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Cardiovascular Research 2005 67(4):655-666; doi:10.1016/j.cardiores.2005.04.016
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Copyright © 2005, European Society of Cardiology

Matrix metalloproteinases and atrial remodeling in patients with mitral valve disease and atrial fibrillation

Wim Annéa, Rik Willemsa, Tania Roskamsb, Paul Sergeantc, Paul Herijgersc, Patricia Holemansa, Hugo Ectora and Hein Heidbüchela,*

aDepartment of Cardiology, University Hospital Gasthuisberg, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium
bDepartment of Pathology, University Hospital Gasthuisberg, University of Leuven, Leuven, Belgium
cDepartment of Cardiac Surgery, University Hospital Gasthuisberg, University of Leuven, Leuven, Belgium

* Corresponding author. Tel.: +32 16 34 42 48; fax: +32 16 34 42 40. Email address: Hein.Heidbuchel{at}uz.kuleuven.ac.be

Received 26 November 2004; revised 11 April 2005; accepted 15 April 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Limitations
 6. Conclusions
 References
 
Background: Atrial fibrillation (AF) is associated with extracellular matrix remodeling involving atrial fibrosis and atrial dilatation. Angiotensin II mediated pathways and matrix metalloproteinases (MMPs) have been implicated in these processes. Our aim was to study atrial structural remodeling and the expression of the angiotensin receptor subtypes and MMPs and their inhibitors (TIMPs) in patients with mitral valve disease with and without AF.

Methods and results: Biopsies from right and left atrial appendages (RA and LA) were taken from patients undergoing CABG (n = 9, all in sinus rhythm (SR)) or mitral valve surgery (MVS; n = 19; 9 with permanent AF and 10 in SR). Patients with MVS and AF had significantly larger atria (versus MVS and SR: p = 0.02; versus CABG: p<0.01). The MVS patients had significantly more fibrosis than the control CABG group. Fibrosis was increased in both the AF and SR MVS groups in the LA, but only in the MVS–AF group in the RA. These AF patients had significantly more tricuspid regurgitation than SR patients. MMP-1 was down-regulated in LA of MVS patients (p = 0.02) independent of the underlying rhythm (SR or AF; p = 0.95). In RA biopsies, MMP-1 was down-regulated only in the MVS and AF group. MMP-9 was down-regulated in the MVS patients compared to CABG both in the RA and LA, and without a difference between the SR and AF groups. Protein expression of AT-1, AT-2, MMP-2, TIMP-1, -2 and -4, TNF-{alpha}, and TNF-{alpha}-converting enzyme did not differ significantly between the 3 groups.

Conclusions: Concordant changes between MMP-expression and fibrosis during mitral valve disease, both in LA and RA, suggest involvement of MMPs in structural atrial remodeling. AF itself did not contribute to altered fibrosis or MMP-expression in the LA. The association between AF and RA changes may be precipitated by greater hemodynamic load due to tricuspid regurgitation in these patients.

KEYWORDS Arrhythmia; Valve disease; Fibrosis; Matrix metalloproteinases; Remodeling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Limitations
 6. Conclusions
 References
 
Atrial fibrillation (AF) is the most common cardiac arrhythmia. Clinical risk factors for the development of AF are coronary artery disease, congestive heart failure, valvular heart disease and older age [1]. These risk factors cause structural atrial changes, including dilatation and fibrosis [2–5]. Moreover AF by itself induces electrical and structural changes in the atrium favoring its persistence [6,7]. On an electrical level the main feature is shortening of the refractory period, caused by changes in the calcium currents [6,8]. On a structural level cellular hypertrophy and degeneration have been described [9].

The renin–angiotensin system has been shown to be involved in this structural remodeling due to AF. There is an increased expression of the angiotensin-converting enzyme (ACE) and angiotensin II dependent signal transduction pathways are activated in atrial tissue of patients with AF [10]. Also in animal models of AF increased plasma levels of angiotensin II have been demonstrated [11]. The effect of angiotensin II is mainly mediated by 2 receptor subtypes. Stimulation of AT-1 induces myocardial hypertrophy and the accumulation of extracellular matrix proteins whereas AT-2 stimulation inhibits proliferative effects [12,13]. It has been reported that the latter is up-regulated in disease states like myocardial infarction and heart failure and may play a role in counterbalancing the effects of AT-1 activation [14].

The renin–angiotensin system is likely not the only system involved in atrial fibrosis and atrial dilatation. Homeostasis of the extracellular matrix is a balance between synthesis and degradation. By breaking down the extracellular matrix, matrix metalloproteinases (MMP) contribute to matrix turnover, to dilatation and to structural remodeling in general [15]. MMP-expression is tightly regulated at different levels. Different cytokines affect their transcription. Most of the MMPs are secreted as inactive zymogens that require subsequent activation to the active protease [15]. Finally their activity is blocked by endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMP) [15]. TNF-{alpha} is one of the cytokines that can induce transcription of MMPs [15]. It is also capable of activating MMPs through activation of different proteases in a paracrine manner [16]. TNF-{alpha} is processed into a membrane-bound form and a soluble form by the TNF-{alpha}-converting enzyme (TACE) [16].

The aim of this study was to assess atrial structural remodeling and the expression of the angiotensin receptor subtypes (AT-1 and AT-2), MMPs and their inhibitors (TIMPs), TNF-{alpha} and TACE in patients with mitral valve disease with or without AF. Patients operated for coronary artery disease without AF served as controls. This enabled us to differentiate the effect of the underlying substrate–mitral valve disease–from a direct effect of AF.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Limitations
 6. Conclusions
 References
 
2.1. Patients
Biopsies from the right and left atrial appendage were taken from patients undergoing coronary artery bypass surgery (CABG; n = 9, all without a history of AF) or mitral valve surgery (MVS; n = 19; 9 with permanent AF (MVS and AF) and 10 without AF history (MVS and SR)). Patients with endocarditis or acute mitral valve regurgitation were excluded. The majority of patients suffered from mitral valve regurgitation (n = 15; 8 in sinus rhythm, 7 in AF). One patient (in SR) was operated for mitral valve stenosis and 3 patients for mixed mitral valve disease (1 in sinus rhythm, 2 in AF). Before surgery all patients gave informed consent and underwent echocardiographic evaluation for determination of left and right atrial size, left ventricular end-diastolic volume and ejection fraction. Left and right atrial diameters were assessed by measuring the long axis in an apical view and the left ventricular diameters in a parasternal view. Mitral and tricuspid valve regurgitation was scored on a scale from 0 until 4. Part of the tissue samples was prepared for pathology (cfr. infra); another part was immediately frozen in liquid nitrogen and stored at –80 °C before western blotting and zymography were performed. This study was approved by the local Ethical Committee of University Hospital Gasthuisberg, Leuven, Belgium and the investigation conforms with the principles outlined in the Declaration of Helsinki.

2.2. Western blotting, zymography and ELISA
Atrial tissue was homogenized in a buffer containing imidazole 10 mmol/l, sucrose 300 mmol/l, dithiotreitol 1 mmol/l, sodium metabisulfite 1 mmol/l and a protease inhibitor cocktail ("Complete-Mini" Roche Molecular biochemicals, Indianapolis, USA). Equal amounts of homogenates were separated on 4–12% Bis–Tris gradient gels (NuPageTM electrophoresis system, Invitrogen, Carlsbad, USA) and blotted onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated overnight with monoclonal (TIMP-1 (Oncogene Research Products, Boston, USA); TIMP-2 (Oncogene Research Products, Boston, USA)) or polyclonal antibodies (AT-1 (Santa-Cruz Biotechnology, Santa Cruz, USA), AT-2 (Santa-Cruz Biotechnology, Santa Cruz, USA), MMP-1 (Calbiochem, San Diego, USA); TIMP-1 (Abcam, Cambridge, UK), TIMP-2 (Abcam, Cambridge, UK), TNF-{alpha} (kindly provided by Prof. J. Anné, Laboratory of Bacteriology, KULeuven, Leuven, Belgium), TACE (R and D Systems, Minneapolis, USA), TIMP-4 (Affinity Bioreagents, Golden, USA)). Detection was performed with alkaline phosphatase-labeled secondary antibodies, using Vistra ECF system (Amersham, Buckinghamshire, UK). After scanning the blots, the number of pixels of each positively stained band (antigen–antibody interaction) was calculated; subsequently they were normalized to a group of 4 samples, which were put on every blot and used as an intern control, and expressed as a percentage of the mean of the whole of the left atrial CABG group.

MMP-2 and MMP-9 activity was detected by zymography. Equal amounts of proteins were separated on a 10% gelatin gel (Invitrogen, Carlsbad, USA) with electrophoresis. Afterwards the gels were washed and incubated for 12 h in an MMP-substrate buffer at 37 °C. After incubation the gels were stained with Coomasie blue and destained in alcohol. The zymograms were digitized and the size-fractioned banding pattern, which indicated MMP-2 and MMP-9 proteolytic activity, was determined by quantified image analysis.

2.3. Histology and immunohistochemical staining
All samples were fixed in 6% buffered formalin, embedded in paraffin and stained with hematoxylin and eosine for routine histological examination. In each section the cross-sectional cell size was measured in an average of 40 different cells using the Leica Quantimet 600 image analysis system (Leica, Heidelberg, Germany).

Sections were stained with Sirius Red for fibrosis quantification. All sections were graded on a scale ranging from 1 (no fibrosis) to 4 (severe fibrosis). The scoring was based on the thickness of fibrotic septa, the degree of pericellular fibrosis, the amount of perivascular fibrosis, and the extent of confluent zones of fibrosis [17].

Immunohistochemistry was performed on 4 µm cryostat sections. Cryostat sections were dried overnight, subsequently fixed in acetone for 10 min, and finally washed in PBS, immediately before use. All samples were incubated with the primary antibody (MMP-1, TIMP-1, TIMP-2) for 30 min at room temperature. Secondary antibodies consisted out of peroxidase-labeled rabbit anti-mouse (monoclonal) or anti-rabbit (polyclonal) IgG. All incubation steps were followed by 3 times washing in PBS for 5 min. The reaction product was developed with the use of 3-amino-9-ethylcarbazole and H2O2 (0.01%) and finally the sections were counterstained with haematoxylin.

Sections stained with the different antibodies were quantified by evaluation of 10 randomly chosen fields. For TIMP-1 the different groups were compared by counting the number of different interstitial cells that stained positively. For TIMP-2 both positively stained interstitial cells and myocytes were counted.

2.4. Statistical analysis
Summary values are given as mean ± standard error of the mean (sem). Statistical significance was defined as a p-value<0.05.

Comparisons between groups for categorical variables were based on {pi}2-test, comparisons between groups for continuous variables were made with one-way analysis of variance, and in the case of significant difference this was further analyzed with Tukey–Kramer test.

Results of Western blotting, zymography and immunohistochemistry were compared between the 3 groups of patients using a Kruskal Wallis test. In the event the p-value<0.05 the different groups were mutually compared using a Mann–Whitney test. Correlations between the expression of the MMPs and the degree of fibrosis were done with the non-parametric Spearman Rank Correlation test.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Limitations
 6. Conclusions
 References
 
3.1. Patient characteristics
Patient characteristics are summarized in Table 1. There was no statistical difference in age (mean 68 ± 2 years) and gender (61% male) between the 3 groups. Mitral valve disease was known for a mean of 4.3 ± 0.9 years whereas AF was known for a mean of 3.9 ± 1.3 years in the group of MVS and AF patients.


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  		Table 1 Clinical characteristics of patients

 
The majority of patients of the CABG group belonged to New York Heart Association functional class (NYHA) 1, versus NYHA class 2 for patients from the MVS and SR group, and NYHA class 3 for patients from the MVS and AF group (Table 1; p<0.01). Five patients had a history of congestive heart failure (2 in SR, 3 in AF).

Four patients who underwent mitral valve surgery received simultaneous aortic valve replacement (2 in SR, 2 in AF), and 7 patients with mitral valve surgery were operated on the tricuspid valve (1 in SR, 6 in AF). Patients were operated on the mitral valve due to myxomatous degeneration (6 in SR, 1 in AF), calcified valves (1 in SR, 4 in AF), rheumatic heart disease (1 in SR, 1 in AF), ischemia dependent mitral valve disease (1 in SR, 1 in AF), or other reasons (1 in SR, 2 in AF).

The majority of patients were treated with beta-blockers (57%), diuretics (57%) and half of them also with ACE-inhibitors (Table 1). There was no statistical difference in the use of these drugs between the 3 groups. As could be expected, nitrates were predominantly used by patients with coronary artery disease (55%), and digoxin in the MVS and AF group (67%). The mean heart rate of the patients in AF the day before surgery was 89 ± 5 bpm.

Left and right atrial diameters were significantly greater in patients from the MVS and AF group versus the other groups (p<0.01). Patients with MVS and AF had also a significantly lower ejection fraction (Table 1). The mean mitral valve regurgitation was 3.1 ± 1.2 in the group of patients who were operated on their mitral valve. MVS and AF patients had significant more tricuspid regurgitation (2.7 ± 1.4) than the other 2 groups (CABG: 0.5 ± 0.5; MVS and SR: 1.4 ± 0.5; p<0.01).

3.2. Pathology
Myoyctes of patients with MVS and AF were significantly larger compared to those of patients with MVS and SR (cross-sectional diameter 24 ± 5 versus 19 ± 2 µm; p<0.01) (Fig. 1) and patients with CABG (17 ± 2 µm; p<0.01). There was no significant difference between left and right atrial cell size.


Figure 1
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  		Fig. 1 A, B, C: Representative Sirius Red stained paraffin sections from the CABG group (A), MVS and SR group (B), MVS and AF group (C); magnification x 10. In the CABG group (A) almost no interstitial fibrosis was present. Patients with MVS and SR (B) showed thicker fibrotic septa delineating groups of myocytes (arrow). In the MVS and AF group (C), fibrosis was even more pronounced with individual myocytes separated by fibrotic tissue (arrow). D: Sum of mean fibrosis score of the left and right atria (mean ± sem). E: Cross-sectional myocyte diameter in the 3 groups (mean ± sem). F, G, H: Representative hematoxyline–eosin stained paraffin sections from the CABG group (F), MVS and SR group (G), MVS and AF group (H); magnification x 20. Cells of patients belonging to the MVS and AF group were larger, had enlarged nuclei (arrow) and a prominent nucleolus, and showed more signs of degeneration such as vacuolization of the cytoplasm.

 
Patients undergoing mitral valve surgery had significantly more left atrial fibrosis than patients undergoing CABG (p<0.01). The degree of fibrosis was not significantly different between patients in SR or in AF in the left atrium. However, in the right atrium, an increased degree of fibrosis was seen only in AF patients (p<0.01). There was a positive correlation between the degree of atrio-ventricular insufficiency (graded 0–4) and the degree of fibrosis (Spearman's Rho: 0.36; p = 0.02) The overall degree of fibrosis (left and right atrium combined) was greater in patients from the MVS and AF group (p<0.01).

Patients with MVS had thicker fibrotic septa forming isolated islands of myocytes, more pericellular fibrosis separating individual myocytes, more perivascular fibrosis and more and greater confluent patches of fibrosis (Fig. 1A, B, C). Especially in the group of MVS and AF, individual myocytes were separated by fibrotic tissue.

Myocytes from MVS and AF patients showed pronounced degenerative characteristics (depletion of contractile elements often leading to a total vacuolization of the cell, markedly enlarged nuclei with prominent nucleoli), whereas patients with CABG had significantly less degenerative signs but more signs of ischemia (shrinking of cells with hypereosinophilia of the cytoplasm, picnotic nuclei). Patients with MVS and SR formed a stage between the 2 other groups. (Fig. 1D, E, F). Aschoff bodies were not noted in any biopsy, and there was no difference in the number of sections of each group showing slight signs of inflammation.

3.3. MMP-expression
Western blotting for MMP-1 showed bands at 53 kDa (pro-MMP-1), at 57 kDa (the glycolysated form of pro-MMP-1) and around 70 kDa (complex of active MMP-1 with TIMP-1) (Fig. 2). Free active MMP-1 could not be detected in our samples. The expression pattern of MMP-1 was different in the left and right atria and was concordant with the degree of atrial fibrosis. Left atrial biopsies of patients with mitral valve disease had a significantly lower total amount of MMP-1 compared to patients undergoing CABG (p<0.01; Table 2) while no difference was present whether mitral valve surgery patients were in SR or in AF (p = 0.95). On the other hand, in right atrial biopsies there was no difference between patients from the CABG group and the MVS and SR group, but there was a trend to a decreased expression of MMP-1 in biopsies of patients with AF (p = 0.07). An inverse relation between the extent of MMP-1 expression and the degree of fibrosis was present (p = 0.04, Spearman's Rho=–0,41) (Fig. 2). MMP-1 expression also correlated inversely with atrial diameter (p = 0.01, Spearman's Rho=–0.38). Immunohistochemistry revealed a clear interstitial staining: MMP-1 was distributed throughout the whole interstitial region (Fig. 2).


Figure 2
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  		Fig. 2 A: Representative Western blot of MMP-1. B: Bar graph depicting MMP-1 expression. The MMP-1 expression in the left atria was decreased in both groups of patients with MVS compared to the control CABG group while no difference was present between patients from the MVS and SR and MVS and AF groups. C: There was an inverse relation between degree of fibrosis and the MMP-1 expression. (Spearman's Rho: –0.41). D: Immunohistochemistry for MMP-1. A clear interstitial staining pattern could be observed (arrow); magnification x 10.

 

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  		Table 2 Quantification of the atrial fibrosis, AT-receptors, MMPs, TIMPs, TNF-{alpha} and TACE

 
Gelatin zymography showed both the pro-forms of MMP-9 (92 kDa) and MMP-2 (72 kDa) as well as a band around 125 kDa (complex of pro-MMP-9 with a microglobuline) (Fig. 3). Active forms of MMP-9 and MMP-2 could hardly be seen. Scanning densitometry revealed a significant decrease in MMP-9 in both MVS groups compared to the CABG group. There was no difference between patients with MVS who were in sinus rhythm compared to those in AF (Table 2). The same pattern could be observed on both sides of the atria (Fig. 3). Spearman's correlation test showed a significant inverse relationship between the degree of fibrosis and the MMP-9 expression (p = 0.03, Spearman's Rho=–0.40) (Fig. 3). To exclude that ischemic heart disease would lead to an increased expression of MMP-9 we compared the patients with MVS with concomitant CABG (8 patients, 5 in SR, 3 in AF) versus those without concomitant CABG (11 patients, 5 in SR, 6 in AF). There was no difference in the expression of MMP-9 between both groups (p = 0.27).


Figure 3
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  		Fig. 3 A: Gelatin zymogram showing lytic bands due to MMP-9 (92 kDa), MMP-2 (72 kDa) and the complex of MMP-9 with a microglobuline (125 kDa). B, C: Quantification by scanning densitometry. D: Relation between the expression MMP-9 and the degree of fibrosis. (Spearman's Rho: –0.40).

 
The expression of MMP-2 was not different among the 3 groups (Table 2; Fig. 3).

3.4. TIMP-expression
No reliable Western blot for TIMP-1 and TIMP-2 could be obtained. Immunohistochemistry for TIMP-1 showed that this MMP-inhibitor was confined to interstitial cells and not expressed by myocytes (Fig. 4). Quantification of the immuno-stained cells did not reveal a difference between the 3 groups (p = 0.21) nor between the left and right atrial biopsies (p = 0.88) (Table 2; Fig. 5). Staining for TIMP-2 showed positively in interstitial cells but also at the surface of the myocytes (Fig. 4). Compared to patients from the CABG group those from the MVS and SR group had a trend to a lower expression of TIMP-2 in interstitial cells. Similar down-regulation was observed concerning positively stained myocytes. In the CABG group 66% of the sections were positively stained whereas in the MVS and SR group this was reduced to 25% and in the MVS and AF group this was 54% (p = 0.14). Overall, TIMP-2 was more expressed in the right then in the left atrium (p = 0.04) (Table 2, Fig. 5).


Figure 4
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  		Fig. 4 A: Sections stained with TIMP-1 antibody belonging to representative patients from the CABG group, MVS and SR group, MVS and AF group; magnification x 20. Only interstitial cells stained positive for TIMP-1 (arrow). B: Sections stained with TIMP-2 antibody in patients from the CABG group, MVS and SR and MVS and AF groups; magnification x 20. In the CABG group both interstitial cells (arrowhead) and myocytes (arrow) were positively stained.

 

Figure 5
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  		Fig. 5 A: There was no difference in the expression of TIMP-1 between the 3 groups (p = 0.57). B: There was a trend for decreased expression of TIMP-2 in the interstitial cells between the CABG group and the MVS and SR group (p = 0.06). C: There was a trend for decreased expression of TIMP-2 in the myocytes without reaching statistical significance.

 
Western blotting for TIMP-4 revealed bands at 29 kDa (glycolysated form) and 46 kDa (complex). Their expression was not significantly different among the 3 groups (Table 2).

3.5. TNF-{alpha}- and TACE-expression
TNF-{alpha}- and TACE-expression was only evaluated on tissue of the left atrium. Western blotting for TNF-{alpha} showed a weak signal at the 17 kDa region (soluble TNF-{alpha}) and no signal at the 26 kDa region (membrane-bound TNF-{alpha}) (not shown). There was no difference in the expression between the different groups.

Also the expression of TACE, which was found around 90 kDa, was not different between the 3 groups (Table 2).

3.6. AT-1 and AT-2 receptor expression
Western blots from left and right atrial tissue revealed both proteins in the 45–55 kDa region. Quantification revealed no different expression between patients from the CABG group, MVS and SR group and MVS and AF group or between the left and the right atria (Fig. 6 and Table 2). Analysis of the patients without ACE-inhibitors or Ang II receptor blockers (CABG n = 5; MVS and SR n = 3; MVS and AF n = 3) did not show a different expression between the 3 groups (Kruskal Wallis AT-1 p = 0.59, AT-2: p = 0.13).


Figure 6
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  		Fig. 6 Representative Western blots of atrial AT-1 (A) and AT-2 (B) receptors and bar graphs depicting their expression normalized to CABG patients (mean ± sem).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Limitations
 6. Conclusions
 References
 
4.1. Main findings
Patients who underwent mitral valve surgery had profound structural changes of the atria compared to a control group who underwent CABG. The atria were larger, cells were hypertrophied, there was more interstitial fibrosis and there were clear signs of cellular degeneration. These structural changes were more pronounced in patients who had persistent AF. Left atrial biopsies showed increased fibrosis in patients undergoing mitral valve surgery compared to those undergoing CABG independent of the underlying rhythm. Increased fibrosis in the right atrium however was only seen in AF patients and was associated with a higher degree of tricuspid regurgitation. MMP-1 and MMP-9 are down-regulated in MVS patients and these changes are concordant with the extent of fibrosis seen on the biopsies. The changes were similar in mitral valve patients with sinus rhythm or persistent AF implicating no direct effect of the arrhythmia itself on this down-regulation. We did not find changed expression of the angiotensin-receptor AT-1 and AT-2, nor of TIMP-1, -2 and -4, TNF-{alpha} and TACE in patients with MVS, with or without AF.

4.2. Structural remodeling
Structural changes underlying the atrial substrate for permanent AF include atrial fibrosis and atrial dilatation. Atrial fibrosis can change a homogeneously activated syncytial atrium into a discontinuous and branching structure susceptible for multiple wavelet re-entry [2]. On the other hand dilated atria will also help to sustain AF since larger atria can harbour more re-entrant wavelets at the same time.

The structural differences that we observed in MVS patients, compared to those with CABG, confirm previous findings in animal models of AF due to heart failure and mitral regurgitation [2,5]. In these studies increased atrial fibrosis, cellular hypertrophy and atrial dilatation have been observed. Also in human biopsies the link between atrial fibrosis and AF has been established. In a large study on atrial biopsies from patients with chronic AF a clear relation between atrial fibrosis and AF was found [18]. Boldt et al. later showed that both in lone AF and AF associated with mitral valve disease there is an increased expression of collagen type I, while in the latter group also collagen type III was increased [4].

We observed more pronounced structural changes in MVS patients with AF than in those with SR. Due to the nature of our experiments we cannot demonstrate whether AF developed because of the more profound remodeling or whether the arrhythmia acted as a positive feedback for it. There are however ample data suggesting that AF by itself may result in structural remodeling [19].

4.3. Signaling pathways
4.3.1. Matrix metalloproteinases
MMPs have been shown to be involved in the ventricular matrix turnover process post myocardial infarction and heart failure [20]. An increase in MMP activity can induce extracellular matrix remodeling leading to dilatation, while a decrease can reduce the extracellular matrix breakdown and lead to fibrosis.

We observed a marked decrease in the expression of MMP-1. This is in agreement with observations on a ventricular level in patients with dilated cardiomyopathies as in animal models of chronic pressure and volume overload [20,21].

We also found an inverse relationship between the atrial diameter and the expression of MMP-1 and between the extent of atrial fibrosis and MMP-1. This suggests that atrial volume overload and accompanying atrial dilatation due to mitral regurgitation may lead to a decreased atrial MMP-1 expression which in turn will lead to an increased presence of atrial fibrosis. MMP-1 is mainly responsible for degrading structural or fibrillar collagens (types I and III) which is known to be up-regulated in patients with mitral valve disease and AF. Moreover the expression of TIMP-1, a potent inhibitor of MMP-1, did not change. This will bring the MMP/TIMP balance further in the direction of diminished MMP-1 activity which would potentially favor extracellular matrix accumulation and hence fibrosis. Our results are in accordance with those from Marin et al. who found a decreased level of MMP-1 and an increased level of TIMP-1 in plasma samples from patients suffering from AF compared to a control group without AF [22].

We also observed a decreased expression of MMP-9 and no changes in MMP-2. This contrasts with previous studies from Nakano and Xu [23,24]. The former reported an up-regulation of MMP-9 [23], the latter found an increased expression of MMP-2 and down-regulation of TIMP-2 with no changes in MMP-9 levels in end-stage heart failure patients with AF [24]. The differences between these studies and ours could be explained by the fact that they studied a heterogeneous AF population. It is known that different cardiac pathologies lead to another activation pattern of MMPs. Heart failure for instance induces ventricular MMP-9 expression whereas hypertension is associated with a decrease in MMP-9 [25]. In addition time-dependent MMP activation is well known during the progression of cardiopathies, both on the ventricular as on the atrial level. Acute pressure and volume overload induces ventricular MMP-9 activation that normalizes with the prolongation of the overload [21]. Animal studies looking to atrial remodeling during AF add additional evidence for this time-dependent change: atria of dogs paced in heart failure showed first an increased expression of MMP-9 which normalized once the dogs were in severe heart failure; MMP-2 did not change until the dogs were in severe heart failure and then it was up-regulated [26].

Since all our patients were already suffering for many years from mitral valve disease and/or AF, we cannot exclude that there was a phase of increased expression of these MMPs with later down-regulation. MMP-9 is known to mainly digest denatured collagen (gelatins), elastin, fibronectin, lamin as well as collagens type IV and V. A decreased expression of this enzyme can lead to an enhanced accumulation of extracellular matrix proteins and increased fibrosis as was seen on the biopsies. Moreover a down-regulation of MMP-9 may be a protective mechanism of the atria against progressive uncontrolled dilatation. An up-regulation of MMP-9 due to ischemic heart disease seems less likely since no different pattern could be seen in patients with MVS and concomitant ischemic heart disease versus those without.

4.3.2. Renin–angiotensin system
Activation of the renin–angiotensin system induces fibrogenesis in a variety of pathologic conditions including hypertensive heart disease, myocardial infarction and chronic heart failure [27]. During the last decade, it has become clear that besides the circulating renin–angiotensin system there is also a local cardiac production of its different components [27]. Angiotensin II may be a key messenger promoting cell degeneration, fibrosis and uncoupling. Brilla et al. showed in cultured rat cardiac fibroblasts that increased Ang II levels diminished MMP-1 expression indicating a relationship between the renin–angiotensin and MMP pathways [28].

Both in animal models and in biopsies from patients with AF, activation of the renin-angiotensin II pathway has been shown [10,11,29]. Angiotensin II induces proliferation of fibroblasts and extra-cellular matrix protein accumulation via the activation of the mitogen-activated protein kinases (MAPK). Inhibition of this angiotensin II pathway attenuates the formation of fibrosis and diminishes the incidence of AF [29–31].

In contrast to previous published papers, we did not find any difference in the expression of the angiotensin II receptor subtypes AT-1 and AT-2. Goette et al. described an increased expression in AT-2 in patients with persistent AF [32], while Boldt et al. found an increased expression of AT-1 and no changes of AT-2 [33]. However in the latter study no difference could be found between patients without mitral valve disease versus those with mitral valve disease. The difference between these studies and ours is that we assessed a very specific subgroup of the AF-population (those who were suffering from mitral valve disease). It cannot be excluded that the AT-1 and AT-2 expression pattern is dependent on underlying disease.


   5. Limitations
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Limitations
6. Conclusions
 References
 
We used patients undergoing CABG as a control group. We cannot exclude that MMP-expression was modified by ischemia in these patients. However, patients belonging to the CABG group neither had dilated atria nor substantial fibrosis.

We cannot exclude that due to the limited numbers our experiments may have missed the power to detect subtle changes in protein expression.

Most of our patients suffered from mitral regurgitation, however a minority (3 patients) had mixed mitral valve disease and one patient had mitral stenosis. Individual values of the MMP-expression did not differ from the rest of the group, but the number of patients is too low to exclude different pathological remodeling in these situations.

Since our study is done in patients with long-lasting AF the time course of changes and the potential impact of AF on atrial fibrosis cannot be assessed in detail. It might be possible that the arrhythmia itself has an impact on the development of atrial fibrosis for a specific time period only. This impact may vanish over time and might be overruled by other factors like changed hemodynamic parameters due to valve regurgitation. Future animal research should address this issue.

The renin–angiotensin pathway and the MMP/TIMP system are only 2 pathways which are involved in extracellular matrix remodeling. Other pathways such as TGF-β, the ADAMS/integrins, bradykinin and endothelin system have also been implicated in this process [34,35]. Their contribution was not assessed in this study.


   6. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Limitations
 6. Conclusions
References
 
Mitral valve disease results in manifest structural atrial remodeling, most pronounced in patients with AF. Matrix metalloproteinases parallel this remodeling suggesting their involvement, while the arrhythmia itself did not alter MMP-expression.


   Acknowledgements
 
This work has been supported by the Institute for the Promotion of Innovation by Science and Technology, Flanders, Belgium (WA) and the 3M Pharma Prize 2003 for Cardiology, Belgium. HH is a fundamental Clinical Investigator of the Fund for Scientific Research, Flanders, Belgium.


   Notes
 
Time for primary review 15 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Limitations
 6. Conclusions
 References
 

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