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Cardiovascular Research 1999 43(1):165-172; doi:10.1016/S0008-6363(99)00111-X
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Copyright © 1999, European Society of Cardiology

Angiotensin II receptor blockade during gestation attenuates collagen formation in the developing rat heart

Steffen Lamparter, Yao Sun and Karl T Weber*

Division of Cardiology, Department of Internal Medicine, University of Missouri Health Sciences Center, Columbia, MS, USA

* Corresponding author. Tel.: +1-573-882-8580; fax: +1-573-884-4691 karl_t_weber{at}muccmail.missouri.edu

Received 7 August 1998; accepted 12 January 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Fetal cardiac development includes rapid formation of a three-dimensional collagen network, composed mainly of type I and III fibrillar collagens. Collagen fibrils have been found in cardiac jelly at very early stages of cardiac development and are thought to have structural and functional properties. In adult rat cardiac tissue, angiotensin II (AngII) via AT1 receptor binding and AngII-regulated expression of transforming growth factor beta-1 (TGF-β1) each upregulate collagen transcription. AT1 and AT2 receptor subtypes are developmentally regulated; both have been localized in fetal tissue where the AT2 receptor is considered a determinant of morphogenesis. We sought to determine whether blockade of either receptor would result in attenuation of collagen mRNA expression and fibrillar collagen accumulation and alter TGF-β1 mRNA expression in the developing fetal heart examined at birth. Methods: Pregnant rats were treated either with an AT1 receptor antagonist losartan or an AT2 receptor antagonist PD123319 and compared with untreated age-matched controls. Offspring were studied within 24 h of birth. Type I and type III collagen mRNA expression, as well as TGF-β1 mRNA expression, were examined by in situ hybridization. Collagen concentration was determined spectrophotometrically by picrosirius red staining and type I and III collagens were detected by immunoblotting. Results: We found: (1) comparable birth weights in control and PD123319-treated animals, but reduced body weight in newborn losartan-treated animals; (2) compared to untreated animals, type I collagen and TGF-β1 mRNA expression in cardiac tissue were each equally reduced in both losartan and PD123319-treated animals; (3) increased type III collagen mRNA expression in both PD123319- and losartan-treated groups; and (4) a significant decrease in total soluble cardiac collagen concentration in both losartan and PD123319-treated groups, confirmed by attenuated immunoreactivity of type I and III collagens in whole heart extracts by Western blotting. Conclusions: The results of these pharmacologic interventions suggest AngII receptors are expressed in cardiac tissue during gestation, where both AT1 and AT2 receptors are involved in the regulation of type I and III collagen expression and structural protein accumulation. These effects appear to be mediated, in part, by attenuated cardiac TGF-β1 levels. The marked decrease in newborn cardiac collagen content has yet undefined functional consequences.

KEYWORDS Cardiac development; Type I and III collagens; Losartan; TGF-β1


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The shape and function of the heart is determined during early fetal development. A complex interaction between cardiac myocytes and fibroblasts is likely involved in the formation of extracellular matrix (ECM) – a three-dimensional structure consisting largely of type I and III fibrillar collagens found within atria, ventricles and contiguous chordae tendineae and valve leaflets. These matrix structural proteins provide for cell alignment and organ function.

Although fibrillar collagens are thought to be involved in cardiovascular morphogenesis and serve to accommodate hemodynamic behavior of the beating heart, little is known about the function and regulation of type I and III collagen expression during fetal development. ECM collagen expression is highly regulated in a spatial and temporal manner in keeping with physiologic functions of the fetal heart. In utero, cardiac collagen has been detected at stage 10 (10 somites, 30 h incubation) in cardiac jelly, located between the outer myocardium and the inner endocardial cell layer of the primitive cardiac tube [1].

Among numerous regulatory factors involved in collagen turnover during fetal and perinatal cardiac development, AngII may have an important role. In adult cardiac tissue it is well established that locally generated AngII contributes in an autocrine and/or paracrine manner to a variety of effects involved in collagen turnover. It raises collagen transcription of cultured neonatal and adult cardiac fibroblasts, where AT1 receptors have been identified [2,3]. Cardiac collagen concentration increases rapidly after birth [4] and in 4-week-old normal rats treated with enalapril for 5 weeks, collagen accumulation was attenuated in tissue of each ventricle, aorta and superior mesenteric artery at week 9 compared to age-matched untreated controls [5]. In normal rats with endogenous elevations in circulating AngII secondary to unilateral renal ischemia or rats treated with pathophysiologic concentrations of exogenous AngII by minipump, abnormal interstitial and perivascular collagen deposition is attenuated by ACE inhibitor treatment [6,7]. Collectively, these findings suggest important stimulatory functions of AngII locally produced and/or circulating on cardiovascular connective tissue accumulation.

Most AngII effects in adult rat tissues are mediated by AT1 receptors. AT1 and AT2 receptors are developmentally regulated so that in embryonic and fetal mesenchymal tissues AT2 receptor expression is predominant [8]. In the embryonic heart, AT1 and AT2 receptor mRNA, as well as AngII receptors, have been demonstrated in atrial and ventricular myocardium [9–11]. AngII may exert its influence on ECM morphogenesis in a paracrine and/or autocrine fashion, mediated by the expression of TGF-β [12], a family of fibrogenic peptides which have been investigated and implicated in cardiac organogenesis. For example, TGF-β mRNA expression in endothelial cells has been correlated with mesenchymal TGF-β polypeptide expression in endocardial cushions, suggesting important paracrine functions in the formation of heart valve leaflets [13], an exteriorized portion of the matrix.

The present study was undertaken in recognition of the presence of AngII receptors in fetal cardiac tissue and to test the hypothesis that both AngII and TGF-β1 are involved in cardiac connective tissue formation. We also sought to determine whether blockade of fetal AT1 or AT2 receptors would result in attenuation of cardiac collagen formation. To elucidate the AngII receptor subtype responsible for any observed changes, pregnant rats were treated with either an AT1 receptor antagonist losartan or an AT2 receptor antagonist PD123319. We examined total soluble collagen concentration, fibrillar collagen accumulation by picrosirius red staining and immunoblotting, mRNA expression of type I and III collagens, and TGF-β1 mRNA in the newborn rat heart.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
This study was approved by the University of Missouri-Columbia animal care and use committee and conforms to the Guide to the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.1 Animal model
Six-week-old female Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN, USA). Three experimental groups were studied (n=3 each): untreated female rats; female rats treated with losartan (40 mg/kg/day) in powdered food; and female rats treated with PD123319 given by a subcutaneously implanted minipump (12 mg/kg/day). Treatment of female rats was started 1 day before housing them with male rats and continued throughout pregnancy. Mothers and newborns were sacrificed within 24 h of delivery by halothane inhalation. Adrenal glands of the dams were removed, rinsed in cold saline and frozen in liquid nitrogen for AngII receptor binding studies and indirect assessment of drug delivery. After rapid determination of body weight, neonates were frozen in isopentane–dry ice solution. For immunoblotting and determination of cardiac collagen concentration by picrosirius red, hearts were dissected free after animal thawing on ice (n=8 in each group). Remaining animals were used for in situ hybridization studies.

2.2 Tissue morphology
Coronal cryostat sections of whole rat neonates were cut in heart level. Sections (6-µm thick) were stained with hematoxylin and eosin (H & E) for examination of tissue morphology. Fibrillar collagen accumulation was determined by staining sections with collagen-specific Sirius Red 3BA in saturated picric acid solution [14].

2.3 Angiotensin receptor binding
Cryostat sections (16 µm) of adult female adrenals were cut and thaw mounted onto gelatin-coated slides. Sections were preincubated in sodium phosphate buffer for 15 min followed by a 1-h incubation with 0.2 µCi/ml ({approx}90 pM) 125I-[Sar1, Ile8] AngII (Amersham, Arlington Heights, IL, USA) in sodium phosphate buffer, 2 g/l albumin, 0.4 mM bacitracin and 5 mM Na2-EDTA. Nonspecific binding was determined in the presence of 1 µM unlabeled AngII. Angiotensin receptor subtypes were characterized by 125I-[Sar1, Ile8] AngII in the presence of 10 µM of either AT1 receptor antagonist losartan or the AT2 receptor antagonist PD123319. Sections were washed thereafter, dried and exposed to Kodak NMB-6 film for 3 weeks. AngII binding optical density was analyzed using a computer image analysis system (NIH IMAGE, 1.60).

2.4 In situ hybridization
Serial coronal cryostat sections (16 µm) were cut at the heart level of neonates and mounted on gelatin–chromium potassium sulfate-coated slides. Six animals were studied in each group. Sections were allowed to dry briefly on a 37°C heating pad and were kept thereafter at –70°C. Before hybridization, sections were air dried at room temperature for 10 min and fixed in a 10% formaldehyde–phosphate buffered, diethyl pyrocarbonate (DEPC)-treated water for 10 min. After two washing steps in phosphate buffered solution, they were incubated with 0.25% acetic anhydride in 0.1 M TE–HCl–0.9% NaCl (pH 8.0). Sections were hybridized for 16 h at 45°C with either [35S]-dATP-random primer labeled type I procollagen cDNA probe (rat fibroblast {alpha}1R1-1300bp insert-PstI digest), type III collagen, or TGF-β1 (American Type Culture Collection, ATCC, Manassas, VA, USA). Following hybridization, slides were washed four times each for 15 min in a standard saline citrate (SSC)–formamide solution at 40°C. This was followed by rinsing slides in SSC, distilled water and 70% ethanol. Sections were air dried and exposed to Kodak Biomax (MR) film for {approx}4 weeks. Optical densities of the respective hybridization were measured (in duplicate) at three different sites within each region (i.e., left and right ventricular free walls and interventricular septum) and calculated as the mean of these nine values for the whole heart and separately for the three sites examined at each region. Six animals were included in each group. Quantification of mRNA optical density was performed using a computer image analysis system (NIH IMAGE, 1.60) and the mean calculated.

2.5 Protein extraction and collagen determination
Total cardiac protein from newborn rats was extracted by a modified procedure described by Tidball [15] and eight animals were studied in each group. Briefly, snap-frozen neonates were thawed and hearts were dissected, weight determined and hearts homogenized in 80 mM Tris–HCl (pH 6.8), 0.1 M dithiothreitol, 70 mM sodium dodecylsulfate (SDS). Samples were boiled for 5 min and stored at –20°C for later studies. Protein content was measured using a detergent compatible kit (DC protein assay, BioRad) based on the method of Lowry [16]. Total soluble collagen concentration was measured spectrophotometrically by a technique based on Sirius Red binding to collagen [17] with a commercially available kit (Sircol collagen assay, Biocolor, Westbury, NY, USA) according to manufacturers’ guidelines. Briefly, 150 µg protein was incubated with 1000 µl Sircol dye and incubated for 30 min on a shaker at room temperature. After centrifugation, the precipitation containing collagen-bound dye was dissolved in 1 ml alkali solution and absorbance measured at 540 nm. Collagen concentration was determined from a standard collagen curve.

2.6 Immunoblotting
Aliquots of tissue extract (50–150 µg total protein mixed 3:1 with protein sample buffer 200 mM Tris–HCl, pH 6.8, 4% β-mercaptoethanol, 8% SDS, 0.4% bromphenol blue, 40% glycerol) were electrophoresed on 8% SDS-PAGE at 15 mA during stacking and 20 mA during separation. Following electrophoresis, proteins were transferred electrophoretically onto a nitrocellulose membrane at 0.8 mA/cm2 for 2 h. Protein transfer was confirmed by staining with a 0.1% Ponceau solution in 5% acetic acid and after removing stain by washing phosphate buffered solution, membranes were blocked in a 5% nonfat dried milk solution at room temperature to avoid nonspecific binding of antibodies. The blot was incubated with goat anti-human type I and III (Southern Biotechnology Associates, Birmingham, AL, USA; 1:1000 dilution) and rabbit anti-TGF-β (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:2500 dilution) antibodies, which cross-react with rat tissue. The blot was then incubated with IgG-peroxidase conjugated secondary antibody (Southern Biotechnology Associates; 1:8000 dilution). This was followed by incubation with diaminobenzidine substrate (Sigma Fast Dab) containing 10% CoCl2 (0.3% w/v stock solution) for enhancement of staining.

2.7 Statistics
Data were expressed as mean±S.E.M. Differences between groups were analyzed by one-way ANOVA with Tukey’s post hoc analysis and differences were considered statistically significant at the 0.05 level.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Treatments were well tolerated. Both PD123319- and losartan-treated dams had litters of comparable size (see Table 1). In both treatment groups hearts appeared normal in size, shape and structure, with a trend to lower heart weight in losartan-treated animals. We found a trend for lower body weight at the end of pregnancy in losartan-treated dams. Moreover, offspring from losartan-treated females had a significantly lower birth weight compared to PD123319 or untreated animals (Table 1).


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  		Table 1 Mean body weight of dams and offspring together with heart weight and heart/bodyweight ratio in newborn rats treated with either losartan or PD123319a

 
To address drug delivery in dams, maternal adrenal glands were subjected to angiotensin receptor binding studies. As demonstrated in Fig. 1, total AngII binding reveals high AngII receptor expression in the zona glomerulosa and medulla in untreated controls. Losartan displaced nearly all AngII binding in the zona glomerulosa whereas PD123319-treated animals had no binding in the medulla. In losartan-treated animals, no AngII receptor binding was detected in the outer cortex, suggesting drug delivery to the dam. Moreover, in PD123319-treated pregnant rats, AngII receptor binding was found only in the outer cortex but not the adrenal medulla. Interestingly, losartan was unable to displace AngII binding in the outer cortex in PD123319-treated animals. However, in studying sections of newborn animals we were unable to demonstrate AngII receptor blockade by either treatment. The explanation for same is uncertain. We would speculate this to be related to a rapid decline of AngII binding sites after birth as reported by Grady et al. [8], a short half-life of each agent and/or disrupted treatment after birth.


Figure 1
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  		Fig. 1 Autoradiographic localization of 125I-AngII receptor binding in maternal adrenal glands. Adrenal glands were removed immediately after delivery and 16-µm sections used for AngII binding studies. In untreated control (CTL), high AngII binding is present in the zona glomerulosa and medulla. In losartan-treated animals (LOS), AngII binding in the zona glomerulosa was abolished, whereas PD123319-treated animals (PD) had no AngII receptor binding in the adrenal medulla. AT1 and AT2 denotes displacement studies with losartan and PD123319 respectively. High binding areas appear dark; (TB, total binding).

 
As shown in Fig. 2 for coronal heart sections of neonates, cardiac structures could be easily identified. We sought to determine collagen volume fraction by immunohistochemical staining with picrosirius red (PSR). However, in contrast to bone tissue, which served as an internal control for positive PSR staining, only faint staining was visible in cardiac ventricles and high background staining of the surrounding tissue made a quantitative approach impossible. In normal cardiac tissue of untreated controls, stronger PSR staining was present in the pericardium, the LV outflow tract and proximal aorta, and the mitral valve than myocardium (data not shown).


Figure 2
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  		Fig. 2 Hematoxylin–eosin staining of a coronal section of newborn rat at the heart level. Cardiac structures are denoted as LV, left ventricle; MiV, mitral valve; IVS, interventricular septum; AoV, aortic valve; RV, right ventricle; SP, spine; ES, esophagus. Magnification 40x.

 
Measuring total soluble collagen concentration in whole heart extracts revealed a significant (P<0.05) decrease in both losartan- and PD123319-treated animals (Fig. 3). This finding was further confirmed by immunoblotting, using specific antibodies for rat type I and III collagens. Although overall staining was faint, labeling for both type I and III collagens was more abundant in the untreated control group and was essentially nondetectable in both treatment groups. We also found a trend to lower immunoreactivity for TGF-β in PD123319-treated animals (data not shown).


Figure 3
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  		Fig. 3 Total soluble collagen concentration of newborn rat whole heart extracts. Both losartan-treated and PD123319-treated rats had significantly lower collagen contents compared to untreated control group. Abbreviations as in Fig. 1. *P<0.05.

 
Quantitative data for type I and III collagen mRNA and TGF-β1 mRNA are presented in Fig. 4. Type I collagen expression was significantly reduced in losartan as well as PD123319-treated animals, suggesting that both receptor subtypes are involved in the regulation of collagen expression in the fetus. Moreover, the same pattern was present for TGF-β1 mRNA expression, which is in keeping with our hypothesis that autocrine properties of AngII regulate TGF-β1 expression. Type III collagen mRNA was upregulated in both treatment groups. No regional differences of either type I and type III collagen or TGF-β1 mRNA expression were detected in left ventricle, interventricular septum and right ventricle of either untreated controls or treated animals (data not shown).


Figure 4
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  		Fig. 4 Quantitative analysis of (A) type I collagen, (B) type III collagen and (C) TGF-β mRNA in cardiac tissue of newborn rats. Each value represents the mean of nine different measurements of optical densities in left ventricle, interventricular septum and right ventricle. Abbreviations as in Fig. 1. *P<0.05.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Collagen concentration of right and left ventricles are equivalent at birth [18]. At birth, type I collagen is only present in small amounts associated with the perimysium [19]. Both type I and III collagen mRNA rapidly increase after birth peaking at day 7 and 3 respectively. In situ hybridization studies have shown intense labeling of type I collagen mRNA in heart valve leaflets, perivascular space or adventitia, and within the interstitium of the myocardium [4]. Marked differences between ventricles follow as right ventricular myocyte size declines in keeping with attenuated pulmonary vascular resistance. The functional significance of the network formed by type I and III collagens is underscored by structural disintegration and aneurysmal formation of the primitive heart that appears in response to β-aminoproprionitrile treatment, an inhibitor of collagen crosslinking [20].

The administration of an ACE inhibitor has been demonstrated to attenuate cardiac collagen accumulation in young otherwise normal rats [5] and young genetically hypertensive rats [21]. Important regulatory functions of AngII on ECM formation during development are suggested by the presence of hypoplastic rat skull (calvarium) and defective formation of long bones, vertebrae and ribs in offspring with ACE inhibitor administration given during gestation [22–24]. Reduced ventricular development and cardiac dilatation has been demonstrated by in vitro treatment of cultured embryos with either AT1 receptor antagonist losartan or AT2 receptor antagonist PD123319 [25], indicating that the developing heart is target tissue of AngII. Although AngII receptors in mesenchymal fetal tissue are predominantly of the AT2 receptor subtype, knockout mice lacking the AT2 receptor develop normally except for abnormal drinking behavior and blood pressure response to AngII challenge [26]; alterations in connective tissue formation have not been addressed. Since AT1 and AT2 receptors are each expressed in embryonic cardiac tissue and AngII raises collagen transcription, we sought to address which AngII receptor subtype regulates collagen formation in the developing fetal heart.

We found total soluble cardiac collagen concentration to be significantly reduced in both treatment groups. This finding was confirmed by immunoblotting using specific antibodies against type I and III collagens. Both type I and III fibrillar collagens are present in cardiac tissue of untreated newborn animals, whereas type I collagen was almost nondetectable in PD123319-treated animals. Type III collagen was reduced in both treatment groups. These findings suggest both receptor subtypes are involved in the regulation of collagen expression in the fetal heart. In earlier studies, AngII receptors have been identified in atrial and ventricular myocardium on embryonic day 14 and in the aorta and pulmonary artery on embryonic day 19 [11]. Due to the small size of the heart, however, whole heart extracts may have contained fragments of large vessels, thus reflecting collagen concentration of both atrial and ventricular myocardium and great vessels. Nevertheless, attenuated collagen formation by losartan and PD123319 observed in the present study support the hypothesis of Price et al. [25] that functional AngII receptors are present in fetal tissues, including cardiovascular connective tissue.

The TGF-β family of fibrogenic peptides augment collagen accumulation by stimulating collagen synthesis and altering protease/protease inhibitor balance thereby inhibiting matrix degradation [27]. AngII may exert its biological influence on connective tissue homeostasis, at least in part, by autocrine secretion of TGF-β1 [28]. For example, AngII induces TGF-β1 mRNA expression in cultured neonatal fibroblasts by transient expression of transcription factors c-fos, c-jun and Egr-1 [29]. Furthermore, losartan treatment results in attenuated TGF-β1 expression in rat models of myocardial infarction [30] and renal injury [31]. TGF-β is implicated in cardiac development and is expressed in cardiac jelly. Antisense oligonucleotides which interfere with TGF-β3 [32] or antibodies directed against TGF-β [33] result in the disruption of endocardial cushion formation. It is thought that TGF-β mediates epithelial-mesenchymal transformation of embryonic cardiac endothelial cells to form primitive valve and septal structures [32,33].

In the present study, type I collagen and TGF-β1 mRNA expression were both attenuated in each treatment group, suggesting AngII effects may in part be mediated by TGF-β1. In keeping with our hypothesis that local AngII is important in regulation of collagen expression [14], no differences were detected in either type I and type III collagen or TGF-β1 mRNA expression in the right ventricle compared to interventricular septum and left ventricle.

Our present findings do not distinguish between circulating and local AngII-mediated effects. All components necessary for de novo AngII generation have been demonstrated in adult cardiac tissue [34]. It is uncertain, however, whether de novo AngII generation is also present in fetal cardiac tissue. For example, ACE binding is not present in either atrial or ventricular myocardium until after birth [11], whereas transcripts for renin in fetal human heart [35] and ACE, angiotensinogen and AT1 and AT2 receptor mRNA were expressed in fetal rat cardiac tissue on embryonic day 10.25 [25]. In fetal cardiac tissue, AT1A receptor mRNA has been demonstrated in the interventricular septum, the bundle of His and the pericardium [36,37]. High AngII receptor density is present in fetal cardiac tissue [38,39], suggesting the developing heart is the target tissue for local and/or circulating AngII. The developmental regulation of AngII receptor expression in cardiac tissue suggests important regulatory functions of AngII during gestation. AngII is an important regulatory peptide during periods of high collagen turnover, as further demonstrated by AT2 receptor reexpression in cardiac tissue repair in the adult heart [40].

Several study limitations should be mentioned. Based on spatial differential expression of AngII receptors, we studied drug delivery in dams by AngII receptor binding studies of maternal adrenal glands. AT1 receptors are predominant in the zona glomerulosa and AT2 receptors in the adrenal medulla [41] and this distribution is not altered in the pregnant rat [42]. As demonstrated by complete inhibition of AngII receptor binding at the respective anatomic site, drug delivery to dams was present in both treatment groups. In newborn rats however, we were unable to demonstrate AngII receptor blockade in either treatment group. Explanations for this finding could be severalfold: interruption of drug delivery to the offspring after birth together with a known rapid decline in AngII binding sites min after birth [8,43]; and/or short plasma half-life of drugs used (e.g., 3 h for losartan) [44]. Since placental uptake and fetal transfer of losartan, as well as its active metabolite EXP3174, is detectable in later gestation (days 15–20) [44], we are of the opinion that sufficient drug delivery occurred, particularly in the late gestational period when AT1 receptors are expressed. Information as to placental transfer of PD123319 is not available. Although we were unable to demonstrate AngII receptor blockade in the fetuses, attenuated collagen formation in both losartan and PD123319-treated animals strongly suggests effective AngII receptor blockade during gestation.

Finally, the present study was not designed to determine whether attenuation of collagen formation in the developing heart may have functional consequences. It further remains to be determined whether valve malformations or membranous septal defects are associated with AngII receptor blockade during fetal development.

In summary, this study investigated regulatory functions of AngII, expressed via either AT1 or AT2 receptors, on connective tissue formation during cardiac development in utero. We have shown that both receptor subtypes are involved in this regulatory process. AngII receptor blockade results in attenuated cardiac collagen formation, which may be mediated in part by TGF-β1. Whether such alterations in collagen formation in response to AngII receptor blockade have functional and/or structural consequences requires further study.

Time for primary review 21 days.


   References
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 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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