Cardiovascular Research

Cardiovascular Research 1997 35(2):223-232; doi:10.1016/S0008-6363(97)00130-2
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Effect of AT₁ receptor blockade on cardiac collagen remodeling after myocardial infarction

Haisong Ju, Shufang Zhao, Davinder S Jassal and Ian M.C Dixon^*

Molecular Cardiology Laboratory, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Faculty of Medicine, University of Manitoba, 351 Tache Avenue, Winnipeg, Man. R2H 2A6, Canada

^* Corresponding author. Tel.: +1 (204) 235-3419; fax: +1 (204) 233-6723; e-mail: iand@sbrc.umanitoba.ca

Received 1 November 1996; accepted 1 May 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Previous work has shown that cardiac fibrosis occurs after myocardial infarction (MI) in non-infarcted ventricular tissue and that this event is associated with abnormal cardiac function. Our aim was to investigate the effect of AT₁ receptor blockade on cardiac collagen remodeling in post-MI rat heart remote from the infarct site by addressing collagen mRNA abundance, posttranslational hydroxylation of collagen monomers, and mature collagen deposition. Prolyl 4-hydroxylase (PH) mediates hydroxylation of procollagen -chains in the endoplasmic reticulum of cardiac fibroblasts and thus regulates the downstream formation and secretion of helical procollagen molecules. Methods: The effects of losartan (15 mg/kg/day) on collagen deposition and mRNA abundance were monitored in viable left and right ventricles in sham-operated (control) and experimental groups in the presence or absence of losartan. Immunoreactive PH concentration in viable tissues as well as cardiac function in control and experimental groups was determined by ELISA. Results: Immunohistochemical staining and 4-hydroxyproline assays confirmed that losartan treatment attenuates fibrosis in experimental hearts. Northern analysis revealed that losartan treatment of 1, 2, or 4 week experimental groups had no effect on collagen mRNA abundance compared to untreated post-MI rats. On the other hand, immunoreactive PH concentration was significantly decreased in the post-MI group treated with losartan. Determination of cardiac mass and cardiac function revealed that losartan treatment was associated with attenuated cardiac hypertrophy and improved left ventricular (LV) function in experimental animals. Conclusions: AT₁ blockade is associated with a significant decrease in cardiac fibrosis in treated post-MI rats, and this trend is positively correlated to a significant decrease in immunoreactive PH compared to untreated experimental animals. The expression of cardiac PH may be regulated by angiotensin via AT₁ receptor activation, and the suppression of PH with losartan treatment may be an important mechanism for modulation of collagen deposition in the post-MI rat heart.

KEYWORDS Collagen remodeling; Gene expression; Myocardial infarction; Prolyl 4-hydroxylase; Immunohistochemistry; Losartan; Rat, ventricle

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Myocardial infarction (MI) is associated with cardiac fibrosis not only in the scar but also in the viable tissues remote from the infarct. Results of either experimental studies from this laboratory [1] and others [2, 3], or those from clinical investigation [4], have provided evidence for increased deposition of total collagen proteins in left ventricle (LV) remote from the infarct. It has been suggested that cardiac fibrosis plays a role in the development of congestive heart failure in post-MI heart [5–7]. It is known that excessive deposition of collagen proteins may impair heart function due to morphological and functional separation of myocytes with subsequent inhibition of electrical coupling and increase of oxygen diffusion distance which leads to hypoxia [8, 9]. Increased deposition of collagens contributes to increased cardiac muscle stiffness and may affect diastolic function [10]. Little information is available regarding regulation of posttranslational collagen metabolism in cardiac remodeling after MI [11]. The posttranslational regulation of fibrillar collagens is complex; prolyl 4-hydroxylase (PH) is a key enzyme for the posttranslational processing of procollagen -chains, as it catalyzes the hydroxylation of specific proline residues on the -monomers, thereby facilitating the formation of the triple-stranded helical procollagen molecules and their subsequent secretion to the extracellular space [12–14]. Fibroblasts and phenotypically transformed fibroblast-like cells (i.e., myofibroblasts) exist within both remote surviving myocardium and scar tissues; while the latter type may be the primary contributor for mediation of wound healing, both cell types have been shown to express angiotensin converting enzyme (ACE) after MI and are thereby equipped to generate angiotensin II (AII) from angiotensin I [3, 15, 16]. Furthermore, AT₁ receptor represent the predominant AII receptors subtype in cardiac fibroblasts, which are known to be responsive to hormonal signals mediated by AII [17]. Cardiac fibroblasts, composed of various phenotypes, account for 70% of the total cell population and are the exclusive sites for synthesis of fibrillar collagens, which made up 90% of the total matrix proteins in the heart [18]. Previous findings have shown that there is a transient increase in DNA synthesis in the non-myocyte cell population within surviving myocardium after MI [19]. Some recent evidence supports the hypothesis that AII is involved in the stimulation of cardiac fibrosis in post-MI hearts. Morphological studies indicate that administration of both captopril (ACE inhibitor) [19, 20]and losartan (an AT₁ receptor antagonist) [21, 22]were associated with the inhibition of cardiac fibrosis in post-MI hearts. However, little information is available to address the effect of losartan on steady-state collagen mRNA abundance or posttranslational regulation of collagen synthesis in post-MI heart. Therefore, we investigated the effect of losartan on cardiac fibrillar collagen profile, fibrillar collagen mRNA abundance, and immunoreactive cardiac PH concentration in both LV and RV at sites remote from the infarct. To provide some information about the relationship between cardiac collagen deposition and LV function in these post-MI hearts, we assessed the effect of losartan on several hemodynamic parameters in experimental animals.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Experimental model
All experimental protocols for animal studies were approved by an appointed Animal Care Committee located at the University of Manitoba, Canada, following guidelines established by the Medical Research Council of Canada. MI was produced in male Sprague-Dawley rats (weighing 200–250 g) by surgical occlusion of the left coronary artery as described previously with minor modifications [23]. The mortality of all animals operated upon in this fashion was about 45% within 48 h. Post-operated animals were divided into 3 groups: group 1, sham-operated animals; group 2, MI; and group 3, MI rats treated with losartan (15 mg/kg/day) [21]. All losartan treatment regimens were initiated 1 day following coronary occlusion by implanting an osmotic mini-pump and continued for 1, 2, and 4 weeks. To achieve the 4-week treatment, 2-week duration Alzet osmotic mini-pumps (Alza Corporation, La Jolla, CA; model 2002) were implanted consecutively. For comparative purposes, sham-operated controls (group 1) and MI animals were administered vehicle (0.9% saline) using the same method. After 1, 2, and 4 weeks drug or vehicle infusion, the animals underwent LV functional assessment and infarct size determination; then the viable left and right ventricular tissues were used to assess collagen protein profile, fibrillar collagen steady-state mRNA abundance, and immunoreactive PH concentration.

2.2 Hemodynamic measurements
LV function and mean arterial blood pressure (MAP) of sham-operated control, MI, and MI+losartan groups were measured at 2 and 4 weeks following induction of MI, as described previously [1, 23]. Briefly, rats were anesthetized by intraperitoneal injection of a ketamine/xylazine mixture (100 mg/kg: 10 mg/kg). A micromanometer-tipped catheter (2-0) (Millar SPR-249) was inserted into the right carotid artery. The catheter was advanced into the aorta to determine MAP, and then further advanced to the left ventricular chamber to record left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and rate of contraction and relaxation (±dP/dt). Hemodynamic data were computed instantaneously and displayed using a computer data acquisition workstation (Biopac, Harvard Apparatus Canada). A total of 48 rats were included in the hemodynamic assessment.

2.3 Infarct size
After heart function was recorded, the LV was fixed by immersion in 10% formalin and embedded in paraffin. Six transverse ‘breadloaf’ slices were cut from the apex to the base. Serial sections (5 µm) were made and mounted; the percentage of infarcted left ventricle was estimated at 1, 2, and 4 weeks after coronary ligation by planimetric techniques as described previously [24]. Only animals (n = 62) with large infarcts (40% of the left ventricular free wall) were used in the current study.

2.4 Determination of cardiac total collagen
Viable left and right ventricles (n = 54) from sham-operated, MI, and MI treated with losartan groups were separated, and the tissue was ground into powder in liquid nitrogen. Then 100 mg (wet weight) cardiac tissue was dried to constant weight. Tissue samples were digested in 6 M HCl (0.12 ml/1 mg dry weight) for 16 h at 105°C. Hydroxyproline was measured according to the method of Chiariello et al. [25], and modified by Pelouch et al [1]. A stock solution containing 40 mM of 4-hydroxyproline in 1 mM HCl was used as a standard. Collagen concentration was calculated multiplying hydroxyproline levels by a factor of 7.46, assuming that interstitial collagen contains an average of 13.4% hydroxyproline. The data were expressed as µg collagen per mg dry tissue [25].

2.5 Immunohistochemistry
A total of 28 rats at 2 and 4 weeks after induction of MI were used in this assay; 4 sham, 5 post-MI and 5 MI+losartan. The viable left ventricle remote from the infarct and right ventricle were immersed in OCT compound and stored frozen at –80°C. Serial cryostat LV and RV sections of 7 µm thickness were mounted on gelatin-coated slides, prefixed in 1% paraformaldehyde, and allowed to air-dry. A minimum of 6 sections from each ventricle of each group were processed and representative sections were chosen. Immunohistochemical staining was performed by the indirect immunofluorescence technique [26]. In brief, the tissue section was incubated with goat polyclonal anti-types I and III collagen (Southern Biotechnology Associates Inc., Alabama, USA), diluted 1:100 with 1% BSA in PBS and applied as the primary antibodies. After incubation overnight at 4°C, the sections were incubated with biotinylated anti-goat IgG secondary antibody for 90 min. Sections were then incubated with Texas-Red-labeled streptavidin and FITC-labeled streptavidin to detect collagens I and III, respectively. Finally, the slides were mounted and coverslipped. The results were recorded by photography on Kodak T-MAX 400 black and white film. Quantification of resulting image data from immunohistochemical staining was performed using an automated image analysis software (SigmaScan Pro).

2.6 RNA extraction and Northern blot analysis
Total RNA was isolated from viable left and right ventricle by the method of Chomczynski and Sacchi [27] at 1, 2, and 4 weeks after operation (n = 52). Recovered RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water and the concentration of nucleic acid was calculated from the absorbance at 260 nm prior to size fractionation. Twenty micrograms of total RNA was electrophoresed in a 1.2% agarose/formaldehyde gel and the fractionated RNA was transferred to a 0.45 µm positive charge-modified nylon membrane (NYTRAN Plus, Schleicher and Schuell). RNA species were covalently crosslinked to the membrane using UV radiation (UV Stratalinker 2400, Stratagene). Blots were prehybridized at 42°C for 6–16 h. Each membrane was hybridized with cDNA probes labeled (³²P) using a random primer labeling kit (specific activity >10⁹ cpm per mg DNA) at 42°C for 16–20 h. After washing, the membranes were exposed to X-ray film (Kodak X-OMAT) at –80°C with intensifying screens. cDNA fragment for human procollagen type a1(I) (Hf 677) [28], human type a1(III) (Hf 934) [29], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [30]were obtained from the American Type Culture Collection. Rat 18S rRNA (5'- ACGGTATCAGATCGTCTTCGAACC-3') [31] was synthesized using the Beckman Oligo 1000 DNA synthesizer. Results of autoradiographs from Northern blot analysis were quantified by densitometry (Bio-Rad imaging densitometer GS 670). The signals of specific mRNAs were normalized to those of GAPDH and 18S to normalize for differences in loading and/or transfer of mRNA.

2.7 Enzyme immunoassay for prolyl 4-hydroxylase
Non-infarcted LV (n = 36) were ground into powder under liquid nitrogen. Powdered tissues (20 mg/1 ml) were homogenized in 10 mM Tris-HCl buffer (pH 7.8) containing 0.1 M NaCl, 0.1 M glycine, 0.1% Triton X-100, 20 mM ethylenediaminetetraacetic acid (EDTA), 10 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM p-hydroxymercuribenzoic acid, 1 mM dithiothreitol (DTT). The homogenized samples were centrifuged 20 000xg at 4°C for 30 min. The supernatants were transferred to fresh Eppendorff tubes and then used for PH assay, employing an ELISA kit (Fuji Chemical Industries, Ltd., Toyama, Japan) [32]. Briefly, this assay employs two monoclonal antibodies wherein the first is used as a capture antibody in solid phase and the other antibody is linked to horseradish peroxidase. Myocardial samples were diluted 1:20 in distilled water prior to the total protein concentration assay using the bicinchoninic acid solution (BCA) kit (Sigma, St. Louis, USA) [33].

2.8 Statistical analysis
All values are expressed as mean±s.e.m. One-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test was used for comparing the differences among control, MI and MI treated with losartan at each time point (SigmaStat). Significant differences among groups were defined by a probability of less than 0.05. The Northern blot data in each figure were expressed as a percentage of control according to the method of Fisher and Periasamy [34].

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Effect of AT₁ receptor blockade on infarct size and cardiac hypertrophy
There was no significant increase in left ventricular weight (LVW) or right ventricular weight (RVW) in 1-week post-MI hearts. Thus, the increased ratios of LVW and RVW to body weight (BW) observed in experimental animals 1 week after MI was due to decreased BW. The 2- and 4-week experimental groups exhibited a significant increase in the mass of RV and non-infarcted LV tissue when compared to values from sham-operated hearts (Table 1, noted by the indices of LVW, RVW, RV/BW ratio, and the LV/BW ratios). Losartan treatment of experimental animals for 4 weeks was associated with the complete prevention of both LV and RV hypertrophy. It should be pointed out that the administration of losartan to infarcted experimental groups had no effect on infarct size.

View this table: [in this window] [in a new window]   Table 1 Effect of losartan on cardiac hypertrophy and infarction size in experimental rats after 1, 2 and 4 weeks treatment

 
3.2 Effect of AT₁ receptor blockade on cardiac function
Loss of normal LV function was apparent at 2 weeks post-MI, as indicated by a decrease in ±dP/dt and an increase of LVEDP in the MI group when compared with sham-operated animals. Treatment with losartan had no effect on heart rate (HR) or on LVSP in either 2- or 4-week experimental groups (Table 2). On the other hand, 2-week treated post-MI animals with were characterized by significantly decreased MAP and LVEDP compared to values from the untreated group. Administration of losartan for 4 weeks was also associated with decreased MAP and LVEDP, as well as significantly increased ±dP/dt vs. untreated post-MI animals (Table 2).

View this table: [in this window] [in a new window]   Table 2 Effect of losartan (15 mg/kg/day) on hemodynamic characteristics in experimental rats after 2 and 4 weeks treatment

 
3.3 Effect of AT₁ receptor blockade on total collagen and immunohistochemical staining of collagen types I and III
Total cardiac collagen concentration in surviving LV at 2 and 4 weeks was increased by 91.3% (P<0.05) and 150.4% (P<0.05), respectively. Similarly, total collagen concentration in right ventricular samples from 2- and 4-week experimental groups were increased by 66.4% (P<0.05) and 77.6% (P<0.05), respectively. Losartan treatment was associated with significantly decreased total collagen concentrations in both viable LV and RV (32.7% and 26.8%, respectively) at 4 weeks after the induction of MI (Fig. 1). Immunohistochemical staining of collagen types I and III and these proteins were visualized by epifluorescence microscopy. We used longitudinal sections to view collagen type I and cross-sectional views to show collagen type III according to the method of Schaper et al. [8]. Relatively low levels of collagen (bright, wavy appearance) are present in the interstitial space in those sections of sham-operated rats in both viable left and right ventricles. In 2- and 4-week experimental groups, the interstitium of viable left ventricles remote from the infarct were characterized by markedly increased deposition of collagen type I (Fig. 2A,B, Fig. 3A,B). The 4-week losartan-treated group was associated with a 47% inhibition of collagen type I accumulation (Fig. 3C) but was associated with no effect after only 2 weeks treatment (Fig. 2C). Similarly, collagen type III was found to be present throughout the extracellular matrix and was arranged in a true network in the extracellular space (Figs. 2 and 3). Collagen type III was increased in viable LV when compared to control at 2 and 4 weeks post-MI (Fig. 2 and Fig. 3D,E). Collagen type III in the viable left ventricle was reduced by 33% with the administration of losartan for 4 weeks vs. untreated experimental animals (Fig. 3F).

View larger version (61K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effect of losartan (15 mg/kg/day) on total collagen concentration in viable left (LV) and right ventricles (RV) at different times after myocardial infarction (MI). All losartan treatment (2 and 4 weeks) regimens were initiated 1 day following coronary occlusion. The data depicted are the mean±s.e.m. of 8–10 experiments and were expressed as µg/mg dry weight. *P<0.05 and #P<0.05 vs. control and MI, respectively.

 

View larger version (108K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Representative immunohistochemical staining of collagen types I and III in viable left ventricular tissues 2 weeks post-MI. A, B, and C are collagen type I from sham-operated animals, myocardial infarction (MI) and MI treated with losartan for 2 weeks respectively. D, E, and F are collagen type III from sham-operated animals, myocardial infarction (MI) and MI treated with losartan for 2 weeks, respectively. All losartan treatment regimens were initiated 1 day following coronary occlusion. Collagen type I appears as wavy fibers between myocytes and collagen type III appears as brightly stained material between myocytes. Magnification x400.

 

View larger version (120K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Representative immunohistochemical staining of collagen types I and III in viable left ventricular tissues 4 weeks post-MI. A, B, and C are collagen type I from sham-operated animals, myocardial infarction (MI) and MI treated with losartan for 4 weeks, respectively. D, E, and F are collagen type III from sham-operated animals, myocardial infarction (MI) and MI treated with losartan for 4 weeks, respectively. All losartan treatment regimens were initiated 1 day following coronary occlusion. Collagen type I appears as wavy fibers between myocytes and collagen type III appears as brightly stained material between myocytes. Magnification x400.

 
3.4 Effect of AT₁ receptor blockade on mRNA abundance of collagen genes
Verification of the integrity of total fractionated RNA samples is provided by visualization of the 28S and 18S rRNA bands in a representative photograph of an agarose gel stained with ethidium bromide (Fig. 4, upper panel). Specific hybridization of cDNA probes revealed characteristic mRNA bands, and these are shown in autoradiographs of representative blots probed with cDNAs of collagen types I, III, GAPDH and 18S (Fig. 4, bottom panel). The calculated ratios for collagen/GAPDH and collagen/18S were performed with similar results. For example, the absolute ratios of collagen type I to GAPDH were 0.188±0.01 for control, 1.633±0.17 for MI, and 1.67±0.24 for MI treated with losartan for 1 week in LV. All subsequent calculated ratios of collagen type I/GAPDH and collagen type III/GAPDH are shown as a percentage of control (control=100%) in Fig. 5. In the viable LV, losartan treatment was not associated with any difference in either collagen type I and III mRNA abundance vs. untreated post-MI values at 1, 2, and 4 weeks (Fig. 5A,B). Similarly, normalized values of RV fibrillar collagen mRNA abundance in experimental animals treated with losartan were not different from untreated experimental animals (Fig. 5C,D). While collagen type III expression was elevated in both LV and RV at all times after the induction of infarction, the relative increases in expression of collagen type III mRNAs were less marked than the increases in collagen type I mRNAs.

View larger version (62K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Upper panel: A representative agarose gel stained with ethidium bromide to visualize the 28S and 18S rRNA bands in total RNA samples extracted from viable left ventricular tissues at 4 weeks after MI. Lower panel: Autoradiograph from Northern blot analysis wherein each lane was loaded with 20 µg total RNA extracted from sham animals (lanes 1–5), myocardial infarction (MI, lanes 6–10) and MI treated with losartan (lanes 11–15) for 4 weeks after MI. All losartan treatment regimens were initiated 1 day following coronary occlusion. Hybridization of fractionated total RNA with cDNA probes for collagen types I and III, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S indicate relative steady-state mRNA levels for each gene tested.

 

View larger version (48K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of losartan (15 mg/kg/day) on collagen mRNA abundance in viable left (LV) and right ventricles (RV) at different times after myocardial infarction (MI). (A) Collagen type I/GAPDH signal ratio in LV. (B) Collagen type III/GAPDH signal ratio in LV. (C) Collagen type I/GAPDH signal ratio in RV. (D) Collagen type III/GAPDH signal ratio in RV. All losartan treatment regimens were initiated 1 day following coronary occlusion. The data were expressed in densitometric units, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) autoradiographic band intensity and noted as a percent value of control expression levels (100%). The data depicted are the mean±s.e.m. of 5–6 experiments. *P<0.05 for each MI value vs. sham-operated values.

 
3.5 Effect of AT₁ receptor blockade on cardiac prolyl 4-hydroxylase concentration
We observed an increase in immunoreactive cardiac PH concentration in viable LV from 2- and 4-week experimental groups of 68.4 and 68.1%, respectively, compared to control animals. Two-week (short-term) losartan treatment of experimental animals was observed to have no effect on PH concentration vs. values from the untreated 2-week post-MI group. On the other hand, 4-week AT₁ blockade was associated with a significant decrease in PH concentration (P<0.05) in the viable LV (Fig. 6).

View larger version (59K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of losartan (15 mg/kg/day) on prolyl 4-hydroxylase (PH) concentration in viable left ventricles in (i) sham-operated animals, (ii) myocardial infarction (MI) and (iii) MI treated with losartan for 2 and 4 weeks. All losartan treatment regimens were initiated 1 day following coronary occlusion. The data depicted are the mean±s.e.m. of 6 experiments and were expressed as µg/mg protein. *P<0.05 and #P<0.05 vs. control and MI, respectively.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Ventricular remodeling in post-MI rat heart has been associated with increased concentration of AII in the infarct zone [35] and with increased AII receptor density in the non-infarcted myocardium [36]. The receptor population for AII is classified into two major subtypes: i.e., either AT₁ or AT₂, based on the binding affinities for the non-peptide antagonists, losartan and PD123177, respectively [37]. A great deal of work to date has shown that the cardiac AT₁ receptors mediate the majority of known physiological effects of AII in heart [38]. The rationale for conducting the current study using AT₁ receptor antagonism is based on findings to support the hypothesis that the AT₁ receptor subtype is the primary AII receptor in cardiac fibroblasts [39], and that this receptor specifically mediates simulation of collagen synthesis by AII in these cells [40]. Furthermore, a recent study has shown that the AT₂ antagonist, PD123319, had no effect on cardiac function or on interstitial fibrosis in post-MI rats with heart failure [41]. The prevention of cardiac hypertrophy in experimental animals from the current study after 4-week losartan treatment is in agreement with previous findings [21]. This result provides further support for the involvement of AII in the hypertrophic process via activation of the AT₁ receptor. Inhibition of cardiac hypertrophy may be related to (i) attenuation of cardiac myocyte growth and/or (ii) to the inhibition of cardiac fibroblast replication [21], as well as to the reduction of extracellular matrix protein accumulation. We have also shown that 4-week losartan treatment of post-MI rats was associated with prevention of the development of LV dysfunction, in agreement with previous studies [24, 42]. Specifically, losartan treatment of post-MI animals was associated with prevention of abnormally elevated LVEDP, possibly due to the preservation of normal cardiac contraction and relaxation.

Administration of losartan for 2 weeks to experimental animals was associated with modest differences in the deposition of total collagens in RV, while this regimen had no apparent effect on collagen concentration noninfarcted LV samples vs. untreated animals. Although others have reported significant inhibition of total collagen deposition in viable LV post-MI with the same dosage and duration of losartan treatment [21], it is possible that this discrepancy at very early points may be due to methodological differences employed for cardiac collagen detection. In contrast to 2-week therapy of experimental animals, 4-week losartan treatment was associated with partial prevention of fibrosis in experimental animals compared to the untreated animals in both LV and RV samples. Our findings with respect to immunohistochemical studies of fibrillar collagens in noninfarcted muscle yielded similar results. Thus, the incidence of fibrosis in this experimental model may be mediated by activation of the AT₁ receptor localized on adult cardiac fibroblasts. The precise mechanism for the antifibrotic action of losartan in RV and LV after induction of MI is unclear. In this regard, inhibition cardiac fibroblast proliferation and/or inhibition the synthesis of collagen may be considered as possible mechanisms. Although AII may directly stimulate DNA, RNA, and protein synthesis in cultured adult cardiac fibroblasts via AT₁ receptor activation [40], previous work has shown that increased cardiac fibroblast proliferation in non-infarcted post-MI heart is a transient event (peak proliferation at 1 week after MI) [19]. Furthermore, losartan treatment of post-MI rats was shown to lead to only slight attenuation of non-myocyte proliferation (including endothelial cells and fibroblasts) 2 weeks after induction of MI [21]. Thus it is unlikely that cardiac fibroblast proliferation plays an major role in the attenuation of cardiac fibrosis losartan in the rat model of chronic MI. As the mRNA abundance of cardiac fibrillar collagens does not appear to be influenced by losartan treatment of any duration in this experimental model, our findings suggest that the partial prevention of collagen accumulation by AT₁ blockade may be mediated at other point(s) within cardiac collagen synthesis. It is pointed out that the current data do not rule out possible involvement of collagen transcriptional events with losartan treatment. Nevertheless, our current results are in agreement with a recent report [43] which demonstrated that treatment of rats with a novel AT₁ receptor antagonist (TCV-116) was not associated with any normalization of elevated collagen mRNA abundance after the induction of MI. In contrast, others have shown that TCV-116 may significantly inhibit cardiac collagen type I and III mRNA abundance in stroke-prone, spontaneously hypertensive rats (SPSHR) [44]. Although the precise mechanism(s) for the differential responses of steady-state mRNA levels in rats with MI and those with spontaneous hypertension to AT₁ blockade remain unclear, they may be linked to the type and stage of disease. Presumably, AII is not the sole stimulus for the induction of cardiac fibrosis. Thus SPSHR and post-MI rat heart may be subject to unique hormonal and mechanical stimuli (i.e., the presence or absence of hypertension), resulting in a disease-specific response to any given therapy. As the dose of TCV-116 associated with inhibition of collagen mRNA abundance is attended by significant reduction of systolic blood pressure [44], the effect of TCV-116 in SPSHR may be related to normalization of blood pressure.

As we have shown a partial prevention of collagen deposition with AT₁ blockade, other factors such as transforming growth factor β1 (TGF-β1) may be involved in the regulation of collagen expression [45] in post-MI heart, although the role of this factor is not well understood in relation to the pathogenesis of heart failure and increased AII generation. Second, as both left and right ventricular chambers are subject to altered preloading and afterloading of post-MI hearts, it was hypothesized that hemodynamic loading is involved in the regulation of collagen mRNA expression [46]. Nevertheless, as losartan treatment has been associated with preservation of normal cardiac preload and afterload without corresponding normalization of collagen mRNA abundance in non-infarcted post-MI hearts, it is possible that other factor(s) may regulate fibrillar collagen expression.

A discrepancy between collagen gene activation and collagen deposition has been reported in aging rats [47]. In a previous study, we also noted that collagen mRNA abundance was at a relative maximum 1 week after MI, but that collagen protein accumulated in a progressive manner in this experimental model [7] and it is suggested that this phenomenon may be due to the involvement of moieties that mediate posttranslational modification(s) of collagen (i.e., hydroxylation and crosslinking steps) required for collagen synthesis. It has been reported that a significant proportion (36–96%) of newly synthesized procollagen monomers are routinely degraded upon their translation in both normal and hypertrophic hearts [12, 48]. Thus, it is possible that the rate of intracellular procollagen degradation is controlled by posttranslational processing and that any alteration in protein modification may affect collagen deposition in vivo. The increase of immunoreactive PH in viable heart muscle may be a major mechanism for increased deposition of collagen in post-MI heart. We found that losartan treatment prevented the elevation of PH protein concentration in LV from experimental animals which correlated with the inhibition of total collagen protein in treated hearts. This finding supports the hypothesis that the antifibrotic effect of losartan is mediated via the inhibition of PH. This study also provided evidence that AII may be involved in modulation of posttranslational regulation of cardiac collagen in post-MI myocardium.

In summary, our results indicated that AII is involved in the development of cardiac hypertrophy and cardiac fibrosis in post-MI rat heart. Secondly, losartan treatment was not associated with any effect on increased steady-state fibrillar collagen mRNA abundance in ventricular tissues after MI. We provide evidence that the partial prevention of fibrosis mediated by losartan in post-MI rat heart may be effected at the posttranslational level of collagen synthesis, and may be due to normalization of increased PH protein concentration in tissues remote from the site of infarction.

Time for primary review 32 days.

	Acknowledgements

 
This study was supported by funding from the Medical Research Council of Canada. Dr. Ian M.C. Dixon is a scholar of the Medical Research Council of Canada/PMAC health program with funding provided by Astra Pharma, Inc. Dr. H. Ju is a recipient of a Manitoba Health Research Council Studentship. We also wish to thank Ms. Tracy K. Scammell-La Fleur and Mr. Jeff P. Werner for their excellent technical assistance. Losartan used in this study was a kind gift from Du Pont Merck.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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