology Information; 2D, two-dimensional; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
Address correspondence to: Dr. Y. James Kang, Department of Medicine, University of Louisville School of Medicine, 511 South Floyd Street, MDR 530, Louisville, KY 40202. E-mail: yjkang01{at}louisville.edu
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