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Cardiovascular Research 2000 46(2):286-297; doi:10.1016/S0008-6363(00)00035-3
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Effect of chronic AT1 receptor blockade on cardiac Smad overexpression in hereditary cardiomyopathic hamsters

Ian M.C. Dixon*, Jianming Hao, Nicole L. Reid and Julie C. Roth

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

* Corresponding author. Tel.: +1-204-235-3419; fax: +1-204-233-6723 iand{at}sbrc.umanitoba.ca

Received 7 October 1999; accepted 2 February 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: As the pharmacological suppression of angiotensin has been associated with cardioprotective effects in cardiomyopathy, our primary aim was to determine whether the expression of Smad protein components of the cardiac TGF-β signaling cascade is modulated by chronic AT1 receptor blockade. Furthermore, we examined the relationship between cardiac Smad protein expression and altered collagen turnover in the cardiomyopathic heart. Methods: Male UM-X7.1 cardiomyopathic (CMP) Syrian hamsters at early (65 days) and late (200 days) stages of cardiomyopathy were subjected to 4 week losartan (15 mg/kg/day) treatment. Expression of left ventricular (LV) receptor-activated (Smad 2) and common-mediator (Smad 4) Smads from control (F1-β strain) hamsters, non-treated cardiomyopathic (CMP), and losartan-treated CMP animals was assessed. Collagen turnover, including fibrillar collagen synthesis/accretion and cardiac MMP activity was assessed. Results: Elevated mRNA abundance of fibrillar collagens and ANF were present in cardiomyopathic hearts and these trends were normalized in the early stage losartan-treated group. 4-Hydroxyproline and zymographic assays confirmed fibrosis and elevated MMP-1 and -2 activities in CMP hearts. Losartan treatment was associated with a modest reduction of cardiac 4-hydroxyproline concentration, and a significant reduction of both MMP-1 and MMP-2 activities. While TGF-β1 mRNAs were elevated in both CMP groups vs. controls, total TGF-β protein content was not different in CMP vs. controls. In LV preparations containing nuclear extract, elevated Smad 2 and Smad 4 protein expression was noted in cardiomyopathic hearts vs. controls. Losartan treatment of late-stage CMP hamsters was associated with a significant reduction in Smad 2 and a modest reduction of Smad 4 protein expression vs. untreated CMP samples. Conclusions: Altered cardiac Smad expression, present in both early and late stage cardiomyopathy, is positively correlated with the occurrence of cardiac fibrosis and elevated collagen turnover in failing CMP hearts. Four week AT1 blockade is associated with normalized expression of cardiac Smad 2 proteins, and these changes occur in parallel with some aspects of collagen turnover in failing cardiomyopathic hearts.

KEYWORDS Angiotensin; Fibrosis; Cardiomyopathy; Heart failure


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Congestive heart failure (CHF) is characterized as a major cause of death in the American population [1]. The cardiomyopathic (CMP) Syrian hamster (UM-X7.1 strain) is a model of hereditary cardiomyopathy that mimics the gradual progression of CHF in humans [2]. In previous experiments we confirmed the presence of increased fibrillar collagen turnover in hearts of cardiomyopathic hamsters including increased cardiac collagen steady-state mRNA abundance and elevation of matrix metalloproteinase (MMP) activity both in early and late stages of this disease [3]. Furthermore, we had previously observed that short-term AT1 receptor blockade therapy was associated with normalization of some aspects of collagen turnover in cardiomyopathic left ventricles [3], supporting an important role for angiotensin II (angiotensin) in cardiac matrix remodeling.

In addition to angiotensin, other factors are known to participate in fibroproliferative events associated with cardiac fibrosis and heart failure. In this regard, TGF-β1 contributes to an array of biological phenomena including regulation of embryogenesis, ECM production and wound repair as well as growth inhibitory effects [4,5]. Furthermore, TGF-β1 is a powerful initiator of the production of fibrillar collagens and other major ECM components by cardiac fibroblasts [4]. The effects of TGF-β1 are mediated through a group of transmembrane receptors with serine/threonine kinase activity referred to as TGF-β receptor type I (TβRI) and type II (TβRII) [6]. These receptor types have been localized in both cardiac myocytes and nonmyocytes, including fibroblasts and myofibroblasts [7]. Crosstalk between angiotensin II and TGF-β signaling systems is suspected to exist in the heart at the level of the ligand itself [8]. The significance of this finding may lie in the powerful anti-fibrotic effects mediated by the chronic blockade of cardiac AT1 receptors as demonstrated in this and other experimental models of congestive heart failure [9,10]. However, it is unknown as to whether AT1 blockade exerts any influence on TGF-β post-receptor signaling. Recently, a major advance in understanding TGF-β signaling has been the identification of Smad proteins as downstream effector molecules of TGF-β. The Smad family includes Smad 2 and Smad 4; the former dimerizes with the latter upon phosphorylation [13–15]. Once phosphorylated, these proteins translocate to the nucleus and initiate gene transcription [11–13] by association with eukaryotic nuclear transcription factors [16] via their specific binding to Smad 2 [17]. We undertook the current study to examine the relationship between cardiac Smad expression and cardiac fibrosis associated with hereditary cardiomyopathy. We also investigated whether AT1 receptor activation may influence the expression of Smad proteins in cardiomyopathic heart.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Experimental model
Breeding male and female UM-X7.1 strain of cardiomyopathic (CMP) hamsters were obtained from the laboratory of Dr G. Jasmin of the Department of Pathology, University of Montreal, Canada. An F1-β strain of Syrian hamster obtained from Charles River served as age matched controls in our experiments. The effect of AT1 receptor blockade was carried out using 4-week losartan infusion in male UM-X7.1 CMP animals (CMP+Los) of 65 and 200 days of age. Losartan (15 mg/kg/day) [10] or vehicle (saline, 0.9% NaCl) were administered by surgical implantation of an osmotic mini pump (Alzet Corporation) to the anterior dorsal region of each animal and delivery was maintained for 28 days by pump implantation. For comparative purposes, age-matched saline-infused F1-β Syrian hamsters were also employed in these studies. Animals were sacrificed by decapitation at 65 and 200 days of age. Each age group represents a distinct stage of the disease; we have observed that animals from this colony expressing cardiomyopathic phenotype may survive until 230±15 (S.E.M.) days. 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 and conforming 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).

2.2 Determination of cardiac total collagen
Left ventricles from each group were 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. [18]. A stock solution containing 40 mM of 4-hydroxyproline in 1 mM HCl was used as a standard. Assuming that interstitial collagen contains an average of 13.4% hydroxyproline, collagen concentrations were calculated by multiplying hydroxyproline levels by a factor of 7.46. The data was expressed as micrograms collagen per milligram dry tissue and thus data was further converted to relative concentration to avoid any variation between different assays.

2.3 Zymography: Detection of cardiac matrix metalloproteinase
Powdered LV cardiac tissue (50 mg) was suspended in 1 ml phosphate-buffered saline (pH 7.4) containing 100 µg/ml phenylmethylsulfonyl fluoride (PMSF) and 2 µg/ml leupeptin and incubated at 4°C with continuous agitation for 20 h to extract a protein fraction enriched with MMP activity [19]. The addition of PMSF and leupeptin to the sample buffer was necessary to rule out nonspecific gelatin lysis due to serine proteinases. Samples were centrifuged at 10 000 rpm at 4°C for 10 min, and the supernatant was employed for total protein assay and zymographic analysis. Total protein content in each sample was determined using the bicinchoninic acid (BCA) protein assay kit (Sigma) [20]. MMP activity was detected using gelatin zymography (final gelatin concentration 1 mg/ml) in a 7.5% standard SDS—PAGE [19]. Gelatin is readily cleaved by MMPs and is easily incorporated into polyacrylamide gels. Thirty µg/lane of non-reduced protein was loaded and samples were run at 15 mA/gel. After electrophoresis, gels were washed 2x15 min in 25 mM glycine (pH 8.3), 2.5% Triton X-100 with gentle shaking at 4°C to eliminate SDS from the gels. Once rinsed, gels were incubated at 37°C for 18 h in substrate buffer (50 mM Tris—HCl, pH 8.0, 5 mM CaCl2). After incubation, gels were stained in 0.05% Coomassie blue (R-250) for 30 min, and then destained in acetic acid and methanol. Gels were dried and scanned using a CCD camera densitometer (Bio-Rad imaging densitometer GS 670). MMP activity was verified using 1,10-phenanthroline, an MMP inhibitor.

2.4 RNA extraction and Northern blot analysis of cardiac mRNAs
Myocardial total RNA was isolated from left ventricles taken from saline-infused (untreated) F1-β control, saline-infused (untreated) CMP and losartan-treated CMP animals by the method of Chomczynski and Sacchi [21] at 65 and 200 days. RNA pellets were dissolved in diethyl pyrocarbonate (DEPC)-treated water and the concentration of RNA was calculated from the absorbance at 260 nm prior to size fractionation. Northern blot analysis for detection of steady-state mRNA abundance was carried out according to standard procedures [22]. Briefly, 20 g of total RNA was denatured in 50% formamide, 7% formaldehyde, 20 mM MOPS pH 7.4, 2 mM EDTA, pH 8.0, 0.1% sodium dodecyl sulfate (SDS) and electrophoresed in a 1.2% agarose/formaldehyde gel to size fractionate the mRNA transcripts. The fractionated RNA was transferred (capillary) to a 0.45-µm positive charge-modified nylon filter (Nytran Plus positively charged membrane, Schleicher & Schuell). Each membrane was hybridized with specific 32P-labeled cDNA probes at 43°C for 16–20 h. Membranes were exposed to X-ray film (Kodak X-OMAT) at –80°C with intensifying screens. 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 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA to normalize for differences in loading and/or transfer of mRNA, as previously described [23].

2.5 Western blot analysis of Smad 2, Smad 4 and TGF-β1
Western blot analysis was carried out under reducing conditions as described previously [24,25]. For total cardiac Smad protein assays, LV tissues obtained from untreated F1-β control, untreated CMP and losartan-treated CMP animals were homogenized with homogenization buffer containing 0.1% Triton X-100. This homogenate was sonicated for 5x5 s to disrupt nuclear membranes. The samples were allowed to lyse for 15 min on ice. After centrifugation at 10 000xg for 20 min at 4°C, the supernatant was retained for determination of Smad proteins. Total protein concentration of all samples was measured using the bicinchoninic acid (BCA) method [20]. Primary antibodies were diluted in TBS-T (Smad 2, 4 and TGF-β1 at 1:500). The specific bands of target proteins were visualized by enhanced chemiluminescence (ECL) or by ECL Plus according to the manufacturer’s instructions (Amersham Life Science Inc. Arlington Heights, IL), and these bands were quantified using a CCD camera imaging densitometer (Bio-Rad, model GS 670).

2.6 Immunofluorescent localization of Smad 2, Smad 4 and TGF-β1
LV tissue from untreated control, untreated CMP and losartan-treated CMP animals were immersed in OCT compound (Immunocor Canada Inc.). Serial cryostat sections (7 µm) of ventricular tissue were mounted on gelatin coated slides. A minimum of six sections from hearts of each group were processed. Indirect immunofluorescence was performed as described in detail previously [23]. Briefly, sections were fixed in 4% paraformaldehyde for 15 min. Polyclonal antibodies against Smad 2 and Smad 4 as well as total TGF-β1 were diluted 1:100–1:500 with 1% BSA in PBS and applied as primary antibodies. After 12–16 h at 4°C, sections were washed with PBS and incubated with biotinylated, anti-goat (or rabbit) IgG secondary antibody and subsequently incubated with FITC-labeled streptavidin for 90 min. Hoechst dye # 33342 was used for staining of cellular nuclei in frozen cardiac sections at a final concentration of 10 µg/ml. Tissue sections were examined under a Nikon Labophot microscope equipped with epifluorescence optics and appropriate filters. The results were recorded by photography on Kodak TMAX 400 black and white film. Sigmascan Pro3 pixelation analysis software was used to quantitate the intensity of TGF-β1 immunofluorescence in frozen sections.

2.7 Reagents
Primary polyclonal antibodies against Smad 2 and 4 as well as TGF-β and HRP-labeled anti-goat secondary antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Complementary DNA probes for TGF-β1 and GAPDH were obtained from the American Type Culture Collection (Rockville, MD). The cDNA fragments for human procollagen type {alpha}1(I) (Hf 677) and human type {alpha}1(III) (Hf 934) were obtained from the American Type Culture Collection.

2.8 Statistical analysis
All values are expressed as mean±S.E.M. One way analysis of variance (ANOVA) followed by Student—Newman—Keuls method was used for comparing the differences among multiple groups (SigmaStat). Significant differences among groups were defined by a probability of less than 0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 General characteristics of UM-X7.1 cardiomyopathic and F1-β control animals
Penetrance of the cardiomyopathic phenotype in UM-X7.1 animals was assessed using standardized techniques, and animals displaying classic markers of cardiomyopathy were used [3]. LV mass at 65 and 200 days (sexually mature and old animals, respectively) (Table 1) revealed an early relatively atrophic state (65 days vs. controls) with a trend to normalization in 200 day vs. control values. Absolute CMP LV mass increased significantly from 65 to 200 days (65 vs. 200 day CMP values: 0.23±0.01 vs. 0.34±0.01 g, P<0.05 — as seen in Table 1), whereas a comparison of mass values from the 65 and 200 day F1-β control groups (65 vs. 200 day F1-β values: 0.38±0.04 vs. 0.40+0.01 g) revealed no significant difference. Hypertrophy, as determined by heart/body weight ratio comparisons between control and CMP groups, were not carried out as the mean body mass in CMP animals from both ages is significantly decreased compared to control values, yielding erroneous estimates. Based on the data generated by intra-group comparison (above) we suggest that relative cardiac hypertrophy occurs in CMP animals with advancing age.


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  		Table 1 Body weight, and ventricle weights of cardiomyopathic (UM-X7.1) and control (F1-β) Syrian hamsters at 65 and 200 days of agea

 
Early overexpression of atrial natriuretic factor (ANF) in hearts of CMP animals precedes the development of pathological changes associated with cardiomyopathy [3,26] and is considered to be a qualitative marker of cardiac hypertrophy [27]. We confirmed the presence of elevated ANF mRNA abundance at 65 days (Fig. 1A and E) which is prior to the onset of hemodynamic dysfunction in these animals [27]. Furthermore, ANF expression may reflect the extent of necrosis in CMP hearts, and is generally regarded as a useful predictor of heart failure [3]. Four week treatment of CMP animals (15 mg/kg/day losartan) of early stage CMP animals was associated with complete normalization of cardiac ANF mRNA abundance vs. untreated 65 day controls (Fig. 1), and a modest reduction of ANF mRNA abundance in the 200-day CMP vs. untreated CMP animals (Fig. 1B and E). The constant-infusion losartan dose used in the current study was based on previous work by Smits et al. [10] and that from our laboratory [3].


Figure 1
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  		Fig. 1 Total RNA and mRNA abundance in left ventricular tissue at 65 and 200 days in control, cardiomyopathic (CMP), and losartan-treated CMP (CMP+Los) hamster hearts. (A and B) Representative total RNA extract from left ventricular tissue of 65 day (A) and 200 day (B) F1-β strain control (lane 1 and 2), UM-X7.1 cardiomyopathic strain (CMP; lanes 3 and 4) and 4 week losartan (15 mg/kg/day) treated CMP (CMP+Los; lanes 5 and 6) hamsters. (C) Histogram of steady-state mRNA abundance of fibrillar collagen type I in left ventricle tissue of control, CMP, and CMP+Los hamsters at 65 and 200 days. (D) Histogram of steady-state mRNA abundance of fibrillar collagen type III in left ventricle tissue of control, CMP, and CMP+Los hamsters at 65 and 200 days. (E) Histogram of steady-state ANF mRNA expression in left ventricular tissue of control, CMP, and CMP+Los hamsters at 65 and 200 days. Data depicted as mean±S.E.M. from total sample sizes of four to six (C and D) and nine to twelve (E). *P<0.05 vs. control value; #P<0.05 vs. CMP value.

 
3.2 Collagen turnover: The effect of losartan on cardiac collagen mRNA abundance, hydroxyproline deposition and MMP activity
Fig. 1 shows representative Northern blots with autoradiographic signals for collagen types I and III as well as that for GAPDH mRNA in 65 and 200 day CMP and F1-β control groups. Calculation of target gene/GAPDH signal ratios were carried out [23,28] for the different stages of cardiomyopathy. CMP hearts were characterized by an increase in fibrillar collagen mRNA abundance in samples of LV tissue vs. control values. Chronic (4 week) losartan therapy in 65 day CMP animals (Fig. 1C and D) was associated with a decrease in fibrillar collagen mRNAs when compared to untreated CMP hearts. In addition, the collagen type III/GAPDH ratio in hearts of 200 day animals was significantly decreased vs. untreated CMP samples while an assay of collagen type I/GAPDH mRNA ratios revealed no difference between treated and untreated 200 day groups. These results indicate that the stage of cardiomyopathy i.e. that associated with prefailure (65 day) or overt heart failure (200 day), has a bearing on the responsiveness of cardiac collagen gene transcription to AT1 receptor blockade. To quantitate fibrillar collagen concentrations in CMP LV tissues, 4-hydroxyproline assays were performed in 65 and 200 day groups. A significant increase in total cardiac 4-hydroxyproline was observed in late stage CMP vs. control values (Fig. 2) whereas values from the early stage CMP hearts were unchanged vs. controls. No significant difference between LV untreated CMP and 4-week losartan-treated CMP samples was noted in either early or late stage cardiomyopathy, although a modest trend toward control values was noted in the treated 200 day samples.


Figure 2
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  		Fig. 2 Relative collagen concentration in left ventricular tissue at 65 and 200 days in control, cardiomyopathic (CMP), and losartan-treated (CMP+Los) hamster hearts. Histogram of relative collagen concentration (4-hydroxyproline concentration) in left ventricle samples from F1-β control strain, UM-X7.1 cardiomyopathic strain (CMP), and 4 week losartan (15 mg/kg/day) treated cardiomyopathic hamsters (CMP+Los) at 65 and 200 days. Data depicted as mean±S.E.M. from a total sample size of four to six. *P<0.05 vs. control value; #P<0.05 vs. CMP value.

 
The gelatinolytic activities of MMP-1 and MMP-2 in control and cardiomyopathic LV samples were determined and the results are depicted in Fig. 3. We observed that MMP-1 and MMP-2 activities were increased in CMP LV samples in both cardiomyopathic groups vs. age-matched control values. Four-week losartan treatment was associated with a significant attenuation of gelatinolytic MMP-1 activity in both groups when compared to saline-infused cardiomyopathic and control samples. MMP-2 activity in the treated late-stage CMP group was significantly reduced vs. untreated CMP values, while this treatment was not associated with any effect vs. the untreated CMP group at 65 days. Thus losartan treatment was associated with general modulation of cardiac MMP activity toward control levels in failing cardiomyopathic hearts.


Figure 3
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  		Fig. 3 Cardiac matrix metalloproteinase activity in 65 and 200 day left ventricular tissue from control, cardiomyopathic (CMP) and losartan treated CMP (CMP+Los) hamster hearts. (A and B) Representative zymographic gel of cardiac matrix metalloproteinase (MMP) activity in 65 day (A) and 200 day (B) left ventricular (LV) tissue from F1-β control strain (lanes 1 and 2), UM-X7.1 cardiomyopathic strain (CMP; lanes 3 and 4), and 4 week losartan (15 mg/kg/day) treated cardiomyopathic hamsters (CMP+Los; lanes 5 and 6). Gelatinolytic activity was taken to represent cardiac collagenase (MMP-1; 54 kDa) and major cardiac gelatinase A (MMP-2; 65 kDa) activity. The 72-kDa band represents the latent form of MMP-2. (C) Histogram representing densitometric scans of zymographic gel analyses for cardiac collagenase (MMP-1) activity in 65 and 200 day LV tissue from control, CMP, and CMP+Los hearts. (D) Histogram representing densitometric scans of zymographic gel analyses for major cardiac gelatinase A (MMP-2) activity in 65 and 200 day LV tissue from control, CMP, and CMP+Los hearts. Data depicted as mean±S.E.M. from a total sample size of six to eight. *P<0.05 vs. control value; #P<0.05 vs. CMP value.

 
3.3 Alteration of TGF-β1 mRNA abundance
Steady-state mRNA abundance of cardiac TGF-β1 was assessed in LV tissues. Fig. 4 shows representative Northern blots with autoradiographic bands specific for TGF-β1 and GAPDH mRNAs from 65 and 200 day LV samples of control, CMP, and losartan-treated CMP tissues. Target gene mRNA abundance was calculated using the ratio of TGF-β1/GAPDH signals. These ratios were significantly increased in both 65 and 200 day CMP samples when compared to controls; TGF-β1 mRNA levels from the 4 week losartan-treated CMP group remained elevated vs. controls but unchanged vs. values from untreated CMP samples.


Figure 4
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  		Fig. 4 TGF-β1 mRNA expression in control, cardiomyopathic (CMP), and losartan treated cardiomyopathic (CMP+Los) hamster hearts. (A and B) Representative Northern blots for 65 (A) and 200 day (B) TGF-β1 and GAPDH in F1-β control (lanes 1 and 2), UM-X7.1 cardiomyopathic (CMP; lanes 3 and 4), and 4 week losartan (15 mg/kg/day) treated (CMP+Los; lanes 5 and 6) hamster left ventricle (LV). (C and D) Quantified data of 65 day (C) and 200 day (D) TGF-β1/GAPDH expression in F1-β control, CMP, and CMP+Los hamster LV. Data depicted as mean±S.E.M. from a total sample size of six to eight. *P<0.05 vs. control value.

 
3.4 Localization and quantitation of cardiac TGF-β1 in tissue sections
Total TGF-β1 was localized using immunofluorescent staining of frozen serial sections of untreated F1-β control, untreated CMP and 4 week losartan-treated CMP LV samples and the same tissue slices were double-stained for cardiac cellular nuclei using Hoechst 33342 dye (Fig. 5). Immunoreactive TGF-β1 was localized to the cardiac interstitium and the staining pattern intensity was quantified using computer pixelation analysis (SigmaScan Pro3 software — Fig. 6). Total TGF-β1 staining was unchanged between sections from F1-β control, untreated CMP, and losartan-treated CMP sections from the 65 and 200 day groups.


Figure 5
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  		Fig. 5 Representative immunofluorescent (IF) staining of TGF-β1 in left ventricle tissue sections from control, cardiomyopathic (CMP), and losartan treated CMP (CMP+Los) hamster hearts. Immunofluorescent stained tissue sections showing 65 (A) and 200 (B) day TGF-β1 patterns in F1-β control, UM-X7.1 cardiomyopathic (CMP), and 4 week losartan (15 mg/kg/day) treated (CMP+Los) left ventricular tissue as well as Hoescht nuclear staining for the identical field of view. Immunoactive TGF-β1 protein appears as brightly stained material. Magnification: x400.

 

Figure 6
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  		Fig. 6 Semiquantitative estimation of total immunoreactive TGF-β1in left ventricular tissue sections from control, cardiomyopathic (CMP), and losartan-treated CMP (CMP+Los) hamster hearts. The histograms are the result of computer pixelation analysis of frozen tissue slices stained for total immunoreactive TGF-β1 in 65 and 200 day F1-β control, CMP, and CMP+Los hamster left ventricular (LV) tissue sections. Fluorescence was quantified using SigmaScan Pro 3.0 software and digitized imaging. Data is depicted as the mean±S.E.M. from a total sample size of four to six for each group.

 
3.5 Western analysis of cardiac TGF-β1 in CMP hearts
In reducing conditions, cardiac 12 and 25 kDa TGF-β1 protein bands in 65 and 200 day control and CMP were detected in nuclei-containing cytosolic samples (Fig. 7). Densitometric scanning analysis revealed no change in the 12-kDa band and a significant decrease of the 25-kDa band in 65 day CMP samples vs. controls. Westerns of 200 day samples revealed a significant increase in the 12 kDa band in the CMP group and a decrease in the 25 kDa band of TGF-β1 vs. controls. Losartan treatment of CMP hearts was associated with normalization of altered density in both autoradiographic bands at both stages of CMP. On the other hand, estimation of total TGF-β1 (12 plus 25 kDa values) revealed no differences between groups. Thus, total TGF-β1 protein expression was unchanged in CMP vs. controls and is nonsynchronous in with respect to changes in steady-state mRNA abundance.


Figure 7
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  		Fig. 7 Western analysis of TGF-β1 protein expression in left ventricular (LV) tissue from control, cardiomyopathic (CMP), and losartan-treated CMP (CMP+Los) hamster hearts. (A and B) Representative Western blots for 65 (A) and 200 (B) day TGF-β1 in F1-β control (lanes 1 and 2), UM-X7.1 cardiomyopathic (CMP; lanes 3 and 4), and 4-week losartan (15 mg/kg/day) treated (CMP+Los; lanes 5 and 6) hamster left ventricle (LV). (C and D) Quantified data of 65 (C) and 200 (D) day TGF-β1 protein expression in F1-β control, CMP, and CMP+Los hamster LV. Data depicted as mean±S.E.M. from a total sample size of four to six. *P<0.05 vs. control value.

 
3.6 Quantification of Smad 2 and Smad 4 in cardiomyopathic heart and the effect of losartan
Western analysis was used to determine cardiac Smad 2 and Smad 4 protein concentrations in control, CMP, and CMP+Los groups of 65-day (Fig. 8) and 200-day cardiomyopathic tissues (Fig. 9). Smad 2 protein levels were significantly increased in 65-day CMP and losartan-treated CMP LV samples vs. controls, and the Smad 4 protein expression pattern was similar. In hearts of 200 day animals, cardiac Smad 2 and Smad 4 protein expression was increased in CMP tissues vs. controls. Four-week losartan treatment of these animals was associated with a significant decrease (Smad 2) and modest reduction (Smad 4) in protein expression, respectively, vs. untreated values.


Figure 8
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  		Fig. 8 Sixty-five-day Smad 2 and Smad 4 protein expression in left ventricular samples from control, cardiomyopathic (CMP), and losartan-treated CMP (CMP+Los) hamster hearts. (A and B) Representative Western blots for 65 day Smad 2 (A) and Smad 4 (B) in F1-β control (lanes 1 and 2), UM-X7.1 cardiomyopathic (CMP; lanes 3 and 4), and 4-week losartan (15 mg/kg/day) treated (CMP+Los; lanes 5 and 6) hamster left ventricle (LV). (C) Quantified data of 65 day Smad 2 and Smad 4 protein expression in F1-β control, CMP and CMP+Los hamster LV. Data depicted as mean±S.E.M. from a total sample size of four to six. *P<0.05 vs. control value.

 

Figure 9
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  		Fig. 9 Two-hundred-day Smad 2 and Smad 4 protein expression in left ventricular samples from control, cardiomyopathic (CMP) and losartan-treated CMP (CMP+Los) hamster hearts. (A and B) Representative Western blots for 200 day Smad 2 (A) and Smad 4 (B) in F1-β control (lanes 1 and 2), UM-X7.1 cardiomyopathic (CMP; lanes 3 and 4), and 4-week losartan (15 mg/kg/day) treated (CMP+Los; lanes 5 and 6) hamster left ventricle (LV). (C) Quantified data of 200 day Smad 2 and Smad 4 protein expression in F1-β control, CMP and CMP+Los hamster LV. Data depicted as mean±S.E.M. from a total sample size of four to six. *P<0.05 vs. control value; #P<0.05 vs. CMP value.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The UM-X7.1 strain of cardiomyopathic Syrian hamster first described by Bajusz [2] provides a good model of congestive heart failure (CHF) and is a paradigm of hereditary dystrophic cardiomyopathy and myopathy. This disease is linked to an autosomal recessive gene expressed in all homozygous animals, and is characterized by progressive myocardial damage out to ~150 days of age with subsequent dilatation of the ventricles (dilated cardiomyopathy) and rapid augmentation in LVEDP thereafter [27,29]. This hamster model of cardiomyopathy is associated with only partial activation of the hypertrophic cascade, and offers the opportunity to study expression patterns of specific components of hypertrophy [27]. The incidence of cardiomyopathy in this model is marked by the early appearance of focal necrosis (~30 days) which may continue for several months, resulting in considerable replacement fibrosis within the myocardium [3,30], which closely resembles the human form of this condition [26,31,32]. Notably, interstitial fibrosis and attendant decreases in compliance of the surviving myocardium are believed to contribute to the occurrence of cardiac dysfunction [33]. In experimental cardiomyopathy, expansion of the cardiac extracellular matrix associated with healing after myocytolysis and of the cardiac interstitium; these changes provide an index for the progression of this disease [3]. The current study is the first to provide a positive correlation Smad overexpression and altered collagen metabolism in experimental cardiomyopathy, and also to provide evidence of crosstalk between AT1 receptors and the Smad post-receptor signaling pathway of TGF-β1.

Smads (Smad 2 and Smad 4 among others) are a class of post-receptor effectors known to be expressed in heart that have been associated with TGF-β1 signaling [34]. TGF-β1 contributes to an array of biological functions including regulation of ECM production, wound repair, and growth inhibition [4,5]. TGF-β receptor activation occurs upon the binding of TGF-β1 to the type II receptor (TβRII), which then recruits and autophosphorylates TβRI [12]. Activated TβRI then phosphorylates cytosolic Smad 2 which is a critical step for activation of this pathway [11,13]. Phosphorylated Smad 2 proteins then bind Smad 4 [15] in the cytosol and this complex translocates to the cellular nucleus and associates with [13,14] FAST-1, a eukaryotic nuclear transcription factor [35], leading to the activation of target gene expression [13–15]. It was reported that the mRNA level of TGF-β was increased in 20–150-day-old CMP hamster although the corresponding protein levels were not investigated [27]. We confirmed the presence of elevated steady-state TGF-β mRNA levels in both 65 day and 200 day CMP hamster hearts, however total TGF-β1 protein expression was unchanged in cardiomyopathic hamster hearts. Although steady-state TGF-β1 mRNA is elevated in CMP, TGF-β1 ligand overexpression may not be a primary mechanism for stimulation of fibrotic events in this model of cardiomyopathy. Nonsynchronous expression of constituitive cardiac target genes has been previously described, and may be due to altered intracellular processing of immature proteins prior to secretion [23,36].

We investigated the protein expression of Smad 2 and Smad 4 in CMP hearts to assess the relationship of these post-receptor effectors to incidence of fibrosis in cardiomyopathy. Increased Smad expression in cardiomyopathic hearts and normalization of Smad 2 expression in the presence of losartan suggests a putative link between angiotensin and Smad 2 expression in experimental cardiomyopathy. We have shown that this may occur even in the absence of elevated total TGF-β1 ligand expression. The parallel in elevated expression patterns of Smad 2 and aspects of collagen turnover in CMP hearts, and their responsiveness to angiotensin suppression suggests that Smads may potentiate the activation of fibroblasts and myofibroblasts in these tissues. Furthermore, angiotensin-mediated elevation of Smad 2 protein expression in cultured primary adult rat cardiac fibroblasts (unpublished data from this laboratory) provides further support for angiotensin-mediated modulation of Smad 2 expression. In this regard, crosstalk among the signaling pathways of the AT1 receptor and those of cytokines has been suggested to determine the ultimate response of the cardiac fibroblast [37].

Cardiac fibroblasts and myofibroblasts are key players in fibroproliferative events in various types of heart disease [25,33] and express key genes supporting angiotensin expression [37]. In frozen tissue slices of CMP heart, Smad 2 and Smad 4 proteins were localized to nonmyocytes in the interstitial space (cardiac fibroblasts and derivative myofibroblasts), confirming previous evidence [34]. Concomitant angiotensin and Smad 2 expression in cardiac (myo)fibroblasts supports the suggestion that Smad 2 may participate in angiotensin-mediated activation of these cells.

We found that despite a general trend to normalization of elevated collagen mRNA expression as well as MMP-1 and -2 activities in losartan-treated hearts, only a modest decrease in total LV 4-hydroxyproline was observed in this group vs. untreated CMP animals. With respect to the latter, our findings are in general agreement with those of Davison et al. [38] who found that 3 months of captopril treatment was not associated with a reduction in average collagen concentrations in these experimental hearts. Nevertheless, as entire left ventricles from each group were homogenized for the preparation of samples for the biochemical detection of total 4-hydroxyproline concentration (taken to reflect total cardiac collagen concentration), we suggest that the apparent modest decrease may be due to dilution of significant changes that occur within the fibrosed microdomains of focal infarcts in CMP heart tissue. This suggestion is borne out by our preliminary characterization of 4 week losartan-treated animals through microspectroscopic mapping of intact tissue slices using high-intensity infrared light from a synchrotron beam source (NSLS, Brookhaven, NY; data not shown).

Cardiac collagen turnover may be defined as a balance between collagen synthesis and processes effecting collagen removal. Our results show that MMP-1 and MMP-2 were increased in CMP hamster. With regard to the latter, collagen types I and III may be cleaved by a 54-kDa interstitial collagenase (MMP-1) and further degradation of the collagen breakdown products may occur via cardiac MMP-2 activity [39]. As elevated MMP activity occurs in the presence of overt fibrosis (e.g. 200-day CMP hearts), we suggest that increased cardiac collagen in CMP hamster is due to the increased collagen synthesis rather than decreased collagen removal. Thus we have demonstrated increased collagen turnover occurs in CMP hamster heart with an imbalance favoring collagen accretion. It has been suggested that increased MMP-1 and MMP-2 activities in cardiomyopathic hearts may potentiate the remodeling of cardiac matrix proteins in these experimental animals, thereby altering the nature of the cardiac collagen weave [40]. Furthermore, areas characterized by a marked absence of connective tissue in the myocardium in these animals may be associated with ventricular wall thinning [41], and elevated enzymatic removal of collagen is a contributing factor to the pathogenesis of experimental cardiomyopathy [42]. In this regard, it has been suggested that increased MMP activities may be responsible for cardiac dilatation and the development of heart failure [40,43]. As AT1 receptor blockade was associated with a significant attenuation of gelatinolytic collagenase activity in both early and late stages of CMP, it is suggested that this therapy may attenuate collagen matrix remodeling in these hearts. Thus, this effect may contribute to the cardioprotective effect of losartan.

In conclusion, increased Smad 2 and Smad 4 protein expression in cardiomyopathic hearts is positively correlated with elevated cardiac collagen turnover in failing cardiomyopathic hearts. Four-week suppression of angiotensin AT1 receptor function is associated with normalization of Smad 2 protein expression, and this change is associated with normalization of aspects of collagen turnover. Smad 2 overexpression in (myo)fibroblasts is implied in extracellular matrix remodeling of the failing cardiomyopathic heart. Finally, our results provide support evidence of crosstalk between AT1 receptor activation and Smad protein expression in heart.

Time for primary review 33 days.


   Acknowledgements
 
This study was supported by funding from the Heart and Stroke Foundation of Canada (IMCD). IMCD is a scholar of the Medical Research Council of Canada/PMAC health program with funding provided by Astra-Zeneca. JH is a current recipient of Manitoba Health Research Council of Graduate Studentship.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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