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Departments of 1 Pharmacology & Toxicology2 Pathology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, the Netherlands3 Laboratory of Protein Biochemistry and Protein Engineering, University of Ghent, Ghent, Belgium
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(Received 21 February 2005;
accepted after revision 11 April 2005; first published online 15 April 2005)
Corresponding author T. De Celle: Department of Pharmacology & Toxicology, Universiteit Maastricht, PO Box 616, Maastricht, 6200 MD, the Netherlands. Email: tijldecelle{at}yahoo.com
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Introduction |
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Proteomic studies of human heart tissue may provide new insights into the specific early molecular mechanisms that underlie the responses to ischaemia or ischaemia–reperfusion injury and therefore may have important implications for the specificity and efficacy of diagnosis and treatment. In relation to this, a 2-dimensional (2-D) electrophoresis technique on myocardial biopsies from patients undergoing coronary artery bypass surgery has been described (McDonough et al. 2002). However, these studies are complicated by factors such as small size of sample, availability, disease state, tissue heterogeneity, genetic variability, medical history and therapeutic interventions (McGregor & Dunn, 2003).
Alternatively, standardized animal models of myocardial infarction and ischaemia–reperfusion are available (Lutgens et al. 1999; De Celle et al. 2004a). To our knowledge in three studies, 2-D gel electrophoresis was used to identify changes in protein levels after myocardial infarction (Schwertz et al. 2002; Sakai et al. 2003; Sawicki & Jugdutt, 2004). Sawicki et al. (2004) found, in an in vivo dog model of myocardial ischaemia–reperfusion, changes in the level of four metabolic enzymes (NAD+-isocitrate dehydrogenase, 
subunit; creatine kinase, chain M; subunit ATP synthase isoforms precursor; and ATP synthase D chain, mitochondrial) and a contractile protein (ventricular myosin light chain 1). Also, in an in vivo rabbit model of cardiac ischaemia–reperfusion, Schwertz et al. (2002) found 10 protein spots that were differentially expressed. Two could be characterized as the protective proteins superoxide dismutase and B-crystallin. In addition, Sakai et al. (2003) found in an in vitro rat model eight protein spots with altered expression after cardiac ischaemia or ischaemia–reperfusion. Five protein spots were identified as the endoplasmic reticulum enzyme protein disulphide isomerase A3, one as 60 kDa heat shock protein and two as mitochondrial elongation factor Tu. These data indicate that the classes of proteins that are differentially expressed after myocardial infarction vary considerably between studies. The fact that different species were used and myocardial tissue was harvested at different time points (i.e. between 60 and 240 min after initiation of ischaemia) may contribute to this.
The mouse has been increasingly used to study the early molecular mechanisms inducing injury to the heart following myocardial infarction. To date, 2-D gel electrophoresis combined with mass spectrometry has not been applied to identify alterations in protein expression in myocardial tissue after in vivo myocardial infarction in mice. In addition, only the study of Sakai et al. (2003) compared changes in protein expression after both ischaemia and ischaemia–reperfusion, although they used an in vitro rat model and not an in vivo model.
Thus, the goal of the present study was to identify changes in cardiac protein expression after in vivo myocardial infarction in the mouse. A model of permanent ischaemia (PI) and a model of ischaemia–reperfusion (IR) were used to identify and distinguish between common and specific changes in protein expression induced by in vivo PI and IR. We studied early (210 min) changes in protein expression in order to identify potential new targets for cardioprotection that are beneficial in the first few hours of myocardial infarction. In addition, by separately analysing the soluble cytosolic fraction and the membrane fraction we were not only able to increase the resolution but also to detect pathology-related protein translocations between both compartments.
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Methods |
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Male outbred Swiss mice (7–9 weeks old; body weight, 35–40 g) were purchased from Charles River (Maastricht, the Netherlands). Experiments were performed according to the guidelines of the University of Maastricht and were approved by the institutional animal ethics committee. The animals were kept on a 12 h light–12 h dark cycle in a temperature-controlled (21 ± 2°C) room. After surgery, animals were housed individually with ad libitum access to water and standard food pellets (type Ssniff, Soest, Germany).
In vivo ischaemia–reperfusion (IR) and permanent ischaemia (PI)
Mice were anaesthetized with ketamine (100 mg kg–1 intramuscularly) and xylazine (5 mg kg–1 subcut-aneously). Body temperature was monitored with a rectal probe and maintained at 37°C using a warming pad and heating lamp. The trachea of each mouse was intubated per orally with a stainless steel tube connected to a respirator (Hugo Sachs Electronic rodent ventilator type 845, March-Hugstetten, Germany) set at a stroke volume of 250 µl and a rate of 210 strokes min–1. After left thoracotomy and exposure of the heart, the left anterior descending coronary artery (LAD) was ligated with a 6–0 polypropylene suture (Surgipro, CT, USA) directly proximal to its main branching point. The suture was tied around a 3-mm long polyethylene tube (PE-10) to induce ischaemia.
After 30 min of ischaemia, in the IR group, the ligature around the LAD was removed and the occurrence of reperfusion was assessed by the observation of blood flow in the epicardial coronary arteries through a surgical microscope. The ischaemic myocardium was reperfused for 180 min. In the PI group, the suture was tied permanently for 210 min. Separate sham groups for both IR and PI models were made following an identical procedure but without the actual tying of the polypropylene suture. The chest was closed with 5–0 silk sutures. The animals were then weaned from the respirator, and the intratracheal tube was removed once they were breathing spontaneously.
Cardiac tissue was harvested 210 min after initiation of ischaemia. At this point, mice were re-anaesthetized with pentobarbital (120 mg kg–1 intraperitoneally) and the thorax was re-opened. In the IR animals the LAD was re-occluded and 500 µl 2.5% trypan blue (Merck, Darmstadt, Germany) was injected into the jugular vein to delineate the non-ischaemic tissue from the ischaemic tissue. This ischaemic area is defined as the area at risk (AAR). Immediately afterwards the heart was excised and cleared of blood by rinsing in isotonic saline (0°C). In the PI and IR group, the AAR was cut out whilst in the sham-operated animals the corresponding left ventricular region was taken. Only these tissue samples (AAR and corresponding region in the sham animals) were snap frozen in liquid nitrogen, stored at –80°C and used whole for further study.
Sample preparation
The frozen tissue samples were pulverized to fine powder under liquid nitrogen using a mortar and pestle and homogenized using a hand-held homogenizer in 40 mM Tris buffer (pH 10.0) containing 300 U DNase and RNase and a protease inhibitor mix (Complete Mini, Merck, Darmstadt, Germany). We used a technique of high-speed sedimentation/centrifugation that separates the total membrane fraction from all soluble cytosolic proteins as described by Pasquali et al. (1997). To ensure reliability, all samples from the IR group (or PI group) and its respective sham group were processed simultaneously. After 30 min on ice, cytosolic fractions were separated by centrifugation at 100 000 g for 60 min at 4°C and stored at –80°C for further analysis. To avoid contamination with remaining cytosolic proteins, the 100 000-g pellet fraction was additionally dissolved in 40 mM Tris buffer and precipitated under the same conditions. After the supernatant was removed, the remaining membrane pellet was dissolved in 40 mM Tris buffer (pH 7.5) containing 7 M urea, 2 M thiourea, 1% DTT, 1% 3-(3-(cholamidopropyl)-dimethylammonio)-1-propanesulfonate (CHAPS) and a protease inhibitor mix (Complete Mini) and stored at –80°C. The final protein concentration of both homogeneous fractions was determined using a protein assay (Bio-Rad, Veenendaal, the Netherlands).
Two-dimensional gel electrophoresis
For the cytosolic fraction as well as the membrane fraction, 400 µg protein was dissolved in rehydration solution (8 M urea, 2% CHAPS, 0.5% immobilised pH gradients (IPG)-buffer, pH 4–7 linear and a trace of Coomassie Brilliant Blue) up to a volume of 450 µl. Only for the cytosolic fraction, a 2-D Clean-Up kit (Amersham Pharmacia, Uppsala, Sweden) was used in order to remove Tris buffer and to dissolve only the protein fraction in the rehydration solution (pH 7.5). Immobiline DryStrips (Amersham Pharmacia), 24 cm, pH 4–7 linear, were allowed to rehydrate (14 h, 30 V) in the protein solution under low viscosity oil in strip holders. Then, isoelectrical focusing was performed at 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h and 8000 V for 16 h (approximately 128 kVh in total). The rehydration step and isoelectric focusing (IEF) steps were accomplished using an IPGphor unit (Amersham Pharmacia) with the temperature being maintained at 20°C.
After the first dimensional run, the individual strips were equilibrated by gently shaking twice for 15 min in a solution containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% v/v glycerol, 2% SDS and a trace of Coomassie Brilliant Blue. Additionally, the first equilibration step contained 1% DTT and the second contained 2.5% iodoacetamide.
After equilibration, the IPG strips were placed on top of a 12% polyacrylamide gel (37: 5: 1) and proteins were then separated according to their molecular weight (Mr) using an electrophoresis system (Ettan Dalt, Amersham Pharmacia). The run was completed when the Coomassie Brilliant Blue front reached the bottom of the gel. We used six animals per group and the material from each animal corresponded with one gel for the cytosolic fraction and one gel for the membrane fraction. The system allowed a maximal run of 12 gels simultaneously. We therefore completed both the cytosolic and the membrane fraction separately using a run for the IR group with its respective sham group and a run for the PI group with its respective sham group. The gels were then washed for 1 h in a 10% v/v methanol and 7% v/v acetic acid solution and were stained overnight (12 h) with Sypro Ruby fluorescent stain (Bio-Rad). Before scanning the gels, they were destained for 1 h in a 10% v/v methanol and 7% v/v acetic acid solution.
Spot detection and analysis
Gel images were scanned at a resolution of 100 µm (Molecular Imager FX, Bio-Rad) and further analysed using PDQuest 7.1 software (Bio-Rad). The gels were ordered in the appropriate matchset and protein spots were manually matched according to the manufacturer's guidelines. Four different matchsets were used to compare separately the IR and PI group with their respective sham group for both the cytosolic fraction as well as the membrane fraction. The quantity of each spot was normalized by total quantity in valid spots using PDQuest 7.1. This normalized value was used to calculate the mean quantity of each spot per group. The fold change (density ratio) for a protein spot that was increased in quantity after IR (or PI) was calculated by dividing the mean quantitative value of that spot in the IR (or PI) group with the mean quantitative value of that spot in the respective sham group. For a protein spot that was decreased in quantity after IR (or PI) the inverse was taken. Using the PDQuest 7.1 software, a correlation coefficient was measured between the quantities of all spots in a gel and its quantities in another gel from the same matchset. The reproducibility of the 2-D gels was expressed as the mean correlation coefficient between all gels in a matchset.
Statistical analysis
A statistical comparison of the relative abundance of each matched protein spot between the IR group (n
= 6) or PI group (n
= 6) and their respective sham group (both n
= 6) was accomplished using a two-tailed t test (PDQuest). Between groups, qualitative difference means that the spot is present in the samples from one of the groups (IR or PI) and not in its respective sham group or vice versa. Between group quantitative or qualitative differences were 1.6 times, and a P-value 
0.05 was considered statistically significant. All values are expressed as mean ±
S.D.
In-gel digestion
Protein spots of interest were excised from Sypro Ruby-stained gels with an automatic spot cutter (Proteome Works Spot Cutter system, Bio-Rad). Each spot was pooled out of three to six gels, washed twice with 200 mM ammonium bicarbonate in 50% acetonitrile/water (20 min at 30°C), and allowed to dry at room temperature. The tubes were then chilled on ice and 8 µl digestion buffer (50 mM ammonium bicarbonate, pH 7.8) containing 150 ng modified trypsin (Promega, Madison, WI, USA) was added. The samples were incubated on ice for 45 min and 15 µl digestion buffer was then added and the samples were incubated overnight at 37°C. The supernatant was recovered, and the remaining peptides were extracted from the gel piece by washing twice with 60% acetonitrile/0.1% formic acid in water. The extracts were combined and the samples were dried in a Speedvac. The samples were resuspended in 10 µl 0.1% formic acid.
Protein identification mass spectrometry
The peptide mixture was analysed on a 4700 Proteomics Analyser, a matrix assisted laser desorption ionisation time of flight (MALDI TOF-TOF) mass spectrometer (Applied Biosystems, Framingham, CA, USA). We applied 1 µl digest mixture, mixed with 1 µl 100 mM
-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoracetate (TFA) onto a MALDI target plate. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis of the peptides was performed. MS spectra were acquired after accumulation of 2000 consecutive laser shots and MS/MS spectra were obtained after 3000 laser shots and air used as collision gas (1.2 x 10–7 Torr). This approach could not unambiguously identify all protein spots. Thus, a second strategy was used to identify the remaining 2-D spots and to confirm the identification of the MALDI analysis. Hence, the samples were loaded on an automated nano-HPLC system (Dionex-LC Packings, Amsterdam, the Netherlands) and separated peptides were then detected on-line by an ESI-Q-TRAP mass spectrometer (Applied Biosystems, Framingham, CA, USA). In this method, an automated MS to MS/MS switching protocol was used for on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the peptides (Sule et al. 2004). For identification, MASCOT v1.9 software was used for searching the MS and MS/MS data, obtained by either MALDI or liquid chromatography-electrospray ionisation (LC-ESI) analysis (Perkins et al. 1999). Searches were performed against the National Center for Biotechnology Information (NCBI) protein database. For the LC-MS/MS analyses, MASCOT was used with the following parameters: peptide tolerance, 0.6 Da; fragment tolerance, 0.8 Da; trypsin, specificity two possible missed cleavage sites, variable modifications carbamidomethylation and methionine oxidation and ESI-TRAP selected as the instrument.
Western blot analysis
Equal amounts of sample protein (10 µg) were separated by 10% SDS-PAGE. After transfer to polyvinylidene difluoride membranes, they were blotted with rabbit anti-annexin 5 polyclonal (Hyphen BioMed, France) or rabbit anti-annexin 3 polyclonal (gift from F. Russo-Marie, Bionexus Pharmaceuticals SA, Gif sur Yvette, France) and scanned. Densities were determined arbitrarily by ImageQuant (Molecular Dynamics, Sunnyvale, CA, USA).
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Figure 1 indicates the protein spots on the 2-DE spot patterns that significantly changed in expression level after IR and/or PI compared with their respective sham group. These protein spots are numbered in separate colours to distinguish between protein spots that changed significantly in expression level after IR alone (blue), after PI alone (red) or after IR and PI (green). The spot numbers in Fig. 1 refer to the numbers in Table 1 (1A–1D) which lists these protein spots and their protein identification. The experimental and theoretical mass and isoelectric point (pI) of the proteins and the density ratio (with P value) of expression level after IR and PI compared with its respective sham group are also listed in Table 1.
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As indicated in this Venn diagram, 22 protein spots (three spots after IR alone, 12 spots after PI alone, seven spots after both PI and IR) could be identified by MS while 10 protein spots (one spot after IR alone, eight spots after PI alone, one spot after both PI and IR) could not. Thus about 70% of the protein spots could be identified by mass spectrometry. In Table 1, the MASCOT score of the identified peptides is given together with (parts of) the deduced amino acid sequences. The unidentified proteins were relatively low in abundance and probably fell, despite pooling, below the detection limit of the mass spectrometry technique.
Protein spots present on the 2-D gels showing no significant change in expression level after IR or PI or with < 1.6-fold change were not identified by mass spectrometry.
As indicated in Fig. 2, proteins (or protein spots) that significantly changed in expression level after IR but not after PI were annexin-A3 (spot 10,24) and dual specificity phosphatase-3 (DSP-3, spot 11). Proteins (or protein spots) that significantly changed in expression level after PI but not IR were cardiac troponin T (cTnT, spot 1–4), -tropomyosin (spot 5), -myosin heavy chain (-MHC, spot 31), serum amyloid P-component precursor (SAP, spot 6), Zn-alcohol dehydrogenase (Zn-ADH, spot 13), prohibitin (spot 32), histidine triad nucleotide binding protein (Hint, spot 12), HSP-27 (spot 26) and HSP-20 (spot 14). The proteins (or protein spots) that significantly changed in expression level in both models were heterogeneous nuclear ribonucleoprotein K (hnRNP K, spot 8), pyruvate dehydrogenase E1-ß (PDHE1-ß, spot 9), catechol-O-methyltransferase (COMT, spot 23), adenylate kinase 1 (AK1, spot 28,29), HSP-20 (spot 7) and HSP-27 (spot 27). In general these identified proteins can be classified into functional groups; anticoagulant proteins, structural proteins, inflammatory-related proteins, transcription- and translation-related proteins, heat shock proteins, metabolism-related proteins and miscellaneous.
Translocation of annexin A3 and annexin A5
2-DE analysis showed that the quantity of annexin A3 in the cytosolic fraction was 4 times lower (P = 0.003) in the IR group than in the respective sham group. In contrast, in the membrane fraction of the IR group, the amount of annexin A3 was 5.8 times higher (P = 0.001) than in the sham group. These findings were verified using Western blots for annexin A3 as well as annexin A5 (Fig. 3). Annexin A5 was significantly reduced in the cytosolic fraction (P < 0.001) and significantly increased in the membrane fraction after IR (P < 0.001). It is remarkable that for both annexin A3 and annexin A5, such changes were specific for IR and did not occur after PI. is As indicated in the Venn diagram (Fig. 2), annexin A3 is significantly changed in expression level at two spot locations: one in the cytosolic fraction (decreased) and one in the membrane fraction (increased). Annexin A5 was not included in the Venn diagram (Fig. 2) because this protein was not found on the 2-DE gels.
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Four of the identified proteins that changed in expression level were found at multiple spot locations, characterized by a difference in pI on the gels (Fig. 2). This suggests that these are isoforms or proteins that underwent post-translational modifications under IR and/or PI. Mass spectrometry was not able to accurately distinguish between isoforms or to determine potential post-translational modifications. Figure 4 represents detailed 2-DE patterns of Sypro Ruby-stained 2-DE gels showing these protein spots. In the cytosolic fraction of the PI group but not in the IR group, HSP-20 shifted to a lower pI with a difference in pI of approximately 0.43. Cardiac troponin T was identified at four spot locations of which three spots had the same apparent molecular weight but had a pI difference of approximately 0.07. These four spots were significantly up-regulated only in the PI group and not in the IR group. In the membrane fraction of the PI group, adenylate kinase 1 and HSP-27 were significantly up-regulated at two spot locations separated by a pI range of 0.27 and 0.60, respectively. For adenylate kinase 1, the spot at the lowest pI and for HSP-27, the spot at the highest pI were not present in the sham group. The same phenomenon was observed for the IR group although the HSP-27 spot at the highest pI was not significantly up-regulated compared to the respective sham group.
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Anticoagulant proteins
The significant decrease of annexin A3 in the cytosolic fraction and the significant increase in the membrane fraction was specific for the IR model. This was also verified by Western blot analysis and suggests translocation of annexin A3 to the membrane fraction triggered by reperfusion. This phenomenon was also observed for annexin A5, as verified by Western blot analysis and correlates with the fact that the C-terminal protein core, which is responsible for the calcium-mediated membrane recognition and binding, is highly conserved between annexins (Barton et al. 1991). An explanation for the fact that annexin A5 was not detected on 2-D gels could be that other proteins masked the corresponding spot. In vivo functional properties for annexins are diverse and include membrane trafficking, endo- and exocytosis, membrane–cytoskeleton interactions, regulation of membrane protein activity, cell signalling and roles in inflammatory and coagulation processes (for review see Gerke & Moss, 2002). The fact that this phenomenon occurred after IR only and not after PI suggests a specific related mechanism that triggers translocation of annexins to the membrane fraction during reperfusion. These findings may give new impetus for further studies in relation to annexins and early diagnosis of reperfusion injury in connection with earlier studies from our laboratory (Dumont et al. 2001).
Structural proteins
Cardiac troponin T (cTnT) and another myofilament protein, -tropomyosin, were significantly increased in the cytosolic fraction after PI but not after IR. cTnT is known to be an -tropomyosin binding protein, and both proteins are involved in the process of muscle contraction (Bacchiocchi & Lehrer, 2002). Several animal studies have shown that the appearance of cTnT in blood is a highly sensitive and specific marker for myocardial injury and is directly correlated with infarct size (Remppis et al. 2000; Metzler et al. 2002). We have recently described that in mice after PI, infarct size is almost 4 times greater than after IR (De Celle et al. 2004a). With respect to this, the data suggest that the increased cytosolic levels of cTnT and -tropomyosin are markers for high breakdown of the contractile apparatus after PI compared to IR.
Inflammatory-related proteins
Myocardial infarction is associated with an early inflammatory response, which is a prerequisite for wound healing and scar formation (see Frangogiannis et al. 2002).
In relation to this we observed that serum amyloid P-component precursor (SAP) was significantly increased in the cytosolic fraction after PI but not after IR. SAP is a precursor for serum amyloid P component which is an acute-phase reactant produced by the liver and able to activate the complement system (Steel & Whitehead, 1994). SAP does not only exist in plasma but also in peripheral tissues where it is associated with extracellular matrix proteins (Dyck et al. 1980; Breathnach et al. 1981, 1983; Zahedi, 1996, 1997). To our knowledge, this study is the first to describe SAP as a potential specific marker for hypoxic cardiac tissue after PI.
Transcription- and translation-related proteins
Prohibitin (membrane fraction) and histidine triad nucleotide binding protein 1 (cytosolic fraction) were both significantly reduced after PI but not after IR. Prohibitin is known to inhibit cell proliferation (Nuell et al. 1991), to regulate transcriptional activity (Fusaro et al. 2003) and to stabilize mitochondrial proteins (Nijtmans et al. 2000). In addition, both proteins have been reported to function as tumour suppressers (Fong et al. 2000; Zanesi et al. 2001; Manjeshwar et al. 2003). However at present, nothing is known about a functional role of these proteins in relation to cardiovascular diseases. In our study, heterogeneous nuclear ribonucleoprotein K (hnRNP K, cytosolic fraction), and in the study of Sakai et al. (2003), elongation factor Tu, showed increased expression after PI and IR. Both proteins are associated with serine/threonine protein kinase C
, a protein complex with a critical role in protecting the myocardium against IR injury (Edmondson et al. 2002). Elongation factor Tu promotes amino-acyl tRNA binding to the ribosome (Cai et al. 2000) and hnRNP K is a nucleic acid-binding protein involved in RNA processing (Shnyreva et al. 2000), translocation (Mattaj & Englmeier, 1998) and translation (Ostareck et al. 2001). The finding that proteins belonging to this functional group changed in expression level suggests possible extensive control over protein expression in part by regulation of mRNA. This partially depends on the model, PI or IR.
Heat shock proteins
In accordance with the previous studies by Schwertz et al. (2002) and Sakai et al. (2003), we observed a change in expression level of heat shock proteins (HSPs) after myocardial infarction. These proteins are considered as ‘molecular chaperones’, the expression of which is increased by cellular stress (Knowlton, 1995). HSP-27 (membrane fraction) and HSP-20 (cytosolic fraction) were increased both after PI and IR. Differential changes in HSP-27 levels have been described in rat (Tanonaka et al. 2001) and human (Knowlton et al. 1998; Scheler et al. 1999) cardiac tissues exhibiting heart failure. HSP-27 acts as a protective agent against hypoxic injury in cultured adult rat (Martin et al. 1997) and canine cardiomyocytes (Vander Heide, 2002). However, this protective effect has not been shown in an in vivo animal model to date. Because of its increased expression level, our study suggests a possible role for HSP-27 in the cardioprotective mechanism after in vivo PI and IR.
HSP-20 was identified at two spot locations after myocardial infarction. The spot at the lowest pI increased in expression level after IR and PI, while the spot at the highest isoelectric point decreased after PI alone. Phospho-isoforms for HSP-20 have been described in the literature (Rembold et al. 2001) and additional research is necessary to investigate whether one or both of these spots in our models represents phospo-isoforms of HSP-20. Recently Chu et al. (2004) showed in mouse cardiomyocytes that HSP-20 increases in expression and phosphorylation after prolonged activation of the ß-adrenergic signalling pathway, which has consequences for the regulation of cardiac contractility and Ca2+ handling. The same group also suggested that HSP-20 and its phospho-isoform may provide cardioprotection against ß-agonist-induced apoptosis (Fan et al. 2004). Extrapolation of these results to our study could mean that HSP-20 may have a role in cardiac myocyte function and cell death after myocardial infarction in which ß-adrenergic stimulation occurs to compensate for a reduced cardiac output.
Myocardial ischaemia is associated with a marked interstitial accumulation of catecholamines (Lameris et al. 2000). Under physiological conditions part of the catecholamines are broken down by catechol-O-methyltransferase (COMT) (Mannisto & Kaakkola, 1999). The finding that COMT levels were significantly decreased in the cytosolic fraction after PI and IR points to a myocardial adaptation of the metabolism of catecholamines or related substrates under these conditions (Ball & Knuppen, 1980; Mannisto et al. 1999).
Metabolism-related proteins
Adenylate kinase 1 (AK1) in the membrane fraction and pyruvate dehydrogenase E1 component beta subunit (PDHE1-ß) in the cytosolic fraction were both significantly increased after IR and PI. AK1 reversibly catalyses (high-energy) phosphotransfer between ADP, ATP, and AMP, and serves as an integral component of phosphotransfer networks (Dzeja et al. 1985). During metabolic challenges, AK1 has been reported to be an essential enzyme in maintaining myocardial energy homeostasis (Dzeja et al. 1999; Pucar et al. 2000) and in metabolic signal transduction to ATP-sensitive K+ channels (Carrasco et al. 2001). Deletion of the AK1 gene revealed that adenylate kinase phosphotransfer supports myocardial function after initiation of ischaemic stress and safeguards intracellular nucleotide pools in post-ischaemic recovery (Pucar et al. 2002). Simultaneously PDHE1-ß, which is a subunit of pyruvate dehydrogenase that largely controls the rate of entry of pyruvate into the Krebs cycle, was up-regulated. Both indicate a regulatory mechanism during IR and PI to achieve energy balance for the increased energy demands that occurs during these stress periods and to achieve cellular energetic economy and metabolic signal transduction. In relation to this, the previous study of Sawicki et al. (2004) showed an increase in isocitrate dehydrogenase, a key enzyme in the Krebs cycle, after IR.
Miscellaneous
At this moment it is not possible to formulate a hypothesis about the relevance of a few proteins: -myosin heavy chain (membrane fraction); dual specificity phosphatase (cytosolic fraction); and a protein ‘similar to Zn-alcohol dehydrogenase’ (cytosolic fraction) which were differentially expressed after PI and/or IR.
Conclusion
This study demonstrates that mouse myocardial infarction models are suitable to study in vivo changes in the proteome early after induction of PI or IR. By analysing selectively membrane and cytosolic fractions we were able to detect a substantial number of proteins that were differentially expressed in either compartment after PI, after IR or both. In addition, our approach allowed the identification of translocation of particular proteins such as annexins, from cytosolic to membrane compartments. Additional research is necessary to identify the functional relevance of these proteins in these pathologies and to determine the character of possible pathology-related post-translational modifications. The specific alterations in protein expression that took place after PI and IR may stimulate the search for new tools to diagnose myocardial infarction and to characterize specific pathology-related changes in protein expression.
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1.6). Protein spots changing in expression level after IR alone, after PI alone or after both IR and PI are numbered in blue, red and green, respectively. Spot numbers refer to the numbers given in 
