It is believed that mitochondria are intimately involved in the pathogenesis of heart failure. Several human disorders are linked to mitochondrial dysfunction,1–3 and the most affected tissues are those that have a high demand for energy such as heart, brain, muscle, and kidney.4 Even though mitochondrial diseases are well known in humans, little is known about mitochondrial function in canine myocardial diseases. In the heart, mitochondria are mainly involved in energy production. Mitochondria also participate in cellular homeostasis, signaling, metabolism, and apoptosis.5
The canine mitochondrial genome is a circular, double-stranded molecule of 16,728 bp. Mitochondrial DNA encodes for 2 ribosomal RNAs (12S and 16S), 22 transfer RNAs, and 13 subunits of the respiratory chain.6 Approximately 90 protein subunits of the respiratory chain complex exist, of which only 13 subunits are encoded by mitochondrial DNA. However, mitochondrial DNA subunits are disproportionately represented because of the multiplicity of mitochondria copies per cell.7 In humans, it is estimated that approximately 1,000 to 2,000 mitochondrial proteins exist.8,9 When researching the pathophysiologic mechanisms responsible for altered mitochondrial function in DCM, proteomics offers several advantages over genomic techniques, such as the ability to detect the presence of isoforms and posttranslational modifications in cells or tissue and to directly quantify protein expression. This may be important in studies of mitochondria, as this organelle has been shown to be altered by fluctuating protein turnover and release10 as well as by transport of proteins into mitochondria from the cytosol.
Dilated cardiomyopathy is a myocardial disease characterized by ventricular dilation and reduced cardiac function.11 Several causes have been implicated in DCM involving different metabolic and signaling pathways. Dogs with rapid ventricular pacing have been used to study the pathophysiologic changes involved in DCM and congestive heart failure. Dogs with experimentally induced rapid ventricular pacing have compromised cardiac function that induces myopathy leading to increased chamber dilation and ventricular stress.12 To our knowledge, a comparison of canine mitochondria protein expression to determine how well the induced form of DCM mimics the naturally occurring disease has not been done.
On the basis of what is known from studies in various species, we anticipate that energy metabolism related to oxidative phosphorylation, TCA cycle, and proteins associated with programmed cell death will present altered protein expression.13–17 The purposes of the study reported here were to map the canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced DCM. Our hypotheses are that qualitative and quantitative differences in mitochondrial protein expression from the left ventricle will be found between control dogs and dogs with naturally occurring or induced forms of DCM. In this study, canine mitochondrial proteins from the left ventricle were mapped at narrow pH gradients by 2dimensional electrophoresis. In addition, mitochondrial protein expression was compared between control dogs, Doberman Pinschers with naturally occurring DCM, and dogs subjected to rapid ventricular pacing.
Matrix-assisted laser desorption/ionization time-of-flight
Quadrupole selecting, quadrupole collision cell, time-of-flight
Manganese superoxide dismutase
Augmenter of liver regeneration
Steroidogenic acute regulatory
3-hexaprenyl-4, 5-dihydroxybenzoate methyltransferase
Inner membrane translocase 22
Voltage-dependent anion channel-2
MITO-ISO 1, Sigma Chemical Co, St Louis, Mo.
BCA protein assay reagent kit, Pierce Biotechnology, Rockford, Ill.
Criterion system, Bio-Rad, Hercules, Calif.
ProteoSilver Plus, Sigma Chemical Co, St Louis, Mo.
Pdquest, version 7.1, Bio-Rad, Hercules, Calif.
Spot picker plus, PDM 1.5, The Gel Co, San Francisco, Calif.
ProteoProfile trypsin in-gel digestion kit, Sigma Chemical Co, St Louis, Mo.
Mass spectrometry laboratory, School of Chemical Sciences, University of Illinois at Urbana-Champaign, Urbana, Ill.
A9650, Sigma Chemical Co, St Louis, Mo.
I5500, Sigma Chemical Co, St Louis, Mo.
Smeitink J, Sengers R & Trijbels F, et al. Human NADH:ubiquinone oxidoreductase. J Bioenerg Biomembr 2001;33: 259–266.
Triepels RH, Van Den Heuvel LP & Trijbels JM, et al. Respiratory chain complex I deficiency. Am J Med Genet 2001;106: 37–45.
Cesseli D, Jakoniuk I & Barlucchi L, et al. Oxidative stressmediated cardiac cell death is a major determinant of ventricular dysfunction and failure in dog dilated cardiomyopathy. Circ Res 2001;89: 279–286.
Kim KS, Lee SE & Jeong HW, et al. The complete nucleotide sequence of the domestic dog (Canis familiaris) mitochondrial genome. Mol Phylogenet Evol 1998;10:210–220.
Dykens JA, Davis RA, Moos WH. Introduction to mitochondrial function and genomics. Drug Dev Res 1999;46: 2–13.
Augustin S, Nolden M & Muller S, et al. Characterization of peptides released from mitochondria. J Biol Chem 2005;280: 2691–2699.
Armstrong PW, Stopps TP & Ford SE, et al. Rapid ventricular pacing in the dog: pathophysiologic studies of heart failure. Circulation 1986;74: 1075–1084.
Knecht M, Regitz-Zagrosek V & Pleissner KP, et al. Characterization of myocardial protein composition in dilated cardiomyopathy by two-dimensional gel electrophoresis. Eur Heart J 1994;15 (suppl D):37–44.
Marin-Garcia J, Goldenthal MJ. Mitochondrial cardiomyopathy: molecular and biochemical analysis. Pediatr Cardiol 1997;18: 251–260.
McCutcheon LJ, Cory CR & Nowack L, et al. Respiratory chain defect of myocardial mitochondria in idiopathic dilated cardiomyopathy of Doberman Pinscher dogs. Can J Physiol Pharmacol 1992;70: 1529–1533.
Sharov VG, Goussev A & Lesch M, et al. Abnormal mitochondrial function in myocardium of dogs with chronic heart failure. Mol Cell Cardiol 1998;30:1757–1762.
MASCOT meptide mass fingerprint [database online]. Boston: Matrix Science Inc.. Available at: www.matrix science.com/search_form_select.html. Accessed Oct 7, 2004.
ExPASy PeptIdent [database online]. Geneva: Swiss Institute of Bioinformatics. Available at: us.expasy.org/tools/peptident.html. Accessed Oct 7, 2004.
ProteinProspector Tool MS-Fit [database online]. San Francisco: USCF Mass Spectrometry Facility, 1995. Available at: http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm. Accessed Oct 7, 2004.
Swiss-Prot [database online]. Geneva: Swiss Institute of Bioinformatics. Available at: us.expasy.org/sprot. Accessed Oct 7, 2004.
MitoProteome Protein List [database online]. San Diego: National Center of Biotechnology Information, 2004. Available at: www.mitoproteome.org/database/index.cgi?MENU_0=Production&S_NAME=Protein_List. Accessed Oct 7, 2004.
Weiller GF, Caraux G, Sylvester N. The modal distribution of protein isoelectric points reflects amino acid properties rather than sequence evolution. Proteomics 2004;4: 943–949.
Pitkanen S, Roninson BH. Mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase. J Clin Invest 1996;98: 345–351.
Rakhit R, Cunningham P & Furtos-Matei A, et al. Oxidationinduced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis. J Biol Chem 2002;277: 47551–47556.
Bahamonde I, Valverde MA. Voltage-dependent anion channel localizes to the plasma membrane and peripheral but not perinuclear mitochondria. Pflugers Arch 2003;446: 309–313.
Choksi KB, Boylston WH & Rabek JP, et al. Oxidatively damaged proteins of heart mitochondrial electron transport complexes. Biochim Biophys Acta 2004;1688: 95–101.
Ide T, Tsutsui H & Kinugawa S, et al. Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing myocardium. Circ Res 1999;85:357–363.
Suleiman MS, Halestrap AP, Griffiths EJ. Mitochondria: a target for myocardial protection. Pharmacol Ther 2001;89:29–46.
Van Remmen H, Williams MD & Guo Z, et al. Knockout mice heterozygous for Sod2 show alterations in cardiac mitochondrial function and apoptosis. Am J Physiol Heart Circ Physiol 2001;281:H1422–H1432.
Kurland CG, Anderson SGE. Origin and evolution of the mitochondrial proteome. Microbiol Mol Biol Rev 2000;64:786–820.
Borgarelli M, Tarducci A & Tidholm A, et al. Canine idiopathic dilated cardiomyopathy. Part II: pathophysiology and therapy. Vet J 2001;162: 182–195.
Tidholm A, Haggstrom J & Borgarelli M, et al. Canine idiopathic dilated cardiomyopathy. Part I: aetiology, clinical characteristics, epidemiology and pathology. Vet J 2001;162:92–107.
Satoh M, Nakamura M & Saitoh H, et al. Aldosterone synthase (CYP11B2) expression and myocardial fibrosis in the failing human heart. Clin Sci 2002;102: 381–386.
Lange H, Lisowsky T & Gerber, et al. An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins. Eur Mol Biol Org 2001;2: 715–720.
Heddi A, Stepien G & Benke PJ, et al. Coordinate induction of energy gene expression in tissues of mitochondrial disease patients. J Biol Chem 1999;274: 22968–22976.
Heinke MY, Wheeler CH & Chang D, et al. Protein changes observed in pacing-induced heart failure using two-dimensional electrophoresis. Electrophoresis 1998;19: 2021–2030.
Casal AJ, Silvestre JS & Delcayre C, et al. Expression and modulation of steroidogenic acute regulatory protein messenger ribonucleic acid in rat cardiocytes and after myocardial infarction. Endocrinology 2003;144:1861–1868.
Levillain O, Hus-Citharel A & Garvi S, et al. Ornithine metabolism in male and female rat kidney: mitochondrial expression of ornithine aminotransferase and arginase II. Am J Physiol Renal Physiol 2004;286:F727–F738.
Wischmeyer PE, Vanden Hoeck TL & Li C, et al. Glutamine preserves cardiomyocyte viability and enhances recovery of contractile function after ischemia-reperfusion injury. JPEN J Parenter Enteral Nutr 2003;27: 116–122.
Glorieux FH, Scriver CR & Delvin E, et al. Transport and metabolism of sarcosine in hypersarcosinemic and normal phenotypes. J Clin Invest 1971;50: 2313–2322.
Otto A, Stahler I & Klein R, et al. Anti-mitochondrial antibodies in patients with dilated cardiomyopathy (anti-M7) are directed against flavoenzymes with covalently bound FAD. Clin Exp Immunol 1998;111: 541–547.
Franchi M, Vullo D & Gallori E, et al. Carbonic anhydrase inhibitors: inhibition of human and murine mitochondrial isozymes V with anions. Bioorg Med Chem Lett 2003;13: 2857–2861.
Vullo D, Franchi M & Gallori E, et al. Carbonic anhydrase inhibitors. Inhibition of mitochondrial isoenzyme V with aromatic and heterocyclic sulfonamides. J Med Chem 2004;47:1272–1279.
Koc EC, Burkhart W & Blackburn K, et al. The large subunit of the mammalian mitochondrial ribosome. J Biol Chem 2001;276: 43958–43969.
Karlberg EOL, Anderson SGE. Mitochondrial gene history and mRNA localization:is there a correlation? Nat Rev Genet 2003;4: 391–397.
Kurz M, Martin H & Rassow J, et al. Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway. Mol Biol Cell 1999;10: 2461–2474.
Poon WW, Barkovich RJ & Hsu AY, et al. Yeast and rat Coq3 and Escherichia coli UbiG polypeptides catalize both o-methyltransferase steps in coenzyme Q biosynthesis. J Biol Chem 1999;274: 21665–21672.
Tsujimoto Y, Shimizu S. The voltage-dependent anion channel: an essential player in apoptosis. Biochimie 2002;84: 187–193.
Feliciello A, Gottesman ME, Avvedimento EV. The biological functions of A-kinase anchor proteins. J Mol Biol 2001;308: 99–114.
Affaitati A, Cardone L & De Cristofaro T, et al. Essential role of A-kinase anchor protein 121 for cAMP signaling to mitochondria. J Biol Chem 2003;278: 4286–4294.