Proteomic Analysis of Left Ventricular Remodeling ... - ACS Publications

Proteomic Analysis of Left Ventricular Remodeling in an Experimental Model of Heart Failure Caroline Cieniewski-Bernard,†,‡,§ Paul Mulder,| Jean-Paul Henry,| Herve´ Drobecq,‡,§,⊥ Emilie Dubois,†,‡,§ Gwe¨nae¨l Pottiez,# Christian Thuillez,| Philippe Amouyel,†,‡,§ Vincent Richard,| and Florence Pinet*,†,‡,§ INSERM, U744, Lille, France, Institut Pasteur de Lille, Lille, France, University of Lille 2, IFR141, Lille, France, INSERM, U644, & Institute for Biomedical Research, IFRMP 23, Rouen University Hospital, Rouen, France, CNRS, UMR8525, Lille, France, and E.A.2465, IMPRT-IFR114, University of Artois, Lens, France Received June 5, 2008

The development of chronic heart failure (CHF) following myocardial infarction is characterized by progressive alterations of left ventricle (LV) structure and function called left ventricular remodeling (LVR), but the mechanism of LVR remains still unclear. Moreover, information concerning the global alteration protein pattern during the LVR will be helpful for a better understanding of the process. We performed differential proteomic analysis of whole LV proteins using an experimental model of CHF in which myocardial infarction was induced in adult male rats by left coronary ligation. Among 1000 protein spots detected in 2D-gels, 49 were differentially expressed in LV of 2-month-old CHF-rats, corresponding to 27 different identified proteins (8 spots remained unidentified), classified in different functional groups as being heat shock proteins, reticulum endoplasmic stress proteins, oxidative stress proteins, glycolytic enzymes, fatty acid metabolism enzymes, tricarboxylic acid cycle proteins and respiratory chain proteins. We validated modulation of selected proteins using Western blot analysis. Our data showed that proteins involved in cardiac metabolism and oxidative stress are modulated during LVR. Interestingly, proteins of stress response showed different adaptation pathways in the early and late phase of LVR. Expression of four proteins, glyceraldehyde-3-phosphate dehydrogenase, alphaB-crystallin, peroxiredoxin 2, and isocitrate dehydrogenase, was linked to echographic parameters according to heart failure severity. Keywords: proteomics • 2D GEL • MALDI-TOF • Western blot • myocardial infarction • ventricles • remodeling

Introduction Left ventricular remodeling (LVR) after myocardial infarction (MI) is a dynamic and complex process that occurs in response to myocardial damage.1 Progressive left ventricule (LV) dilation after MI has been documented to be a strong predictor of both chronic heart failure (CHF) and death.2 LVR is the process by which LV undergoes complex short- and long-term pathological changes in size, architecture and function. It describes the compensatory responses of the cardiovascular system when faced with an acute loss of myocardial contractile function.3 Postinfarction remodeling could be divided in two phases. The early phase (occurring few hours to several weeks after infarction) involves the expansion of the infarct zone, including * To whom correspondence should be addressed. Dr. Florence Pinet, INSERM U744-IPL, 1 rue du professeur Calmette, 59019 Lille cedex, France. Tel: (33) 3 20 87 72 15. Fax: (33) 3 20 87 78 94. E-mail: [email protected]. † INSERM, U744. ‡ Institut Pasteur de Lille. § University of Lille 2. | INSERM, U644, & Institute for Biomedical Research, Rouen University Hospital. ⊥ CNRS, UMR8525. # University of Artois.

5004 Journal of Proteome Research 2008, 7, 5004–5016 Published on Web 10/16/2008

dilatation and thinning of the infarct zone.4 Late remodeling (months, years) involves globally the LV and is associated with time-dependent dilatation, distortion of ventricular shape, mural hypertrophy, as well as structural tissue changes.1 Initially, LV dilatation could be considered as a protective mechanism to maintain systolic flow and cardiac “pump” function. However, this phenomenon ultimately leads to alterations of global function of the LV and ultimately aggravates heart failure. Several mechanisms implicated in LVR have been identified, including hypertrophy, fibrosis, apoptosis and proteolytic activation.1,2 However, the exact molecular determinants of LV dilation after MI are not completely understood. Moreover, the severity of LVR cannot be fully predicted based on its known determinants such as the extent of MI.5 Following MI, around 30% of patients will develop HF 2 years after the acute event, despite current therapeutics treatments.6 A better understanding of the complex molecular events that initiate and perpetuate the process of LVR would facilitate the development of more selective and efficient therapies.7 Current research on the molecular mechanisms of LVR has been limited to the evaluation of factors or pathways already believed to contribute to its physiopathological evolution, for 10.1021/pr800409u CCC: $40.75

 2008 American Chemical Society

Proteomic Analysis of Left Ventricle Tissue in Heart Failure example, those involved in neurohumoral activation, contractile dysfunction or changes in extracellular matrix.8 An understanding of the physiopathological significance of LVR leading to CHF is therefore fundamental, and requires the evaluation of corresponding protein translation as well as the integration of these findings into the overall disease context. Proteomic technology allows the examination of global alterations in protein expression in the diseased heart, and can provide new insights into the cellular mechanisms involved in cardiac dysfunction.9 The use of proteomic analysis to investigate heart disease should result in the generation of new diagnostic and therapeutic markers.10 To search for cardiac specific biomarkers, the analysis of cardiac tissue remains necessary11 and experimental animal models allow to study serial changes in cardiac tissue proteins over time, and relate these changes to LVR and cardiac dysfunction. Our objective was thus to identify heart tissue markers of LVR in CHF after MI. For this purpose, we performed differential proteomic analysis of LV in rats in which MI is induced by left coronary ligation.12 We observed that post-MI LVR is associated with the modulation of numerous heat shock proteins, together with proteins involved in cellular protection against oxidative stress, as well as metabolic enzymes which probably reflect the metabolic changes that occurs in remodeled hearts.

Experimental Procedures Animals. All experiments are conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised in 1996). French law allowed to perform animal experiments under supervision of person with habilitation to perform experiment of alive animals (F. Pinet: 59-350126 expiration date: 20 February 2011) and there is no ethical approval delivered. CHF was induced in 10-week-old male Wistar rats (n ) 24) (Charles River, France) by left coronary ligation according to the method of Pfeffer et al.13 and modified by Mulder et al.14 constituting the CHF group. The occlusion was permanent and no reperfusion occurred. Twenty-one rats were subjected to the same protocol excepted that the snare was not tied, constituting the sham-operated group. All rats were allowed standard rat chow and drinking water ad libitum and maintained on a 12-h/12-h light/dark cycle. Sham-operated and CHF rats were then further separated in two subgroups that were sacrificed at 7 days post-MI (acute LVR), or 2 months post-MI (chronic LVR). In all cases, hemodynamic measurements and echocardiographic studies were performed before sacrifice. In some analyses, the 2 month-CHF rats were further separated in two other subgroups according to lung weight (LW) and hemodynamic parameters: CHF (n ) 12, LW range: 0.84-1.11 g) and severe CHF (CHF+++) groups (n ) 5, LW range:1.18-2.05 g). Echocardiographic Studies. Transthoracic Doppler echocardiographic studies were performed in sodium methohexitalanesthetized rats (50 mg/kg ip) using a system (HDI 5000, ATL) equipped with an 8.5-MHz transducer as previously described.14 Posterior end-diastolic and end-systolic LV posterior wall thicknesses and diameters were measured by the American Society of Echocardiology leading-edge method from at least three consecutive cardiac cycles.

research articles LV outflow velocity was measured by pulse-wave Doppler, and cardiac output was calculated as CO ) aortic VTI × [π × (LV outflow diameter/2)2] × heart rate, where VTI is velocitytime integral. Hemodynamic Measurements. Systolic blood pressure and heart rate were also determined in anesthetized rats. The right carotid artery was cannulated with a micro manometer-tipped catheter (SPR 407, Millar Instruments) and advanced into the aorta for the recording of LV pressures, their maximal and minimal rate of rise (dP/dtmax and dP/dtmin), and LV relaxation constant Tau. Heart Preparation and Protein Extraction. After assessment of the hemodynamic measurements, the heart was excised and incubated in ice-cold Krebs-Henseleit buffer to wash out blood. Each cardiac compartment was then carefully dissected to remove all the necrotic/scarred zones to keep only the viable myocardium. Left ventricles were then immediately frozen in liquid nitrogen and kept at -80 °C until analysis. LV proteins were extracted using Dounce-Potter homogenization on ice in 40 mmol/L Tris-HCl, pH 9.5, containing antiproteases (one tablet for 10 mL buffer, Complete EDTAfree, Roche Diagnostics, Meylan, France). Soluble fraction was transferred into a 1.5 mL Eppendorf tubes and protein concentrations were determined using Bradford assay. Two-Dimensional (2-D) Gel Electrophoresis. Two-dimentional gel electrophoresis (2DE) was performed as previously described in detail.15 The first dimension (IEF) was performed on a Protean IEF Cell System (Bio-Rad, Hercules, CA) as follows: 100 µg (for analytical gels) or 500 µg (for preparative gels) of proteins was mixed in 250 µL of urea solubilization/rehydratation buffer (8 mol/L Urea, CHAPS 2%, 97 mmol/L DTT and 5% ampholytes) for IPG strips (Genomic Solutions, Ann Arbor, MI), and loading buffer (8 mol/L Urea, CHAPS 4%, 65 mmol/L DTT and 40 mmol/L Tris) for IPG strips (Genomic Solutions) for a final volume of 450 µL. The mixture was applied onto a dry IPG strip (length 24 cm, pH 3-10 linear gradient, GE Healthcare, Orsay, France). Complete sample uptake onto strip was achieved after 9 h at 20 °C without any current. Focusing was carried out at 20 °C under a current limit of 50 µA per strip and performed at 50 V for 9 h (active rehydratation step), 200 V for 1 h (linear progression), 1000 V for 1 h (linear progression) followed by a slow ramping to 10 000 V for 6 h (linear progression), and was completed at 10 000 V for 4.5 h (fast progression). After IEF, the IPG strip gels were equilibrated by two consecutive incubations for 15 min each at room temperature with buffer containing 6 mol/L urea, 37.5 mmol/L Tris-HCl, pH 8.8, 30% glycerol (v/v) and 2% SDS (w/v) with 2% DTT (w/v) for the first incubation, and 2.5% IAA (w/v) for the second one. The equilibrated IPG gels were applied to the top of a 12% Duracryl (Genomic Solutions) gel and sealed with low melting temperature agarose (GE Healthcare). Electrophoresis was carried out at 10 °C with the Ettan Daltsix large vertical system (GE Healthcare) in running buffer (25 mmol/L Tris, 192 mmol/L glycine, 0.1% SDS (w/v) at 70 V overnight. 2D-Gel Staining. The analytical gels were silver stained according to the protocol previously described by Shevchenko et al.16 with minor modifications. Briefly, gels were fixed overnight in 30% ethanol (v/v) and 5% acetic acid (v/v) followed by 4 washes of 18 MΩ-H2O. Gels were sensitized for 1 min in 0.02% sodium thiosulfate (w/v), followed by two 1-min washes in 18 MΩ-H2O, and then incubated in 0.2% silver nitrate (w/v) for 30 min. Proteins were then visualized using developing Journal of Proteome Research • Vol. 7, No. 11, 2008 5005

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Cieniewski-Bernard et al.

solution (0.028% formalin (v/v), 0.0125 sodium thiosulfate (w/v), 2.4% sodium carbonate (w/v)) until an appropriate level of staining was achieved, after which development was stopped by adding 10% acetic acid (v/v).

autolysis products ([M H ] 842.5100; [M+H+]1045.5642 and [M+H+] 2211.1046) and externally with lyozyme and then to pick peaks, with a threshold that depended on the background, a resolution >10 000, and contaminant ions not excluded.

Preparative gel was colloidal blue stained. Briefly, gel was fixed in 50% ethanol (v/v) containing 2% orthophosphoric acid (v/v) overnight, followed by a wash of 1 h in 2% orthophosphoric acid (v/v). Gel was incubated for 20 min in 15% ammonium sulfate (w/v), 2% orthophosphoric acid (v/v), 17% ethanol (v/v) and finally incubated in 15% ammonium sulfate (w/v), 2% orthophosphoric acid (v/v), 17% ethanol (v/v) containing 0.1% Coomassie Brilliant Blue G-250 (w/v) for 2-3 days. Gel was then rapidly washed with 18 MΩ-H2O. Image Acquisition and Bioinformatic Analysis. Silverstained 2D gels were digitized at 200 dpi resolution using an Imagescanner scanner (GE Healthcare). A calibration filter using different shades of gray was applied to transform pixel intensities into optical density units. The images were exported in Tagged Image File format and imported into ImageMaster 2D Platinum 6.0 gel image analysis software (GE Healthcare). Spots were detected automatically according to three parameters (smooth, 2; area, 5; saliency, 1). The background was removed from each gel and the images were edited manually, for example, adding, splitting and removal of artifacts. One gel was chosen as the master gel, and used for the automatic matching of spots in the other 2D-gels. To report data analysis, two classes of gels were considered: sham-operated rats (n ) 4) and CHF rats (n ) 6) at 2 months. Total spot volume was calculated for each image and each spot assigned a normalized spot volume as a proportion of this total value. After editing and manual matching, the images were analyzed for protein spot differences. Polypeptidic spots were considered to have significant different normalized spot volume between shamand CHF-rats according to the threshold (1.4-fold) (p < 0.05) and two criteria: (1) presence of the spot on all gel used for the bioinformatic analysis and (2) reproducible modulation of the spot detected. Protein Identification by Mass Spectrometry. Protein identification of the selected spots was performed from two preparative 2D-gels by an in-gel digestion method. Briefly, the gel plugs excised were washed with ultrapure water until totally destained. Gel pieces were then rinsed in acetonitrile (ACN)/ Tris 50 mmol/L, pH 9.0, and dried in a SpeedVac evaporator before rehydratation with 50 mmol/L Tris, pH 9, containing 10 µg/mL trypsin (Trypsin Gold, Promega, Madison, WI). After digestion overnight at 37 °C, the supernatant was removed, and the gel pieces were washed with 45% ACN (v/v)/0.1% TFA (v/v), followed by a wash with 95% ACN (v/v)/0.1% TFA (v/v). The collected supernatants were pooled, concentrated, and resuspended in 10 µL of 0.1% TFA (v/v) just prior to be desalted using Zip-Tips C18 (Millipore, Bedford, MA). Peptides were directly eluted with R-cyano-4-hydroxy-cinnamic acid (CHCA) matrix, 10 µg/mL in a solution of ACN/0.1% TFA (v/v) and spotted onto the MALDI-TOF target.

Protein identification was also performed using the Proteineer workflow from Bruker Daltonics (Bremen, Germany) for spots not identified in MALDI and also to confirm some identifications performed by MALDI. Colloidal coomassie blue stained spots were excised from preparative 2D-gels with a spot picker (PROTEINEER sp) and placed into 96-well microtiter plates. In-gel digestion and sample preparation for MALDI analysis were performed according to the manufacter’s instructions using a digester/spotter robot (PROTEINEER dp) and a digest kit (DP 96 standard kit, Bruker Daltonics). The MALDI target plate (AnchorChip, Bruker Daltonics) was covered with CHCA matrix (0.3 mg/mL in acetone/ethanol, 3:6 (v/v)). Extracted peptides were directly applied onto the CHCA matrix thin layer and recrystallized after drying with ethanol/acetone/ 0.1% TFA-acidified water (6/3/1 (v/v/v)). The molecular mass measurements were performed in automatic mode using FlexControl 2.2 software on an Ultraflex TOF/TOF instrument (Bruker Daltonics) and in the reflecton mode for MALDI-TOF peptide mass fingerprint (PMF). External calibration was performed using the peptide calibration standard kit (Bruker Daltonics). Peak lists were generated from MS spectra using Flexanalysis 2.4 software (Bruker Daltonics). Database searches using Mascot (Matrix Science Ltd., London, U.K.) and PMF data sets were performed via ProteinScape 1.3 (Bruker Daltonics).

Protein identification was carried out by peptide mass fingerprinting using a MALDI-TOF mass spectrometer Voyager DE-STR PRO (PerSeptive Biosystems, Framingham, MA) equipped with a 337.1 nm nitrogen laser and the delayed extraction facility (125 ms). Peptide mass fingerprint spectra were registered in reflection positive mode under 20 kV voltage, 61% grid. Typically, 200 laser shots were recorded per sample. DataExplorer Software version 4.0 (PerSeptive Biosystems) was used to calibrate the resultant spectra internally with trypsin 5006

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Tryptic monoisotopic peptide masses were identified by peptide mass fingerprint with Profound (http://Prowl.rockefeller.edu/ prowl-cgi/profound.exe) software against the NCBI nr (2008/03/ 01) (18 900 sequences) and MS-Fit (http://prospector.uscf.edu) software against Swiss-Prot (SwissProt.2007.10.10) (6670 entries) with the following parameters: rat species, one missed cleavage site and a mass tolerance setting of 25 ppm. Partial chemical modifications such as oxidation of methionine and carbamidomethylation of cysteine were considered for the queries. The criteria used to accept identifications included the extent of sequence coverage (>20%), the number of peptides matched (minimum of four), the Mowse probability score (minimum of 70), the mass accuracy and whether rat protein appeared as the top candidate in the first-pass search with no species restriction. Identifications were accepted when peptides matched multiple members of a protein family only when top candidates were obtained from the fractions of at least 2 mass spectra of trypsin digest of spots from two 2D gels, and theroretical and experimental Mr and pI were expected to be similar. Otherwise, the identification was not considered valid. Western Blot Analysis. Proteins (5-50 µg) from LV were separated by SDS-PAGE (12% acrylamide gel) and transferred to 0.45 µm nitrocellulose or PVDF membranes (GE Healthcare). Equivalent total protein loads were confirmed visually by Ponceau red staining of the nitrocellulose membrane. The blots were then subsequently washed in TBS-Tween 20, saturated in 5% nonfat dry milk or BSA in TBS-Tween and blotted overnight in blocking solution with antibodies against RBcrystallin (Stressgen, 5 µg proteins/lane, 1/2000), HSPB6 (Abcam, 25 µg, 1/2000), protein disulfide isomerase (Abcam, 20 µg, 1/1000), ubiquitin C-term hydrolase (AbD Serotec, 50 µg, 1/1000), glutathione peroxidase 1 (Abcam, 50 µg, 1/1000), ATP synthase alpha subunit (Molecular Probes, 20 µg, 1/2000), ATP synthase D chain (Mitosciences, 20 µg, 1/1000), GAPDH (Novus biologicals, 25 µg, 1/1000), phosphoglycerate kinase 1 (Santa

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Proteomic Analysis of Left Ventricle Tissue in Heart Failure a

Table 1. Echocardiographic and Hemodynamic Parameters

CHF+++

CHF

LVEDP, mm Hg LVESP, mm Hg LVEDD, mm LVESD, mm dP/dtmax, 103 mm Hg/s dP/dtmin, 103 mm Hg/s FS, % SV, mL/beat CO, mL/min E/A Tau DBP, mm Hg

Sham (n ) 16)

7-days (n ) 5)

2-months (n ) 13)

2-months (n ) 5)

0.73 ( 0.29 119.5 ( 6.1 6.24 ( 0.08 3.17 ( 0.09 8.82 ( 0.49 -7.75 ( 1.21 49.28 ( 1.29 0.346 ( 0.01 143.4 ( 5.7 1.23 ( 0.12 3.38 ( 0.11 91.9 ( 5.3

3.74 ( 0.63†b 96.6 ( 11.7 8.98 ( 0.15†b 7.28 ( 0.19†b 6.92 ( 0.88 -5.89 ( 0.58 18.97 ( 1.15†b 0.313 ( 0.01 129.5 ( 4.6 1.38 ( 0.11 3.96 ( 0.13†b 80.5 ( 11.7

4.50 ( 0.85†b 110.85 ( 7.5 9.61 ( 0.28†b 8.05 ( 0.24†,‡b 6.94 ( 0.73†b -4.28 ( 1.34 16.22 ( 0.96†b 0.303 ( 0.01†b 122.6 ( 4.7†b 1.99 ( 0.21†,‡b 7.17 ( 0.97†,§b 86.7 ( 6.5

5.93 ( 1.09‡b 112.5 ( 10.3 10.62 ( 0.19†,$,&b 9.16 ( 0.32†,#,&b 6.56 ( 0.52 -5.64 ( 0.58 13.80 ( 2.15†b 0.304 ( 0.02 119.9 ( 12.01 2.91 ( 0.84 6.18 ( 0.63*,¶b 90.7 ( 5.2

a LVEDP indicates left ventricle (LV) end diastolic pressure; LVESP, LV end systolic pressure LVDD, LV end diastolic diameter; LVSD, LV end systolic diameter; dP/dtmax, cardiac contractility; dP/dtmin, cardiac relaxation; FS, fractional shortening; SV, stroke volume; CO, cardiac output; E/A, ratio between LV E and A waves; Tau, the LV relaxation constant; DBP, diastolic blood pressure. b *p < 0.05 and †p < 0.01 versus sham; ‡p < 0.05 and §p < 0.01 2-month CHF versus 7-day- CHF; #p < 0.05 and $p < 0.01 2-month CHF+++ versus 2-month CHF; ¶p < 0.05 and &p < 0.01 2-month CHF+++ versus 7-day CHF.

Cruz Biotechnology, 50 µg, 1/200), triose phosphate isomerase (Abcam, 5 µg, 1/10 000), phosphoglucomutase 1 (Abnova Corporation, 10 µg, 1/1000), acyl-coenzyme A thioesterase 2 (Abnova Corporation, 50 µg, 1/1000), enoylCoA hydratase (ProteinTech Group, 25 µg, 1/1000), isocitrate dehydrogenase (AbD Serotec, 5 µg, 1/10 000), superoxide dismutase 2 (Abcam, 5 µg, 1/2000), and peroxiredoxin 2 (Abcam, 50 µg, 1/2000). The blots were then washed five times in TBS-Tween and incubated with the appropriate horseradish peroxidase labeled secondary antibody (anti-mouse (GE Healthcare), 1/5000; anti-rabbit (GE Healthcare), 1/5000; anti-goat (Abcam), 1/5000) for 1 h in blocking solution. The membranes were washed five times in TBS-Tween and incubated with enhanced chemiluminescence (ECL) reagents (GE Healthcare). Detection was carried out using an Ettan DIGE Imager (GE Healthcare) using the 480 nm excitation and 530 nm emission wavelength. The intensity of the bands was quantified with Quantity One Image analyzer software (Bio-Rad). Data was presented as means ( SEM. Differences between means were considered significant when p < 0.05, according to Student’s t test.

Results Echocardiographic, Hemodynamic, and Histomorphometric Parameters. Out of the 24 rats with MI, 2 died during the study and were excluded; thus, a total of 43 rats (21 sham, 22 CHF) were finally included in the analysis. Table 1 illustrates the echocardiographic and hemodynamic parameters measured in anesthetized 7-day and 2-month sham-, 2-month CHF- and 2-month CHF+++- rats. Sham-rats (7 days and 2 months) have identical parameters summarized in the same column. Two-month CHF-rats were divided in two groups: CHF (n ) 13) and CHF+++ (n ) 5) as described in Experimental Procedures. CHF significantly decreased fractional shortening (FS) at 2 months but also at 7 days, and reduced stroke volume and cardiac output both at 7 days and 2 months. CHF also increased E/A at 2 months and the relaxation constant Tau at both times. CHF significantly increased LV end-diastolic pressure (LVEDP) at 7 days and 2 months, without significantly affecting LV endsystolic pressure (LVESP). CHF also significantly decreased dP/ dtmax, at both times, and nonsignificantly increased dp/dtmin.

Figure 1. Correlation of LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD) with degree of heart failure determined as described in Experimental Procedures. Trend curves were calculated from the mean values for each groups of rats: 7 day-CHF, 2-month CHF, 2 month-CHF+++. Data are expressed as mean values (plain line) and also represented by polynomial trend curves (dashed line) indicated on the figure. X-axis represents each CHF rat groups and Y-axis represents the level of LVESD (A) and LVEDD (B).

Echocardiographic evaluation also demonstrated significantly marked increases in LV end-systolic (LVESD) and enddiastolic (LVEDD) diameters at both times, demonstrating a correlation between LV dilation and severity of CHF as shown in Figure 1. Table 2 shows the values for histomorphometric parameters. At 2 months, CHF was associated with significant increases in total heart weight, total heart/body weight ratio, right ventricular weight, LV weight and atrial weight. At 7 days, only atrial weight was increased, with no changes in ventricular or total heart weight. LW was already elevated at 7 days, and further increased at 2 months. Journal of Proteome Research • Vol. 7, No. 11, 2008 5007

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Table 2. Histomorphometric Parameters in Sham- and CHF-Ratsa CHF+++

CHF Sham (n ) 16)

7-days (n ) 5)

2-months (n ) 13)

2-months (n ) 5)

BW, g 440 ( 11 450 ( 16 433 ( 9 455 ( 15 HW, g 0.95 ( 0.02 1.08 ( 0.07 1.15 ( 0.02†b 1.35 ( 0.09†,¶b HW/BW, 2.16 ( 0.05 2.40 ( 0.10 2.66 ( 0.08†b 2.97 ( 0.20†,¶b mg/g RVW, mg 160 ( 5 190 ( 13 193 ( 6†b 252 ( 66 981 ( 4†,$b LVW, mg 741 ( 17 795 ( 45 888 ( 17†b AW, mg 46 ( 5 95 ( 20 69 ( 7*b 115 ( 25 LW, g 0.94 ( 0.03 1.08 ( 0.05*b 1.02 ( 0.02*b 1.60 ( 0.24 a BW indicates body weight; HW, heart weight; RVW, right ventricle weight; LVW, left ventricle weight; AW, atrial weight; LW, lung weight. b *p < 0.05 and †p < 0.01 versus sham; ‡p < 0.05 and §p < 0.01 2-month CHF versus 7-day- CHF; #p < 0.05 and $p < 0.01 2-month CHF+++ versus 2-month CHF; ¶p < 0.05 and &p < 0.01 2-month CHF+++ versus 7-day CHF.

The 2-months CHF-group was subdivided in two subgroups according to the degree of heart failure: CHF-rats and severeCHF rats annotated CHF+++ classified by lung weight. LV Proteome of 2-Month Sham- and CHF-Rats. Figure 2 represents patterns of LV proteome for 2-month sham- (Figure 2A) and CHF-rats (Figure 2B) with, respectively, 1021 ( 52 and 1020 ( 29 polypeptidic spots well resolved on 2D-gels. The differential proteomic analysis performed from four sham-rats and six 2-month CHF-rats. revealed 49 spots with differential abundance levels and a statistically reproducible difference over the two groups. Five were downregulated (spots 25-28, 46), 5 induced (spots 1, 2, 6, 36 and 41), and 39 upregulated in CHFrats (Figure 2C). Interestingly, 22 differentially abundant spots (spots 1-22) were located in a restricted zone of the 2D-gel, comprised between 45 and 70 kDa and pH range of 3.5-5 (inset Figure 2C). We could hypothesize that several spots represent the same protein with post-translational modifications. The differentially abundant proteins were identified using mass spectrometry according to the recent guidelines.17 Of the 49 polypeptidic spots selected to be differentially expressed (i.e., fold change of at least 1.4), 8 were not identified (spots 10-16 and 49) due to the low intensity signal on mass spectrum or a low probability score. Detailed mass spectra, peak list and peptides identified are presented as supplemental data. Table 3 summarizes the identity and factor of variation of LV proteins in 2 month-CHF rats of the 27 nonredundant identified proteins. These proteins could be classified in 9 classes according to their functional significance, as being molecular chaperones, in particular small heat shock proteins (HSP), HSP-beta-2 (spot 41), HSP-beta-6 (spot 47), HSP-beta-7 (spot 46) and alpha-B crystallin (spots 43 and 48); proteins of endoplasmic reticulum (ER) stress and degradation pathways, protein disulfide isomerase (PDI) (spots 20-21), isozyme L3 of ubiquitin C-terminal hydrolase (spot 32) and beta 3 subunit of proteasome (spot 34); proteins of oxidative stress, peroxiredoxin-2 (spot 40), peroxideroxin-6 (spot 33), glutathione peroxidase 1 (spot 36) and superoxide dismutase 2 (spot 44); glycolytic enzymes, phosphoglycerate kinase 1 (spots 27-28), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (spot 29), triose phosphate isomerase (spots 30 and 35), phosphoglycerate mutase 1 (spot 31); proteins of fatty acid metabolism, acylcoenzyme A thioesterase 2 (spot 26) and enoyl-coenzyme A hydratase (spot 38); enzymes of tricarboxylic acid cycle, alpha 5008


subunit of isocitrate dehydrogenase which comigrated on 2Dgels with phosphoglycerate kinase 1 (spots 27-28), fumarate hydratase (spot 25) and dihydrolipoyl dehydrogenase (spots 23-24); protein of respiratory chain, NADH dehydrogenase [ubiquinone] flavoprotein 2 (spot 39); proteins implicated in ATP synthesis, ATP synthase D chain (spots 42 and 45), ATP synthase alpha-subunit (spot 37) and beta-subunit (spot 22); proteins implicated in kinin pathway, T-kininogen 1 (spots 7-9) and 2 (spots 1-6) precursors and serine protease inhibitor A3K (spots 17-19). Some of the identified proteins were highly expressed in heart tissue as being acyl-CoA thioesterase 2 (spot 26) and glyceraldehyde-3-phosphate dehydrogenase (spot 29), or specifically expressed in heart as HSP-beta-7 (spot 46). Modulation of LV Proteins Differentially Expressed According to HF Severity. To validate the proteins selected to be differentially abundant in 2-month CHF-rats by proteomic analysis, we compared protein expression level by Western blotting in LV of 7-day and 2-month rats after MI. The 2-month CHF-group was subdivided in two subgroups according to the degree of heart failure: CHF-rats and severe CHF-rats annotated CHF+++ classified by LW. All results are presented (Figure 3 and 4) except for proteins with Western blots of poor quality (i.e., dihydrolipoamide dehydrogenase, HSP-beta-2, HSP-beta-7) or with high unspecific signals (i.e., peroxiredoxin-6 and proteasome subunit beta3). Figures 3 and 4 presented, respectively, classes of proteins linked to protection system against stress and to metabolic pathways, Modulation of all the proteins selected by 2D was confirmed by Western blot except for phosphoglycerate mutase 1 and peroxiredoxin-2. We confirmed the increase of two small HSPs, alphaBcrystallin and HSP-beta-6 (Figure 3A). The increase in HSPbeta-6, particularly marked in 2-month severe CHF-rats, was proportional to the degree of HF, while the expression level of alphaB-crystallin is similar in CHF- and severe CHF-rats. Increase of alphaB-crystallin was detected in 7-day rats, but to a lower extent than in 2-month rats. We also tested proteins implicated in ER stress. Protein disulfide isomerase was only increased in 2-month severe CHFrats (Figure 3B), while ubiquitin C-term hydrolase increased significantly in 7-day CHF-rats (Figure 3B), and to a lesser extent in 2-month CHF- and severe CHF-rats. We validated modulation of expression of oxidative stress proteins (Figure 3C), except for peroxiredoxin-2, whose expression only increased in 7-day CHF-rats. In contrast, superoxide dismutase 2 increased (by 2-fold) only in 2-month CHF-rats. The immunoblot analysis of glutathione peroxidase 1 revealed two bands, one corresponding to glutathione peroxidase 1 monomer while the other band had a high molecular weight (>100 kDa), probably corresponding to homotetramer of glutathione peroxidase 1. As for superoxide dismutase 2, there was an increase in glutathione peroxidase 1 content in 2-month CHF-rats (Figure 3D), while there was a significant decrease in 7-day CHF-rats. Interestingly, the modulation of glutathione peroxidase 1 was inversely proportional to that observed for the high molecular complex (Figure 3D). Modulation of metabolic enzymes was also considered (Figure 4A). In particular, we validated the increase of triose phosphate isomerase in 2-month CHF-rats, as well as of GAPDH, but this increase was not statistically significant. We observed a decrease in phosphoglycerate kinase expression in both CHF- and severe CHF-rats, as well as for phosphoglycerate

research articles

Proteomic Analysis of Left Ventricle Tissue in Heart Failure

Figure 2. Representative 2D-gel of rat LV proteins. Whole proteins (100 µg) were separated on a linear pH gradient (3-10) followed by a 12% SDS-PAGE. An average image was established for LV proteome of 2-month sham- (n ) 4) (A) and CHF-rats (n ) 6) (B). The positions of MW are indicated on the left and the pI on the bottom of the gels. Differentially polypeptidic spots selected using Platinum6.0 software are indicated by a number (C) and identified by mass spectrometry. The corresponding identifications are listed in Table 3. The inset on panel C represents 2D-gel for a pI between 3.5 and 5 and a MW between 45 and 70 kDa with differentially expressed spots indicated by a number.

mutase content, in contradiction with the data of bioinformatic analysis of 2D-gels. Nevertheless, we cannot exclude a possible post-translational modification of phosphoglycerate mutase, which could interfere in the detection of this protein by the antibody. We also confirmed the decrease in acylCoA thioesterase 2 expression in CHF-rats and observed a tendency for an increase in enoylCoA hydratase in 2-month CHF-rats (Figure 4B). Bioinformatic analysis of 2D-gels revealed a decrease in expression of enzymes of tricarboxylic cycle, validated for isocitrate dehydrogenase in 2-month CHF-rats (Figure 4C), being proportional to the severity of CHF. The last group considered was that of proteins implicated in ATP synthesis (Figure 4D). ATP synthase D-chain level increased in 7-day and 2-month CHF-rats, the modulation being proportional to the degree of CHF, although it was only statistically relevant in severe CHF-rats. No statistical variation was measured at 7-days post-MI for alpha subunit of ATP synthase, but its expression was higher in CHF- and severe CHF-rats.

Moreover, in order to more precisely analyze the relationship between the severity of the disease and the proteomic changes, we performed trend curves of correlation for proteins involved in ATP synthesis, fatty acid metabolism, RE stress and stress response (Figure 5). Interestingly, when trend curves were used, we observed a correlation between two echographic parameters (LVEDD and LVESD) and levels of four proteins depending on the CHF-rat groups (Figure 6): GAPDH and alphaB-crystallin have a level of expression proportional to LVEDD and LVESD levels and peroxiredoxin 2 and isocitrate deshydrogenase had a level of expression inversely proportional to LVEDD and LVESD.

Discussion This study was undertaken to investigate cardiac protein expression changes in postinfarction LVR using an experimental model of CHF and proteomic technology. In particular, we showed that a large number of proteins involved in cellular stress response and cardiac metabolism are modulated in LVR Journal of Proteome Research • Vol. 7, No. 11, 2008 5009

5010

accession number

P08932

P01048

P05545

P04785

P10719

Q6P6R2

P14408

O55171

P16617

Q99NA5

P04797

P48500

P25113

Q91Y78

spot number

1

2 3 4 5 6 7

8 9 10 11 12 13 14 15 16 17

18 19 20


21 22

23

24 25

26

27

28

29

30

35 31

32

Ubiquitin carboxyl-terminal hydrolase isozyme L3

Phosphoglycerate mutase 1

Triose phosphate isomerase

Glyceraldehyde-3-phosphate dehydrogenase

Isocitrate dehydrogenase [NAD] subunit alpha

Phosphoglycerate kinase 1

Acyl-coenzyme A thioesterase 2

Fumarate hydratase

Dihydrolipoyl dehydrogenase

ATP synthase subunit beta

Protein disulfide-isomerase 1

NI NI NI NI NI NI NI Serine protease inhibitor A3K

T-kininogen 1

T-kininogen 2

protein name

Carbohydrate metabolism process Thiol protease, involved in ubiquitin conjugation pathway

Carbohydrate degradation. Functions as a killing protein in apoptosis when overexpressed Carbohydrate biosynthesis and degradation

Tricarboxylic acid cycle enzyme

Carbohydrate metabolism; tricarboxylic acid cycle Catalyzes hydrolysis of acylCoA to free fatty acid and coenzyme A Carbohydrate degradation

Produces ATP from ADP in the presence of a proton gradient across the membrane Component of the glycine cleavage

Catalyzes the formation, breakage and rearrangement of disulfide bonds

Binds to and inhibits kallikreins

Plasma glycoprotein. Inhibitor of thiol proteases

Plasma glycoprotein. Inhibitor of thiol proteases

functions

26.1

28.5

26.7

35.6

39.4

43.1

49.7

54.4

54.0

56.3

56.9

46.5

47.7

47.7

theo MW

4.9

6.2

6.5

8.2

6.5

6.2

7.7

9.1

8.0

5.2

4.8

5.3

6.1

5.9

theo pI

Table 3. Detailed List of Differentially Expressed Proteins in Left Ventricle after Myocardial Infarction

22.9

21.5 23.3

22.9

25

34.0

34.6

38.5

49.2 44.4

49.2

51.7 46.2

53.2 52.7 52.2

55.7 55.6 53.5 53.5 53.5 53.4 53.4 53.3 53.4 53.4

56.5 56.4 56.2 56.2 55.9 55.9

56.6

exp MW

4.3

6.6 6.8

6.03

7.1

5.7

5.4

7.03

7.67 7.5

7.28

4.14 4.26

3.73 3.88 4.0

4.4 4.5 3.81 3.87 3.92 3.98 4.07 4.15 3.62 3.67

3.85 3.90 3.98 4.05 4.2 4.3

3.78

exp pI

31.9 33.7 24.9 26.3 27 24.7 31.9 23.5

8.62 × 107 4.65 × 1010 1.48 × 107 1.67 × 105 1.54 × 107 2.14 × 106 1.17 × 108 1.09 × 1011

3.31 × 10 5.51 × 109 2.52 × 109

56.2

6 × 1010

8/52

13/82 8/84

16/60

1.79 × 105

9

34.8

63.1 40.2

49.9 34.7 31.5

1.58 × 1014 7.44 × 1010 1.75 × 107 20/78 16/78 9/91

2.02 × 10 4.29 × 105

60.7 31.4

1.29 × 1019 1.04 × 107 27/89 10/89

20/87

40.4

8

39.5 34.3

47

1.42 × 108 1.42 × 10 3.85 × 108

26.7 40.3

31.5 36.1 38.3 1.18 × 107 3.20 × 1014

6

16.3

23.7

3.61 × 106

3.85 × 103

sequence coverage (%)

probability score on protein Prospector

1.62 × 1012

15/61 15/68

17/68

15/59 21/79

12/43 15/75 18/80

7/56

12/86 14/83

13/69 16/71 11/55 12/76 14/64 9/54

9/56

number of matched peptides/total peptides

2.9

5.6 1.7

2

1.6

0.62

0.72

0.62

2.7 0.62

2.2

2.5 1.6

4.5 1.6 2.8

3 1.4 5 2.9 3 2 2.4 5 4 2.2

*a 1.7 3.4 1.5 *a 4.5

*a

fold-change (CHF vs sham)

research articles Cieniewski-Bernard et al.

P14604

P19234

P35704

O35878

P31399

P23928

P07895

38

39

40

41

42

45 43

48 44

49

NI

Heat shock protein beta-6


Superoxide dismutase 2

Alpha-Crystallin, B chain

ATP synthase subunit D


NADH-ubiquinone oxidoreductase 24 kDa subunit Peroxiredoxin-2

Enoyl-CoA hydratase

ATP synthase subunit alpha

Proteasome subunit beta type-3 Glutathione peroxidase 1

Peroxiredoxin-6

protein name

Destroys radicals which are toxic to biological systems. Manganese superoxyde dismutase Interacts with C-terminal domain of Actin-binding protein 280 Belongs to the small heat shock protein (HSP20) family

Belongs to the small heat shock protein (HSP20) family

Involved in redox regulation of the cell Multicatalytic proteinase complex Protects the hemoglobin in erythrocytes from oxidative breakdown Produces ATP from ADP in the presence of a proton gradient Fatty acid beta oxidation cycle. Short chain specific Transfer of electrons from NADH to the respiratory chain Involved in redox regulation of the cell Belongs to the small heat shock protein (HSP20) family. Stress response One of the chains of the F(0) subunit of ATPase complex

functions

17.5

9.8

24.6

20.1

18.6

20.3

21.6

27.3

31.5

25.6

22.3

22.9

24.8

theo MW

6.1

4.8

9.0

6.8

6.2

5.3

5.3

6.2

8.4

7.0

7.7

6.1

5.6

theo pI

16.1

17.3

16.6

17.8 19.8

17.1 18.4

18.9

18.5

18.5

20.5

22.7

21.8

20.8

21.0

20.8

exp MW

7.68

6.2

6.07

7.07 8.78

5.5 7.1

5.85

4.9

4.7

4.3

7.6

6.9

6.7

6.0

5.3

exp pI

7/56

4/53

15/61 10/55

11/85 10/76

7/47

6/64

9/65

10/71

13/86

13/53

12/62

8/95

14/86

number of matched peptides/total peptides

54.4

8.15 × 10

9.86 × 10 1.98 × 106

47.8 50.6

3.73 × 105

68.6 49.5

56.5 52

2.14 × 103

2.92 × 108 1.04 × 107

5

44.1

40.4 5

1.57 × 10

3.06 × 105

39.9 6

1.11 × 10

37.2

3.49 × 106 8

19.2

77.1

5.93 × 10

8.49 × 105

33.7 9

1.42 × 10

51.8

7.95 × 107 3

sequence coverage (%)

probability score on protein Prospector

2.3

1.5

0.21

2.7 1.5

3.1 9.2

3.3

*a

4

2.3

1.5

4.7

*a

2.7

1.5

fold-change (CHF vs sham)

No. spot, number assigned to polypeptidic spots identified on gel from Figure 2; theo, theorical; exp, experimental. Fold change, induced indicates that the spot is only detected in 2D-gel from left ventricule of CHF-rats. *, Spots were not detected in 2D-gel performed form sham-rats and fold-change could not be calculated Spots in italic were not identified by mass spectrometry. NI, not identified. Assignments were made according to UniProtKnowledgebase Release 13.1 which consists of: UniProtKB/Swiss-Prot Release 55.4 of 20-May-2008 (385 721 entries) and to UniProtKB/TrEMBL Release 38.4 of 20-May-2008 (5 814 087 entries).

a

P15999

37

P97541

P04041

36

47

P40112

34

Q9QUK5

O35244

33

46

accession number

spot number

Table 3. Continued


research articles

Journal of Proteome Research • Vol. 7, No. 11, 2008 5011

research articles


Figure 3. Analysis of HSPs (A), proteins of RE stress (B) and proteins of oxidative stress (C and D) in LV from 7-day and 2-month sham- and CHF-rats by Western blot. Quantification of proteins was performed from 7-day (n ) 5) and 2-month (n ) 16) sham-rats (white boxes), and 7-day (n ) 5) CHF-rats (hatched boxes), 2-month CHF- (gray boxes) (n ) 12) and severe CHF(CHF+++) (black boxes) (n ) 5) rats. Data are expressed as percentage ( SEM of sham-rats (arbitrary units). *p < 0.05; #p < 0.01.

(Figure 7). Interestingly, the proteins identified and belonging to the same biological pathways have been shown to be modulated in different animal models of heart disease. But as the authors mentioned, investigating the contribution of these proteins’ abundance to altered cellular function underlying cardiac dysfunction will be a major challenge.10 We used the well-characterized model of rat MI, together with echocardiographic and hemodynamic studies to assess the changes in LV geometry and function, in parallel to cardiac proteomic changes. As expected, induction of MI was associated with the development of severe long-term LV dysfunction (e.g., a marked progressive decrease in LV FS) and dilation (e.g., marked progressive increase in LVEDD and LVESD). To differentiate the changes associated with early post-MI LV remodeling to those attributable to late LV dilation and CHF, we studied two time points: • an early time point (7 days), which we verified to correspond to moderate cardiac remodeling with no detectable cardiac hypertrophy and only limited LV dilation, as well as modest cardiac dysfunction (especially maintained fractional shortening (FS)), 5012


Figure 4. Analysis of glycolytic enzymes (A), proteins of fatty acid metabolism (B), tricarboxylic acid cycle proteins (C) and proteins implicated in ATP synthesis (D) in LV from 7-day and 2-month sham- and CHF-rats by Western blot. Quantification of proteins was performed from 7-day (n ) 5) and 2-month (n ) 16) shamrats (white boxes), and 7-day (n ) 5) CHF-rats (hatched boxes), 2-month CHF- (gray boxes) (n ) 12) and severe CHF- (CHF+++) (black boxes) (n ) 5) rats. Data are expressed as percentage ( SEM of sham-rats (arbitrary units). *p < 0.05; #p < 0.01.

• a late time point (2 months), which we also verified to correspond to severe adverse remodeling, characterized by marked cardiac hypertrophy and LV dilation, as well as severe LV dysfunction, as demonstrated by the markedly reduced FS and the increased LW. Cellular Stress Response. We found 4 small HSPs as being modulated, 3 of them being up-regulated (alphaB-crystallin, HSP-beta-2/MKBP and HSP-beta-6/HSP20), while the cardiovascular HSP-beta-7 was down-regulated, as it was already described in a model of LV hypertrophy following aortic banding.18 In particular, we showed that the development of


research articles

Figure 5. Correlation of protein level expression with the degree of heart failure determined as described in Experimental Procedures by lung weight and hemodynamic parameters in 2 month-CHF rats. Trend curves from Western blot data were calculated for proteins involved in ATP synthesis, fatty acid metabolism, reticulum endoplasmic stress and oxidative stress. Data were expressed as percentage of the highest value for each protein and represented by a trend polynomial curve. Y-axis represents the level of expression for each protein and X-axis represents each rat classified from the lowest to the highest degree of heart failure with gray box corresponding to the severe CHF rats group.

CHF was associated with a modulation of HSP-beta-6 and thus could be considered as a tissue marker of the severity of this disease. In contrast, in a model of early LV hypertrophy secondary to hypertension (SHR rats), HSP-beta-6 was shown to be down-regulated compared to normotensive rats (WKY rats).19 Interestingly, HSP-beta-6/HSP20 is biochemically associated with alpha B-crystallin, suggesting that these two small HSPs, after their association with actin, may be involved in modulating cytoskeletal or contractile dynamics of cardiac myocytes.20 In contrast, we did not find high molecular HSPs to be modulated as previously described.21,22 HSPs, also implicated in protein-folding machinery, could be involved in protein quality control, including proteasome and ubiquitinylation systems. We have shown that isozyme L3 of ubiquitin C-terminal hydrolase and beta 3 subunit of proteasome were up-regulated in the LV of CHF-rats, the increase of ubiquitin C-term hydrolase being particularly marked in the early phase of LVR. It was shown that an increase in ubiquitin C-term hydrolase could lead to inappropriate ubiquitin conjugation and thereafter contribute to loss of function in an experimental model of dilated cardiomyopathy.23 We could also postulate that increase of the beta 3 subunit of proteasome could alter specificity and selectivity of proteasome against various substrates,24 as it was recently demonstrated in a model of pressure overload.25 Protein disulfide isomerase (PDI), a molecular chaperone implicated in endoplasmic reticulum (ER) stress response, was

shown to be up-regulated in severe CHF-rats. Interestingly, the synthesis of collagen by prolyl 4-hydroxylase, a multienzymatic complex including PDI,26 is a key event in LVR,27 as suggested by the fact that inhibition of this enzyme reduces LVR after aortic banding28 and MI29 leading to increased survival in the latter model.27 From our data, we could speculate that the specific increase of PDI in severe CHF-rats could participate in the folding and assembly of procollagen, allowing collagen accumulation and thus fibrosis, known to play a deleterious role in LVR. Oxidative stress is enhanced in CHF and participates in cardiac hypertrophy and remodeling processes.29 In particular, mitochondrial electron transport complex I was shown to be a source of reactive oxygen species (ROS) in failing myocardium30 due to a decrease in complex I activity. We revealed that NADH dehydrogenase [ubiquinone] flavoprotein 2, the 24 kDa subunit of complex I, increased in 2-month CHF-rats, while its expression level decreased in the severe CHF-rats, and this may result in an increase in ROS. Interestingly, in this model, Mulder et al. have shown induced myocardial ROS at 7 day- and 90 dayCHF rats compared to sham rats.31 While the modulation of the mitochondrial SOD isoform is a critical determinant in the tolerance of the heart to oxidative stress,32 our results indicate an increase of SOD2 expression only in CHF-rats. Moreover, the increase in mitochondrial ROS produced enzymes was not counterbalanced by SOD2 in severe CHF-rats. Our results also showed an increase in glutathione peroxidase 1 in CHF- but Journal of Proteome Research • Vol. 7, No. 11, 2008 5013

research articles


Figure 6. Correlation of four protein level expression with LVESD and LVEDD. Trend curves from Western blot were calculated for GAPDH, alphaB-crystallin, peroxiredoxin 2 and isocitrate dehydrogenase and represented by a trend polynomial curve. Y-axis represents the level of expression for each protein and X-axis represents the level of LVSED (left panel) and LVEDD (right panel). Data are expressed as percentage of sham-rat values.

not in severe CHF-rats. Since overexpression of glutathione peroxidase was recently shown to prevent LVR after MI,33,34 it is possible that the observed increase in glutathione peroxidase 1 contributes to the less severe dysfunction observed in CHFversus severe CHF-rats. Interestingly, we detected that glutathione peroxidase 1 is included in a covalent complex which decreased in CHF-rats, probably leading to an increase of glutathione peroxidase 1, while the complex increased in severe CHF-rats inversely correlated with the glutathione peroxidase 1 mononer content (Figure 5). Taken together, this data argue in favor of a more marked oxidative stress in severe CHF, due to an increase in ROS-producing enzymes associated with a defect of antioxidant systems. Metabolism. We showed a decrease of two enzymes of tricarboxylic acid cycle: fumarate hydratase and alpha subunit of isocitrate dehydrogenase, the latter being correlated with the development of HF. We also showed a decrease of acylCoA thioesterase 2, specific of very long chain fatty acid metabolism, particularly marked in 2-month CHF-rats, which is in agreement with the switch of failing heart from oxidative metabolism 5014


to a more fetal glycolytic metabolism.35,36 We revealed an increase of short chain enoylCoA hydratase, suggesting that the decrease of fatty acid utilization involved essentially long chain, rather than short chain fatty acid. Several subunits of ATP synthase, the mitochondrial respiratory chain complex V, were shown to be differentially modulated during the development of CHF. The alpha subunit and the D chain, which belongs to the F0 subunit, were respectively only increased in 2-month CHF- and severe CHF-rats. Interestingly, this protein also increased in an early model of hypertrophy secondary to hypertension.19 Concerning the glycolytic pathways, triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase were increased and phosphoglycerate kinase and phosphoglycerate mutase significantly decreased in the late phase of LVR. Our results argue in favor of, respectively, an increase and a decrease of glycolytic flux upstream and downstream the formation of 1,3-bisphosphoglycerate. Recently, it was shown that 1,3-bisphosphoglycerate could increase activity of cardiac sarcolemmal K+ATP channel,37 to which triose phosphate


research articles

Figure 7. Classification tree of the proteins selected to be modulated in viable LV after MI. Proteins were categorized by cellular function, intracellular localization and modulation: proteins down-regulated in 2-month CHF-rats (black boxes) proteins up-regulated in 2-month CHF- and severe CHF-rats (dark gray boxes), proteins up-regulated only in 7-day CHF-rats (right striped gray boxes), proteins upregulated in 7-day and 2-month CHF-rats (white boxes) and proteins down-regulated in 7-day CHF-rats and up-regulated in 2-month CHF-rats (vertical striped gray boxes). Proteins in boxes with dotted line were only analyzed by 2D gel and those in boxes with plain line were analyzed by 2D gel and Western blot.

isomerase and GAPDH belong38 as described by Surber et al.,39 who demonstrated that K+ATP channel current increased in remodeled cardiomyocytes after MI. Contractile Proteins. Surprisingly, we did not find any contractile proteins to be modulated in viable LV in our model of HF. First, one explanation could be that we did not perform organellar protein fractions as did, for example, Gramolini et al., who detected modulation of contractile proteins.40 Second, preliminary studies allow to postulate that the contractile dysfunction that occurs during the development of CHF could be linked to a modulation of the activity and function of the proteins through post-translational modifications rather than by a modulation of the protein expression (personal communication).

Conclusions One goal of functional proteomics is to assemble and integrate protein information in order to elucidate the functional role of proteins in normal and diseased organs. An important question is the compartmentalization of protein changes between tissue and plasma and in our case heart/ plasma. One limitation of our study is that we did not correlate to plasma levels our protein changes, despite the fact that Zhang et al.41 suggest that plasma is a rich source of biomarkers that could indicate the status of the different organs. The other important thing to do should be the integration of data from analysis of various sources as heart is constituted of a diversity of cell types (myocytes, fibroblasts, smooth muscle cells, endothelial cells), circulating cells (monocytes, leukocytes) as suggested in a review.10

In this study, we correlated changes in LV proteome with different mechanisms implicated in the development of CHF, notably cardiac metabolism and oxidative stress. We have shown that some proteins are modulated in the early phase of LVR (i.e., peroxiredoxin-2), while others are late phase tissue markers (i.e., PDI) and that modification of metabolic enzymes occurs in the late phase of LVR. Our results strongly suggest different adaptation pathways and responses against myocardial damage according to the severity of CHF (CHF vs. severe CHF rats). We hypothesize that the increase in proteins assisting the protection of myocardium against stress (especially oxidative stress) may be overcome in the context of more severe CHF. Recently, Gramolini et al.40 performed proteomic analysis of ventricule from a mouse model of cardiomyopathy, corresponding to the end-stage human dilated cardiomyopathy. Using another proteomic technique, they also found modulation of proteins mapped to the same biological pathways such as ER stress response, chaperone-mediated protein folding or activation of apoptosis. Indeed, we found expression of four proteins (GAPDH, alphaB-crystallin, isocitrate deshydrogenase and peroxiredoxin 2) linked to echographic parameters according to heart failure, that may be promising candidates as new markers or new mediators of LVR and dysfunction in heart failure.

Acknowledgment. This work was supported by the Agence Nationale de la Recherche 2005 (ANR), and the Fondation de France. C. Cieniewski-Bernard is a recipient of a fellowship from Fondation Lefoulon-Delalande. Journal of Proteome Research • Vol. 7, No. 11, 2008 5015

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