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European Journal of Heart Failure 2008 10(8):749-757; doi:10.1016/j.ejheart.2008.06.003
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© 2008 European Society of Cardiology

Using peripheral blood mononuclear cells to determine proteome profiles in human cardiac failure

Alessia Mazzolaa,d, Riccardo Ciantic, Luca Binic, Alessandro Arminic, Ivano Eberinia, Gerarda Pompellab, Pier Leopoldo Capecchib, Mariarita Nataleb, Maria P. Abbracchioa,d and Franco Laghi-Pasinib,*

a Department of Pharmacological Sciences, University of Milan Italy
b Department of Clinical Medicine and Immunological Sciences, University of Siena Italy
c Department of Molecular Biology, University of Siena Italy
d Centro Cardiologico Monzino Milan, Italy

* Corresponding author. Department of Clinical Medicine and Immunological Sciences, Policlinico "Le Scotte", University of Siena, 53100 Siena, Italy. Tel.: +39 0577 585743/585741; fax: +39 0577 44114. E-mail address: laghi{at}unisi.it


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Background: In chronic heart failure (CHF), peripheral blood mononuclear cells (PBMC) might undergo structural and/or functional alterations as a consequence of the development and progression of the disease.

Aims: This study was aimed at: (1) assessing the proteome profile of PBMC from Controls and CHF subjects, (2) identifying differentially-expressed proteins in healthy subjects and patients, and (3) analysing the expression of these proteins in patients after heart transplantation.

Methods and results: Proteome changes were assessed in PBMC from 8 healthy and 11 end-stage CHF (6 Ischaemic Heart Failure [IHF], 5 Dilated CardioMyopathy [DCM]) subjects by gel electrophoresis, PD-Quest analysis and mass spectrometry. Eighteen proteins were differentially expressed in Controls and CHF patients. However, among CHF patients, these proteins were equally expressed in IHF and DCM subjects. Eleven proteins were found to belong to 4 functional classes (3 cytoskeletal, 4 cell-cycle progression, 2 stress response and DNA repair, 2 energetic metabolism proteins). Changes in three of the differentially-expressed proteins were also confirmed by Western blot and were reversed after heart transplantation.

Conclusion: Results demonstrate an altered protein expression profile in PBMC of CHF patients compared to Controls, thus providing a basis for further diagnostic and prognostic tests for CHF.

Key Words: Blood cells • Heart failure • Proteins

Received December 5, 2007; Revised April 28, 2008; Accepted June 3, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Chronic heart failure (CHF), as the result of both Dilated CardioMyopathy (DCM) and Ischaemic Heart Failure (IHF), remains a major cause of ill health in industrial societies [1,2]. The molecular mechanisms of CHF are not fully understood, but are likely to result from specific alterations in gene and protein expression. Advances in sequencing of the human genome and gene microarrays have made it possible to study gene expression in small tissue samples in humans, allowing thousands of gene to be analyzed simultaneously and serially [3,4], thereby opening up the possibility of undertaking rapid, global transcriptomic profiling of mRNA expression in healthy and diseased tissues. However, at least in eukaryotes, the number of functional gene products expressed by an organism far exceeds the number of genes that encode them. Moreover, there is often a poor correlation between mRNA abundance and the quantity of the corresponding functional protein [5]. Furthermore, co- and post-translational modification events result in a diversity of protein products from a single open reading frame. Finally, transcriptomic analysis does not give any information on protein maturation and degradation, which are critical processes controlling the amount of functionally active protein within a cell. Thus, the identification of the leading causes of human diseases may greatly benefit not only from the transcriptomic approach, but also from proteomics, i.e., the direct and large-scale analysis of protein expression in healthy and diseased tissues [6]. Thus, global mapping of human "proteome" in a given target tissue will allow us not only to elucidate the functional role of gene products, but also to shed light on the alterations contributing to the onset and progression of a disease. In heart diseases, proteomic investigations have so far focused on acute coronary syndromes [7], and DCM [6-10]. Using electrophoresis, as many as 100 cardiac proteins have been observed to have significantly altered expression in DCM, with the majority of these proteins being less abundant in the diseased heart [8-10]. In cardiovascular diseases, peripheral blood mononuclear cells (PBMC) may be used to examine disease-related modifications in gene and protein expression [11,12], and changes in specific receptor proteins in PBMC have been proposed as biomarkers of the haemodynamic profile in CHF [11]. Moreover, PBMC are known to modulate inflammatory responses in CHF, with relevant implications in terms of adverse prognosis [13,14]. The demonstration of specific CHF-related alterations in PBMC may help to identify useful markers for predicting response to treatment, and also for monitoring the effectiveness of both pharmacological and non-pharmacological interventions and, possibly, disease progression or regression. On this basis, the present work was undertaken to assess proteome changes in PBMC from healthy control subjects and from patients with end-stage CHF awaiting heart transplantation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Subject selection
Eleven consecutive patients with Dilated CardioMyopathy and end-stage CHF (6 Ischaemic Heart Failure [IHF], 5 Idiopathic Dilated CardioMyopathy [DCM]), admitted to our Institution for evaluation for cardiac transplantation, were enrolled in the study. All patients had a left ventricular ejection fraction <40%. Five of these 11 end-stage CHF patients were also studied following heart transplantation. Blood sampling was performed 3-6 months after transplantation when haemodynamic parameters were completely normalized. Eight healthy volunteers acted as Controls.

The study was conducted in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the local ethics committee of the Siena University. All patients gave their written informed consent.

2.2. Isolation of blood peripheral cells, differential blood cell count, CRP levels
Venous blood was collected and aliquoted for PBMC isolation by density gradient centrifugation over Ficoll-Histopaque (Sigma Chemical Co., St. Louis, MO, USA), differential blood cell count, and measurement of CRP levels (Syncron Chemical System Lx 20, Beckman, Fullerton, CA, USA).

2.3. Two-dimensional gel electrophoresis
A conventional 2-D lysis buffer, containing 8 M urea, 4% w/v CHAPS, and 1% w/v DTE (Sigma Aldrich, Italy) was used. 2-D was performed as described previously [10]. The IEF was run on a non-linear wide range immobilized pH gradient IPG (pH 3-10, 18 cm long IPG strips) and achieved using the EttanTM IPGphorTM system. Analytical-run IPG strips were rehydrated with 60 µg of protein in 350 µl of lysis buffer and 0.2% v/v carrier ampholyte for 1 h at 0 V and for 8 h at 30 V, and then focused at 200 V for 1 h, from 300 V to 3500 V in 30 min, 3500 V for 3 h, from 3500 V to 8000 V in 30 min and 8000 V until a total of 80,000Vh was reached. MS-preparative runs were obtained using the MultiphorTM II electrophoresis system. Gel strips were rehydrated with 1000 µg of sample in 600 µl of lysis buffer and 2% v/v carrier ampholyte for 12 h at room temperature. The following IEF voltage steps were applied: 200 V for 12 h, 300 V for 30 min, 600 V for 30 min, 1100 V for 30 min, 1700 V for 30 min, 2500 V for 30 min, 3500 V for 30 min, 5000 V for 20 h. After focusing, analytical and preparative gel strip equilibration was performed in 6 M urea, 2% w/v SDS, 2% w/v DTE (Sigma Aldrich, Italy), 30% v/v glycerol and 0.05 M Tris-HCl pH 6.8 for 12 min, and for further 5 min in 6 M urea, 2% w/v SDS, 2.5% w/v iodoacetamide, 30% v/v glycerol, 0.05 M Tris-HCl, pH 6.8 bromophenol blue (unless indicated differently, all reagents were from Amersham Biosciences, Sweden). The second dimension was run on 9-16% polyacrylamide linear gradient gels (18 cmx20 cmx1.5 mm). SDS-PAGE was carried out at 40 mA for gel constant current, until the dye front reached the bottom of the gel. Analytical gels were stained with ammoniacal silver nitrate as previously described [15], whereas the MS-preparative gels were stained according to the protocol compatible with MS [16]. Electrophoretogram images were obtained with a computing densitometer (Molecular Dynamics 300S; Sunnyvale, CA, USA), and image analysis was carried out with PD-Quest 2-D software version 7.3.1.07 (Bio-Rad, Hercules, CA). For quantitative analysis, for each spot, the volume relative to the sum of volumes of all spots in each gel was calculated, in order to correct for silver staining-related variability.

2.4. Protein identification by mass spectrometry
Protein identification was carried out as previously described [17]. After visualization with silver staining, electrophoretic spots were excised, destained [18] and then dehydrated with acetonitrile (Baker). Proteins were rehydrated in trypsin solution (Sigma Aldrich, Italy) and in-gel protein digestion was performed by an overnight incubation at 37 °C. 0.75 µl of each supernatant was directly spotted onto the target disk and air-dried. Then 0.75 µl of matrix solution (a saturated solution of alpha-cyano-4-hydroxycynnamic acid (Laser Bio Labs) in 50% v/v acetonitrile (Baker) 0.5% v/v TFA (Merck)) was added to the dried samples and again allowed to dry. Spectra were obtained using an Ettan MALDI-TOF/Pro mass spectrometer (Matrix-assisted-laser-desorption-ionization) (Amersham Biosciences). Peptide mass fingerprinting database searching was carried out in NCBInr and Swiss-Prot database using ProFound (http://prowl.rockefeller.edu/). Protein identification was performed using MASCOT (Matrix Science, London, UK; http://www.matrixscience.com) to search the human protein in the NCBInr and Swiss-Prot databases. The functional proteomic strategy used in the study is illustrated in Fig. 1.


Figure 01
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  		Fig. 1 Flow diagram showing the functional proteomic strategy used in the study. PBMC: Peripheral blood mononuclear cells, IEF: isoelectric focusing, SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

 
2.5. Western blotting analysis
To substantiate the proteomic findings with a conventional analysis, we performed Western blotting of 3 protein candidates. PBMC were washed twice in ice-cold PBS and lysed on ice with lysis solution (1% Triton X-100, 150 mM NaCl, 20 mM Tris pH 8 and protease inhibitors). The protein concentration of the samples was determined by the Bio-Rad protein assay (Bio-Rad Quick Start). Cell lysates were boiled at 100 °C for 5 min in SDS gel Loading Buffer and later separated on a Bis-Tris gels 4-12% (Invitrogen). After 1 h, the proteins were electrotransfered to nitrocellulose membranes (Pierce) for 2 h. The filters were blocked for 1 h at room temperature in 5% milk dissolved in Tris-Buffered Saline Tween (TBS-T), and then incubated with specific antibodies against HSP27 (Calbiochem, dilution 1:500), TCP1{varepsilon} (Abcam, dilution 1:250) and CAPZA1 (Abcam, dilution 1:2000) overnight at 4 °C. After 2 washes of 20 min each in TBS-T, the membranes were incubated with specific horseradish peroxidase (HRP)-conjugated second antibodies for 1 h at room temperature and washed twice in TBS-T. Colorimetric detection was achieved by using 4-chloro-1-naphthol as substrate for horseradish peroxidase (Opti-4CN, Bio-Rad Laboratories). β-actin was also determined in each filter in order to normalize slight variations in protein loading.

2.6. Statistical analysis
Differences between spots were computed for each protein using SPSS 1.1 software for Macintosh. Data are expressed as mean±SD. A p value <0.05 was considered statistically significant (t-test and Student-Neuman-Keuls's test).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
Demography and clinical characteristics of the 11 end-stage CHF patients are shown in Table 1. The Controls comprised 8 healthy volunteers (5 males, 3 females; mean age 51.81±7.12 years). CRP and differential blood cell counts were in the normal range in both CHF groups (Table 1).


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  		Table 1 Demography and clinical characteristics of the 11 end-stage CHF patients

 
3.1. Protein expression profile
Individual gels from the 8 Control and 11 CHF patients were analyzed and corresponding images were obtained from the software. Matching of proteomic maps was checked manually, and showed several differences between Control and CHF subjects (Fig. 2A and B, respectively). Of the quantitative variations, 2 spots displayed an increased abundance in CHF patients compared to Controls (indicated as spots N. 1 and 6 in Fig. 2), whereas 4 spots showed a signal intensity decrease (indicated as N. 2, 3, 4, and 5 in Fig. 2). Qualitative differences included proteins found in the Control but not in the CHF subjects (indicated as N. 7-12) and proteins found in PBMC from CHF patients only (indicated as N. 13-18). Among the CHF patients, proteins were equally expressed in the IHF and DCM subjects. Of the 18 differentially-expressed proteins, 11 spots were clearly identified in preparative gels, and were thus subjected to unambiguous identification by MALDI-TOF MS. The characteristics of all identified proteins are reported in Table 2.


Figure 02
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  		Fig. 2 Protein expression profile in PBMC from Control and CHF subjects by 2-D electrophoresis. Representative proteomic maps from one single Control subject (panel A) and one CHF patient (panel B) are shown. Numbers from 1 to 6 ({Delta}) mark the protein spots differentially expressed in PBMC from Control subjects and CHF patients. Numbers from 7 to 12 ({square}) mark the protein spots which were found in the PBMC from Control subjects but not in CHF patients, whereas numbers from 13 to 18 ({circ}) mark the protein spots which were only found in the PBMC from CHF patients.

 


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  		Table 2 Characteristics of all 11 identified proteins

 
3.2. Identification of differentially-expressed proteins
Five of the six differentially-expressed proteins were identified by MALDI-TOF MS. Protein N. 1, which was the only one to be significantly increased in CHF subjects (Fig. 3) was identified as the translationally controlled tumor protein (TCTP or p23), a cytoplasmic protein which dynamically interacts with tubules during the cell cycle [19]. The second spot which appeared increased in CHF versus Controls did not reach statistical significance. The other 4 differentially-expressed proteins which were down-regulated in CHF patients with respect to Controls (Fig. 3), were identified as follows. Protein N. 2 was the nucleoside diphosphate kinase A (NDK A or nm23-H1), a protein involved in energetic metabolism and ATP synthesis. Protein N. 3 was identified as the 14-3-3 protein zeta/delta (KCIP-1, protein kinase C inhibitor protein-1), a protein involved in cell-cycle progression which acts as a scaffold protein and a molecular chaperonine in regulating the activity of Raf, Cdc25 e BAD [20]. The "sigma" isoform of this protein induces cell-cycle block in the G2 phase. Protein N. 4 was identified as CAP-Z (F-actin capping protein alpha 2-subunit), a cytoskeletal protein which facilitates actin polymerization. Protein N. 5 was the T-complex protein 1 (epsilon subunit: TCP-1-epsilon), a molecular chaperonine-like protein involved in actin and tubulin folding and in cell-cycle progression. Expression of the different subunits of this protein has been related to cell growth. Interestingly, in CHF patients, besides the reported down-regulation of the epsilon subunit (Figs. 3, 6Go), expression of the beta subunit of this protein was completely abolished (see also below and Fig. 4).


Figure 03
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  		Fig. 3 Differentially-expressed proteins in PBMC from Control (C) and CHF subjects. A, Enlargements of areas of representative 2-D gel images of the five identified proteins in one Control and one CHF subject/protein. B, Histograms showing the semi-quantitative analysis of spots. For each protein, data are expressed as mean±SD of relative volume from 8 Control subjects and 11 CHF patients. N. 1: Translationally controlled tumor protein; N. 2: Nucleoside diphosphate kinase A; N. 3: 14-3-3 protein zeta/delta; N. 4: F-actin capping protein alpha 2-subunit; N. 5: T-complex protein 1-epsilon. *p<0.05 with respect to Control subjects, t-test for Equality of Means.

 


Figure 04
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  		Fig. 4 Proteins whose expression is abolished in CHF patients. A, Enlargements of areas of representative 2-D electrophoretic maps showing the two identified proteins (N. 7: Heat-shock protein 27, and N. 9: T-complex protein 1-beta) found in the PBMC from Control subjects but not in those from CHF patients. 2-D images from one single Control and one CHF subject/protein are shown. B, Semi-quantitative analysis of relative expression of protein N. 7 and 9 in the 8 analyzed Control subjects. For each spot, data are expressed as mean+SD of relative volume.

 
3.3. Proteins whose expression is abolished in CHF patients
Two proteins were present in PBMC from Controls but not from CHF patients (Fig. 4). Protein N. 7 was identified as HSP27 (heat-shock protein 27), a protein involved in stress response and DNA repair, which associates with tubulin and microtubules [21]. Protein N. 9 was identified as T-complex protein 1-beta subunit, which belongs to the same family of cytoskeletal proteins already found to be altered in CHF subjects (see Fig. 3). In particular, mutations of the beta subunit of this protein have been associated with cytoskeletal disorganization and aberrant chromosomal segregation [22].

3.4. Proteins induced in CHF patients
Four proteins which were not found in PBMC from Controls were expressed at significant levels in PBMC from CHF patients (Fig. 5). Protein N. 13 was identified as the Elongation factor 1-delta (EF-1-delta), a radiation-inducible gene that has been hypothesized to participate in the G2-M cell-cycle checkpoint [23]. Protein N. 15 was identified as Talin 1, a protein which binds to vinculin and integrins and is likely involved in connections of major cytoskeletal structures to the plasma membrane [24]. Protein N. 16 was identified as NADH-ubiquinone oxidoreductase, a multiprotein complex located in the inner mitochondrial membrane involved in the transport of electrons from NADH to ubiquinone [25]. Protein N. 18 was identified as PRP19/PSO4, a ubiquitary protein involved in stress response and DNA repair, which is induced 15 to 30-fold in cells by gamma radiation and chemical mutagens [26]. Loss of hPRP19/PSO4 expression induced by siRNA results in accumulation of double-strand breaks, apoptosis, and decreased cell survival after DNA damage [26].


Figure 05
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  		Fig. 5 Proteins induced in CHF patients. A, Enlargements of areas of representative 2-D electrophoretic maps showing the four identified proteins (N. 13: Elongation factor 1-delta; N. 15: Talin 1; N. 16: NADH-ubiquinone oxidoreductase; N. 18: PRP19/PSO4) exclusively found in the PBMC from CHF patients but not in those from Control subjects. 2-D images from one single Control and one CHF subject/protein are shown. B, Semi-quantitative analysis of relative expression of protein N. 13, 14, 16 and 18 in the 11 analyzed CHF patients. For each protein, data are expressed as mean±SD of relative volume.

 
3.5. Western immunoblotting analysis of differentially-expressed proteins
Two of the six differentially-expressed proteins and one of two proteins absent in CHF patients, each representative of one functional class and chosen on the basis of specific antibody availability, were also detected by immunoblotting. As expected, immunoblotting analysis showed that the expression of HSP27 (a stress response and DNA repair protein) was abolished, whereas CAP-Z and TCP-1-epsilon, cytoskeletal, and cell-cycle-progression proteins, respectively, were down-regulated in CHF patients with respect to Controls (Fig. 6). Very interestingly, the expression of these proteins was restored in all patients after heart transplantation, together with the normalization of haemodynamic conditions.


Figure 06
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  		Fig. 6 Western immunoblotting analysis of differentially-expressed proteins from Controls, CHF and transplanted patients. (A) Heat-shock protein 27, (B) F-actin capping protein alpha 2-subunit and (C) T-complex protein 1-epsilon were detected by immunoblotting analysis after electrophoretic separation by SDS-PAGE of post-nuclear supernatants in PBMC from Control subjects, CHF patients and patients after heart transplantation. Lane 1 is representative of 5 Control subjects, lane 2 of 5 CHF patients and lane 3 of 5 patients after heart transplantation. β-actin was measured in parallel in order to monitor equal protein loading on gels. Statistical evaluation was performed with the Student-Neuman-Keuls's test for multiple comparisons. ***= p<0.001; n=5.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
This study was aimed at assessing proteomic changes in PBMC from 11 end-stage CHF patients in comparison with 8 healthy subjects. Of the proteins separated by 2-D electrophoresis from PBMC in the two groups of subjects, the expression of some proteins was found to be specifically and significantly altered in CHF patients with respect to healthy subjects. Differences in protein expression levels between the two groups of subjects were quite obvious, reproducible, and consistently found in all the subjects of each group; moreover, the protein expression profile was the same in those patients with Dilated CardioMyopathy (DCM) and those with Ischaemic Heart Failure (IHF). The relative abundance of the differentially-expressed proteins was sufficient to enable us to unambiguously identify eleven of them by MALDI-TOF MS. As summarized in Table 2, these proteins belong to 4 main functional classes: (i) cytoskeletal proteins, (ii) cell-cycle-progression proteins, (iii) stress response and DNA repair proteins, and (iv) energetic metabolism proteins. Concerning class (i), changes of cytoskeletal and myofibrillary proteins are consistent with the contractile dysfunction and defects in excitation-contraction coupling in CHF. In our study, we also found that the expression of the beta subunit of T-complex protein 1, a chaperonine-like protein involved in actin and tubulin folding, was completely abolished in CHF patients. Interestingly, mutations of this protein have been shown to be associated with cytoskeletal disorganization and increased sensitivity to microtubule destabilizing agents [22]. Conversely, Talin 1, a membrane cytoskeletal adaptor protein involved in the assembly of actin filaments, which is not normally expressed in PBMC from healthy subjects, was consistently induced in CHF patients. In contrast to the data available from proteomic studies in human DCM, PBMC of CHF patients also showed alterations in the four proteins involved in cell-cycle progression (class (ii)). In particular, while 14-3-3-protein zeta/delta and T-complex protein 1-epsilon were down-regulated, translationally controlled tumor protein was increased, and elongation factor 1-delta was induced in PBMC of CHF patients. Although formally included in protein functional class (ii), some of these proteins may be also considered stress response proteins and changes in their expression may reflect a general unspecific response of circulating blood cells to disease. Among the proteins in class (iii) (involved in stress response and DNA repair), HSP27, a protein involved in actin organization, was undetectable or markedly down-regulated in PBMC of CHF subjects, as confirmed by immunoblotting analysis, despite its presence in Controls. This result is highly consistent with previous data in human myocardial tissue. In fact, in samples from failing explanted hearts from patients with DCM or Ischaemic Heart Failure (IHF), 2-D electrophoretic separation of proteins showed a marked decrease of 28 kDa HSP27-immunostained spots [27]. On the other hand, in the failing hearts, HSP27-immunostained spots with a lower molecular mass increased in intensity, suggesting proteolysis and that some form of HSP27 degradation occurs during heart failure. HSP27 expression is reported to be elevated in PBMC and polymorphonuclear leukocytes from patients with an inflammatory condition together with a concomitant increase in CRP levels. [28,29]. In our study, HSP27 expression in PBMC from CHF patients was significantly reduced with respect to Controls, whereas CRP was in the normal range in all the study groups, thereby reasonably ruling out the possibility that a different inflammatory pattern in CHF and Control subjects might influence the different expression in HSP27 in the two groups. The expression of HSP70 in the PBMC of CHF patients was similar to Control levels following heart transplantation, in parallel with haemodynamic normalization. Thus, at least for HSP27, changes in peripheral blood circulating cells seem to mirror changes occurring in the disease target organ. This finding is particularly relevant for the identification of new prognostic/diagnostic peripheral markers of disease.

In PBMC from CHF patients we also detected PRP19/PSO4, another stress response protein which is not normally expressed in PBMC, but which has been shown to be induced following DNA damage in other cell systems [26]. CHF induced expression of this protein may reflect an attempt to increase cell survival, since in different tumour cell lines (HL60, HeLa, Jurkat, Molt4) abolition of this protein by RNA interference was associated with impaired ability to survive DNA damage [26]. In terms of class (iv) proteins (energetic metabolism proteins), PBMC of CHF patients showed induction of NADH-ubiquinone oxidoreductase, and a down-regulation of Nucleoside diphosphate kinase A, a protein with a major role in the synthesis of nucleotide triphosphates other than ATP. Interestingly, both these enzymes were also found to undergo changes in an "in vitro" model of hypertrophic cardiomyopathy [30]. However, in the latter study, opposite changes in the proteins were detected. We speculate that this qualitative difference may be due to the fact that the "in vitro" model likely reproduces an earlier stage of the disease. To confirm the validity of the strategy and techniques utilized in this study, CHF-related changes were also confirmed by Western blot analysis for 3 of the 18 differentially-expressed proteins. These 3 proteins (one cytoskeletal protein, one cell-cycle-progression protein, and one stress response and DNA repair protein) were either down-regulated or absent in CHF patients with respect to Controls by MALDI-TOF-MS analysis, and similar changes were also found by Western blot analysis. Most importantly, Western blotting analysis of proteins from heart transplanted patients also suggests that recovery of the haemodynamic profile after transplantation is associated with a normalization in the expression of these 3 reference proteins. As regards the potential effect of current therapy on changes detected in CHF patients, protein expression profile was identical in all patients despite some differences in their therapeutic schedule. This finding strongly suggests a main role for haemodynamic alterations in determining the detected protein profile.

In conclusion, proteomic profiling of PBMC has enabled the identification of several proteins which are differentially expressed in patients with CHF. The main study limitation is that there is no comparison with differentially-expressed myocardial tissue proteins from biopsies. However, although it cannot be assumed that in all cases the changes observed actually mirror the alterations occurring in the disease target organ, it is likely that protein expression in PBMC may be an expression of the generalized alterations associated with cardiovascular disease, thereby providing new important information for the development of novel prognostic/diagnostic biomarkers.


   Acknowledgement
 
This work was sponsored by the Italian Ministry for University and Research, Prot. #2004062711.


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

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