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European Journal of Heart Failure 2008 10(8):740-748; doi:10.1016/j.ejheart.2008.06.001
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© 2008 European Society of Cardiology

A proteomic study of the effects of ramipril on post-infarction left ventricular remodelling in the rabbit

Ching-Yi Chena, Bai-Chin Leeb, Hsiu-Ching Hsub, Hung-Ju Linb, Chia-Lun Chaob, Yen-Hung Linb, Yi-Lwun Hob and Ming-Fong Chenb,*

a Department of Animal Science and Technology, National Taiwan University 50 Lane 155, Sec. 3, Keelung Rd., Taipei, Taiwan
b Department of Internal Medicine, National Taiwan University Hospital 7 Chung-Shan S Rd, Taipei, Taiwan

* Corresponding author. Department of Internal Medicine, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, Taiwan. Tel.: +886 2 2312 3456x5059; fax: +886 2 3322 3937. E-mail address: mfchen{at}ntu.edu.tw, austinr3{at}yahoo.com.tw (M.-F. Chen).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objectives: In this study, we used a proteomic approach to investigate the potential proteins regulated by ramipril in post-infarction left ventricular remodelling in the rabbit.

Methods and results: Myocardial infarction (MI) was induced in male New Zealand White rabbits (2.5–3 kg) by ligation of the left anterior descending coronary artery. Two months later, the rabbits were either left untreated (MI group) or were treated daily for one month with 0.1 mg/kg wt of ramipril (ramipril group), then sacrificed. One month of ramipril treatment resulted in a significant improvement in the LV ejection fraction (LVEF) and a decrease in hydroxyproline content. The protein profiles of LV tissue showed that ramipril caused upregulation of glutathione peroxidase, superoxide dismutase (SOD), and heart-type fatty acid binding-protein (h-FABP) and downregulation of HSP27 and cyclophilin A. Ramipril treatment caused an increase in catalase, glutathione peroxidase, and SOD activity in the LV tissue. Oxidized glutathione levels and the GSSG/GSH ratio in the heart tissue were lower in the ramipril group than in the MI group.

Conclusions: Ramipril increased antioxidative protein expression and enzyme activity, which could partly explain the role of ramipril in attenuating LV remodelling. In addition, the present study identifies several potential protein targets which may help to explain the mechanism by which ramipril exerts its effect in post-infarction LV remodelling in the rabbit.

Key Words: Rabbit • Myocardial infarction • Ramipril • Antioxidative defence • Coronary ligation

Received November 23, 2007; Revised March 20, 2008; Accepted June 4, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Reactive oxygen species (ROS), by-products of normal cellular aerobic metabolism, are generated during oxidative phosphorylation in the mitochondria and are involved in various normal tissue reactions. However, excess ROS production caused by oxidative stress damages normal cellular functions, resulting in protein nitration, lipid peroxidation, and modulation of redox signalling [1-3]. Myocardial infarction (MI) is one of the most important aetiologies of heart failure. Cardiac remodelling after MI consists of a series of changes in the left ventricular myocardium, leading to a decline in left ventricular performance. There is evidence that ROS are involved in the processes underlying cardiac remodelling. Increased ROS production and oxidative stress are observed in animal models of ischaemia-reperfusion and surgically induced myocardial ischaemia and in patients with chronic heart failure secondary to MI [2]. Studies in which antioxidative enzymes were over-expressed in order to decrease ROS production have demonstrated significant protection against cardiac remodelling in a rodent model [4,5].

Ramipril, an angiotensin converting enzyme inhibitor (ACEI), has a well-established beneficial role in the treatment of patients with diabetes, hypertension, and congestive heart failure [6-8]. It decreases blood and tissue levels of apolipoprotein AI and serum levels of oxidized low density lipoprotein (LDL)-cholesterol [6], improves left ventricular remodelling [7], attenuates blood pressure [8], reduces aortic lesion size [9], and increases the survival rate of patients with MI [10].

In addition to the functions mentioned above, ramipril also acts as an antioxidant by increasing the resistance of LDL to CuSO4-induced oxidation in mice [9], decreasing plasma malondialdehyde (MDA) concentrations [8], and suppressing LDL-cholesterol oxidation in patients with type-2 diabetes [6]. Although ramipril plays a crucial role in the treatment of cardiovascular diseases, its precise mechanism of action is complex and requires further evaluation. In the present study, we used a rabbit model for the proteomic study of the effects of ramipril on post-infarction LV remodelling. The antioxidative effects of ramipril in LV tissues were also evaluated. In addition, we explored the potential proteins regulated by ramipril. These proteins could be a target for further evaluation of the effect of ramipril on heart failure progression.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animal model and experimental protocol
Male New Zealand White rabbits weighing 2.5-3 kg were subjected to myocardial infarction (MI) by ligation of the left anterior descending coronary artery as previous described [11]. Six rabbits were subjected to sham operation, and underwent a similar procedure except for ligation of the left coronary artery and served as controls. Two months later, the rabbits with coronary ligation were either left untreated (MI group) (n=9) or were treated with ramipril (ramipril group, 0.1 mg/kg wt daily; Sanofi-Aventis, Gouda, the Netherlands) (n=9) for one month. Since the clinically recommended dose of ramipril for acute MI patients is 0.04-0.16 mg/kg/day (body weight 60 kg), the dosage for the rabbits was set at 0.1 mg/kg/day in this study.

At the end of the study period the rabbits were anaesthetized with 10 mg/kg of xylazine and 50 mg/kg of ketamine given intramuscularly, and haemodynamic parameters were measured. A polyethylene Millar catheter was inserted into the right carotid artery and connected to a transducer (Model SPR-407, Miller Instruments, Houston, TX) to measure the aortic systolic and diastolic pressures as the mean of five consecutive pressure cycles. After the haemodynamic measurements, the anaesthetized rabbits were sacrificed with a pentothal overdose. The non-infarcted region of the left ventricle from each rabbit was cut into small pieces and stored in liquid nitrogen until analysis.

The study conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.2. In vivo myocardial function measurements by transthoracic echocardiography
Echocardiography was performed before ligation, two months after ligation, and at the end of the study. The rabbits were anesthetized with xylazine (5 mg/kg, intramuscular injection) and ketamine (16 mg/kg, intramuscular injection), and the left ventricular ejection fraction (LVEF) was measured using an echocardiography system (SONOS 2500, Philips, Andover, MA, USA) equipped with a 12 MHz phased-array probe. The LV end-diastolic diameter (LVEDD) and the LV end-systolic diameter (LVESD) at the level of the papillary muscles were measured in the parasternal short-axis view. The LVEF was calculated using the equation:


Formula

2.3. Measurement of hydroxyproline and thiobarbituric acid reactive substances (TBARS)
Hydroxyproline was measured following the procedure of Reddy and Enwemeka [12]. The tissue homogenate (non-infarcted part of left ventricle) was hydrolyzed in alkali. The hydrolyzed sample was then mixed with a buffered chloramines-T reagent, and the oxidation was allowed to proceed for 25 min at room temperature. The chromophore was developed with the addition of Ehrlich's reagent. The absorbance of reddish purple complex was measured at 550 nm. TBARS were measured by fluorometric assay (excitation at 515 nm, emission at 552 nm) using 1,1,3,3-tetraethoxypropane as the standard, as described by Chen et al. [13].

2.4. Measurement of superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase enzyme activities in the non-infarcted part of left ventricle
Glutathione peroxidase and glutathione reductase activity was measured based on the consumption of nicotinamide adenine dinucleotide phosphate following reduction of t-butyl hydroperoxide or oxidized glutathione, respectively [14]. The catalase assay was based on its peroxidative activity as described by Johansson and Borg [15]. The superoxide dismutase (SOD) assay was based on the reduction of cytochrome c [16]. These procedures were performed in an Epos 5060 analyzer (Eppendorf Corp, Hamburg, Germany). All biochemical measurements were performed in triplicate and the mean values recorded.

2.5. Determination of reduced (GSH) and oxidized (GSSG) glutathione levels in the non-infarcted part of left ventricular tissue
The procedure for determining the total (GSH+GSSG) glutathione content in the non-infarcted part of the left ventricle has been described previously [13]. All procedures were performed at 30 °C. Twenty milligrams of tissue was lysed in 2 mL of 5% trichloroacetic acid, then 50 {varepsilon}L of the lysate was added to 150 {varepsilon}L of the glutathione assay mixture, which consisted of 0.6 nM 5,59-dithiobis-(2-nitrobenzoic acid), 0.4 U of glutathione reductase, and 0.2 nM reduced nicotinamide adenosine dinucleotide phosphate, and the mixture incubated for 1.5 min. The absorbance at 410 nm was then measured and the concentration calculated from a standard curve prepared using dilutions of a known GSSG or GSH concentration. For the GSSG assay, N-ethylmaleimide (0.02 N final concentration) was added to the lysate to remove GSH by forming a stable complex to prevent it from interfering with the assay. Following incubation for 60 min at 25 °C, the solution was extracted with 10 volumes of ether to ensure complete removal of the unreacted sulfhydryl reagent which would inhibit glutathione reductase activity in the assay mixture. After the extraction process, the solution was added to the assay mixture for GSSG determination. GSH levels were calculated by subtracting the GSSG value from the GSSG+GSH value.

2.6. Two-dimensional electrophoresis and image analysis
Tissue lysate from the non-infarcted part of the left ventricle was prepared and quantized as described by Chen et al. [17]. Isoelectric focusing (IEF) was carried out on an Ettan IPGphor isoelectric focusing system (Amersham Bioscience) using 13 cm IPG strips (pI 3-10). Protein (130 µg) was added to rehydration solution (7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 0.2% DTT, 0.5% Triton X-100, and 0.4% carrier ampholytes). The strips were rehydrated, and then IEF was performed for a total of 24500 Vh. Sodium dodecylsulfate polyacrylamide electrophoresis on 12% gels was used for the second dimension. After the second dimension, the gel was silver stained according to manufacturer's instruction (Amersham Bioscience).

Image analysis was performed using ImageMaster 2D Elite 3.10 software (Amersham Bioscience) as described previously [17]. Briefly, protein spots were detected automatically and confirmed manually, and then the volume of each spot was normalized as a fraction of the volume of all the spots on the gel and expressed relative to the same value for the sham group. Each LV sample was performed in triplicate and the mean values recorded, and each inter-group comparison of samples was carried out on 6 replicate LV samples.

2.7. In-gel digestion and mass spectrometry
The protein of interest was cut, destained and dehydrated. The dried gel was then rehydrated and incubated in trypsin solution (0.1 mg/mL in 25 mM ammonium bicarbonate) for 20 h at 37 °C. The peptides were eluted in 0.8 mL of matrix solution (a-cyano-4-hydroxycinnamic acid, 8 mg/mL in 70% v/v acetonitril/1% formic acid) directly onto a target plate and subjected to mass spectrometric analysis. A QStar hybrid quadruple time-of-flight (QqTOF) mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a matrix-assisted laser desorption ionization (MALDI) source and a nitrogen laser (337 nm) was used to acquire MALDI-MS and -MS/MS spectra. The MS/MS spectra were used to automatically search for the Oryctolagus cunniculus (Rabbit) and other mammals on the NCBInr protein database using MASCOT software (http://www.matrixscience.com/). Multiple peptides from each protein were generally detected, generating confidence in the protein identifications. Identification parameters included peptide mass accuracy within 0.1 Da, and one possible missed tryptic cleavage per peptide. Carbamidomethylation of the cysteine and oxidization of methionine were specified as variable chemical modifications. Monoisotopic mass value was used for peptide search. The m/z tolerance was set as 0.1 Da for both parent and fragment ions. A default setting was used for all other variables. Identifications were based on the observed pI and MW (kDa), the number of matching peptide masses and the total percentage of the amino acid sequence that those peptides covered, in comparison to other database entries.

2.8. Real-time quantitative reverse-transcription polymerase chain reaction
RNA was isolated from the non-infarcted part of the left ventricle using RNAzol (TEL-TEST, Inc., Friendswoods, TX). For reverse transcription, 1 µg of total RNA was used for reverse transcription with PowerScript reverse transcriptase (Clontech, Palo Alto, CA) using oligo-dT and random primers. One-twentieth of reverse-transcription products were used as template for real-time PCR. PCR of each sample were run in triplicate using the TaqMan PCR Core Reagent Kit and the ABI Prism 7700 Sequence Detection System and software (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Sequence specific PCR primers and TaqMan probes were designed using the Primer Express software version 1.5 (Applied Biosystems). The primer sequences for h-FABP were: forward primer 5'-TGCGGGAGCTCAGTGATG -3', reverse primer 5'-CGT AAG TGC GTG TGC TAA CTG-3', and the probe 5'-FAM-ACT CAT CCT GAC GCT CAC C-TAMRA-3'. The primer sequences for GADPH were: forward primer 5'-GAT GGT GAA GGT CGG AGT GA-3', reverse primer 5'-CAA CAT CCA CTT TGC CAG AGT TAA AA-3', and the probe 5'-FAM-ACG GAT TTG GCC GCA TTG-TAMRA-3'. Each pair of oligonucleotide primers was designed to span an intron to avoid amplification of genomic DNA. For all specific mRNA amplified linear inverse correlations were observed between amount of RNA and CT value (number of cycles at threshold lines). Gene expression was normalized to GADPH and expressed relative to the same value for the sham group.

2.9. Western blotting
Tissue samples were homogenized in RIPA lysis buffer (Santa Cruz, Santa Cruz, CA), and protein concentrations were measured using the protein assay kit (Bio-Rad, Hercules, CA). Protein suspension from the tissue lysate (20 µg) was loaded onto a SDS-PAGE gel for electrophoresis and then transferred to a PVDF membrane (Amersham Bioscience). The membrane was then incubated for 1-2 h at room temperature with primary antibodies against h-FABP and actin (Santa Cruz), respectively. After washing with PBS-T, the membrane was incubated with secondary antibody against goat or mouse IgG and the immunoblotting was visualized using QUANTITY ONE (Bio-Rad).

2.10. Statistics
All data are expressed as the mean±SD. Differences among groups were assessed by analysis of variance followed by a Tukey's test. Statistical analyses were performed using SAS (version 9.1; SAS Institute Inc., Cary, NC). A P value<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Physiological parameters
Thirty rabbits underwent coronary ligation, 12 died during the surgical operation or the treatment period, leaving only nine rabbits in each of the surgery groups. We found LV hypertrophy and scar formation in the LV two months after induction of MI (data not shown), indicating that cardiac remodelling was induced in the rabbit heart.

The sham, MI, and ramipril groups had similar body weights, heart weights, haemodynamic parameters, and heart weight/body weight ratios (Table 1). The higher concentration of hydroxyproline in the non-infarcted part of the left ventricle seen in the MI group compared to the ramipril group demonstrates that ramipril improved the fibrosis of the left ventricle following MI. LVEF was used to evaluate heart function. The net change in LVEF before ligation and at two months after ligation was similar in the MI and ramipril groups, indicating that MI was induced similarly in all rabbits. There were no significant differences in heart function among the groups at the beginning of the study. However, heart function in the MI group deteriorated continually following coronary ligation, whereas ramipril treatment resulted in a significant improvement in heart function.


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  		Table 1 Physiological parameters in the three study groups

 
3.2. Protein profiles and expression in the left ventricle
Fig. 1 shows the protein profiles of the non-infarcted part of the left ventricle. Identified proteins are numbered in the Figure and listed in Table 2. Sixty-eight spots were cut from the gels for identification, but only 25 were confidently identified because of the limited information on rabbit protein sequences in available databases. Nine of these proteins involved in the oxidation system are listed in Table 3 and include three redox-related proteins (glutathione peroxidase, Mn-SOD, and Cu-Zn-SOD) and two inflammation-related proteins [heat shock protein 27 (HSP27) and cyclophilin A (CyPA)].


Figure 01
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  		Fig. 1 Protein profile for the rabbit left ventricle. Tissue lysate protein (130 µg) was loaded on pI 3-10 linear IPG strips (13 cm), with 12% SDS-PAGE as the second dimension, and then the gel was silver stained. The numbers refer to the identified proteins listed in Table 2. MI: adult rabbits with myocardial infarction for three months. Ramipril: MI rabbits treated with ramipril (0.1 mg/kg daily) for the final month.

 


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  		Table 2 Protein profiles for the rabbit left ventricle by mass spectrometry

 


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  		Table 3 Regulation of proteins in the left ventricle by ramipril

 
Table 3 shows the data for each of these proteins in the MI and ramipril groups calculated as the spot volume divided by the sum of the volumes of all spots on the gel expressed relative to the corresponding value in the sham group. Ramipril treatment upregulated the expression of heart-type fatty acid binding-protein (h-FABP), glutathione peroxidase, Cu-Zn-SOD and Mn-SOD in comparison with the MI treatment (P<0.05). Expression of CyPA and HSP27 in the left ventricle was higher in the MI group than in the ramipril group (P<0.05).

3.3. Western blotting and mRNA expression in the left ventricle
Western blotting and RT-PCR were used to corroborate the proteomic results. No differences were observed between the groups for the mRNA level of h-FABP in the rabbit LV (Fig. 2A). However, coronary ligation (MI and ramipril group) decreased the protein expression of h-FABP in the left ventricular tissue compared to sham group (Fig. 2B). In addition, ramipril treatment caused an increase in h-FABP of rabbit LV with MI, which was concordant with the proteomic results, and these results indicate that ramipril may modulate the expression of h-FABP by post-translational modification.


Figure 02
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  		Fig. 2 Representative mRNA level of FABP in the rabbit left ventricle (A). Total RNA of samples was extracted and mRNA levels were analyzed by quantitative real-time RT-PCR. Each RNA sample was analyzed in triplicate. The values shown are the ratio of gene expressed of each treatment relative to the same value for the sham group. (B) Representative Western blots, confirming the proteomic results that h-FABP expressions were higher in the ramipril group than in the MI group. Bar chart showing the relative density measurements of bands obtained from Western blots of all treatments. Data represent the means±SD (n=6).

 
3.4. Antioxidative defence in the left ventricle
TBARS levels in left ventricle, a well-established method for screening and monitoring lipid peroxidation (formation of MDA), were higher in the ramipril group than in the MI group (Table 4). We therefore presume that ramipril increased fatty acid utilization, thus increasing the likelihood of lipid peroxidation and TBARS production.


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  		Table 4 Effect of ramipril on the antioxidative system in the rabbit left ventricle

 
Table 4 shows the effect of ramipril on the antioxidative system in the rabbit left ventricle. Oxidized glutathione levels were significantly higher in the MI group than in the ramipril group. The GSSG/GSH ratio in left ventricular tissue was decreased by ramipril. Ramipril treatment caused a significant increase in catalase, glutathione peroxidase, and SOD (total SOD or Mn-SOD) activity. The decrease in the oxidized glutathione levels and the increase in antioxidative enzyme activity demonstrate that ramipril caused a better antioxidative defence.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In the present study, we demonstrated that ramipril increased antioxidative protein expression and enzyme activity in the left ventricular tissue, thus increasing antioxidative defence in the heart. Using a proteomic approach, we found that ramipril upregulated the expression of fatty acid metabolic-related protein (h-FABP) and downregulated the expression of two inflammation-related proteins (HSP27 and CyPA). These results suggest that ramipril is associated with the promotion of fatty acid utilization and the attenuation of inflammation on post-infarction LV remodelling.

4.1. Effect of ramipril on antioxidative defence
Reactive oxygen species (ROS) have several different effects on heart tissue. Increased ROS production is observed in the heart with MI [2,3,18]. ROS release has recently been linked to the development of left ventricular hypertrophy and heart failure progression [3,18]. Reducing ROS production in heart failure improves NO bioavailability, endothelial dysfunction, and left ventricular function, and prevents left ventricular remodelling [3,18,19]. Over-expression of either SOD or glutathione peroxidase provides significant protection from post-ischaemic injury and results in a significant decrease in infarct size in rodents [4,5].

Ramipril has a well-established beneficial role in the treatment of cardiovascular diseases [6-8]. In addition, it has been shown to be an antioxidant. A recent study showed that ramipril protects cells from oxidation by decreasing levels of apolipoprotein AI and serum oxidized LDL-cholesterol [6]. It also increases the resistance of mouse LDL to CuSO4-induced oxidation [9], increases SOD and glutathione peroxidase activity [19,20], and decreases renal nitrotryosine production and NADPH oxidase gene expression [21].

Using two-dimensional electrophoresis and mass spectrometry, we found that redox-related proteins (SOD and glutathione peroxidase) in the left ventricular tissue were upregulated by ramipril, and redox-related enzyme activity (SOD, glutathione peroxidase, and catalase) in the heart showed a concordant pattern. In addition, oxidized glutathione levels and the GSSG/GSH ratio were decreased by ramipril. Surprisingly, TBARS levels in the left ventricle were higher in the ramipril group than in the MI group, while plasma TBARS levels were similar between treatments. TBARS are the products of lipid peroxidation, but their role as a biomarker of oxidative stress is controversial [22-24]. Some researchers have found that serum TBARS levels are a biomarker for patients with stable coronary artery disease and stroke [22,24], while others found plasma TBARS levels were similar in patients with dilated cardiomyopathy and controls [22,23]. Although the role of TBARS as a biomarker of oxidative stress is controversial, reduced glutathione levels and the GSSG/GSH ratio are widely used to evaluate oxidative stress in congestive heart failure animals [25,26]. In another study in rabbits, the myocardial GSH/GSSG ratio in the MI group decreased in a time-dependent manner [25]. Our present results show that ramipril caused a decrease in oxidized glutathione levels and the GSSG/GSH ratio in the left ventricle. Taken together, these results show that ramipril decreases myocardial oxidative stress by increasing antioxidative enzyme activity and thereby attenuates left ventricular remodelling.

4.2. Effect of ramipril on fatty acid utilization
In this study, we found that ramipril treatment upregulated the expression of h-FABP in the rabbit heart following induction of MI. h-FABP, a cytosolic protein, facilitates the intracellular translocation of long-chain fatty acids (LCFA), which are the major energy source in the heart [27]. h-FABP knock-out mice have a markedly lower (50%) LCFA uptake rate and oxidation [27,28]. In addition, h-FABP is involved in signal transduction pathways by translocating fatty acids into the nucleus and modulating peroxisome proliferator-activated receptor signalling [29,30].

Malondialdehyde, generated by the peroxidation of unsaturated fatty acids, is monitored and screened by the method of TBARS. MDA levels can change with time following an acute MI. Erythrocyte MDA levels in patients increase immediately during an acute MI, peak on day 1 post-MI, and then decline to 57% of the peak value by day 45 post-MI [31]. In the present study, the different treatments started at 60 days post-MI, so we expected TBARS levels in the ramipril group to be as low as, or lower than, those in the MI group. However, we found that the ramipril group had higher TBARS production and higher h-FABP expression in the left ventricle than the MI group. We therefore presume that ramipril improves fatty acid utilization in the heart of rabbits with MI by increasing h-FABP expression, resulting in more fatty acid transport into cells, and increasing the chance of lipid peroxidation, thereby increasing TBARS production.

4.3. Functions of other proteins modulated by ramipril
The present study showed that ramipril activated antioxidative defence not only by increasing SOD and glutathione peroxidase activity, but also by modulating the expression of other redox proteins which have previously received little attention.

Heat shock protein 27, which acts as a molecular chaperone or protease, is induced by environmental stress, including heat shock, oxidative stress, ischaemia, and reperfusion. HSP27 expression in the rat heart increases soon after coronary artery ligation, then decreases 8 weeks after the operation [32]. However, higher HSP27 expression is seen in both humans and animals with end-stage heart failure [33,34]. The higher HSP27 expression observed in the MI group therefore suggests end-stage heart failure.

Cyclophilin A, a peptidyl-prolyl isomerase, is secreted by cells for protection from oxidative stress-induced apoptosis and therefore modulates extracellular signalling pathways [35,36]. However, high CyPA levels promote the secretion of inflammatory cytokines and matrix metalloproteinases-2 and -9, thus causing endothelial cell dysfunction and contributing to cardiovascular diseases [37-39]. The lower CyPA expression in the ramipril group suggests that ramipril attenuates the inflammatory response in rabbit with MI, thus decreasing the risk of cardiovascular dysfunction.

4.4. Limitations of the study
The rabbit is an ideal subject for studying cardiovascular disease, since it is convenient for surgery and observation; however, information on rabbit protein sequences in databases is limited. To corroborate proteomic results, either gene expression or immunoblotting can be used, but mRNA levels do not fully represent protein expression due to post-translation modifications, and the lack of availability of specific antibodies to rabbit proteins excluded Western blotting. However, using a combination of enzyme activity assays, 2D-electrophoresis, and mass spectrometry, we successfully set up a rabbit model for the study of post-MI left ventricular tissue. Although information on rabbit protein sequences is currently limited, this will be less of a problem in the future, since proteomics is developing rapidly and therefore it is likely that the rabbit model will be able to be widely applied in the future.

In the protein profiles of rabbit cardiomyocytes, the expression of aconitase and calsarcin-1 tends to be increased on ramipril treatment compared with the sham group. However, since no sham plus ramipril treatment group was included in this study; it is hard to distinguish the effect of ramipril between MI- and sham-rabbits. Accordingly, the effect of ramipril in sham animals needs further elucidation.

4.5. Conclusion and clinical implication
To conclude, ramipril promotes antioxidative defence in post-MI rabbit hearts, and this could partly explain the role of ramipril in attenuation of cardiac remodelling. Of 11 identified proteins, 7 were upregulated and 4 were downregulated by ramipril in the post-MI rabbit hearts. The role of CyPA and h-FABP in LV remodelling has previously received little attention; however, this study identifies the potential functions of these proteins in the progression of heart failure, these possibilities require further study.


   Acknowledgements
 
The study was partially supported by NSC grant 95-2314-B002-004 from the National Science Council of the Republic of China. The authors are grateful to Mr. Che-Hui Chen and Miss Mai-Jun Lai for technical assistance.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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