Cardiovascular Research

Cardiovascular Research 2001 52(2):265-273; doi:10.1016/S0008-6363(01)00398-4
© 2001 by European Society of Cardiology

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Copyright © 2001, European Society of Cardiology

Cardiac remodeling after long term norepinephrine treatment in rats

Wilfried Briest^a,*, Alexander Hölzl^a, Beate Raßler^a, Alexander Deten^a, Monika Leicht^a, Hideo A. Baba^b and Heinz-Gerd Zimmer^a

^aCarl-Ludwig-Institute of Physiology, University of Leipzig, Liebigstr. 27, D-04103 Leipzig, Germany
^bGerhard-Domagk-Institute of Pathology, University of Münster, Münster, Germany

^* Tel.: +49-0341-971-5500; fax: +49-0341-971-5509 barw{at}medizin.uni-leipzig.de

Received 15 November 2000; accepted 15 June 2001

	Abstract

Top Abstract 1 Introduction 2 Material and methods 3 Results 4 Discussion Acknowledgments References

 
Objective: In this study we have tested the hypothesis that degradation of collagen by matrix metalloproteinase 2 (MMP-2) precedes the deposition of extracellular matrix (ECM) after long term norepinephrine (NE) treatment. Methods: Female Sprague–Dawley rats received continuous i.v. infusion of NE (0.1 mg/kg·h) for 1, 2, 3, 4 and 14 days. Heart function and weight as well as expression of cardiac colligin and of collagen I and III were examined. Furthermore, we have assessed the degradation pathway of collagen by measuring the mRNA and activity of myocardial MMP-2 and tissue inhibitor of metalloproteinase 2 (TIMP-2) as well as the protein level of TIMP-2. Results: NE induced hypertrophy predominantly of the left ventricle (LV) in a time-dependent manner. It increased the mRNAs of colligin, collagen I and III, and of MMP-2 and TIMP-2 as well as MMP-2 activity in two phases: In the initial phase, at 3 and 4 days, the mRNA of colligin and of collagen I and III was elevated predominantly in the LV, MMP-2 and TIMP-2 mRNA, as well as TIMP-2 protein and MMP-activity were increased in both ventricles. The second phase, after 14 days, was characterized by a less pronounced increase in colligin, collagen I and III and in MMP-2 activity which occurred exclusively in the LV. Finally, long-term treatment with NE induced a 37% increase in interstitial fibrosis which was shown to occur exclusively in the LV after 14 days. Conclusion: NE treatment induced fibrosis exclusively in the LV which was associated with hypertrophy predominantly of the LV. The elevated MMP-2 activity seems to be necessary for the ECM to adapt to the enlargement of myocytes and to reduce overproduction of collagen.

KEYWORDS Adrenergic (ant)agonists; Extracellular matrix; Fibrosis; Gene expression; Hemodynamics; Hypertrophy; Receptors

	1 Introduction

Top Abstract 1 Introduction 2 Material and methods 3 Results 4 Discussion Acknowledgments References

 
Impaired functional performance despite hypertrophic enlargement characterizes the decompensated failing heart. Manifestations of decompensated function include ventricular dilatation, reduced fractional shortening, diminished ejection fraction, and decreased myocardial force production. Cardiac fibrosis is a common and well recognized feature of heart failure [1] and of other pathological conditions [2,3], as well as of normal aging [4]. It is absent from hearts in which the stimulus for hypertrophy is exercise training [5] or hyperthyroidism [6]. Fibrosis increases the stiffness of the myocardium leading to diastolic dysfunction and exacerbation of heart failure [7]. Fibrosis results, in part, from increased expression of genes encoding extracellular matrix (ECM) proteins that make up the scaffolding that provides the framework in which myocytes function.

In previous experiments we have shown hypertrophy of the left ventricle (LV) after norepinephrine (NE, 0.1 mg/kg·h) infusion in rats which was accompanied by an increased collagen mRNA expression predominantly in the LV [8]. In this study we have tested the hypothesis that the increased collagen mRNA expression leads to morphologically detectable cardiac fibrosis. Another purpose was to examine whether ECM proteins are also subjected to degradation after NE treatment. Therefore we have analyzed the expression and gelatinolytic activity of matrix metalloproteinases (MMPs) and the protein level of their tissue inhibitor (TIMP-2). The MMPs and TIMPs play the predominant role in the turnover of ECM. A more prolonged observation period was necessary for this investigation to document NE-induced cardiac fibrosis.

	2 Material and methods

Top Abstract 1 Introduction 2 Material and methods 3 Results 4 Discussion Acknowledgments References

 
All experiments were performed on female Sprague–Dawley rats weighing 210–260 g at the beginning of the study. They were supplied by Charles River (Sulzfeld, Germany). Animals used in this study were maintained in accordance with the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). The animals were allowed to move freely in their cages with access to tap water and rat chow diet (Altromin C 100, Altromin GmbH, Lage, Germany). All substances were given as constant intravenous infusion via a catheter (Vygon, Aachen, Germany) positioned in the left jugular vein. When infusion for 14 days was done, the catheter was connected to an alzet mini-osmotic pump (alza corporation, Palo Alto, CA, USA), which was implanted subcutaneously. The catheter was connected to a 20-ml syringe placed in an infusion pump (Infors AG, Basel, Switzerland) when infusion was limited to 4 days. In this set-up, the infusion rate was 4 ml/kg·h. Norepinephrine (NE) was administered at a dose of 0.1 mg/kg·h dissolved in 0.9% NaCl; sodium chloride-infused animals served as controls. To prevent oxidation, 100 mg/l ascorbic acid was added to the solutions, and the syringes were protected from light. Carvedilol (0.5 mg/kg·h; Boehringer Mannheim, Mannheim, Germany), a β-adrenergic antagonist and vasodilator with ₁-blocking activity [9] was infused alone and in combination with NE for 3 days. For continuous application of the calcium-channel blocker nisoldipin (0.1 mg/kg·h; Bayer, Leverkusen, Germany, dissolved in absolute ethanol) for 3 days, alzet mini-osmotic pumps (alza corporation, Palo Alto, CA, USA) were implanted intraperitoneally. NE was purchased from Sigma (Deisenhofen, Germany). L-(+)-Ascorbic acid was obtained from Merck (Darmstadt, Germany).

Hemodynamic measurements were performed as previously described [8]. After the hemodynamic measurements were obtained, the hearts were rapidly excised, and the RV free wall was trimmed away. Both ventricles were weighed after freezing in liquid nitrogen.

2.1 Histological changes
In another series of 14 days of NE treatment, the hearts were excised, the atria were cut off, the ventricles were weighed, and the hearts were divided parallel to the valve level. On the basic part of the heart, the RV free wall was trimmed away and the residues of the ventricles were frozen in liquid nitrogen. The apical part of the hearts was fixed in 4% buffered formaldehyde, embedded in paraffin, and 5 µm sections were stained with Hematoxylin-Eosin, PAS (periodic acid Schiff) and Sirius red (0.5% in saturated aqueous picric acid). The sections were morphometrically quantified as previously described [10] to quantify cardiac fibrosis.

Frozen sections of 5 µm were mounted on silan-coated glass slides and fixed in 4% ice-cold acetone for 90 s. For visualization of collagen I and III, antibodies against collagen type I and III (Southern Biotechnology) were used. The respective antibody was applied in a humidified chamber at 4°C overnight in a concentration of 1:50, followed by an alkaline phosphatase conjugated rabbit anti-goat antibody (1:50 in RPMI; 30 min at room temperature; Dianova) and an alkaline phosphatase conjugated goat anti-rabbit antibody (1:100 in RPMI; 60 min at room temperature; Dianova). The enzyme reaction was developed for 25 min at room temperature in a freshly prepared new fuchsin solution containing levamisole. Finally, the sections were counterstained with hematoxylin and mounted with Kayser’s glycerine gelatine. Omission of the primary antibody served as negative control.

To obtain another independent measure of cardiac hypertrophy at the cellular level, the mean cardiomyocyte diameter was calculated by measuring 100 cells per specimen in the region of the cell nucleus using the two point distance function of the analysis system [10]. The sections were stained with PAS to discriminate the cell borders.

2.2 RNase protection assay
Total RNA isolation was performed according to a modified phenol/guanidiniumthiocyanat method of Chomczynski and Sacchi [11] using TRIZOL^TM (GIBCOBRL^TM, Karlsruhe, Germany). A total of 5 µg RNA was used in the RNase protection assay (RPA) with the probe template set labeled with RiboQuant^® In Vitro Transcription Kit (Pharmingen, Hamburg, Germany; final probe concentration: 4x10⁵ cpm/µl) and [-³²P] UTP (3000 Ci/mmol, Amersham, Freiburg, Germany), as described by the manufacturer. After hybridization (56°C; 12–16 h), the unhybridized riboprobe was digested with a mixture of RNases A and T1 (RiboQuant^® RPA Kit, Pharmingen, Hamburg, Germany), according to the manufacturer’s instructions. Protected probes were displayed by electrophoresis on a denaturing gel containing 5% polyacrylamide/8 M urea followed by visualization with the Molecular Imager (BioRad, München, Germany). The densitometric quantification of the individual bands of the RPA assays were performed by the Multi-Analyst program version 1.1 (BioRad, München, Germany). The probe template set contained the following cDNA (probe length in bp/protected; GenBank Accession No., position): collagen I (504/449; Z78279 [GenBank] , 3480-3928), TIMP-2 (404/294; AJ409332 [GenBank] , 1362-1656), MMP-2 (360/269; U65656 [GenBank] , 708-976), collagen III (286/211; X70369 [GenBank] , 110-320), colligin (225/161; M69246 [GenBank] , 224-386), ARPP (143/113; X15096 [GenBank] , 559-671) and GAPDH (128/82; M17701 [GenBank] , 673-755).

2.3 Gel electrophoresis and immunostaining
SDS–PAGE and Western Blotting were performed as previously described [8] using 15% acrylamide gels. For electrophoresis, minigels of 1.5 mm thickness were used. Gels were loaded with 100 µg protein per lane. The concentration of the anti-TIMP-2 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was 1 µg/ml. TIMP-2 bands from Western Blots on X-ray films were quantified by laser scanning densitometry.

2.4 Preparation of cardiac tissue extract and zymography
Extracellular proteins of approximately 25 mg frozen tissue were extracted with 20-fold volume of extraction buffer (10 mM Tris–Cl pH 7.5, 150 mM NaCl, 20 mM CaCl₂, 1 µM ZnSO₄, 0.01% (v/v) Triton X-100, 1.5 mM NaN₃, 0.5% PMSF) over night at 4°C. These protein extracts contained approximately 1.5 mg/ml protein (BioRad Protein assay, BioRad, München, Germany).

Myocardial matrix metalloproteinase activity in the gel was measured as described by Tyagi et al. [12]. Gelatin (0.1% (w/v), Merck, Darmstadt, Germany) was added to standard Laemmli acrylamide polymerization mixture. Tissue extract was mixed 1:4 with substrate gel sample buffer (10% (w/v) SDS, 4% (w/v) sucrose, 0.25 mM Tris–Cl pH 6.8 and 0.1% (w/v) bromphenol blue), ca. 4 µg of extracted protein were loaded immediately without boiling. Gels were run at 20 mA at 4°C. Following electrophoresis, the gels were soaked in 2.5% (w/v) Triton X-100, incubated overnight at 37°C in substrate buffer (50 mM Tris–Cl pH 8, 5 mM CaCl₂ and 0.02% (w/v) NaN₃), stained for 15–30 min in 0.05% Coomassie Blue R-250 in acetic acid:methanol:water (1:4.5:4.5 by volume), destained in 10% acetic acid 5% methanol and scanned for lysis band intensity. The lysis band intensity was used to estimate the collagenase activity (semiquantified by reverse-image densitometry). A gelatinase zymography standard (human MMP-2 and -9, Chemicon, Hofheim, Germany) was used to detect the correct band.

2.5 Statistical analysis
All data were analyzed and expressed as mean±S.E.M. For evaluation of a statistical significance, one-way ANOVA was used with the multiple comparison procedure according to Tukey (using SigmaStat 2.0^®, Jandel Corporation). A value of P<0.05 was considered significant.

	3 Results

Top Abstract 1 Introduction 2 Material and methods 3 Results 4 Discussion Acknowledgments References

 
3.1 Hemodynamic measurement and cardiac hypertrophy
Hemodynamic parameters were measured between 1 day and 14 days during continuous i.v. infusion of NE. The changes of heart function occurred immediately after the start of infusion of NE and remained constant until 14 days of NE-infusion. The hemodynamic parameters of the RV changed more dramatically than those of the LV. The RV systolic pressure (RVSP) was more than twice as high as the time-corresponding controls (Table 1). RV dP/dt_max was nearly doubled during NE treatment (Table 1). In contrast to the RV, LVSP was only moderately and non-significantly elevated after NE-infusion (Table 2). LVSP was higher at 14 days in controls, and the elevation induced by NE was more, but also not significantly pronounced. Heart rate (HR) increased by 30%, LV dP/dt_max was more than doubled, and the mean aortic pressure (MAP) was not significantly increased (Table 2). Total peripheral resistance (TPR) was elevated after 4 days of NE stimulation by 46% from 0.425 to 0.664 mmHg·kg·min/ml. After 14 days, there was a non-significant elevation by 5% from 0.395 to 0.413 mmHg·kg·min/ml (Table 2).

View this table: [in this window] [in a new window]   Table 1 Changes in functional parameters of the right ventricle (RV) after 4 and 14 days of continuous intravenous infusion of norepinephrine (NE, 0.1 mg/kg·h)a

 

View this table: [in this window] [in a new window]   Table 2 Changes in functional parameters of left ventricle (LV) and of circulation after 4 and 14 days of continuous intravenous infusion of norepinephrine (NE, 0.1 mg/kg·h)a

 
NE caused a significant increase in the LV weight/body weight ratio (LVW/BW, Table 2). After 4 days there was a significant elevation by 27% and after 14 days by 47% (Table 2). Despite the pronounced elevations in RVSP and RV dP/dt_max, the development of hypertrophy of the RV was significantly delayed: After 14 days the RVW/BW ratio was significantly elevated by 24% (Table 1).

LV hypertrophy after 14 days of NE treatment was also confirmed by morphometric analysis of the myocyte diameter on histological sections (Fig. 1). The diameter of the control myocytes from the LV was 22% greater than that obtained from the RV. After NE stimulation, the diameter of myocytes from the LV was elevated by 29%, i.e. 43% greater than that from the RV. In the RV, there were no differences in the diameter of myocytes after NE stimulation.

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Mean myocyte diameter determined morphometrically in the left (filled bars) and right ventricle (open bars) of rats infused for 14 days with norepinephrine (NE, 0.1 mg/kg·h). Means±S.E.M.; *P<0.05 vs. the time-corresponding controls (CTRL); P<0.05 vs. right ventricle; number of measurements in parentheses.

 
3.2 mRNA expression in the right and left myocardium
The ratio between collagen III and I in controls and after NE stimulation was unchanged and was approximately 1.5 (Fig. 2 top and second from top). We observed a time-dependent and ventricle-specific elevation of both collagens after NE treatment. Controls were unchanged within the observation period of 14 days. After 4 days of NE infusion, the expression was increased in both ventricles, but more pronounced in the LV (for collagen I in LV: 23-fold, in RV: 9-fold; for collagen III in LV: 19-fold, in RV: 9-fold). After 14 days, the expression level of collagen I in both ventricles was nearly the same (LV: 0.33, RV: 0.31). Since, however, expression was lower in the control LV, there was a significant elevation in the LV after NE treatment. Collagen III mRNA was increased in both ventricles after 14 days of NE stimulation (LV: 3-fold, RV: 2-fold). The expression level of colligin was around half that of collagen I in controls (Fig. 2 bottom). There was a 5-fold increase in the LV and a 3-fold increase in the RV after 4 days NE-infusion. After 14 days, colligin was elevated only in the LV (2-fold).

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Relative abundance of left (filled bars) and right ventricular (open bars) collagen I (top), collagen III (second from top) and colligin (bottom) mRNAs at different time periods of norepinephrine infusion (NE, 0.1 mg/kg·h) obtained by RPA detection. Data are expressed as mRNA abundance relative to GAPDH mRNA expression. Means±S.E.M.; *P<0.05 vs. the time- and ventricle-corresponding controls (CTRL); P<0.05 vs. right ventricle; number of measurements in parentheses.

 
The mRNA expression of TIMP-2 was elevated in both ventricles after 3 and 4 days of NE-infusion (Fig. 3 top), but only in the LV after 14 days (1.6-fold). MMP-2 mRNA was increased after 3 days in both ventricles (LV: 3.1-fold, RV: 2.8-fold; Fig. 3 bottom), and was further elevated after 4 days (LV: 5.8-fold, RV: 4.4-fold). After 14 days, this mRNA was significantly higher only in the LV (2.2-fold). MMP-9 mRNA was hardly detectable with 5 µg total RNA and RPA. However, there was no increase between 1 day and 14 days (data not shown).

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Relative abundance of left (filled bars) and right ventricular (open bars) tissue inhibitor of metalloproteinase 2 (TIMP-2, top) and matrix metalloproteinase 2 (MMP-2, bottom) mRNAs at different time periods of NE infusion (0.1 mg/kg·h) from RPA detection. Data are expressed as fold stimulation over NaCl-infused controls, normalized to GAPDH mRNA. Means±S.E.M.; *P<0.05 vs. the time-corresponding controls; P<0.05 vs. the right ventricle after NE treatment; number of measurements in parentheses.

 
3.3 Cardiac MMP-2 activity and TIMP-2 protein abundance
The MMP-2 activity was elevated moderately. However, it was increased significantly after 3 and 14 days. The increase was more pronounced in the LV (Fig. 4). In the RV, the MMP-2 activity was elevated after 3 days, however, this increase was not statistically significant.

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 (A) Representative zymography from cardiac tissue extracts obtained after 3 days of norepinephrine infusion (NE, 0.1 mg/kg·h) in the left (LV) and right ventricle (RV). Each lane was loaded with 15 ng protein extract from the LV and RV from NaCl-infused control rats (NE; –) and animals with NE (+; 0.1 mg/kg·h) treatment. The matrix metalloproteinase (MMP)-standard contained human MMP-2 and MMP-9. (B) Relative abundance of LV (filled bars) and RV (open bars) MMP-2 activity at different time periods of NE infusion (0.1 mg/kg·h) obtained from zymography. The lysis band intensities were compared with the protein concentration. Data are expressed as fold stimulation over NaCl-infused controls, normalized to GAPDH mRNA. Means±S.E.M.; *P<0.05 vs. the time-corresponding controls; P<0.05 vs. the RV after NE treatment; number of measurements in parentheses.

 
The expression of TIMP-2 protein in the LV was not altered after 3 days, but was significantly increased after 4 days (Fig. 5).

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Representative Western Blotting analysis of immunoreactivity against tissue inhibitor of metalloproteinase 2 (TIMP-2, top) of heart extracts of the left (LV) and right (RV) ventricle from rats at 3 and 4 days of NaCl infusion and of norepinephrine infusion (NE, 0.1 mg/kg·h). Relative abundance of LV (filled bars) and RV (open bars) TIMP-2 protein level (bottom). Data are expressed as percent over NaCl-infused controls. Means±S.E.M.; *P<0.05 vs. controls; number of measurements in parentheses.

 
3.4 Cardiac fibrosis
Parallel sections were stained with Sirius red and with polyclonal antibodies to type I or III collagen (Fig. 6). After NE treatment, the LV had developed collagen protein deposition shown with Sirius red staining. Colocalization of types I and III collagen fibers was observed in all areas of collagen deposition. The ratio between the collagen types was not changed after NE stimulation, which was confirmed morphometrically. Morphometric analysis of fibrosis showed an elevation after NE treatment only for interstitial collagen deposition (Fig. 7). NE increased collagen deposition significantly from 0.7 to 1.1% in the LV after 14 days. The interstitial collagen fraction in the control RV was more pronounced than in the control LV. It was not changed in the RV after NE stimulation. The cardiac perivascular collagen content was greater than the interstitial collagen. However, it was not changed by NE treatment (1.7% vs. 1.2% in controls).

View larger version (150K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Representative staining of various collagen isoforms after norepinephrine (NE) treatment. Sections of hearts from a control rat and from a rat that had received continuous i.v. infusion of norepinephrine (NE, 0.1 mg/kg·h) for 14 days were stained with sirius red (total collagen) or with specific anti-collagen I or anti-collagen III antibodies. Magnification x100.

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Interstitial collagen content in the myocardium estimated by morphometry at a final magnification of x100 in the left (filled bars) and right ventricle (open bars) of control rats (CTRL) and of rats infused with norepinephrine (NE, 0.1 mg/kg·h) for 14 days. Means±S.E.M.; *P<0.05 vs. the time-corresponding controls; P<0.05 vs. the right ventricle; number of measurements in parentheses.

 
3.5 Effects of - and β-adrenergic and calcium-channel blockade
Expression of TIMP-2 and MMP-2 mRNA after adrenergic blockade was measured after 3 days. Both mRNAs showed a significant elevation after NE infusion at that time point. This was prevented when the - and β-receptor antagonist carvedilol was infused together with NE (NE-C, Fig. 8). Fig. 8 presents the results as abundance of the mRNA relative to GAPDH, in contrast to Fig. 3, where the x-fold stimulation over control was shown. There was no difference between the different controls in the mRNA level. Simultaneous calcium-channel blockade with nisoldipin for 3 days reduced the TPR in NE-infused rats to control levels. Despite normalization of TPR, LV hypertrophy was still present to a degree similar to that elicited by NE infusion alone [8]. In accordance with these results, nisoldipin did not affect the NE-induced increase in TIMP-2 and MMP-2 mRNA (Fig. 8).

View larger version (55K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Relative abundance of left (filled bars) and right ventricular (open bars) tissue inhibitor of metalloproteinase 2 (TIMP-2, top) and matrix metalloproteinase 2 (MMP-2, bottom) mRNAs in control rats (CTRL) and in rats with norepinephrine infusion (NE, 0.1 mg/kg·h) for 3 days. NE was combined with carvedilol (NE-C, 0.5 mg/kg·h; respective control: CTRL-C). or with nisoldipine (NE-Nis, 0.1 mg/kg·h; respective control: CTRL-Nis). Data were obtained from RPA detection and are given as mRNA abundance relative to GAPDH mRNA expression. Mean values±S.E.M. *P<0.05 vs. the time-corresponding controls; P<0.05 vs. the NE treatment; number of measurements in parentheses.

 

	4 Discussion

Top Abstract 1 Introduction 2 Material and methods 3 Results 4 Discussion Acknowledgments References

 
The results of this study show that 14 days of NE infusion induced differential changes in LV and RV function which were similar to those shown previously for 3 days of NE administration [8]. The different inotropic sensitivity of both ventricles to catecholamines was already found in isolated canine hearts [13]. In this study the maximal hemodynamic effect was obtained with 340 µg of NE. The elevation of RVSP was more pronounced (248%) than that of the LVSP (61%). The different response of systolic pressure was also found in intact dogs by sympathetic nerve stimulation [14]. RVSP was elevated by 62% and LVSP by 10% during right cardiac sympathetic nerve stimulation. This difference was also seen in rats with acute intravenous NE infusion [15]. RVSP was increased by 139% and LVSP by 10% with infusion of 0.1 mg/kg·h NE. A possible explanation may be seen in the different ⁴⁵Ca uptake which was induced by isoproterenol. It was more pronounced in the RV than in the LV [16].

The differential hypertrophic response of the LV and RV was also found in dogs [17]. LVW/BW was elevated by 21% after 28 days of subcutaneous (s.c.) application of NE (0.03 mg/kg·h) by osmotic mini pumps. RVW/BW was non-significantly elevated by 10%. Similar results were obtained in rats. Chronic treatment with NE caused LV, but not RV hypertrophy at different doses for varying time periods of NE application [18–21]. The elevation of the LVW/BW ratio was not the result of NE-induced edema, since the ratio was increased to the same level by NE when the dry weight of the heart was used for the calculation of the LVW/BW ratio [19]. When the NE concentration or the treatment time were increased, there was also an elevation in the RVW/BW ratio to a degree similar to that found in the present study (Table 1) [19,21].

A second independent parameter for the detection of the NE-induced hypertrophy of the LV was the morphometric measurement of the myocyte diameter after 14 days of NE treatment (Fig. 1). In agreement with the results of previous studies [22] the LV myocyte diameter was significantly greater than that of RV myocytes in control hearts. After 14 days of NE stimulation, the LV myocyte diameter was elevated to a similar degree (Fig. 1) as the increase in the LVW/BW ratio (Table 2).

In a previous study, the collagen mRNA expression was analyzed by northern blotting up to 3 days of NE stimulation [8]. Now we have measured the mRNA abundance exclusively by RPA. The results from both measurements are comparable. However, the advantage of the RPA method is that mRNA concentration can be determined as abundance relative to GAPDH (Fig. 2). Therefore it was possible to determine the ratio of type III/type I collagen. This was unchanged in both ventricles after 4 and 14 days of NE stimulation and amounted to 1.5. The ratio was also unchanged on the protein-level, which was confirmed on collagen type I and III stained sections (Fig. 6). However, the collagen message of both ventricles was different. The mRNA was lower in the LV in comparison to the RV, especially of collagen I from 14 days controls (Fig. 2). This is in accordance with the lower morphometrically measured extent of collagen fraction in the LV of control hearts (Fig. 7). A lower collagen content of interstitial collagen area in the LV or fewer pepsin-insoluble collagenous protein in the LV was obtained also by other authors [23,24].

After NE treatment, the most dynamic phase of ECM remodeling on the mRNA level was confined to the first 3 to 4 days (Fig. 2). Colligin mRNA showed a similar time course. It is also known as heat shock protein 47 (Hsp47) and is the chaperone for various collagen types, which is involved in the intracellular folding and/or assembly of procollagen [25]. Previously we have shown that the expression of colligin after NE treatment started earlier than that of collagen I and was more pronounced on the protein level [8]. The elevated collagen mRNA level after NE infusion was followed by the histologically detected increased collagen fraction in the LV, but not in the RV (Fig. 7). The NE-induced fibrosis is not comparable with that found in experimental hypertension due to aldosterone or renovascular hypertension. In these experimental models there was a rise in the interstitial collagen volume fraction and in the perivascular collagen area. It was found not only in the hypertrophied left ventricle, but also in the normotensive, non-hypertrophied right ventricle [26]. In contrast, the NE-induced fibrosis was confined to the LV and affected predominantly the interstitial collagen.

The mRNA expression of TIMP-2 and MMP-2 was increased in a time-dependent fashion after NE treatment (Fig. 3), and was accompanied by an elevation of MMP-2 activity. This could be a result of initial induction of remodeling of the ECM, which adapted after 14 days. The increase of MMP-2 activity in the RV after NE treatment could also have prevented the overproduction of collagen in the RV (Fig. 2), so that fibrosis did not develop in the RV (Fig. 7). The attenuated activity of MMP-2 after 4 days of NE stimulation seems to be the result of elevated protein level of its inhibitor TIMP-2 at this time point (Fig. 5). Several studies have demonstrated increased MMP expression and abundance in experimental models of chronic heart failure and with end-stage cardiomyopathic disease in humans [27]. Recently it was shown that increased MMP-2 expression in patients with dilated cardiomyopathy is associated with elevated plasma NE level [28]. The increased MMP-2 expression seems to be a sign for the remodeling process which is accompanied by fibrosis of the failing heart.

The NE-induced elevation of mRNA levels of MMP-2 and TIMP-2 seems to be due to a direct stimulation of cardiac adrenoceptors. This conclusion is strengthened by the finding that the expression of MMP-2 and TIMP-2 was prevented by - and β-receptor blockade with carvedilol and was still elevated when the NE-induced afterload increase was normalized by nisoldipin (Fig. 8). This result is in accordance with the previous finding that carvedilol normalized the NE-induced functional changes and prevented the development of LV hypertrophy, while nisoldipin did not influence the NE-induced LV hypertrophy despite normalization of TPR.

In summary, long term NE treatment induced fibrosis exclusively in the LV which was associated with hypertrophy predominantly of the LV. The fibrosis after 14 days of NE infusion seems to be the result of an elevated mRNA level of collagen I and III which was observed after 4 days. The collagen type III to type I ratio was unchanged at 1.5. This study has shown for the first time, that NE induced elevated MMP-2 and TIMP-2 mRNA expression as well as increased MMP-2 activity in rat hearts by stimulation of cardiac adrenergic receptors. These results indicate the significance of MMPs for the regulation of remodeling of the ECM after NE treatment.

Time for primary review 42 days.

	Acknowledgments

Top Abstract 1 Introduction 2 Material and methods 3 Results 4 Discussion Acknowledgments References

 
This work was supported by the Deutsche Forschungsgemeinschaft (ZI 199/10-3). The GAPDH-probe was supplied by Dr K. Eschrich (Leipzig, Germany), and the ARPP probe by Dr G. Sparmann (Rostock, Germany). The technical assistance of Grit Marx is gratefully appreciated.

	References

Top Abstract 1 Introduction 2 Material and methods 3 Results 4 Discussion Acknowledgments References

 

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