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    Figure 1—

    Diagram of complex I expression of mitochondrially and nuclear-encoded protein subunits altered in tissue specimens from dogs with natural and induced forms of DCM. Notice subunits altered in the natural form of DCM (black arrows), subunits altered in the induced form of DCM (underlined subunits), and subunit altered in both forms of DCM (outlined subunit). FP = Flavoprotein segment. IP = Iron-protein segment. HP = Hydrophobic protein segment. ND = Natural disease. IM = Inner mitochondrial membrane. ↓/⇓ = Protein expression downregulated. ↑/⇑ = Protein expression upregulated.

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Correlation of mitochondrial protein expression in complexes I to V with natural and induced forms of canine idiopathic dilated cardiomyopathy

Rosana LopesDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802.

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Philip F. SolterDepartment of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802.

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D. David SissonDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802.

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Mark A. OyamaDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802.

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Robert ProsekDepartment of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802.

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Abstract

Objective—To identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with natural or induced dilated cardiomyopathy (DCM).

Sample Population—Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with DCM induced by rapid right ventricular pacing.

Procedures—Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by 2-fold or greater between groups were analyzed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry.

Results—A total of 22 altered mitochondrial proteins were identified in complexes I to V. Ten and 12 were found in complex I and complexes II to V, respectively. Five were mitochondrial encoded, and 17 were nuclear encoded. Most altered mitochondrial proteins in tissue specimens from dogs with naturally occurring DCM were associated with complexes I and V, whereas in tissue specimens from dogs subjected to rapid ventricular pacing, complexes I and IV were more affected. In the experimentally induced form of DCM, only nuclear-encoded subunits were changed in complex I. In both disease groups, the 22-kd subunit was downregulated.

Conclusions and Clinical Relevance—Natural and induced forms of DCM resulted in altered mitochondrial protein expression in complexes I to V. However, subcellular differences between the experimental and naturally occurring forms of DCM may exist.

Abstract

Objective—To identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with natural or induced dilated cardiomyopathy (DCM).

Sample Population—Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with DCM induced by rapid right ventricular pacing.

Procedures—Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by 2-fold or greater between groups were analyzed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry.

Results—A total of 22 altered mitochondrial proteins were identified in complexes I to V. Ten and 12 were found in complex I and complexes II to V, respectively. Five were mitochondrial encoded, and 17 were nuclear encoded. Most altered mitochondrial proteins in tissue specimens from dogs with naturally occurring DCM were associated with complexes I and V, whereas in tissue specimens from dogs subjected to rapid ventricular pacing, complexes I and IV were more affected. In the experimentally induced form of DCM, only nuclear-encoded subunits were changed in complex I. In both disease groups, the 22-kd subunit was downregulated.

Conclusions and Clinical Relevance—Natural and induced forms of DCM resulted in altered mitochondrial protein expression in complexes I to V. However, subcellular differences between the experimental and naturally occurring forms of DCM may exist.

Idiopathic DCM is the result of myocardial injury induced by a variety of mechanisms.1,2 It is the most commonly acquired myocardial disease of humans, and it occurs naturally in several dog breeds, including Doberman Pinschers, Boxers, Cocker Spaniels, Newfoundlands, and Great Danes. It is characterized in dogs and humans by dilation of the ventricular chambers and reduced cardiac function.2 The study of DCM is possible in dogs with experimentally induced congestive heart failure that is induced by rapid ventricular pacing; the induction of rapid ventricular pacing results in impaired systolic and diastolic left ventricular function and considerable chamber dilatation in the absence of left ventricular hypertrophy.3

The mitochondrion was discovered more than 100 years ago.4 Since that time, dysfunction of mitochondria in electron transfer and energy metabolism associated with disease has become well established.5,6 Moreover, there is much evidence supporting impaired energy metabolism in heart diseases of humans and other mammals.7–12

Oxidative phosphorylation, a process by which ATP is formed through a series of oxidation-reduction reactions in the ETC, occurs in the mitochondria and is essential for energy production in the heart and other organs.5,13 Energy deficits caused by oxidative phosphorylation dysfunction have also been linked to cardiomyopathies. The ETC is comprised of a series of protein complexes labeled I through V. Dysfunction of the ETC has been observed in several forms of heart failure in humans.5,6,14,15 Complex I, the biggest complex in the ETC, is composed of peripheral and membrane arms, giving the complex a distinct L-shaped appearance.16–18 A deficiency of NADH-ubiquinone oxidoreductase (complex I) has been implicated as the most common site for alteration in the ETC in human diseases of the nervous and muscular systems, heart, and kidney and also in cancer.5,6,14,15 In contrast, succinate ubiquinone oxidoreductase (complex II) deficiency is rare in humans, responsible for only 2% of altered energy metabolism.19

Complex I contains 46 known protein subunits, of which 7 are encoded by mitochondrial DNA and 39 are encoded by nuclear DNA.20,21 Complex I has the following 3 segments: a flavoprotein, an iron-protein, and a hydrophobic protein. The flavoprotein and ironprotein segments are located in the peripheral arm, whereas the hydrophobic protein segment is in the membrane arm. Electrons are transferred from the flavoprotein to the iron-protein segment, then to the hydrophobic protein segment, and subsequently transferred to a noncomplex protein, ubiquinone.20,21 Complex II contains the following 4 nuclear-encoded subunits: 70-kd flavoprotein, 27-kd iron-sulfur protein, cytochrome b, and a succinate dehydrogenase membrane anchor protein that is anchored to the matrix side of the inner mitochondrial membrane.19 Cytochrome c reductase (complex III) contains 10 nuclear and 1 mitochondrially encoded subunits. Active redox groups are formed by cytochromes b and c1 and also by an ironsulfur protein. Complex III transfers electrons from ubiquinone to cytochrome c.19 Cytochrome c oxidase (complex IV) is the last enzyme complex in the ETC and is formed by 10 nuclear-encoded and 3 mitochondrially encoded subunits. This complex contains 2 heme centers (cytochrome and a3) as well as 2 copper atoms, and it is responsible for the transfer of electrons from ferrocytochrome c to molecular oxygen.22

Adenosine triphosphate synthase (complex V) catalyzes the synthesis of ATP from ADP and inorganic phosphate by use of the proton-motive force generated during electron transfer from complexes I, III, and IV. Complex V is composed of the following 2 segments: the F1 unit, which contains 5 types of polypeptide chains (α3, β3, γ, δ3, and ε), and an FO unit responsible for pumping protons out of the matrix side into the intermembrane space and driving ATP synthesis. This complex has 14 nuclear and 2 mitochondrially encoded subunits.19,22,23

Proteins encoded by mitochondrial DNA make part of 4 of 5 complexes in the ETC. These mitochondrial polypeptides are represented by subunits ND1 to ND6 and ND4L of complex I; cytochrome b of complex III; COX polypeptides I, II, and III of complex IV; and ATP6 to ATP8 of complex V.19,22 The purpose of the study reported here was to identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with naturally occurring or induced DCM. In this study, we describe alterations in mitochondrial protein expression from the left ventricle in complexes I to V of the ETC by comparing tissue specimens from healthy control dogs to that of Doberman Pinschers with the natural form of DCM and to dogs subjected to rapid ventricular pacing.

Materials and Methods

Collection of tissue specimens—Left ventricular free wall was obtained from 7 clinically healthy mixed-breed dogs immediately after euthanasia. These dogs were part of a terminal study unrelated to this project and ranged in age and body weight from 4 to 8 years old and 20 to 35 kg, respectively. Left ventricular free wall samples were also obtained from 7 Doberman Pinschers with spontaneously occurring DCM at the time of euthanasia. All dogs in this group were at the end stage of disease with a mean percent fractional shortening of 13.4%. The age and weight of this group ranged from 4 to 11.5 years old and 29.5 to 48 kg, respectively.

Left ventricular free wall was also obtained from the experimental group dogs, which included 7 adult male hounds weighing between 21 to 35 kg. Three dogs were subjected to rapid ventricular pacing by use of an endocardial lead system placed in the apex of the right ventricle. The other 4 dogs were induced to rapid ventricular pacing by use of an epicardial lead system placed in the apex of the right ventricle. Initially, dogs were paced at 180 beats/min for 10 days. Subsequently the pacing rates were increased to 200, 210, 220, and 240 beats/min, respectively, in 7-day increments. In this group, the mean percent fractional shortening decreased from 30.5% to 12.5% at the time of euthanasia. The heart muscle specimens were either stored at −70°C in liquid nitrogen immediately after euthanasia or used immediately for mitochondrial fractionation. All dogs in this study were euthanatized by IV administration of standard pentobarbital-based euthanasia solution.

Mitochondrial isolation—Heart tissues were cleaned of connective tissue and fat and minced into 0.2- to 0.3-mmwide pieces. One hundred-milligram aliquots were homogenized in ice-cold mitochondrial isolation buffera with a dounce tissue grinder, transferred to 1.5-mL microcentrifuge tubes, and centrifuged at 600 × g for 5 minutes. The supernatant was transferred to new microcentrifuge tubes and centrifuged at 11,000 × g for 10 minutes. The mitochondria pellets were collected. The total protein was measured by use of bicinchonic acidb with bovine serum albumin standards, and pellets were stored in liquid nitrogen.

Two-dimensional gel electrophoresis and imaging analysis—The 2-dimensional electrophoresis protocol included the use of a commercial systemc and isoelectric point 3 to 10 linear immobilized pH gradient strips for isoelectric focusing. Polyacrylamide electrophoresis gels (11 cm in length; 12%) were used for second dimension electrophoresis, allowing up to 12 gels to run simultaneously. Each mitochondria sample was run in triplicate, applying 100 μg of protein sample to each gel, along with 2-dimensional gel electrophoresis standards. All gels were stained with silver.d Gels were scanned and resulting protein expression patterns analyzed with 2-dimensional gel analysis software.e Molecular mass and isoelectric point values were automatically calculated for the remaining protein spots. The quantitative analysis of protein spots was determined. Decreases or increases of 2fold or greater of mitochondrial protein expression in tissue specimens from diseased dogs, compared with control dogs, were considered substantial and evaluated. Statistical analysis was performed to compare mitochondrial protein expression between tissue specimens of control and diseased dog groups by use of a Mann-Whitney test. Significance was set at a value of P < 0.05. All mitochondrial protein spots found to differ significantly and by at least 2-fold were visually matched to confirm differences in their protein expression from control dogs. Mitochondrial protein samples from 3 control dogs were run in triplicate within narrow pH gradients of 3 to 6, 5 to 8, and 7 to 10 by use of the protocol already described.

Trypsin digestion and analysis by mass spectrometry—Only spots that differed significantly between control and diseased dog groups and by at least 2-fold were selected for mass spectrometry analysis. Protein in-gel digestion for each selected spot was processed after manual protein spot excision with a 1.5-mm-diameter spot cutterf into a siliconized microcentrifuge tube. Each selected spot was visually assessed to compare the altered mitochondrial protein expression between control and diseased dog groups. A protein in-gel digestion kitg was used for digestion of protein spots according to recommendations of the manufacturer. Prepared samples were analyzed by MALDI-TOF mass spectrometry with the use of a biospectrometry workstation.h External calibration of the biospectrometry workstation was performed with human angiotensin Ii that had a mass average of 1,297.50 daltons and bovine insulinj that had a mass average of 5,734.60 daltons.

Each protein spot was run 3 times in the MALDI-TOF spectrometer. Mass spectral data for each protein were compared with protein databases to achieve protein identification. For protein identification, 3 Web sites were searched.24–26 The monoisotopic peptide masses were searched in a protein database.27 A peptide mass tolerance of 1 dalton was allowed for all Web site databases. Mitochondrial protein spots not identified by MALDI-TOF mass spectrometry were analyzed by QqTOF mass spectrometry with an orthogonal accelerator.h

Results

For tissue specimens from each dog, triplicate gels were run resulting in a mean ± SD for matched mitochondrial protein spots of 345 ± 29 in control dogs, 355 ± 29 in dogs with naturally occurring DCM, and 356 ± 28 in dogs with induced DCM. Within each group, no significant qualitative changes in left ventricular mitochondrial protein expression among gels, among dogs, or between matched fresh and frozen heart tissues were found. However, significant differences in protein expression were found among tissue specimens from each of the 3 groups. Expression analysis revealed 40 protein spots in tissue specimens from the control dogs that had a mean expression value that was significantly different and changed by at least 2-fold, compared with that of tissue specimens from dogs with induced DCM or dogs with naturally occurring DCM. Analysis by MALDI-TOF mass spectrometry was performed on the 40 protein spots. Of these, 10 were identified as subunits of complex I, 9 of which were subunits of NADH-ubiquinone oxidoreductase chain including chains 2, 4, and 5, as well as the 9.6-, 20-, 23-, 24-, 30-, and 22-kd subunits (Table 1).

Table 1—

Complex I (NADH-ubiquinone oxidoreductase) proteins identified by MALDI- TOF mass spectrometry or QqTOF mass spectometry.

Protein name*Subcellular localizationSpecies matchedMolecular mass (kd)Isoelectric pointProtein expression
MeasuredExpectedMeasuredExpectedNatural DCMInduced DCM
Chain 2/P29867IMDrosophila mauritana29.4132.225.999.46IncNC
Chain 4/P92623IMAgkistrodon acutus26.7925.557.018.68IncNC
Chain 5/P03919IMHylobates lar71.4567.327.469.20DecNC
18 kd/45053IMHomo sapiens21.8320.095.3710.62DecNC
22 kd/Q9CQJ8IMMus musculus20.1921.857.007.84DecDec
24 kd/Q9D6J6IMMus musculus29.6327.316.717.00IncNC
9.6 kd/Q10217IMSchizosaccharomyces pombe13.7912.525.535.13NCInc
20 kd/Q42577IMArabidopsis thaliana23.8224.045.869.53NCDec
23 kd/Q12644IMMus musculus24.2024.906.006.22NCInc
30 kd/Q9DCT2IMMus musculus28.7226.485.585.45NCDec

Online database27 or National Center of Biotechnology Information28 accession numbers are shown.

Proteins analyzed by MALDI-TOF mass spectrometry.

Protein identified by QqTOF mass spectrometry.

Dec = Decreased protein expression by 2-fold or greater. Inc = Increased protein expression by 2-fold or greater. IM = Inner mitochondrial membrane. NC = No change.

We were unable to obtain a positive match by MALDI-TOF mass spectrometry for 3 protein spots; however, these spots were identified by QqTOF mass spectrometry as the following: NADH dehydrogenase ubiquinone Fe-S protein 4 18 kd of complex I (Table 1); ATP synthase α-chain, heart isoform, mitochondrial precursor; and ATP synthase β-chain, mitochondrial precursor of complex V (Table 2). All 10 protein subunits of complex I were part of the inner mitochondrial membrane portion. Five of the 22 subunits of complexes I to V that were identified were encoded by the mitochondrial genome, whereas 17 were nuclear encoded. Of the mitochondrial-encoded proteins in complex I, 2 (chain 2 and chain 4) were upregulated only in tissue specimens from dogs with naturally occurring DCM. In contrast, chain 5 had decreased expression in the natural form of DCM. None of the mitochondrially encoded proteins had significant differences in complex I expression in tissue specimens from dogs subjected to rapid ventricular pacing (Figure 1). The mitochondrial-encoded subunit 2 was identified with 2 peptides and 25.9% sequence coverage whereas chains 4 and 5 had 3 matched peptides and sequence coverage of 26% and 18%, respectively. Two nuclear-encoded subunits that were identified in tissue specimens from dogs with naturally occurring DCM were found in the peripheral arm of complex I. A 24-kd subunit was upregulated and was identified with 5 matched peptides, resulting in 26% sequence coverage, whereas an 18-kd subunit was downregulated in tissue specimens from dogs with naturally occurring DCM and identified by QqTOF mass spectrometry analysis. In dogs subjected to rapid ventricular pacing, only 1 subunit was associated with the peripheral arm of complex I, the nuclear-encoded 30-kd subunit, which was downregulated and identified with 3 matched peptides, resulting in 28.1% sequence coverage. In the induced form of DCM, most nuclear-encoded proteins were related to the hydrophobic arm of complex I. Among them, 9.6- and 23-kd subunits were upregulated and identified with 2 and 4 matched peptides resulting in 25% and 23.7% sequence coverage, respectively. The 20and 22-kd subunits were downregulated and matched with 4 and 3 peptides resulting in 47.7% and 30.9% sequence coverage, respectively. Of the mitochondrial-encoded proteins, cytochrome c polypeptide II of complex IV was downregulated in tissue specimens from dogs with naturally occurring DCM and identi sequence coverage, whereas cytochrome b of complex III was upregulated in tissue specimens from dogs with rapid ventricular pacing and identified with 4 matching peptides and 37% sequence coverage. Six nuclearencoded subunits were also associated with the natural form of DCM: the ATP synthase γ- and δ-chains and ATP12 protein of complex V were downregulated and identified with 2, 4, and 2 matching peptides and with 14.8%, 38%, and 29.7% sequence coverage, respectively. In contrast, cytochrome c B6-F iron-sulfur protein of complex III and COX polypeptide Vb of complex IV were upregulated and identified with 3 and 4 matching peptides and 29% and 32% sequence coverage, respectively. The ATP synthase α-chain of complex V was also upregulated. In the rapid ventricular pacing, group 5 nuclear-encoded proteins had altered expression and 3 of them (succinate dehydrogenase iron-sulfur protein of complex II, COX polypeptide Vb, and COX polypeptide IV-1 of complex IV) were upregulated. The first and last protein had 3 and 2 matching peptides with 26.1% and 15.3% sequence coverage, respectively. The COX polypeptide Vb peptide number and sequence coverage were described previously in dogs with naturally occurring DCM. In this same experimental group, only 2 proteins (COX assembly protein COX11 of complex IV and ATP synthase βchain mitochondrial precursor encoded by nuclear DNA) were downregulated. The first protein spot was identified with 3 peptides matched and 21.7% sequence coverage. The nuclear-encoded protein NADH-ubiquinone oxidoreductase 22-kd subunit and COX polypeptide Vb had a similar expression in both forms of DCM. The 22-kd subunit was universally decreased, whereas COX polypeptide Vb was a unique protein that had an increased expression in both forms of DCM. The functional classification of the canine heart mitochondrial proteins identified in our study followed the human mitochondrial protein classification from a protein data list.28

Table 2—

Mitochondrial proteins identified by MALDI-TOF mass spectrometry or QqTOF mass spectrometry in complexes II to V.

Oxidative phosphorylationProtein name*Subcellular localizationSpecies matchedMolecular mass (kd)Isoelectric pointProtein expression
MeasuredExpectedMeasuredExpectedDCMDCM
Complex IISDH ISP/P32420†IMUstilago maydis29.5933.285.558.91Inc
Complex IIICyt b/Q8SGT2†IMRaphus cucullatus28.3929.815.496.81Inc
Cyt B6-F ISP/P08980†IMSpinacia oleracea28.226.254.206.94Inc
Complex IVCOX II/P38596†IMHalichoerus grypus25.1426.035.854.77Dec
COX polypeptide Vb/P00428†IMBos Taurus21.5018.156.208.46IncInc
COX11/Q9Y6N1†IMHomo sapiens31.8031.465.609.22Dec
COX IV-1/O46580†IMHylobates agilis17.4916.796.539.18Inc
Complex Vγ-chain/O01666†IMDrosophila melanogaster32.2832.877.509.29Dec
ATP 12 protein/P22135†IMSaccharomyces cerevisae27.7932.947.157.09Dec
δ-chain/P22479†IMBacillus pseudofirmus21.5020.546.206.19Dec
α-chain/114402‡§IMBos Taurus63.2659.685.359.43Inc
β-chain/114562‡IMRattus novergicus101.3956.326.025.26Dec

Peptide sequences identified with 46-TGTAEVSSILEER-58, 134-TGAIVDVPVGEELLGR-149, 150-VVDALGNAIDGKGPVGSK-167 (V-substitution V for I), 403-GIRPAINVGLSVSR-416, and 442-EVAAFAQFGSDLDAATQQLLSR-463.

Peptide sequences identified with 213-TVLIMELINNVAK-225, 463-FLSQPFQVAAEVFTGHMGK-480.

Cyt = Cytochrome. SDH = Succinate dehydrogenase. ISP = Iron-sulfur protein.

See Table 1 for remainder of key.

Figure 1—
Figure 1—

Diagram of complex I expression of mitochondrially and nuclear-encoded protein subunits altered in tissue specimens from dogs with natural and induced forms of DCM. Notice subunits altered in the natural form of DCM (black arrows), subunits altered in the induced form of DCM (underlined subunits), and subunit altered in both forms of DCM (outlined subunit). FP = Flavoprotein segment. IP = Iron-protein segment. HP = Hydrophobic protein segment. ND = Natural disease. IM = Inner mitochondrial membrane. ↓/⇓ = Protein expression downregulated. ↑/⇑ = Protein expression upregulated.

Citation: American Journal of Veterinary Research 67, 6; 10.2460/ajvr.67.6.971

Discussion

Many of the changes to mitochondrial protein expression observed in our study are anticipated to result in decreased myocardial cell energy production. For example, decreased NADH-ubiquinone oxidoreductase chain 5 expression has been shown in other species to be vital for complex I activity29 and energy metabolism.30 Altered protein expression in complexes I, III, IV, and V are similar to those associated with altered oxidative phosphorylation in neonates and children with dilated and hypertrophic cardiomyopathies.31–33 Dysfunction of complexes III and V induced by ventricular pacing in dogs has been observed by others.34 In addition, a study of respiratory complexes in Doberman Pinschers with naturally occurring DCM found a reduction of 60% in complex I and 50% in complex V activity.10

Several subunits in complex I had decreased expression, suggesting that overall complex I function and assembly is likely diminished in both forms of canine DCM. Experimental evidence suggests that it is not necessary for all proteins within a complex to change for an effect on complex function to be evident. For example, it has been shown that dysfunction in 8-, 15-, 18-, 20-, 30-, and 39-kd subunits can compromise assembly of complex I.35 Others, such as 20- and 23-kd NADH-ubiquinone oxidoreductase subunits, are thought to participate in the last electron transfer through the ETC.36–38 Hence, alterations to various components of each complex likely affect the function of the entire complex and of the entire ETC.

The NADH dehydrogenase (ubiquinone) Fe-S protein 4 was decreased in the natural form of DCM. This protein connects the hydrophobic and peripheral arms of complex I.39 In contrast, the study of mice with mitochondrial cardiomyopathy that was induced through adenine nucleotide translocator-1 gene knockout resulted in upregulation of the nuclear gene encoding NADH dehydrogenase (ubiquinone) Fe-S 18-kd protein 4.40 However, the phosphorylation of serine residues in this protein has been shown to increase the activity of complex I and augment improved ATP synthesis.41–43 Hence, although we did not detect any modified forms of this protein, we cannot rule out that the cause of the decreased expression of this protein in our studies could be the result of posttranslational modification, such as phosphorylation.

The only complex I protein with decreased expression in both forms of DCM was the 22-kd subunit. The function of this subunit is unknown,44–46 although its location in the membrane arm of complex I could imply that it is involved with the ubiquinone-binding site. Because it was decreased in both forms of DCM, it may be an important focus in future studies. In contrast, several of the NADH-ubiquinone oxidoreductase proteins of complex I were increased in both types of experimentally induced DCM, which may be an attempt to improve complex I function. For example, increased expression of NADH-ubiquinone oxidoreductase 24-kd subunit, which is a component of the first site of electron transfer in complex I,47 might signal a response to reduced complex I activity. The NADH-ubiquinone oxidoreductase 9.6-kd subunit promotes cell lipoic acid concentrations, and decreased activity of this enzyme can cause respiratory chain dysfunction.48 Increased expression of this protein may, therefore, imply an attempt at increased respiratory function through the lipoic acid pathway.

Three of the 4 complex V proteins identified in our study had decreased expression. All 3 are believed to be necessary for assembly of complex V,49,50 as well as ATP synthesis.51 Reduced expression of the γ-chain subunit of complex V in our study suggests that assembly of the whole complex and its catalytic activity is impaired. The ATP synthase β-chain of complex V was downregulated in rapid ventricular pacing. Downregulation of the γ-chain subunit of complex V has also been observed by others.52 The upregulation of the α-chain in dogs with naturally occurring DCM suggests that this subunit, although being overexpressed, is not assembled properly in complex V. Interestingly, upregulation of the α-chain subunit occurs in neurons of patients with Alzheimer's disease.53 Whether or not cytosolic accumulation could lead to myocyte degeneration in dogs with naturally occurring DCM is unknown. Studies of bovine mitochondrial ATP synthase have shown that amino-terminal residues of the α-chain are essential for the functional connection between F1 and FO segments.54 In humans, it was found that ATP12 also interacts with the α-chain of the F1segment.55

Alterations to several of the COX assembly proteins were observed in dogs with either naturally occurring or pacing-induced DCM in our study. As others have shown that oxygen concentrations during electron transfer regulate the COX proteins,56–58 several of these observed changes suggest that mitochondria may be responding to a relative hypoxia. The COX polypeptide Vb was the unique mitochondrial protein upregulated in both forms of DCM. The COX polypeptide Vb gene is known to be upregulated under hypoxic conditions,56 suggesting that canine DCM is associated with low mitochondrial oxygen availability. Likewise, increased expression of a protein associated with oxygen sensing,59 succinate dehydrogenase iron-sulfur protein, in dogs with rapid ventricular pacing, is consistent with hypoxia. The downregulation of COX II, a mitochondrial-encoded subunit of complex IV, observed in our study, could also be suggestive of hypoxia. Cytochrome c oxidase assembly protein COX11 of complex IV was downregulated in dogs with rapid ventricular pacing. Whereas COX11 has been speculated to function as a chaperone for the copper B center in complex IV,60 the decreased expression of COX11 suggests that assembly and stability of complex IV is also altered. In dogs with naturally occurring DCM, COX IV-1 expression was increased. Two isoforms, COX IV-1 and COX IV-2, are found in mammals61 and are believed to be involved in regulatory function of complex IV because of the proximity of catalytic subunits I and II to this complex.62

In this study we also found evidence of altered complex III protein expression in dogs with DCM. Increased expression of cytochrome b of complex III was observed in dogs subjected to rapid ventricular pacing. The overexpression of cytochrome b observed in our study may be an attempt of complex III to increase its function. Cytochrome b is known to be essential for complex III catalytic activity because dysfunction of complex III as a result of cytochrome b mutations have been observed in humans with DCM and other forms of cardiomyopathy.31,63,64 Upregulation of mitochondrial proteins in dogs with naturally occurring DCM was also observed in cytochrome B6-F ironsulfur protein of complex III, which interacts with redox subunits cytochrome b and c1, which are essential to complex III function.

As mentioned, in addition to differences between control dogs and dogs with DCM, differences were found in mitochondrial protein expression between the naturally occurring and induced forms of DCM. These findings suggest that there may be inherent pathophysiologic differences in mitochondrial dysfunction between experimentally induced and naturally occurring forms of DCM. Several potential causes of these differences are likely. For example, dogs with naturally occurring DCM were all receiving heart failure treatment that consisted primarily of positive inotropic agents, diuretics, and angiotensin-converting enzyme inhibitors. However, we believe that it is likely that most of the observed changes reflect differences between the experimentally induced disease and the clinical disease state itself. We also do not believe that the differences between the 2 disease groups represent differences in the stages of disease because both groups were at similar stages of heart failure, based on the mean percent ventricular fractional shortening, a measure of cardiac function, which was similar between the 2 disease groups. An additional possibility for the difference between the 2 disease groups is related to the rate of onset of disease, assuming that rapid ventricular pacing brought about heart failure faster than the natural progression of spontaneously developing DCM. An improved experimental form of DCM that would better mimic the natural disease may allow for a more gradual onset of disease.

In summary, results of our study indicate that natural and induced forms of DCM are associated with altered mitochondrial protein expression, which may influence ETC complexes assembly and energy metabolism. In the natural form of DCM, the key subunit affected is NADH-ubiquinone oxidoreductase chain 5, which is involved in the regulation of complex I energy metabolism. Moreover, in dogs with naturally occurring DCM, altered mitochondrial protein expression is mainly associated with complex I and complex V. Mitochondrial protein changes in this group caused by altered expression of ATP synthase α-, γ-, and δ-chains and ATP 12 could impair assembly of F1 subunits, as well as the whole complex, and reduce the activity of FO segment. In addition, complex IV had a divergent response in expression of proteins COX II and COX polypeptide Vb subunits, suggesting that the heart is working under low oxygen concentrations, compromising complex IV catalytic activity. In contrast, in the experimental form of DCM, altered subunits of complex I (9.6-, 20-, and 30-kd subunits) suggest impaired complex assembly. In dogs subjected to rapid ventricular pacing, complex IV was more affected, revealing instability of subunit II and altered assembly caused by COX11 downregulation as well as reduced catalytic activity induced by upregulation of COX polypeptide Vb and COX IV-1 subunits. Hence, there may be subcellular differences between experimentally induced and naturally occurring forms of canine DCM.

ABBREVIATIONS

DCM

Dilated cardiomyopathy

NADH

Reduced form of nicotinamide adenine dinucleotide

ETC

Electron transport chain

COX

Cytochrome c oxidase

MALDI-TOF

Matrix-assisted laser desorption/ionization time-of-flight

QqTOF

Quadrupole selecting, quadrupole collision cell, time-of-flight

a.

MITO-ISO 1, Sigma Chemical Co, St Louis, Mo.

b.

BCA protein assay reagent kit, Pierce Biotechnology, Rockford, Ill.

c.

Criterion system, Bio-Rad, Hercules, Calif.

d.

ProteoSilver Plus, Sigma Chemical Co, St Louis, Mo.

e.

Pdquest, version 7.1, Bio-Rad, Hercules, Calif.

f.

Spot picker plus, PDM 1.5, The Gel Co, San Francisco, Calif.

g.

ProteoProfile trypsin in-gel digestion kit, Sigma Chemical Co, St Louis, Mo.

h.

Mass spectrometry laboratory, School of Chemical Sciences, University of Illinois at Urbana-Champaign, Urbana, Ill.

i.

A9650, Sigma Chemical Co, St Louis, Mo.

j.

I5500, Sigma Chemical Co, St Louis, Mo.

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Contributor Notes

Supported in part by the 2004 Spangler Fund and the 2005 Companion Animal Grants. Rosana Lopes was supported through a research fellowship from Brazilian National Council of Scientific and Technologic Development. The Voyager-DE STR mass spectrometer was purchased in part with a grant from the National Institutes of Health, Division of Research Resources (No. RR 11966). The Q-TOF Ultima mass spectrometer was purchased in part with a grant from the National Science Foundation, Division of Biological Infrastructure (No. DBI0100085).

The authors thank Furong Sun for technical assistance.

Address correspondence to Dr. Solter.