Quantitative Protein Profiling in Heart Mitochondria from Diabetic Rats

Abstract

Quantitative protein profiling based on in vitro stable isotope labeling, two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry is an accurate and reliable approach to measure simultaneously the relative abundance of many individual proteins within two different samples. In the present study, it was used to define a set of alterations caused by diabetes in heart mitochondria from streptozotocin-treated rats. We demonstrated that the expression of proteins from the myocardial tricarboxylic acid cycle was not altered in diabetes. However, up-regulation of the fatty acid β-oxidation favored fatty acids over glucose as a source of acetyl CoA for the tricarboxylic acid cycle. Protein levels for several proteins involved in electron transport were modestly decreased. Whether this may depress overall ATP production remains to be established, since the protein level of ATP synthase seems to be unchanged. Other changes include down-regulation of protein levels for creatine kinase, voltage-dependent anion channel 1 (VDAC-1), HSP60, and Grp75. The mitochondria-associated level of albumin was decreased, while the level of catalase was substantially increased. All of the changes were evident as early as 1 week after streptozotocin administration. Taken together, these data point to a rapid and highly coordinated regulation of mitochondrial protein expression that occurs during the heart adaptation to diabetes. Quantitative protein profiling based on in vitro stable isotope labeling, two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry is an accurate and reliable approach to measure simultaneously the relative abundance of many individual proteins within two different samples. In the present study, it was used to define a set of alterations caused by diabetes in heart mitochondria from streptozotocin-treated rats. We demonstrated that the expression of proteins from the myocardial tricarboxylic acid cycle was not altered in diabetes. However, up-regulation of the fatty acid β-oxidation favored fatty acids over glucose as a source of acetyl CoA for the tricarboxylic acid cycle. Protein levels for several proteins involved in electron transport were modestly decreased. Whether this may depress overall ATP production remains to be established, since the protein level of ATP synthase seems to be unchanged. Other changes include down-regulation of protein levels for creatine kinase, voltage-dependent anion channel 1 (VDAC-1), HSP60, and Grp75. The mitochondria-associated level of albumin was decreased, while the level of catalase was substantially increased. All of the changes were evident as early as 1 week after streptozotocin administration. Taken together, these data point to a rapid and highly coordinated regulation of mitochondrial protein expression that occurs during the heart adaptation to diabetes. Recent advances in quantitative protein profiling have made possible the relative quantification of individual proteins within two different samples (1Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4341) Google Scholar, 2Mirgorodskaya O.A. Kozmin Y.P. Titov M.I. Korner R. Sonksen C.P. Roepstorff P. Rapid Commun. Mass Spectrom. 2000; 14: 1226-1232Crossref PubMed Scopus (332) Google Scholar, 3Yao X. Freas A. Ramirez J. Demirev P.A. Fenselau C. Anal. Chem. 2001; 73: 2836-2842Crossref PubMed Scopus (779) Google Scholar, 4Munchbach M. Quadroni M. Miotto G. James P. Anal. Chem. 2000; 72: 4047-4057Crossref PubMed Scopus (251) Google Scholar, 5Goodlett D.R. Keller A. Watts J.D. Newitt R. Yi E.C. Purvine S. Eng J.K. von Haller P. Aebersold R. Kolker E. Rapid Commun. Mass Spectrom. 2001; 15: 1214-1221Crossref PubMed Scopus (271) Google Scholar, 6Cagney G. Emili A. Nat. Biotechnol. 2002; 20: 163-170Crossref PubMed Scopus (218) Google Scholar, 7Smolka M. Zhou H. Aebersold R. Mol. Cell. Proteomics. 2002; 1: 19-29Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 8Sechi S. Rapid Commun. Mass Spectrom. 2002; 16: 1416-1424Crossref PubMed Scopus (69) Google Scholar, 9Gehanne S. Cecconi D. Carboni L. Righetti P.G. Domenici E. Hamdan M. Rapid Commun. Mass Spectrom. 2002; 16: 1692-1698Crossref PubMed Scopus (48) Google Scholar, 10Goodlett D.R. Yi E.C. Funct. Integr. Genomics. 2002; 2: 138-153Crossref PubMed Scopus (33) Google Scholar). The approaches vary in details but all of them are based on labeling the proteins in two separate samples with isotopically normal and heavy reagents. A relative abundance of a protein in the original samples is determined by mass spectrometry as the ratio of signal intensities for the isotopically normal and heavy forms of a specifically labeled peptide. Several strategies of in vitro stable isotope labeling have been described previously (1Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4341) Google Scholar, 2Mirgorodskaya O.A. Kozmin Y.P. Titov M.I. Korner R. Sonksen C.P. Roepstorff P. Rapid Commun. Mass Spectrom. 2000; 14: 1226-1232Crossref PubMed Scopus (332) Google Scholar, 3Yao X. Freas A. Ramirez J. Demirev P.A. Fenselau C. Anal. Chem. 2001; 73: 2836-2842Crossref PubMed Scopus (779) Google Scholar, 4Munchbach M. Quadroni M. Miotto G. James P. Anal. Chem. 2000; 72: 4047-4057Crossref PubMed Scopus (251) Google Scholar, 5Goodlett D.R. Keller A. Watts J.D. Newitt R. Yi E.C. Purvine S. Eng J.K. von Haller P. Aebersold R. Kolker E. Rapid Commun. Mass Spectrom. 2001; 15: 1214-1221Crossref PubMed Scopus (271) Google Scholar, 6Cagney G. Emili A. Nat. Biotechnol. 2002; 20: 163-170Crossref PubMed Scopus (218) Google Scholar). The most recent approach suggests an alkylation of cysteine residues in proteins with light (non-deuterated) and heavy (deuterated) forms of acrylamide (8Sechi S. Rapid Commun. Mass Spectrom. 2002; 16: 1416-1424Crossref PubMed Scopus (69) Google Scholar, 9Gehanne S. Cecconi D. Carboni L. Righetti P.G. Domenici E. Hamdan M. Rapid Commun. Mass Spectrom. 2002; 16: 1692-1698Crossref PubMed Scopus (48) Google Scholar). In the present report, this method was used for quantifying protein expression in heart mitochondria from diabetic rats. The studies on diabetic mitochondria generally suggest that diabetes causes dysfunctional mitochondria (11Pierce G.N. Dhalla N.S. Can. J. Cardiol. 1985; 1: 48-54PubMed Google Scholar, 12Tanaka Y. Konno N. Kako K.J. Cardiovasc. Res. 1992; 26: 409-414Crossref PubMed Scopus (64) Google Scholar, 13Mokhtar N. Lavoie J.P. Rousseau-Migneron S. Nadeau A. Diabetes. 1993; 42: 682-687Crossref PubMed Scopus (37) Google Scholar, 14Tomita M. Mukae S. Geshi E. Umetsu K. Nakatani M. Katagiri T. Jpn. Circ. J. 1996; 60: 673-682Crossref PubMed Scopus (47) Google Scholar). However, little is known about the nature or extent of mitochondrial dysfunction in diabetes and the relationship between mitochondrial dysfunction and diabetic pathology has yet to be defined. The cardiac function is dependent on a constant rate of resynthesis of ATP by mitochondrial oxidative phosphorylation (OXPHOS). 1The abbreviations used are: OXPHOS, oxidative phosphorylation; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; STZ, streptozotocin; IDDM, insulin-dependent diabetes mellitus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ACTH, adrenocorticotropic hormone; VDAC-1, voltage-dependent anion channel 1.1The abbreviations used are: OXPHOS, oxidative phosphorylation; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; STZ, streptozotocin; IDDM, insulin-dependent diabetes mellitus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ACTH, adrenocorticotropic hormone; VDAC-1, voltage-dependent anion channel 1. Fatty acids and glucose are normally major energy substrates in heart. Both pathways, glucose oxidation and fatty acid oxidation, merge in the mitochondria on the level of acetyl CoA production. Acetyl-CoA then enters the tricarboxylic acid cycle. The last step in ATP production is the entry of NADH and FADH2, derived from the tricarboxylic acid cycle, into the electron transport chain coupled with the mitochondrial ATP synthase. Insulin-dependent diabetes mellitus (IDDM) causes alterations in myocardial metabolism characterized by a decrease in glucose uptake and utilization and an increase in fatty acid oxidation (15Stanley W.C. Lopaschuk G.D. McCormack J.G. Cardiovasc. Res. 1997; 34: 25-33Crossref PubMed Scopus (405) Google Scholar, 16Depre C. Young M.E. Ying J. Ahuja H.S. Han Q. Garza N. Davies P.J.A. Taegtmeyer H. J. Mol. Cell Cardiol. 2000; 32: 985-996Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 17Young M.E. McNulty P. Taegtmeyer H. Circulation. 2002; 105: 1861-1870Crossref PubMed Scopus (399) Google Scholar). As a first step toward profiling the changes in diabetic mitochondria, we have analyzed the pattern of protein expression in heart mitochondria from streptozotocin (STZ)-treated rats. Our primary focus was on proteins involved in three major mitochondrial cycles associated with ATP production, namely on β-oxidation, tricarboxylic acid cycle, and OXPHOS. The diabetes-associated changes in expression levels of several other mitochondrial proteins were also estimated. Animals and Treatment—Male Harlan Sprague-Dawley rats (median body weight 250 g) were from Harlan Sprague-Dawley, Indianapolis, IN. Animals were maintained on standard rat chow and tap water ad libitum. STZ (Sigma) was dissolved in sterile 0.1 m citrate buffer (pH 4.5) and injected intraperitoneally at a dose of 60 mg/kg body weight. Control rats received the corresponding volume of citrate buffer. The blood glucose levels were monitored using Accu-Chek Advantage blood glucose monitor and test strips (Roche Applied Science). The STZ-treated rats that showed fasting blood glucose levels above 350 mg/dl, in addition to loss of weight and polyuria, were considered as insulin-dependent diabetes mellitus. Diabetic rats and age-matched control rats were sacrificed at 1 or 4 weeks after STZ injection. Isolation of Heart Mitochondria—Mitochondria from individual rat hearts were isolated using differential centrifugation (18Hovius R. Lambrechts H. Nicolay K. de Kruijff B. Biochim. Biophys. Acta. 1990; 1021: 217-226Crossref PubMed Scopus (310) Google Scholar) in 5 mm Hepes-KOH (pH 7.4), 0.25 m mannitol, 0.1 mm EDTA, 0.1% bovine serum albumin supplemented with protease and proteasome inhibitors: 1mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.8 μm aprotinin, 50 μm bestatin, 15 μm E-64, 20 μm leupeptin, 10 μm pepstatin A, 20 μm proteasome inhibitor I, 20 μm lactacystin, and 20 μm MG-132. Purified mitochondria were further isolated using 30% Percoll, essentially as described in Ref. 18Hovius R. Lambrechts H. Nicolay K. de Kruijff B. Biochim. Biophys. Acta. 1990; 1021: 217-226Crossref PubMed Scopus (310) Google Scholar. Percoll-purified mitochondria were then sonicated and centrifuged at 100,000 × g for 60 min. The high speed supernatant fraction includes mitochondrial soluble proteins. The high speed pellet includes mainly mitochondrial membrane proteins. Total protein concentrations were determined using the Bio-Rad protein assay with bovine serum albumin as the standard. Labeling of Mitochondrial Proteins—The high speed supernatant and pellet fractions from Percoll-purified heart mitochondria from diabetic (1 week and 4 week of diabetes), and age-matched control rats were alkylated with acrylamide. 1 mg of total protein was dissolved in 1 ml of 2 m thiourea, 7 m urea, 4% CHAPS, 40 mm dithiothreitol, and incubated for 60 min at room temperature. The mitochondrial fractions from normal rats were then alkylated with 400 mm light (non-deuterated) acrylamide, while the mitochondrial fractions from diabetic rats were alkylated with 400 mm heavy (deuterated) acrylamide (Cambridge Isotope Laboratories, Woburn, MA) for 5 h at room temperature. The corresponding normal and diabetic fractions were then mixed in the 1:1 ratio. Two-dimensional Gel Electrophoresis (Two-dimensional PAGE)— The first dimension was performed using the PROTEAN IEF cell (Bio-Rad). The 7-cm pH 3–10 immobilized pH gradient strips were rehydrated with 125 μl of a solution (2 m thiourea, 7 m urea, 4% CHAPS, 0.5% Triton X-100, 0.5% Biolytes 3–10, 1% dithiothreitol, and bromphenol blue) that contained 200 μg of the total protein. Isoelectric focusing was conducted at 250 V for 15 min, linearly increased over 2 h to a maximum of 4,000 V, and then run to accumulate a total of 20,000 V/h. For the second dimension, the immobilized pH gradient strips were equilibrated for 15 min in 50 mm Tris-HCl (pH 8.8), 6 m urea, 30% glycerol, 2% SDS, and bromphenol blue. The strips were then embedded in 0.7% (w/v) agarose on the top of 10% acrylamide slab gels containing a 4% stacking gel. Identification and Quantification of Mitochondrial Proteins—The proteins were identified by matrix-assisted laser desorption ionization/time-of-flight MS analysis (MALDI-TOF) of in-gel tryptic digest of selected Coomassie-stained spots. MALDI-TOF was performed using a Voyager-DE STR BioSpectrometry work station (PerSeptive Biosystems) operated in the delayed extraction and reflector mode with a matrix of α-cyano-4-hydroxy-cinnamic acid. Bradykinin fragment 1–7 (MH+ 757.3997), angiotensin II (MH+ 1046.5423), synthetic peptide P14R (MH+ 1533.8582), and ACTH fragment 18–39 (MH+ 2465.1989) were used for external calibration. All spectra were also calibrated internally on the trypsin peaks at 842.51 and 2211.0968. Peptide mass fingerprinting data were evaluated using the MASCOT software (www.matrixscience.com). For quantifying the relative abundance of a protein, the peak height of the monoisotopic peak of the heavy (deuterated) acrylamide-labeled peptide was divided by the peak height of the monoisotopic peak of the light (non-deuterated) acrylamide-labeled form of the peptide. Six MALDI-TOF mass spectra were acquired for each individual sample, and the results were averaged. All measurements were performed for n = 6 animals, which were handled individually. Data are presented as the mean ± S.E. Principle of the Method— Fig. 1 shows a schematic presentation of the method of quantitative proteomics used in this report. It is based on the use of light (non-deuterated or d 0) and heavy (deuterated or d 3) acrylamide to alkylate cysteine residues in proteins before their separation by two-dimensional PAGE and subsequent analysis by MALDI-TOF MS. To estimate the relative amount of a specific protein in two separate samples, one sample is labeled with the light acrylamide and another sample is labeled with heavy acrylamide. Heavy acrylamide has 3 deuteriums instead of 3 protons and is 3 mass units heavier than light acrylamide. From the chemical point of view, there is no difference between light and heavy forms of acrylamide. This warrants equal efficiency of alkylation in separate samples and, after mixing, the co-migration of the same proteins from separate samples on two-dimensional PAGE (1Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4341) Google Scholar, 7Smolka M. Zhou H. Aebersold R. Mol. Cell. Proteomics. 2002; 1: 19-29Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The areas of interest were then in-gel digested with trypsin and identified using MALDI-TOF MS. The following quantitative profiling was accomplished by comparing a ratio between the intensities of the light and heavy mass components of the cysteine-containing peptide(s). An example in the Fig. 1 shows that the cysteine-containing peptide, YCTQDAFFQV, from dienoyl-CoA isomerase is represented by two isotopic envelopes that are separated by 3 mass units. The first dienoyl-CoA isomerase from normal The second dienoyl-CoA isomerase from diabetic mitochondrial samples were mixed we that the level of dienoyl-CoA isomerase was increased in the diabetic Quantification of in Protein in Heart by a of two-dimensional the mitochondria were first separated in two the high speed supernatant fraction which includes mitochondrial soluble proteins and the which includes mainly mitochondrial membrane proteins. two fractions were then analyzed The Coomassie-stained two-dimensional gel are in the Fig. Our primary focus was on proteins that to β-oxidation, tricarboxylic acid cycle, or to OXPHOS. We have identified many proteins involved in these three major mitochondrial However, it be that the quantitative method used in this has It is not to proteins cysteine The other is the peptide with another then quantification be For these we to of the identified proteins. proteins for which a reliable quantification was are in the Fig. The of these proteins is in the For all of these the from software was than the at which for that proteins also to the mass and of proteins in Coomassie-stained gel in Fig. and chain chain acid 3 NADH 1 NADH NADH protein NADH protein ATP ATP in a Fig. 3 shows of quantification the level of a protein remains between control and diabetic samples. Quantification for these three proteins was made based on containing two cysteine This results in a and a of light and heavy isotopic quantification was possible for a peptide from with 5 peaks in the isotopic Fig. 4 shows of quantification the level of a protein was during the of diabetes. include a increase or a decrease of the relative amount of a protein over the of 1 and 4 weeks of diabetes. The results for all proteins in Fig. 2 are in protein abundance after 1 or 4 weeks of diabetes. isotopic envelopes for selected cysteine-containing from chain catalase and creatine are for and diabetic of proteins in Coomassie-stained gel in Fig. and in ± ± chain ± ± ± ± ± ± ± ± ± ± ± ± acid ± ± ± ± ± ± ± ± ± ± 3 ± ± ± ± NADH 1 ± ± NADH ± ± NADH protein ± ± NADH ± ± ± ± protein ± ± ATP ± ± ATP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± in a The quantitative method for protein profiling used in the present has It is not to proteins that not have cysteine The other to be is a peptide with another then quantification be For these we were not to proteins of as the of ATP synthase cysteine or several proteins involved in electron transport chain mass was not to cysteine-containing we this method to be and reliable for quantification of many other selected proteins and it to profiling of heart mitochondrial proteins during the of in rats. Our primary focus was on mitochondrial involved in ATP production, namely on β-oxidation of fatty tricarboxylic acid cycle, and OXPHOS. in protein levels for these three are in source of energy for heart is fatty acid oxidation, with the from the oxidation of derived from and level of fatty acids and glucose uptake and Our measurements that mitochondrial proteins involved in fatty acid oxidation are in The levels of II chain in the mitochondrial three different that the first of fatty dienoyl-CoA isomerase the first and the second were increased The expression of electron a electron for many mitochondrial was also increased. Our for heart from rat of with a that is based on quantitative of levels in from a of R. S. A. 2002; PubMed Scopus Google Scholar). R. S. A. 2002; PubMed Scopus Google Scholar) demonstrated that the levels for many proteins involved in β-oxidation of fatty acids were increased in the from STZ-treated for the tricarboxylic acid cycle. It that in to β-oxidation, the levels of tricarboxylic acid cycle proteins were not altered in heart from diabetic rats for proteins we not a difference between diabetic and normal at 1 and 4 of Our measurements measurements of tricarboxylic acid cycle in of diabetic rats Diabetes. PubMed Scopus Google Scholar). of that oxidation to phosphorylation in the mitochondrial membrane and most of the ATP studies suggest that this may be in the diabetic heart (11Pierce G.N. Dhalla N.S. Can. J. Cardiol. 1985; 1: 48-54PubMed Google Scholar, 12Tanaka Y. Konno N. Kako K.J. Cardiovasc. Res. 1992; 26: 409-414Crossref PubMed Scopus (64) Google Scholar, 13Mokhtar N. Lavoie J.P. Rousseau-Migneron S. Nadeau A. Diabetes. 1993; 42: 682-687Crossref PubMed Scopus (37) Google Scholar, 14Tomita M. Mukae S. Geshi E. Umetsu K. Nakatani M. Katagiri T. Jpn. Circ. J. 1996; 60: 673-682Crossref PubMed Scopus (47) Google Scholar). Our shows that the protein level for of was A decrease was for from changes are but for a of proteins involved in OXPHOS, with studies (11Pierce G.N. Dhalla N.S. Can. J. Cardiol. 1985; 1: 48-54PubMed Google Scholar, 12Tanaka Y. Konno N. Kako K.J. Cardiovasc. Res. 1992; 26: 409-414Crossref PubMed Scopus (64) Google Scholar, 13Mokhtar N. Lavoie J.P. Rousseau-Migneron S. Nadeau A. Diabetes. 1993; 42: 682-687Crossref PubMed Scopus (37) Google Scholar, 14Tomita M. Mukae S. Geshi E. Umetsu K. Nakatani M. Katagiri T. Jpn. Circ. J. 1996; 60: 673-682Crossref PubMed Scopus (47) Google Scholar) and to be a of the overall of adaptation in diabetic heart. A based on measurements R. S. A. 2002; PubMed Scopus Google Scholar) an to R. S. A. 2002; PubMed Scopus Google Scholar) demonstrated that several from were in the from STZ-treated We also the protein level for two from ATP synthase and them unchanged. Taken measurements of protein levels in diabetic heart mitochondria that adaptation of heart metabolism to includes up-regulation of fatty acid This the source of for the cycle. the same it seems that the efficiency of the cycle to NADH and into the electron transport chain remains unchanged. It is that on the level of and be by an increase in Whether this the overall production of ATP is not since V was unchanged. In the primary difference in mitochondrial metabolism of diabetic heart to be an increased of fatty Whether production of ATP was remains to be We also to that is based on the measurements of protein levels in diabetic mitochondria than protein and we the of oxidative which may of protein involved in ATP proteins involved in other mitochondrial were also level was that decrease the level of creatine an energy source for heart at high decrease of creatine from STZ-treated rats was based on measurements C. Young M.E. Ying J. Ahuja H.S. Han Q. Garza N. Davies P.J.A. Taegtmeyer H. J. Mol. Cell Cardiol. 2000; 32: 985-996Abstract Full Text PDF PubMed Scopus (166) Google Scholar). The protein levels of 2 acid and II were unchanged. anion channel 1 was the one example in the present the protein level 1 week after STZ and then Whether it is to heart adaptation in diabetes further mitochondrial and were decreased. function of is 2001; PubMed Scopus Google Scholar). diabetes causes a in mitochondrial it may mitochondrial and to mitochondrial dysfunction with diabetes. The protein level of albumin was in diabetic For catalase it was an of increase after 1 week and increase after 4 weeks of diabetes. and catalase into a of which are in The of proteins with mitochondria may or may not be E. B. S. S.P. Nat. Biotechnol. PubMed Scopus Google Scholar). However, control and diabetic mitochondria were in and since the protein levels for albumin and catalase were during the diabetes it is not possible to their in mitochondria as In of proteins may be involved in and their with diabetic mitochondria be to the increased oxidative in diabetes. In for the first a quantitative approach was used in the diabetes-associated changes in heart In addition to the and we several of that mitochondrial function in diabetes may be as a of simultaneously altered expression of