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Cardiovascular Research 2002 54(3):559-567; doi:10.1016/S0008-6363(02)00256-0
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Copyright © 2002, European Society of Cardiology

Collagen accumulation after myocardial infarction: effects of ETA receptor blockade and implications for early remodeling

Presented in part at the 72nd Scientific Session of the American Heart Association, Atlanta, GA, USA, November 7–10, 1999, and published in abstract form (Circulation 1999;100(Suppl. 1):562)

Daniela Fraccarollo*, Paolo Galuppo, Johann Bauersachs and Georg Ertl

Medizinische Klinik, Julius-Maximilians-Universität Würzburg, Würzburg, Germany

d.fraccarollo{at}medizin.uni-wuerzburg.de

* Corresponding author. Present address: Medizinische Universitätsklinik, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany. Tel.: +49-931-201-5301; fax: +49-931-201-5302

Received 13 November 2001; accepted 4 January 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objectives: Endothelin A (ETA) receptor blockade started early after myocardial infarction (MI) promotes adverse left ventricular (LV) dilatation. We tested the hypothesis that inhibition of ETA receptors during the early phase of healing affects collagen synthesis and accumulation, and induces expansion of infarcted myocardium. Methods: Starting 3 h after coronary ligation, female Wistar rats were treated with the selective ETA receptor antagonist LU 135252 (30 mg/kg body wt/day) or placebo. A period of 7 days after MI, hemodynamic, morphometric and biochemical studies were performed. Results: ETA receptor blockade enhanced infarct expansion index and decreased LV systolic function. Infarct scar of LU 135252-treated rats displayed decreased gene expression of fibrillar type I/III collagens and of transforming growth factor-β1 (TGF-β1). Collagen content in the infarct scar and border regions was lower after ETA inhibition. In addition, Western blot analysis revealed, after ETA receptor blockade, enhanced matrix metalloproteinases MMP-13, and MMP-2 expression in the infarcted LV myocardium. Conclusions: These data demonstrate that endothelin stimulates collagen accumulation at the site of infarction. Decreased collagen and TGF-β1 gene expression, associated with enhanced infarct expansion and MMP up-regulation likely contributes to ETA receptor blockade–mediated deleterious effects on ventricular remodeling after infarction.

KEYWORDS Endothelins; Extracellular matrix; Gene expression; Infarction; Remodeling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Early left ventricular (LV) dilatation with expansion of the infarcted tissue is associated with increased incidence of heart failure and mortality. Enhanced collagen turnover (breakdown and synthesis) occurs early after myocardial infarction (MI) [1–4]. Preservation of the extracellular matrix and collagen deposition at the site of myocyte necrosis are essential for structural integrity of the infarcted heart. Excessive collagen degradation and impaired fibrous tissue formation may reduce the tensile strength of the necrotic zone and lead to enhanced infarct expansion [5–9].

Studies performed on isolated rat cardiac fibroblasts have shown that ET-1 increases collagen synthesis, and reduces gelatinolytic activity [10]. Long-term treatment with ETA receptor antagonists improves ventricular remodeling [11] and survival in rats with heart failure [12], when administered 7–10 days after MI. In contrast, early ETA receptor blockade promotes adverse left ventricular dilatation [13] and causes thinning of myocardial scar [14] when this is examined 4 weeks after coronary occlusion.

We therefore hypothesized that inhibition of ETA receptor influences scar collagen formation during the early phase of healing. Accordingly, we investigated whether the selective ETA receptor antagonist LU 135252 administered during the acute repair period after experimental MI (I) induces weakening and expansion of infarcted wall and (II) alters collagen expression and accumulation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 MI and study protocols
Left coronary artery ligations were performed in adult female Wistar rats (200–250 g) as previously described [15]. LU 135252 was administered in drinking water starting 3 h after MI at a concentration of 30 mg/kg body wt/day according to the manufacturer instructions (Knoll AG). Drinking volume was measured daily and the concentration of LU 135252 was adjusted according to the water intake and body weight.

Animals were sacrificed 7 days after infarction, at a time when significant collagen deposition has begun [4,16], the increase in MMPs activity peaks [2], and the infarct expansion reaches the plateau phase [16].

2.2 Infarct expansion study
2.2.1 Hemodynamic measurements
Left ventricular systolic (LVSP), end-diastolic pressures (LVEDP) and the maximal rate of rise in LV pressure (dP/dtmax) were measured under light ether anesthesia and spontaneous respiration by micromanometer (Millar Instruments) [15].

2.2.2 Infarct size, expansion index
The hearts were arrested by intravenous KCl injection, excised and placed in ice-cold KCl to achieve uniform diastolic arrest. Then, the LV was infused with a volume of 10% phosphate-buffered formalin corresponding to pressure of 7.5 mmHg and fixed for 2 h. After fixation, the hearts were weighed and 10-µm thin sections were serially cut from apex to base at 1-mm intervals. MI size (fraction of the infarcted LV) was calculated as the average of all slices and expressed as a percentage of length [15].

Expansion index was determined by previously described methods [16,17]. A transverse section representing the middle of LV and with the most marked cavity dilatation was used for each heart (Fig. 1). The expansion index was calculated with the formula: Expansion Index=(Left Ventricular Cavity Area/Total Left Ventricular Area)x(Septum Thickness/Scar Thickness).


Figure 1
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  		Fig. 1 Scar thickness and expansion index 7 days after myocardial infarction. Mean±S.E.M., {dagger} P<0.05, {ddagger} P<0.01 vs. Placebo. (a) Diagram of the transverse section of the heart used for expansion index measurements. C and C' were calculated tracing LV and right ventricle endocardial contours and by computer algorithm as the mean x and y coordinates of all pixels within the contours. Septum thickness: mean of five septum wall measurements, along evenly spaced radiants passing through C with the central radian connecting C and C'. Scar thickness: mean of three scar wall measurements along the central radian connecting C and the thinnest part of the infarct wall and the two radians on either side separated from the central radian by 22.5°.

 
2.3 Biochemical study
A separate group of animals was used for the biochemical study.

2.3.1 Tissue sampling
The heart was divided into right ventricle and LV, including septum in ice-cold saline. After MI size estimation (the LV was pressed flat on glass plates and the boundary lengths of the infarcted and non-infarcted epicardial and endocardial surfaces were traced and digitized) [15], the LV was divided into scarred area, infarct borders (~2 mm tissue) and septum.

2.3.2 Quantification of mRNA expression by competitive RT-PCR
Total RNA was isolated from LV tissue using TRIzol reagent (Life Technologies). Transforming growth factor-β1 (TGF-β1), collagen {alpha}1(I) and {alpha}1(III) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA expression was determined by competitive polymerase chain reaction (PCR) (Table 1) in cardiac samples (placebo, n=5; LU 135252, n=5; and sham rats, n=4). Products of PCR amplification were separated on 2% agarose gel. A given mRNA level was expressed as a ratio with respect to the level of mRNA for GAPDH.


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  		Table 1 Summary of competitive RT-PCR

 
2.3.3 Tissue homogenization, Western blot analysis, zymography, reverse zymography
LV samples (placebo, n=5; LU 135252, n=6; and sham rats, n=3) were homogenized (1:30 w/v) in ice-cold extraction buffer (pH 5.0) containing cacodylic acid (10 mmol/l), NaCl (0.15 mol/l), ZnCl2 (1 µmol/l), CaCl2 (20 mmol/l), NaN3 (1.5 mmol/l), and 0.01% Triton X-100 [2]. The homogenate was then incubated with continuous agitation for 20 h and centrifuged at 8000g for 30 min at 4 °C. Proteins were determined by Bradford assay.

For Western blot analysis myocardial extracts (4 µg protein per-lane) were mixed with sample loading buffer and under reducing conditions separated on 12% SDS–polyacrylamide gel. Proteins were electrotransferred onto PVDF membrane (Immun-Blot® 0.2 µm, Bio-Rad), incubated overnight at 4 °C in Tris buffered saline-Tween (TBS-T) with 5% blocking agent (Amersham), followed by incubation for 2 h with primary antibody in TBS-T with 0.5% blocking agent at a dilution of 1:200. After washing, the blots were incubated for 1 h with horseradish peroxidase-labeled goat anti-mouse IgG antibody (Amersham) prepared in TBS-T with 0.5% blocking agent at a diluition of 1:10 000. The bands were detected using chemiluminescence assay (ECL+Plus, Amersham). Primary antibodies used included mouse anti-rat MMP-13 (clone LICOIID1; MAB 13426) from Chemicon; mouse anti-MMP-2 (clone 42-5D11; IM33L) from Oncogene.

Zymography was performed by previously described methods [18,19]. Scar (4 µg protein per-lane), infarct borders and septum (8 µg protein per-lane) extracts were mixed with loading buffer and electrophoresed on a 10% SDS–polyacrylamide gel containing 1 mg/ml gelatin (type A from porcine skin) under non-reducing conditions. Gelatinolytic bands were quantified by Image software Quantity One (Bio-Rad).

Reverse zymography was performed in a similar manner except that purified MMP-2 (30 µg/ml, Oncogene) was incorporated into a 15% SDS–polyacrylamide gel along with 1 mg/ml gelatin [20].

Human recombinant purified enzymes (MMP-9, MMP-3, MMP-2, TIMP-2, TIMP-1: Oncogene; MMP-13, MMP-1: Chemicon) were used as a positive control and standardization among gels. Molecular weights were determined using pre-stained SDS–PAGE standard and precision protein standards (Bio-Rad).

2.3.4 Hydroxyproline determination
Tissue homogenates were freeze–dried, weighed and hydrolyzed in 6 N HCl at 110 °C for 24 h. Hydroxyproline concentration was measured spectrophotometrically [21]. Collagen content was expressed in µg/mg dry tissue weight assuming that collagen contains an average of 13.4% in hydroxyproline. Assays were performed in triplicate.

2.4 Statistical analysis
All results are reported as mean±S.E.M. Between-group (placebo, LU 135252) comparisons were analyzed by Student's t-test. A one-way ANOVA followed by Newman–Keuls test was used to compare baseline characteristics and for within-group comparisons (LV-Sham versus scar, infarct borders and septum). P<0.05 was considered statistically significant. Correlations were determined by linear regression analysis.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
There were no differences in mortality between placebo and LU 135252 treatment.

3.1 Infarct expansion study
Table 2 shows baseline characteristics of sham-operated and MI rats included in the infarct expansion study. Infarct size was similar in placebo and LU 135252-treated rats. All MI animals developed elevated LVEDP, reduced LVSP and dP/dtmax. In ETA antagonist-treated rats dP/dtmax was further reduced. Moveover, LU 135252-treated rats had a markedly thinner scar and a higher infarct expansion index compared with the placebo-treated group (Fig. 1).


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  		Table 2 Baseline characteristics of infarct expansion study

 
3.2 Biochemical study
Mean infarct sizes of rats included in the biochemical study was similar (Placebo MI: 42.2±2%; LU 135252 MI: 40.5±2%).

3.2.1 Collagen and TGF-β1 gene expression
A period of 7 days after infarction, collagen type I and type III collagen mRNA levels (Fig. 2) were significantly increased in the border zones of infarct, compared to controls and showed the greatest elevation in the scar tissue. An increase in collagen type I and III mRNA was also observed in the surviving LV myocardium remote from the infarct site. ETA receptor inhibition significantly decreased collagen gene expression in the scar region, and the effect was more marked for type I collagen than for type III (Fig. 2).


Figure 2
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  		Fig. 2 Type I and III collagen mRNA levels and TGF-β1 gene expression in the scar, infarct borders and septum of placebo and LU 135252-treated rats, and in the LV of sham-operated rats. Type I/III collagens and TGF-β1 mRNA levels were corrected for GAPDH mRNA levels. Mean±S.E.M., *P<0.05 vs. Sham, {dagger} P<0.05 vs. Placebo.

 
TGF-β1 mRNA levels (Fig. 2) were slightly raised in the septum, markedly increased in the infarct borders and showed the greatest elevation in the scar tissue. TGF-β1 mRNA levels were significantly lowered in ETA antagonist-treated rats in the scar region. Collagen type I mRNA levels closely correlated with TGF-β1 gene expression (r=0.90, P=0.0001). Also collagen type III and TGF-β1 gene expression was highly correlated (r=0.95, P=0.0001).

3.2.2 Collagen content
In the infarct scar and border regions of LU 135252-treated rats, collagen content was significantly lower compared to placebo (Fig. 3).


Figure 3
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  		Fig. 3 Collagen content in the scar, infarct borders and septum of placebo and LU 135252-treated rats, and in the LV of sham-operated rats. *P<0.05 vs. Sham, {dagger} P<0.05 vs. Placebo.

 
3.2.3 MMP and TIMP
In placebo MI rats the intensity of the 72 kDa MMP-2 and the 59 kDa MMP-13 protein bands were enhanced in the infarct borders and showed the greatest increase in the scar extracts (Fig. 4a). After ETA receptor blockade, an increase of MMP-2 (P<0.05) and MMP-13 (P=0.06) protein expression was observed in the scar extracts (Fig. 4b).


Figure 4
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  		Fig. 4 (a) MMP-2 (72 kDa) and MMP-13 (59 kDa) proteins expression as revealed by Western blot analysis in the scar, infarct borders and septum of placebo and LU 135252-treated rats, and in the LV of sham-operated rats (4 µg protein per-lane). MMP-2 and MMP-13 proteins expression were enhanced in the infarcted left ventricular myocardium and were greater after ETA receptor blockade. (b) MMP-2 and MMP-13 proteins expression as quantified by Western blot analysis. Mean±S.E.M., *P<0.05 vs. Sham, {dagger} P<0.05 vs. Placebo.

 
A period of 7 days after infarction, analysis of MMP activity by in vitro gelatin–zymography (Fig. 5) showed the presence of latent MMP-2 (~72 kDa, ~66 kDa), active MMP-2 (~58 kDa), and latent MMP-9 (92 kDa). Infarct scar and infarct borders of LU 135252 treated rats displayed enhanced activity for both latent and active MMP-2 and for MMP-9 compared with placebo MI (Fig. 5).


Figure 5
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  		Fig. 5 Representative zymogram with long exposure for showing differences among bands of MMP-9 and active MMP-2. Gelatin was used as zymografic substrate to characterize specific MMPs activities in the scar, infarct borders and septum of placebo and LU 135252-treated rats, and in the LV of sham-operated rats. Scar (4 µg protein per-lane), infarct borders and septum (8 µg protein per-lane). Active MMP-2 was increased in the infarct borders and in the scar tissue. Further increase in gelatinolytic activity was observed after ETA receptor blockade.

 
Low TIMP-1 (28 kDa) inhibitory activity was found in the scar, whereas TIMP-2 (21 kDa) inhibitory activity was slightly higher in the septum, increased in the infarct borders and in the scar, and were similar in both treatment groups (Fig. 6).


Figure 6
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  		Fig. 6 Reverse zymogram demonstrating inhibitory activity of TIMP-1 (28 kDa) and TIMP-2 (21 kDa) in the scar, infarct borders and septum of placebo and LU 135252-treated rats, and in the LV of sham-operated rats. Scar (4 µg protein per-lane), infarct borders and septum (8 µg protein per-lane). Dark bands represent areas of MMP inhibition. TIMP-1 was only detectable in the scar. TIMP-2 levels were increased in the infarct borders and in the scar.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
This study demonstrates that ETA antagonist treatment early after myocardial infarction reduces collagen accumulation and leads to enhanced expansion of infarcted myocardium.

4.1 Infarct expansion
Infarct expansion, thinning and dilatation of the infarcted myocardial wall, is an event of the early phase after MI. Dilatation and hypertrophy of surviving myocardium occur in parallel and continue even after expansion of the infarcted tissue is complete [22]. Marked thinning of myocardial scars and enhanced infarct expansion index were observed in animals treated with LU 135252. Previously, Nguyen et al. [14] have also shown that LU 135252, administered within the first 24 h after MI, promoted scar thinning and LV enlargement 4 weeks after coronary ligation. Together, these findings indicate that early ETA receptor blockade affects scar formation which in turn worsens infarct expansion leading to adverse remodeling.

4.2 ETA receptor blockade and collagen accumulation
TGF-β1 plays an important role for the transformation of fibroblasts to myofibroblasts, which appear at the site of myocyte necrosis and are responsible for increased collagen synthesis [1,9]. Moveover, TGF-β1 stimulates collagen types I and III gene expression [23]. In the DOCA-salt hypertensive rats, a reduction in procollagen I/III expression by ETA receptor inhibition was associated with a blunted TGF-β1 gene expression [24]. In the present study, TGF-β1 appears to be involved in the decrease in type I/III collagens gene expression after ETA receptor inhibition in the infarct scar. Collagens and TGF-β1 gene expression were highly correlated and similarly reduced after ETA receptor blockade. In addition, as ET-1 has also been shown to activate the procollagen I promoter [25], endothelin may also directly influence collagen type I expression at the site of injury. This may explain the more pronounced effect of ETA receptor inhibition on collagen type I mRNA observed in our study.

The reduction in collagen gene expression at the site of infarction after ETA antagonism and the presence of elevated ET-1 levels in the infarcted tissue [14,26] provide evidence that endothelin is linked to collagen formation and scar stabilization. Low collagen content renders the scar less resistant to distention under conditions of volume and pressure overload and pre-disposes to expansion. In fact, infarct expansion occurs until appreciable amounts of collagen have been deposited in the infarct and only a thick and strong scar is able to withstand expansion [6,27]. Thus, ETA blockade-induced expansion of the infarcted wall and the decrease in infarct collagen accumulation are likely to be causally related. The recent evidence that increased osteopontin expression after MI protects against LV dilation by promoting collagen gene expression and accumulation [5], supports our hypothesis.

Side-to-side slippage of myocardial cell bundles [28] and individual myocytes [29] especially occurs very early post-MI and contributes not only to infarct expansion but also to thinning of surviving myocardium. Future experiments should elucidate whether ETA receptor blockade promotes early fiber slippage.

4.3 Myocardial MMPs and TIMPs
MMPs are synthesized as latent enzymes and an important determinant of matrix degradation is activation of MMPs. We found that latent MMP-2 is activated in the border zones of infarct and in the scar. In addition, our data confirm recent reports on the presence of MMP-13 in the rat heart post-MI [3]. MMPs are inhibited by specific tissue inhibitors of metalloproteinases [30]. We observed that TIMP-2 inhibitory activity increased in the infarct scar and in the myocardium bordering the scar, whereas TIMP-1 inhibitory activity was only detectable in the infarct scar. Down-regulation of TIMPs in presence of increased gelatinolytic activity was observed in the failing heart of patients with end-stage dilated cardiomyopathy [30–32], suggesting that the regulation of TIMPs varies with the type and timing of tissue damage. Peterson et al. [3] recently observed that TIMP-1 and TIMP-2 protein levels remain unchanged during the first week and increase 2 weeks post-MI in the rat heart. These measurements were made on extracts of the entire left ventricle, and localized changes in TIMPs protein in the infarcted myocardium are likely to be obscured by the lack of such changes in surviving left ventricle. Apart from their inhibitory function, TIMP-2 is required for activation [33] of latent MMP-2. Therefore, we cannot exclude that changes in TIMP-2 levels have contributed to activation of latent MMP-2.

We demonstrated MMP up-regulation in the infarcted left ventricular myocardium after ETA receptor blockade, which was not balanced by an increase in TIMP-1 and TIMP-2 inhibitory activity. The imbalance between collagenase/gelatinase and its inhibitor may enhance extracellular matrix degradation and further worsen the expansion of the infarcted wall [34]. Recent studies have been shown that inhibition of MMPs can prevent early LV dilation in mice post-MI [35]. Furthermore, infarcted mice with deficiency of MMP-9 have decreased incidence of early myocardial rupture [36] and attenuated LV dilatation [37]. However, in vitro zymography of myocardial extracts may not exactly reflect in vivo MMP activity and conclusions on interrelations between MMPs and TIMPs in vivo on the basis of our analysis should be drawn with some caution. Therefore, the reduction of fibrillar collagen accumulation after ETA blockade may be explained by a reduction in the expression of the fibrogenic growth factor TGF-β1 and collagen synthesis.

In contrast to early phase of healing, in the late phase post-MI excessive accumulation of collagen remote from the infarct site increases myocardial stiffness and contractile dysfunction, and contributes to the progression of ventricular remodeling and heart failure [9,38]. Moveover, an increase in MMP activity results in increased deposition of poorly structured fibrotic tissue in the myocardium [39]. Podesser et al. [40] recently reported that long-term ETA receptor blockade prevents ventricular dilation and MMP activation in the myocardium remote from the infarct. Similarly, we found that long-term treatment with LU 135252, started after the early phase of healing (7 days post MI) improved LV dilatation in rats with heart failure. This beneficial effect of late ETA receptor inhibition was accompanied by reduced collagen gene expression, fibrosis and MMP activity in the surviving LV myocardium [41]. We hypothesize that indirect mechanisms such as improvement of LV remodeling and a decrease in myocardial wall stress by long-term ETA receptor blockade could have contributed to reduction of MMPs observed late post-MI. Supporting this notion, regional over-expression of MMP-9 correlates with increased mechanical stress in the infarcted LV [42] and the reduction in myocardial MMP-9 levels was related to the attenuation of LV dilation after chronic MMP inhibition in developing LV failure [43]. MMP up-regulation after acute ETA receptor inhibition may be the result of increased wall stress related to enhanced LV wall thinning and/or a direct effects on MMP as shown in cultured fibroblasts [10,44].

In summary, endothelin appears to play a critical role in promoting fibrous tissue formation at the site of injury (early reparative fibrosis) as well as remote from the infarct (late reactive fibrosis), and the cardioprotective effects of ETA receptor antagonists strongly depend on timing of pharmacologic intervention post-MI.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
The present study suggests that endothelin, during the early MI phase, stimulates collagen accumulation at the site of infarction, leading to infarct repair. Decreased collagen and TGF-β1 gene expression, associated with a rise in MMPs activity likely contributes to ETA receptor blockade–mediated infarct expansion.

Time for primary review 27 days.


   Acknowledgements
 
This work was supported by the Deutsche Forschungsgemeinschaft (SFB355, B9).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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L. E. Spieker and T. F. Luscher
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