© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Type 2 angiotensin II receptor is downregulated in cardiomyocytes of patients with heart failure
aDepartment of Clinical Laboratory Medicine, Hiroshima University School of Medicine, Hiroshima, Japan
bFirst Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan
cFirst Department of Surgery, Hiroshima University School of Medicine, Hiroshima, Japan
* Corresponding author. Tel.: +81-82-257-5552; fax: +81-82-257-5554 fwga3144{at}mb.infoweb.ne.jp
Received 12 August 1999; accepted 12 August 1999
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Background: The human heart expresses type 2 angiotensin (AT2) receptor, but the function is poorly defined. Methods: In the present study, we investigated (1) the cellular localization of the AT2 receptor and (2) the relationship between the AT2 receptor protein expression and the cardiac function of patients with ischemic heart disease. The receptor localization was assessed by immunohistochemistry and the protein expression was quantified by Western blotting in atrial tissues freshly obtained from 22 patients undergoing coronary artery bypass graft surgery (63.0±11.0 years old; male ratio, 85%). Prior to the surgery, blood was drawn for determination of atrial-natriuretic hormone level and the left ventricular function was assessed by ultrasound cardiography. Results: The results of immunohistochemistry showed that the AT2 receptor was localized to cardiomyocytes and was not present in fibroblasts, vascular smooth muscles, or vascular endothelium. Atrial tissues showed various degrees of structural remodeling, but the localization of the AT2 receptor was not altered in any tissue sections. The amount of the AT2 receptor was negatively correlated with end-diastolic left ventricular diastolic dimension (r=–0.56, P<0.01), calculated left ventricular mass index (r=–0.51, P<0.02) and the plasma atrial natriuretic peptide (ANP) concentration (r=–0.62, P<0.01) and positively correlated with left ventricular ejection fraction (r=0.48, P<0.05). Conclusions: (1) The AT2 receptor is localized to cardiomyocytes independently of the cardiac function. (2) Left ventricular dysfunction is associated with decreased expression of myocardial AT2 receptor protein.
KEYWORDS Angiotensin; Fibrosis; Ischemia; Heart failure; Receptors; Coronary disease; Myocytes; Ventricular function
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Ischemic heart disease is one of the major causes of human heart failure [1]. Angiotensin II (Ang II) plays a major role in the development of heart failure following a myocardial infarction [2,3]. Two subtypes of Ang II receptors, AT1 and AT2 receptors, are expressed in human hearts [4–10]. The AT1 receptor mediates signals leading to cell growth and proliferation [11], whereas the AT2 receptor is thought to mediate signals that are associated with an inhibition of cell growth and proliferation [12,13]. The AT1 receptor predominates in the heart of most animal species including the rat [14] and the hamster [15]. Radioligand binding studies in humans [6,9,10] suggest that the AT2 receptor subtype predominates in the myocardium of the normal as well as the failing hearts, representing 50–70% of the binding sites identified. Treatment with a selective AT1 receptor antagonist leads to a marked elevation in the circulating Ang II level [16], which selectively binds to the AT2 receptor and cause an exaggeration of the effects mediated by the AT2 receptor. However, the function of AT2 receptors is still poorly defined in the human heart.
The heart consists of myocytes and non-myocytes, mostly fibroblasts. Although the cardiomyocytes, although occupy 75% of the cardiac space, they represent only one-third of the total cardiac cells. In the failing heart, either due to hypertension or myocardial infarction, the proliferation of fibroblasts and the deposition of collagen occur in the interstitial space, whose extent is a major determinant of left ventricular dysfunction [17]. In cultured systems, Ang II causes cardiomyocyte-production of Ang II and growth factors such as TGF-β [11], which may in turn stimulate fibroblasts as well as cardiomyocytes. It is not clear whether the AT2 receptor is involved in the structural remodeling of the failing human heart. To elucidate the role of this receptor, it is important to determine its cellular localization in the failing as well as in the normal myocardium, as recent studies [9,10] indicate that the receptor expression is differentially regulated by different cell-types. Immunohistochemistry currently is the best technique for identifying cellular localization [18]. However, the immunohistochemical localization of the AT2 receptors has not been determined in human heart.
In the present study, using immunohistochemistry and Western blotting, we investigated the cellular localization of AT2 receptors and the relationship between the receptor protein expression and cardiac function in patients undergoing coronary artery bypass graft.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Patients
A relationship between cardiac AT2 receptor expression and cardiac function was investigated in 22 patients with ischemic heart disease (18 males, 4 females; aged 63.4±11.3 years; weight, 58.2±8.5 kg; height, 158.2±8.7 cm) undergoing coronary artery bypass graft surgery. All of these patients had stable hemodynamics, were free of valvular disease and lacked congenital heart disease. Each operation was staged. Specimens of the right atrium were obtained intraoperatively and were snap-frozen in liquid nitrogen. All patients had received medical therapy prior to surgery (Table 1) that consist of two or more drugs of the following categories (ACE in 7, Ca-channel blockers in 14, beta-adrenergic receptor blockers in 6, nitrates in 20 and anti-platelet drugs in all patients). Cardiac function was evaluated by echocardiography few days before surgery. For immunohistochemical studies, in addition to the 22 specimens of these patients, 3 intraoperative specimens of left atrium from patients with mitral valve stenosis and 3 specimens of cardiac ventricles (autopsy cases who had died of cancer.) were obtained. The left atrial tissues had severe interstitial fibrosis. These cases were included to study possible changes in AT2 receptor-localization in fibrotic heart. All subjects agreed to the usage of cardiac tissue for this protocol with written informed consent. Study protocols were approved by the Committee on ethics of the Hiroshima University School of Medicine.
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2.2 Cardiac parameters
The morphology and function of the left ventricle was examined preoperatively by echocardiography. Examinations were performed using a Hewlett Packard (SONOS 5500). The parasternal acoustic window was used to record at least 10 consecutive beats of two-dimensional and M-mode recordings. Left ventricle internal dimension and interventricular septal- and posterior wall thickness were measured at end-diastole and end-systole according to the recommendation of the American Society of Echocardiography [19]. The left ventricular ejection fraction (EF) and left ventricular mass index (LVMI) were calculated as described previously [20].
For endocrinological assessment of left ventricular function, a blood sample was drawn on the morning of the operation for the determination of human ANP (Table 1) using radioimmunoassay.
2.3 AT2 receptor antiserum
Goat anti-AT2 receptor polyclonal antiserum (S-7540), directed toward the carboxyl terminal of the native receptor, was purchased from Santa Cruz International (Santa Cruz, CA). The selectivity of the antiserum against AT2 was evaluated in Western blot analysis.
A single 
55-kDa band was observed in specimens of the right and left atria and lungs, which are both known to express the AT2 receptor [21], whereas no band was observed in gastric tissue, which has not been reported to express the receptor (Fig. 1). The approximate molecular mass of the AT2 receptor was consistent with that previously reported [22]. There was no cross-reactivity with AT1 receptor (information from Santa Cruz Co.)
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2.4 Immunohistochemical analysis
Immunohistochemical analysis was performed using paraffin embedded sections as described previously [18,23,24]. Briefly, each tissue specimen was fixed with 10% Formalin and embedded in paraffin, and 3 µm sections were cut. The endogenous peroxidase and non-specific binding sites of secondary donkey antibody were blocked with 1% H2O2 in phosphate buffered saline (PBS) and 10% normal donkey serum and 1% non-fat dry milk in PBS, respectively. The sections were incubated overnight at 4°C with one of the following: (1) AT2 receptor antiserum, (2) the IgG fraction of the non-immune serum, or (3) the AT2 antiserum preadsorbed with its pure peptide antigen. For preadsorption of the AT2 receptor antiserum, the serum was incubated overnight at 4°C with an equal molar amount of the immunizing peptide (Santa Cruz, sc-7420 p). All antibodies were diluted at 1:1000 in 1.5% normal goat serum and 0.5% non-fat dry milk in PBS. After being washed in PBS, immunostaining was detected and visualized by the avidin-biotin immunoperoxidase method (Santa Cruz ABC kit) using diaminobentizin as a substrate. Tissue sections were lightly stained with hematoxylin, dehydrated, and placed under a coverslip.
2.5 Western blot analysis of AT2 receptor protein expression
Samples were prepared as previously described [18,23,24]. Tissues stored in liquid N2 were homogenized with Polytron in buffer A (10% glycerol, 20 mM Tris–HCl, 100 mM NaCl, 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM EGTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A). The homogenate was centrifuged at 15 000 g for 30 min at 4°C. The pellets were resuspended in buffer B (buffer A with 1% NP 40), stirred for 1 h at 4°C, and centrifuged again at 15 000 g for 20 min at 4°C. The supernatant was frozen at –80°C until use. The protein concentration was measured by the bicinchoninic acid method [24,25]. The solubilized samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE, 10% running gel). The resolved proteins were transferred onto a nitrocellulose membrane (Hi-bond ECL, Amersham) and probed with the anti-AT2 receptor antiserum (1:2500 dilution). The bands were visualized with an ECL Western Blotting Detection Kit (Amersham). For quantification of the AT2 receptor protein, the bands were area-traced and the mean density in the area was analyzed using a NIH Image software program. The product of the mean density by the area was defined as band intensity. To standardize the quantification, an equal amount (24 µg) of protein from subjects and three standard samples with constant amounts of AT2 receptor (a stock sample from the right atrium of one patient, containing 32, 16, and 8 µg protein) were always loaded in the same gel. The standard samples were used for generation of a standard curve. The amount of AT2 receptor in each sample was read from this standard curve.
To confirm that amounts of samples loaded on the gel are equal, the nitrocellulose membrane, after detection of AT2 receptor, was re-probed for actin as an internal standard. The primary and secondary antibodies were removed from the membrane by submerging in buffer containing 100 mM mercaptoethanol and 2% SDS for 30 min at 50°C. The membrane was washed, blocked with 5% skim milk, and incubated with anti-actin antibody (MAB1501, Chemikon International, Temecula, CA), which non-specifically react with all kinds of actin. The bands were visualized as described above.
2.6 Statistical analysis
All data were analyzed with StatView version 4.54 (Abacus) software. The data are presented as mean±S.E.M. Differences at P0.05 were considered statistically significant.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Western blotting of the AT2
A single
55-kDa band was observed in specimens of the right and left atria and lungs, which are both known to express the AT2 receptor [21], whereas no band was observed in gastric tissue, which has not been reported to express the receptor (Fig. 1).
3.2 Immunohistochemistry for the AT2 receptor protein
Positive immunostaining for the AT2 receptor was observed in the cardiomyocytes but not in the regions of perivascular or interstitial fibrosis, the smooth muscle layer, or endothelium of the epicardial and intracardiac arteries (Fig. 2B, D). This localization of the AT2 receptor was similar in the left atrium (data not shown) and the right atrium (Fig. 2) and in the left ventricle (Fig. 3). In cross-section of the myocytes, we observed relatively heavier staining on the surface (Fig. 4B). Individual specimens of the right atria showed various degrees of perivascular and interstitial fibrosis. However, no difference was noted among individuals as to the localization of the AT2 receptor (Fig. 2B, D). The left atrium of the patients with mitral stenosis showed marked interstitial fibrosis, but similar localization of the AT2 receptor (data not shown). The pre-absorption controls for all these immunohistochemical studies were all negative (Fig. 2A, C, Fig. 3A, Fig. 4A).
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), but not in connective tissues (*), fibroblasts and smooth muscle layer of intra-cardiac small arteries (
). No difference in the distribution pattern of the receptor is noted between the two patients. Magnification x200.
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3.3 AT2 receptors and correlation with cardiac parameters
A single
55-kDa band was observed by Western blotting in all samples. The amount of AT2 receptor was analyzed as the band intensity. Patients showed various amounts of AT2 receptor. After the detection of the AT2 receptor, the antibody was stripped off and the same membrane was proved with anti actin antibody. The band intensities of actin in samples were constant, indicating that the observed difference in the amounts of AT2 receptor was not a reflection of the variation of protein amount (Fig. 5). Quantitative analysis of the band revealed that the amount of the AT2 receptor protein was positively correlated with EF (Fig. 6A, r=0.481, p0.05), and was negatively correlated with the left ventricular diastolic dimension (LVDd) (Fig. 6B, r=–0.558, p0.01), the calculated LVMI (Fig. 6C, r=–0.508, P<0.02) and the plasma ANP concentration (Fig. 6D, r=–0.616, P<0.02). However, we observed no significant correlation between the amount of the AT2 receptor and the left ventricular wall thickness, the left atrial dimension, or the diameter of the aortic root. The amount of the AT2 receptor to the patients’ age, gender, or the medications administered was unrelated.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The major findings in the present study are as follows; The AT2 receptor was identified in cardiomyocytes but not in fibroblasts, vascular smooth muscles or vascular endothelium. Left ventricular dysfunction due to ischemic heart disease did not influence this distribution pattern. However, the expression of the AT2 receptor in myocytes decreased with development of the left ventricular dysfunction.
4.1 Importance of cardiac AT2 receptor in development and treatment of heart failure
It is reported that the AT2 receptor, versus AT1 receptor, is a predominant subtype of Ang II receptors in human heart [6,9,10]. Clinical treatment with AT1 receptor antagonists leads to elevation of plasma Ang II level [16], which may selectively stimulate the AT2 receptors. Therefore, it is important to elucidate the function of the cardiac AT2 receptor in humans. The amount of myocardial AT2 receptor was negatively correlated with LVDd, LVMI, and ANP, and was positively correlated with EF in our subjects. Regardless of etiology, presence of left ventricular dysfunction is associated with dilatation of the cardiac chamber, an overall increase in the left ventricular mass, and a reduction in ejection fraction [1,17]. Plasma level of ANP is an established indicator of cardiac dysfunction [25]. Therefore, in the present study, the cardiac AT2 receptor was decreased in parallel with development of heart failure. It is tempting to speculate that the amount of AT2 receptor determines the cardiac function, and selective stimulation of the AT2 receptor may improve cardiac function. Supporting this view, transgenic mice overexpressing AT2 receptor in cardiomyocytes showed an attenuated response to AT1 receptor mediated pressor- and chronotropic effects [26]. In rat model of post-infarcted heart failure, beneficial effect of losartan on myocardial hypertrophy and interstitial fibrosis were all abolished by administration of selective AT2 receptor antagonist [27].
However, it is not possible to tell from whether the change in the AT2 receptor observed in the present study was the cause or the effect of the cardiac dysfunction. A decrease in the AT2 receptor can occur as a consequence of upregulation of protein kinase C [28] or other growth factors [29], which are reported to be increased in the failing heart [30,31]. Also, change in the myocardial AT1 receptor in failing heart needs to be addressed, although most previous studies on this subtype show its downregulation [4–6,8–10].
4.2 Localization of the AT2 receptor
We identified the AT2 receptors only in the cardiomyocytes, but not in the fibroblasts. This finding provide one basis for a hypothesis that AT2 receptors is involved in the myocardial growth or function. Second, if the myocardial AT2 receptors have any effects on the interstitial fibrosis, the effects should be through substances produced in cardiomyocytes. In contrast to our immunohistochemical findings, recent studies by emulsion autoradiography demonstrated the presence of AT2 receptors in the cardiac interstitium (although not prominent in normal hearts). These receptors showed a marked upregulation within the fibrous regions in patients with end-stage heart failure [9,10]. Although the reasons for the discrepancy from our findings are unclear, it may be explained by the differences in the severity of heart failure (cardiac dysfunction in our subjects are not severe compared with those in references [9,10] and the technique to detect the AT2 receptor. It has been reported in murine cell-line that there are distinct sub-populations within AT2 receptor [32]. In human ventricles, Wharton et al. [10] found an expression of two distinct AT2 receptor gene transcripts reflecting alternative splicing. Ligand-binding techniques including emulsion autoradiography may not be able to recognize such subclasses of the AT2 receptors, if any, unless the binding property was not affected. On the other hand, the antibody to the AT2 receptor is sensitive to differences in the molecular sequence. Our present results raise the possibility that the AT2 receptors identified in the fibrous regions only by the ligand binding technique may represent as-yet-unknown subclass of AT2 receptor. Variation of the molecular sequence and the binding property of the AT2 receptor may also come from difference in the glycosylation status of the receptor [22]. Additional studies on the presence of AT2 receptor-like activity in fibrous tissue are needed.
It may be also possible that our antibody to the AT2 receptor, which is directed toward the C-terminus of the protein, is sensitive only to a small population of the AT2 receptor and the amount of the receptor is underestimated. However, this is not likely to be the case because immunohistochemical localization of AT2 receptor in rat heart, examined with antibody directed toward N terminus of the receptor, was completely consistent with that in humans [24]. Furthermore, the immuno-detected AT2 receptor was decreased in hypertrophied left ventricles of spontaneously hypertensive rats [23], again consistent with our present observation.
4.3 Discrepancy
Previously, changes in AT1 and AT2 receptors in patients with heart failure have been studied by use of the ligand binding technique [6,33,34], autoradiography [9,10] and reverse transcription polymerase chain reaction [5,8]. The AT1 receptor was found to be downregulated in most of studies [4–6,8–10] whereas different results have been reported as to the AT2 receptor. Consistent with our observation, downregulation of the AT2 receptor was reported in end-stage heart failure [6] and in hypertrophied ventricles [33,35]. On the other hand, in end-stage heart failure, Haywood et al. [8] reported that the AT2 receptor mRNA was unchanged, and Tsutsumi et al. [9] and Wharton et al. [10] reported that the AT2 receptor-binding sites increased. Although the reason for this discrepancy is unclear, it may be related to differences in the pathophysiology of the disease investigated and its clinical stage and methodology to detect the receptor.
AT2 receptor is upregulated in acute phase of myocardial infarction [36]. Although the mechanism is unclear, change in the AT1 receptor may be involved. It has been reported that AT1 receptor mediates the signal for upregulation of AT2 receptor [36]. In acute myocardial infarction, the AT1 receptor is upregulated, while it is decreased with development of heart failure.
4.4 Right atrium
We investigated the change in the AT2 receptor in the right atrium instead of the left ventricle for the following reasons, (1) AT1 and AT2 receptors are expressed in the myocardium throughout both atria and ventricles with a similar proportion of the two subtypes and their expression is even higher in atria than in ventricles [6,9,10,34]; (2) previous studies [6,9,10,34] have demonstrated parallel changes in the receptor expression in atria and ventricles; (3) histological signs of cardiac remodeling were also observed in atria as well as in the ventricles [10]. Mechanical stress, although smaller than in left ventricle, as well as humoral factors may affect the expression of AT2 receptor in the right atrium in the same way as in ventricles.
In summary, we demonstrated immunohistochemical localization of the human cardiac AT2 receptor. This receptor subtype is localized mainly in cardiomyocytes, not in vessels and regions of fibrosis. Left ventricular dysfunction did not alter the distribution. The expression of the myocardial AT2 receptor decreased with development of heart failure. The myocardial AT2 receptor in human heart may play a key role in the development of left ventricular dysfunction.
Time for primary review 21 days.
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Acknowledgements |
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This study was supported by a Grant-in-Aid for Scientific Research (No. 11470518). The authors wish to thank Katsunari Ogawa, Yuko Omura and Yumi Tsujimura for their secretarial assistance.
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