Proteomic Analysis of Hypertrophied Myocardial Protein Patterns in

Oct 19, 2006 - The cardiac protein profiles of spontaneously hypertensive and renovascularly hypertensive hypertrophy showed a significant alteration ...
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Proteomic Analysis of Hypertrophied Myocardial Protein Patterns in Renovascularly Hypertensive and Spontaneously Hypertensive Rats Si-Gui Zhou,† Shu-Feng Zhou,‡ He-Qing Huang,† Jian-Wen Chen,† Min Huang,§ and Pei-Qing Liu*,† Pharmacology and Toxicology Laboratory and Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510080, China, and Department of Pharmacy, Faculty of Science, National University of Singapore, 117543, Singapore Received December 13, 2005

The cardiac protein profiles of spontaneously hypertensive and renovascularly hypertensive hypertrophy showed a significant alteration compared with normal hearts. Most proteins with significant modulations in their expressions belong to the category of metabolic and stress-related proteins. Among these proteins, glutathione-S-transferase mu2 and short-chain acyl-CoA dehydrogenase may be two candidate proteins associated with left ventricular hypertrophy in spontaneously hypertensive rats. Keywords: left ventricular hypertrophy • spontaneously hypertensive rats • renovascularly hypertensive rats • cardiac metabolism • two-dimensional difference gel electrophoresis • matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Introduction Hypertension is manifested by an increased arterial pressure as well as varied structural and functional alterations in its target organs. Hypertension-induced left ventricular hypertrophy (LVH), the essential clinical criterion of hypertensive heart disease, initially serves as an adaptive ventricular response to pressure overload, but, eventually, it is a major independent risk factor associated with increased cardiovascular morbidity and mortality.1 In addition, LVH is associated with an increased risk of cardiac failure, sudden death, ventricular arrhythmias, and coronary heart disease.2 Although much research has been undertaken to understand the causes of hypertrophy and the methods to regress hypertrophy, the mechanisms explaining LVH have not been well-elucidated. Distinct phenotypes could be associated with LVH and ultimate left ventricle failure due to left ventricle overload. Specific changes in protein expression could provide us with more insight into the molecular mechanisms involved in the development of LVH. Results of proteomic analysis of the failing human and animal heart are available, but most of these studies pertain to the dilated-type of left ventricle failure.3-6 Although the proteomic analysis of left ventricles biopsies from * To whom correspondence should be addressed: School of Pharmaceutical Sciences, Sun Yat-sen University, 74 Zhongshan II Road, Guangzhou, People’s Republic of China, 510080. Phone, +86-20-87334613; fax, +86-2087334718; e-mail, [email protected]. † Pharmacology and Toxicology Laboratory, School of Pharmaceutical Sciences, Sun Yat-sen University. ‡ Department of Pharmacy, Faculty of Science, National University of Singapore. § Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University. 10.1021/pr050456l CCC: $33.50

 2006 American Chemical Society

hypertensive patients would be most favorable, this material is difficult to obtain due to ethical reasons. Furthermore, such a study design would be complicated by difficulties in obtaining adequate control tissues. Both genetically determined and artificially induced hypertension lead to LVH. The spontaneously hypertensive rat (SHR), which develops hypertension and LVH spontaneously as it matures, is the most widely studied animal model of essential hypertension. It provides a simple and accessible model to investigate the genetic mechanisms of hypertension. The renovascularly hypertensive rat (RHR) is commonly used as an experimental model for the study of secondary hypertension. Recent evidence has shown that renovascular hypertension is associated with LVH and re-expression of fetal genes. Both models have been used extensively for the study of the mechanisms, pathophysiology, and therapeutic modalities of hypertension-induced LVH. Presently, the biochemical mechanism for the pathogenesis of hypertension and hypertensioninduced LVH is complicated and not fully elucidated. The aim of this study was to analyze the changes in the left ventricle’s proteome of SHR and RHR and to identify key proteins associated with hypertensive hypertrophy. For this purpose, we established a rat model of LVH due to chronic pressure and volume overload by means of clipping two kidney arteries. Two-dimensional difference gel electrophoresis (DIGE) was used for the separation and quantification of proteins. The proteins that were found to be significantly up- or downregulated in the hypertrophic left ventricles were subsequently analyzed with sensitive and accurate matrix-assisted laser desorption/ionization, time-of flight (MALDI-TOF) and tandem TOF/TOF mass spectrometry, coupled with database interrogation. Journal of Proteome Research 2006, 5, 2901-2908

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research articles Materials and Methods Animals. Male SHR (15-week-old) and Wistar Kyoto (WKY) rats (90-120 g) were obtained from Vital River experimental animal technology limited company (Beijing, China). All rats had free access to standard laboratory food pellets and water and were housed under controlled environmental conditions (12-h light/dark cycle and room temperature 22 ( 1 °C). After 1 week, the 16-week-old SHRs were sacrificed for tissue harvest. The experimental protocol was approved by the Ethical Committee for Animal Research of Sun Yat-sen University, China. 2K2C Goldblatt Rats and Noninvasive Measurement of Systolic Blood Pressure. Hypertension was induced in male WKY rats by clipping the right and left renal arteries with a silver clip (0.30-mm internal gap), as described previously.7 Sham-operated rats, which underwent the same surgical procedure except for the placement of the renal artery clip, served as controls. Body weight, heart rate, and systolic blood pressure were measured once a week. Indirect systolic blood pressure was measured using a MLT125/R NIBP Pulse Transducer/Pressure Cuff with NIBP System and PowerLab Data Acquisition Systems (AD Instruments Pty, Ltd.) as previously described.8 At 12 weeks after surgery, the rats were sacrificed and heart tissues collected. Echocardiography. At the end of the experiment, rats were anesthetized with sodium pentobarbital (45 mg/kg body weight by ip). Two-dimensionally guided M-mode echocardiography was performed using a Technos MPX ultrasound system (ESAOTE, Italy) equipped with a 8.5-MHz imaging transducer as described previously.9 For M-mode recordings, the parasternal short-axis view was used to image the heart in twodimension at the level of the papillary muscles with a depth setting of 3 cm. M-mode recordings were then analyzed at a sweep speed of 150 mm/s with the axis of the probe aligned with the middle of the ventricle. The following parameters were measured: left ventricular internal dimensions (LVID) at both diastole and systole (LVIDd and LVIDs, respectively), left ventricular posterior wall (LVPW) dimensions at both diastole and systole (LVPWd and LVPWs, respectively), and interventricular septal (IVS) dimensions at both diastole and systole (IVSd and IVSs, respectively); percentage of left ventricular fractional shortening (FS) and left ventricular ejection fraction (EF); and heart rate (HR). Tissue Preparation, Morphometry, and Protein Samples Preparation. Some rats were perfusion-fixed and sacrificed. The hearts were excised, and the fixed transverse sections of the hearts were embedded in paraffin, cut into 5-µm cross sections, and stained with hematoxylin-eosin. Other rats were killed by decapitation, body weight (BW) and left ventricular weight (LVW) were determined, and the LVW/BW ratios were calculated. The left ventricles were rinsed for 1 min in 0.9% cold normal saline, then they were frozen immediately in liquid nitrogen for further analysis. Eight left ventricles in each group were homogenized on ice in lysis buffer (7 M urea, 2 M thiourea, 30 mM Tris, and 4% CHAPS, pH 8.5) using a glass homogenizer. One gram of tissue was placed in 10 mL of lysis buffer. The suspended solution was then sonicated at 4 °C. The solubilized proteins were separated from nonsolubilized cellular components by centrifugation (4 °C, 12 000g, 1 h). Protein concentration was determined using detergent-compatible Rc Dc Protein Assay kit (Bio-Rad). The protein samples were stored in aliquots at -80 °C. 2902

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Labeling of Proteins with CyDyes. The rat heart lysates were labeled with CyDyes as previously described.10 To derive statistical and meaningful data on any differences due to different hypertension models, the following experimental design was applied. The 24 left ventricles were randomly assigned to either Cy3 or Cy5 labeling. The random use of Cy3 and Cy5 was done to exclude potential variation in labeling efficiency or fluorescence. In addition, a pool of all 24 left ventricles (pool of SHR, 2K2C, and control rats) was labeled with Cy2. A total of 12 gels was run in a set to obtain statistical analysis of protein expression variation. 2-D DIGE and Image Acquisition and Analysis. The three sets of tripartite-labeled samples were each brought to 450 µL of final volume with rehydration buffer and loaded onto 24 cm 3-10 immobilized pH gradient (IPG) strips (Amersham Biosciences). The first dimension IPG strips were run on an Amersham IPGphor until 65 564 Vh was reached. Prior to SDSPAGE, IPG strips were equilibrated with a dithiothreitol (DTT) (10 mg/mL) SDS equilibration solution followed by treatment with iodoacetamide (25 mg/mL) SDS equilibration solution as described in the Amersham Ettan DIGE protocol. After equilibration, strips were placed on top of 12.5% polyacrylamide gels and sealed with a solution of 0.5% (w/v) agarose containing a trace of bromophenol blue. Gels were run on an Amersham Ettan Dalt 6 at 2.5 W per gel for 30 min followed by 100 W until the bromophenol blue dye front reached the bottom of the gels. Second dimension SDS-PAGE gels were precast with low-flourescence glass plates (Amersham Biosciences), with one glass plate presilanized to affix the polymerized gel to only one of the glass plates. The Cy2, Cy3, and Cy5 components of each gel were individually imaged using mutually exclusive excitation/emission wavelengths of 488/520 nm for Cy2, 532/580 nm for Cy3, and 633/670 nm for Cy5 using a Typhoon 9410 Variable Mode Imager (Amersham Biosciences). After cropping and filtering, images were subjected to automated Difference in-gel Analysis (DIA) and Biological Variation Analysis (BVA) using the Batch Processor of DeCyder software, Version 5.02 (Amersham Biosciences). Difference thresholds were set at 2-fold protein volume change, and spots with a Student’s t-test p value less than 0.05 were included. Preparative 2-D Gels for Spot Picking. Three preparative gels were run for spot picking. First- and second-dimension electrophoresis was performed as described above, except that the IPG strips (pH 3-10) were loaded with 1 mg of proteins from three groups (SHR, 2K2C, and control rats). For preparative gels, Cy-labeling was not used. Instead, after seconddimension electrophoresis, the gels were fixed overnight and stained in a Coomassie Brilliant Blue G-250 staining solution for 4 days.11 Images of the analytical gels of Cy-labeled samples (2-D DIGE and Image Acquisition and Analysis) were matched to the Coomassie Brilliant Blue G-250-stained preparative gels, and matching of pick-assigned proteins of interest was controlled manually. Mass Spectrometry and Database Search. Protein spots with significant changes were excised from the preparative gels and sliced into small pieces using a sterile scalpel. Gel pieces were washed twice for 15 min with 500 µL of water-acetonitrile (Sigma; 1/1 v/v) and placed at 56 °C for 45 min in 0.01 M DTT and 0.1 M ammonium bicarbonate (Sigma). After liquid removal, gel pieces were digested with 10 µL of porcinemodified trypsin protease (Promega) in 20 mM ammonium bicarbonate for 3 h at 37 °C, and the resulting proteolytic

research articles

Proteomic Analysis of Hypertrophied Myocardial Protein Patterns

peptides were extracted in two cycles of 60% acetonitrile and 0.1% triflouroacetic acid and dried. Peptides were reconstituted in 5 µL of 60% acetonitrile and 0.1% triflouroacetic acid, and 0.5 µL of this mixture was applied to a MALDI target and mixed on-target with 0.5 µL of R-cyano-4-hydroxycinnamic acid matrix (5 mg/mL in 60% acetonitrile and 0.1% triflouroacetic acid, supplemented with 1 mg/mL ammonium citrate). MALDITOF MS and TOF/TOF tandem MS were performed on a MALDI-TOF-TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems) in the positive ion reflector mode, using data-dependent tandem MS acquisition on the 10 most abundant ions present in each MALDI-TOF peptide mass map. MALDI-TOF peptide mass maps and accompanying tandem mass spectra were then collectively searched against the SwissProt and NCBInr databases using GPS Explorer software (Applied Biosystems) running the Mascot database search engine (Matrix-Science). MALDI-TOF peptide mass maps were internally calibrated to within 50 ppm mass accuracy using trypsin autolytic peptides (m/z ) 842.51 and 2211.10). Searches were performed without constraining protein molecular weight or isoelectric point, and allowed for carbamidomethylation of cysteine, partial oxidation of methionine residues, and one missed trypsin cleavage. Identifications were accepted based on a tripartite evaluation that takes into account significant molecular weight search (Mowse) scores, spectrum annotation, and observed versus expected migration on the 2D gel.12 Western Blotting. The protein samples were run on 12% SDS-PAGE gels under reducing conditions and transferred onto poly(vinylidene difluoride) [PVDF] membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline Tween 20 (TBST) for 2 h at room temperature and then probed with antibodies. Primary antibodies involved in this study included glutathione-S-transferase (GST) M1-1 (354209, Calbiochem) diluted at 1:1000 and R-tubulin (T6074, Sigma) diluted at 1:8000. After incubation with corresponding secondary antibodies, the bands were detected using an ECL kit (Pierce) and visualized and quantified using a Bio-Rad Image Analyzer densitometry system. Reverse Transcription Polymerase Chain Reaction (RTPCR). Total RNA was extracted from frozen tissue samples using Trizol (Invitrogen) according to the manufacturer’s instruction. Reverse transcription (RT) with random primer (9mers) was performed to generate cDNAs from 5 µg of total RNA using AMV reverse transcriptase following the instructions provided by the manufacturer. DNA amplification was carried out with Taq DNA polymerase (Invitrogen). The reaction was initiated at 94 °C for 5 min, followed by 27-31 cycles at 94 °C for 30 s, 52.0-60.4 °C for 30 s, and 72 °C for 30 s, and final extension at 72 °C for 10 min. The following primers were used: SCAD (496 bp) sense primer, 5′-CGAGATTGGCGAAGATTACAT-3′; antisense primer, 5′-AGGCTTTGGTGCCGTTGA-3′ (31 cycles, and annealing temperature was 60.4 °C); 18S rRNA (419 bp) sense primer, 5′-GTCCCCCAACTTCTTAGAG-3′; antisense primer, 5′- CACCTACGGAAACCTTGTTAC-3′ (27 cycles, and annealing temperature was 52.0 °C). RT-PCR products were analyzed on 2% agarose gels. In Vitro GST and Short-Chain Acyl-CoA Dehydrogenase (SCAD) Activities Determination. Mu class GST activities were measured spectrophotometrically in cytosolic fractions prepared from left ventricles according to Habig et al.13 using the M1/M2 isoenzyme-specific substrate DCNB at 345 nm. The cytosolic protein content was measured using a Bio-Rad Protein Assay kit with BSA as a standard.

Mitochondria were isolated from left ventricles following standard methods.14,15 Determination of SCAD activities in left ventricles mitochondria was performed as described previously.16,17 Butyryl-CoA (Sigma) was used as substrate. Statistical Analysis. Significant differences among the mean values of multiple groups were evaluated by one-way ANOVA. P < 0.05 was used as the threshold for statistically significant differences.

Results Blood Pressure in SHR and 2K2C Rats. The mean systolic blood pressure in male WKY rats weighing 90-120 g was ∼110 mmHg before renal artery stenosis, rose to 147 ( 9 mmHg at the end of 2 weeks, then exceeded 170 mmHg at 4 weeks in 2K2C rats after renal artery stenosis. Afterward, it rose progressively and reached a peak mean value of 221 ( 14 mmHg at 6 weeks and then remained constant until 12 weeks. In shamoperated groups, systolic blood pressure remained at constant levels over the 12 weeks period. Correspondingly, 16-week-old SHRs showed an average systolic blood pressure of 213 ( 11 mmHg, significantly higher (p < 0.01) than the age-matched WKY rats (118 ( 6 mmHg). Characteristics of Left Ventricular Hypertrophy. The ratio of LVW/BW was analyzed as a surrogate marker of hypertension-induced LVH. Compared with control rats, superimposed hemodynamic overload resulted in an increase of LVW/BW in both SHR and 2K2C rats of 61 and 57% (SHR 3.19 ( 0.27 mg/ g, 2K2C 3.11 ( 0.40 mg/g vs control 1.98 ( 0.16 mg/g, p < 0.01), showing that the degrees of LVH were comparable in the two models. Despite the similar degree of LVH in SHR and 2K2C rats with additional hemodynamic overload, the patterns of LVH were markedly different. Microscopic pictures taken of transverse sections of the hearts with HE staining for the SHR, 2K2C, and control rats are shown in Figure 1. 2K2C rats had significantly thinner free walls than control rats. However, hemodynamic overload in SHR resulted in substantial free wall thickening. Correspondingly, the left ventricle’s internal diameter and cavity area were increased in 2K2C rats relative to control rats, resulting in a decrease in the left ventricle’s wall-to-lumen ratio, indicating significant eccentric hypertrophy. In contrast, SHR had a significant increase in the left ventricle’s wall-to-lumen ratio, which is suggestive of concentric hypertrophy. Echocardiographic Studies. In order to differentiate between concentric and eccentric hypertrophy, left ventricles’ dimensions were analyzed for chamber dimensions and wall thicknesses. Typical M-mode echocardiograms are displayed in Figure 2. It demonstrates the changes that occurred in the LVID, IVS, and LVPW dimensions at both diastole and systole. IVS and LVPW were significantly decreased at diastole and systole in 2K2C rats compared with control rats. Correspondingly, LVIDd and LVIDs significantly increased in 2K2C rats, which exhibited chamber dilatation at diastole that reached 158% (146% at systole) of control values (Table 1). Conversely, SHR exhibited significant thickening of the IVS and LVPW at diastole and systole. Also, LVIDd and LVIDs significantly decreased in SHR. Subsequently, SHR exhibited markedly concentric hypertrophy. In SHR and 2K2C rats, reduced systolic function was noted with reduced FS and EF. Heart rate was similar in all experimental groups. Left Ventricular Protein Profiles Are Different among Spontaneously Hypertensive Rats, 2K2C Hypertensive Rats, and Control Rats. Over 2000 proteins were resolved with Journal of Proteome Research • Vol. 5, No. 11, 2006 2903

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Figure 1. Representative hematoxylin-and-eosin-stained gross histology from transverse sections of the hearts in three groups. The appearance was obtained by Sony digital camera (Sony T7). 2K2C rats exhibited eccentric hypertrophy. Conversely, SHR exhibited markedly concentric hypertrophy.

Figure 2. Representative chart of transthoracic M-mode echocardiograms. Note posterior wall and septal thinning and increased luminal dimension in 2K2C rats. Note the diminished luminal diameter in SHR. IVS, interventricular septum; PW, posterior wall; left ventricular internal dimensions (LVID). Table 1. Echocardiographic Parameters of SHR and 2K2C Ratsa parameter

control

2K2C

SHR

LVIDd (mm) LVIDs (mm) IVSd (mm) IVSs (mm) LVPWd (mm) LVPWs (mm) EF% FS% HR (bpm)

5.71 ( 0.69 2.73 ( 0.36 1.89 ( 0.27 3.04 ( 0.31 1.95 ( 0.27 3.76 ( 0.49 91 ( 7 62 ( 5 387 ( 40

7.94 ( 0.82** 4.60 ( 0.39** 0.74 ( 0.68* 1.85 ( 0.26* 0.81 ( 0.10* 2.37 ( 0.32* 79 ( 8* 40 ( 8** 405 ( 49

2.06 ( 0.41** 0.35 ( 0.05** 3.36 ( 0.42* 4.58 ( 0.64* 3.42 ( 0.47* 4.99 ( 0.63* 82 ( 5* 43 ( 6** 414 ( 38

a LVIDd and LVIDs, left ventricular internal dimensions (LVID) at both diastole and systole; IVSd and IVSs, interventricular septal (IVS) dimensions at both diastole and systole; LVPWd and LVPWs, left ventricular posterior wall (LVPW) dimensions at both diastole and systole; EF, left ventricular ejection fraction; FS, left ventricular fractional shortening; HR, heart rate. Values are mean ( SD; n ) 8 in each group. *P < 0.05, **P < 0.01 compared with control rats.

isoelectric points between pH 3 and 10 and MW between 10 and 100 kDa. Upon comparison of DIGE gels of SHR, 2K2C, and control rats, 29 spots were detected which exhibited significantly difference. Among the 29 spots, 19 spots between 2K2C and control groups, 23 spots between SHR and control groups, and 20 spots between 2K2C and SHR groups displayed 2-fold changes in abundance that were consistent across all 12 measurements (Figure 3). In total, accurate protein identifications were achieved for 18 spots. These 18 identified protein spots belong to 16 unique proteins with different potential isoforms and post-translational modifications that have been grouped into four major functional categories (Table 2): contractile proteins, energy metabolism, oxidative stress, and others. GSTM2 and SCAD Expression Were Greatly Reduced in SHR. Glutathione-S-transferase, mu2 (GSTM2, spot 15) and 2904

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short-chain acyl-CoA dehydrogenase (SCAD, spots 4 and 5) were particularly interesting because of their decreases in SHR, but not in 2K2C rats, compared with control rats. Figure 4 displays the amino acid sequence of GSTM2 by MALDI-TOFTOF MS and database search-obtained peptide fragments in bold letters. Expressions of GSTM2 and SCAD were decreased in SHR, which was confirmed by Western blot and RT-PCR. As shown in Figure 5, we observed similar fold changes in mRNA levels of SCAD in SHR and 2K2C rats as measured by DIGE. In addition, similar results in Western blot analysis of GSTM2 were obtained. Enzyme Activities. As shown in Figure 6, the Mu class GST and SCAD activities of SHR were significantly decreased compared with 2K2C and control rats, showing that lower expression of GSTM2 and SCAD resulted in the decreased activities.

Discussion Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. SHR is a genetic model of human essential hypertension. In SHR, pressure overload resulted in the concentric hypertrophy. However, the 2K2C renovascular model of hypertension has some features in common with, but others that are different from, the SHR. Chronic 2K2C hypertension has its primary aetiology in the kidney. In 2K2C rats, the two kidneys were clipped. In this case, the pressure natriuresis can no longer occur, and sodium retention occurs, along with plasma volume expansion. Pressure and volume overload resulted in the eccentric hypertrophy. In this study, we demonstrate for the first time, that the cardiac protein profiles of spontaneously hypertensive and renovascularly hypertensive hypertrophy are different.

Proteomic Analysis of Hypertrophied Myocardial Protein Patterns

Figure 3. DIGE analysis using the mixed-sample internal standard. (A) Representative superimposed image from Cy3-labeled left ventricles of control rats, Cy5-labeled left ventricles of 2K2C rats, and Cy2-labeled internal standard. The red spots show an increased protein expression in 2K2C rats. (B) Representative superimposed image from Cy3-labeled SHR, Cy5-labeled control rats, and Cy2-labeled internal standard. The red spots show an increased protein expression in control rats. (C) Representative superimposed image from Cy3-labeled 2K2C rats, Cy5-labeled SHR, and Cy2-labeled internal standard. The green spots show an increased protein expression in 2K2C rats.

It is well-known that cardiac hypertrophy involves an increase in contractile protein content.18 Consistent with these

research articles data, we found that the altered protein spots belonging to contractile proteins were up-regulated in hypertrophied hearts. β-Myosin heavy chain (β-MHC) was up-regulated in SHR and 2K2C rats, which demonstrated that the pressure overload or combination of pressure and volume overload activate the β-MHC gene and deactivate the R-MHC gene, leading to a slower and more efficient contraction.19 We also observed an increase in myofilament-associated desmin in SHR and 2K2C rats. Altered desmin expression has been observed in other pressure overloaded types of cardiac hypertrophy, as well as familial cardiomyopathies and dilated cardiomyopathy.20-22 An important amount of proteins with significant modulations in their expressions belong to the category of metabolic and stress-related proteins. This is consistent with the increased energy requirements along with the decreased perfusion present in hypertrophied hearts. We found variations in proteins involved in fatty acid oxidation, glycolysis, tricarboxylic acid cycle, electron transport system, and oxidative stress. The heart has a tremendous capacity for ATP generation, allowing it to function as an efficient pump throughout the life of the organism. The adult myocardium uses fatty acid as its main energy source. Studies in a variety of mammalian species, including humans, have demonstrated a reduction in fatty acid oxidation and increased glycolysis in pathologic cardiac hypertrophy, consistent with the fetal energy metabolic program.23,24 SCAD is a member of the highly conserved acyl-CoA dehydrogenase family, which is made up of mitochondrial flavoenzymes containing one molecule of FAD per subunit (1-7).25 It catalyzes the first step of the β-oxidation of fatty acids.26 SCAD was significantly down-regulated in SHR, which may account, in part, for the reduced fatty acid oxidation in hypertrophied hearts of SHR. In addition to a decreased expression level, a significant shift to a lower pI value of SCAD was observed, indicating post-translational addition of an acidic moiety, possibly a phosphate group, in the left ventricles of SHR. In parallel, a decreased expression of electron-transfer flavoprotein R-subunit was also observed in SHR and 2K2C rats, which acts as an electron acceptor from dehydrogenases such as SCAD. Eno1 has been characterized as highly conserved cytoplasmic glycolytic enzyme that catalyzes the formation of phosphoenolpyruvate from 2-phosphoglycerate, the second of the two highenergy intermediates that generate ATP in glycolysis.27 However, recent studies have provided evidence that Eno1 has a transcriptional regulation function. Eno1 and c-myc binding protein originate from a single gene through alternative use of translational starting sites. Both Eno1 and c-myc binding protein can bind to the P2 element in the c-myc promoter and compete with TATA-box binding protein to suppress transcription of c-myc.28,29 Up-regulated Eno1 may result in the increased glycolysis in SHR, which is consistent with the energy substrate switch away from fatty acid utilization to glycolysis in the hypertrophied heart.23,24 Of special interest, Eno1 was down-regulated in 2K2C rats, which may display transcriptional regulation but not glycolytic function, for the increased transcription of c-myc in 2K2C hypertrophied hearts. C-myc, an immediate-early gene, can induce both proliferation and growth in many cell types. In addition, the expression of pyruvate dehydrogenase E1 component β-subunit was increased in SHR and 2K2C rats, which could convert pyruvate resulted from accelerated rates of glycolysis into acetyl CoA for further oxidation via the tricarboxylic acid cycle. Journal of Proteome Research • Vol. 5, No. 11, 2006 2905

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Table 2. Identified Altered Proteins and Change Folds of Their Spot Volumesa spot no.

1

2 3 4

5

6

7

8

9

10 11

12

13 14

15 16 17

18

protein identified

NCBI accession no.

observed MW/pi

theoretical MW/pI

peptide count

sequence coverage

Contractile Proteins 28.4/5.9 12 42%

change fold score

2K2C/controlb

SHR/controlc

2K2C/SHRd

95

7.22 ( 0.61

3.16 ( 0.29

2.28 ( 0.32

124

4.57 ( 0.36

3.49 ( 0.42

1.31 ( 0.11

49.2/6.2 45.4/7.2

53.3/5.2 14 47% Energy Metabolism 47.1/6.2 20 59% 44.7/8.5 17 63%

148 118

-1.57 ( 0.12 -1.42 ( 0.11

1.53 ( 0.13 -9.67 ( 0.82

-2.40 ( 0.21 6.81 ( 0.76

P15651

45.1/6.8

44.7/8.5

15

58%

112

-1.35 ( 0.15

-4.76 ( 0.55

3.53 ( 0.41

AAH93375

44.6/6.6

40.8/7.6

11

45%

113

-2.46 ( 0.26

-6.18 ( 0.79

2.51 ( 0.34

AAH93375

45.2/5.9

40.8/7.6

12

48%

119

-2.19 ( 0.18

-4.93 ( 0.67

2.25 ( 0.19

P13803

28.7/7.9

35.3/8.6

15

52%

135

-5.16 ( 0.74

-3.52 ( 0.39

-1.47 ( 0.23

Q6GSM4

34.0/7.2

35.7/8.9

18

61%

127

5.28 ( 0.69

14.59 ( 1.76

-2.76 ( 0.31

O88989

33.2/6.1

36.5/5.9

13

55%

114

2.58 ( 0.21

3.02 ( 0.41

-1.17 ( 0.19

P49432

44.9/7.3

38.8/5.9

16

64%

121

3.14 ( 0.43

8.02 ( 1.38

-2.55 ( 0.28

Q9D6R2

32.5/6.9

39.6/6.5

11

43%

115

-7.91 ( 0.83

-2.73 ( 0.29

-2.90 ( 0.34

P39069

20.6/7.4

21.6/7.7

14

51%

126

-8.93 ( 0.78

-9.54 ( 0.98

1.07 ( 0.21

P15999

48.1/7.3

55.3/8.2

17

59%

117

-4.62 ( 0.39

-2.16 ( 0.37

-2.14 ( 0.18

Glutathione-Stransferase, mu2 Thiol-specific antioxidant protein Aldehyde reductase 1

NP-803175

25.1/7.7

Oxidative Stress 25.7/6.9 15 69%

129

-1.22 ( 0.09

-10.27 ( 0.95

8.42 ( 0.61

P35704

24.3/6.8

21.8/5.3

13

46%

98

-8.24 ( 0.94

-2.69 ( 0.33

-3.06 ( 0.35

NP-036630

27.4/7.5

35.8/6.3

16

52%

113

-2.17 ( 0.23

-6.33 ( 0.84

2.92 ( 0.36

Similar to myozenin 2

XP-215692

30.9/7.2

29.8/7.0

Others 18

57%

120

3.95 ( 0.34

2.41 ( 0.30

1.64 ( 0.21

β isoform of myosin heavy chain (fragment) desmin

P11778

29.6/7.0

P48675

54.1/5.3

Enolase 1 short-chain acyl-CoA dehydrogenase short-chain acyl-CoA dehydrogenase NADH dehydrogenase 1 alpha subcomplex 10 NADH dehydrogenase 1 alpha subcomplex 10 Electron transfer Flavoprotein alpha-subunit, mitochondrial precursor (Alpha-ETF) Malate dehydrogenase, mitochondrial Cytosolic Malatede hydrogenase Pyruvate dehydrogenase E1 component beta-subunit Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial precursor Adenylate kinase isoenzyme 1 ATP synthase alpha chain, mitochondrial (precursor)

AAH78896 P15651

a Theoretical molecular mass and pI are derived from the amino acid sequences in NCBI. “Peptide count” means the number of peptides matched in MS analysis. b Fold changes from 2K2C to control rats. c Fold changes from SHR to control rats. d Fold changes from 2K2C rats to SHR.

A decreased expression of isocitrate dehydrogenase in loadinduced cardiac hypertrophy of hypertensive rats has been reported before30 and could be confirmed by our data. Downregulation of the ATP synthase alpha chain and NADH dehydrogenase 1 alpha subcomplex 10 (Ndufa10), contribute to the disturbed energy metabolism of hypertrophied hearts in SHR and 2K2C rats. In addition to a decreased expression level, a significant shift to a lower pI value of Ndufa10 was also observed, which suggested that it was possibly post-translationally modified. The modification of this protein was recently re2906

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ported in bovine heart and arguably considered to reduce the catalytic activity of complex I by changing the affinity with NADH.31,32 Conversely, malate dehydrogenase, which catalyzes the conversion of malate to oxaloacetate for the production of ATP by the tricarboxylic acid cycle, was increased in SHR and 2K2C rats. The increase of malate dehydrogenase showed a role in balancing ATP production and regeneration. Adenylate kinase, which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate iso-

Proteomic Analysis of Hypertrophied Myocardial Protein Patterns

research articles

Figure 4. (A) One spectrum of peptide masses obtained by MALDI-TOF-TOF MS after tryptic digestion of spot 15. (B) The amino acid sequence of GSTM2 with peptide fragments obtained by mass spectrometry after tryptic digest in bold font.

Figure 6. (A) Left ventricle’s mitochondrial SCAD activities were represented by butyryl-CoA dehydrogenation [in (µmol/h)/mg of protein]. Bars represent mean ( SD; n ) 8 in each group. **P < 0.01 compared with 2K2C and control rats. (B) GST activities measured by DCNB (mu class M1/M2-specific) in cytosolic fractions of SHR, 2K2C, and control rats.

Figure 5. (A) RT-PCR analysis of mRAN levels of SCAD. mRNA was extracted from SHR, 2K2C, and control rats. Each expression level was quantified by densitometric analysis. The relative levels of SCAD mRNA were calculated with the level of 18S rRNA as an internal control. (B) The total protein extracts from SHR, 2K2C, and control rats were analyzed by Western blotting using antiGSTM2 antibody. R-Tubulin was used as an internal control. Bars represent mean ( SD of three independent experiments. **P < 0.01 compared with 2K2C and control rats.

enzymes, adenylate kinase isoenzyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. The expression of AK1 was also significantly diminished in SHR and 2K2C rats, which very likely contributed to the severe energy deficit in the hypertrophied hearts. Taken together, the observed changes in the expression of energy metabolism proteins resulted in a reduction of ATP synthesis capacity. Besides alterations in the expression of metabolic proteins, proteins involved in the stress response were also altered in our study. GSTM2 belongs to a large gene family encoding glutathione S-transferases, which catalyze the conjugation of electrophilic compounds to glutathione, thus, playing a prominent role in cellular resistance against oxidative stress.33 This is significant in stroke-prone spontaneously hypertension rat, as it was shown that the reduction of GSTM1 expression played a pathophysiological role in hypertension and oxidative stress.34 Our finding showed that the down-regulated expression of

GSTM2 in SHR may result in hypertrophy via induction of chronic oxidative stress. In parallel, the expression of thiolspecific antioxidant protein was also decreased in SHR and 2K2C rats, which maintains the reduction state of the cell and scavenges free radicals.35 Aldehyde reductase 1, which metabolizes lipid peroxidationderived aldehydes (LP-DA) and reduces oxidative injury by LPDA, was found to be decreased in SHR, suggesting that impaired aldehyde reductase 1-mediated metabolism of LPDA might participate in the myocardial injury secondary to oxidative stress.36,37 Interestingly, the expression of aldehyde reductase 1 was up-regulated in 2K2C rats. This finding may account for the aldehyde reductase 1 regulated NF-κB-mediated mitogenic signaling, which is consistent with a growth-regulating role of the enzyme in several tissues.38 In the present study, we showed that GSTM2 and SCAD were significantly down-regulated in SHR. However, we did not observe a significant change in 2K2C rats, which have been further confirmed by enzyme activities determination. Therefore, GSTM2 and SCAD may be two candidate proteins associated with LVH in SHR. This difference may account, in part, for different causes and characteristics of LVH in SHR and 2K2C rats. Despite highly significant changes in expression between SHR and 2K2C rats, we have not excluded the possibility that these could be secondary to blood pressure differences. Further studies in young animals during the development of hypertension may help elucidate primary mechanisms. Although further investigations remain to be performed, this work provides the potential clues to understanding basic pathophysiological mechanisms in cardiac hypertrophy of primary and secondary hypertension. Journal of Proteome Research • Vol. 5, No. 11, 2006 2907

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