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European Journal of Heart Failure 2008 10(9):840-849; doi:10.1016/j.ejheart.2008.06.020
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© 2008 European Society of Cardiology

Increased expression of adrenomedullin 2/intermedin in rat hearts with congestive heart failure

Takuo Hirosea,*, Kazuhito Totsunea,b, Nobuyoshi Moric, Ryo Morimotod, Masahiro Hashimotoa, Yukiko Nakashigea, Hirohito Metokia,e, Kei Asayamab, Masahiro Kikuyaa, Takayoshi Ohkubob,f, Junichiro Hashimotob,f, Hironobu Sasanog, Masahiro Kohzukic, Kazuhiro Takahashib,h and Yutaka Imaia,b

a Department of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences and Medicine 6-3 Aramaki-aza-aoba, Aoba-ku, Sendai 980-8578, Japan
b Tohoku University 21st Center of Excellence Program "Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation" (CRESCENDO) 6-3 Aramaki-aza-aoba, Aoba-ku, Sendai 980-8578, Japan
c Department of Internal Medicine and Rehabilitation Science, Tohoku University School of Medicine 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
d Department of Medicine, Tohoku University School of Medicine 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
e Department of Medical Genetics, Tohoku University School of Medicine 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
f Department of Planning for Drug Development and Clinical Evaluation, Tohoku University Graduate School of Pharmaceutical Sciences and Medicine 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
g Department of Pathology, Tohoku University School of Medicine 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
h Department of Endocrinology and Applied Medical Science, Tohoku University Graduate School of Medicine 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

* Corresponding author. Tel.: +81 22 795 6807; fax: +81 22 795 6839. E-mail address: hirose-t{at}m.tains.tohoku.ac.jp (T. Hirose).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Adrenomedullin 2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. To investigate the pathophysiological role of AM2/IMD in heart failure, we examined the expression of AM2/IMD, adrenomedullin (AM) and receptor complex components (calcitonin receptor-like receptor, three types of receptor activity-modifying proteins) by quantitative RT-PCR and immunohistochemistry in the hearts and kidneys of rats with congestive heart failure (CHF). Significantly increased levels of AM2/IMD mRNAwere found in the atrium, right ventricle, non-infarcted part of the left ventricle and the infarcted part of the left ventricle of CHF rats, compared with sham operated rats (about 2.8-fold, 1.7-fold, 1.7-fold and 2.5-fold, respectively). Expression levels of mRNA encoding AM and the receptor complex components were also increased in the hearts of CHF rats. In a separate experiment, AM2/IMD mRNA levels in the heart did not differ between Wistar—Kyoto and spontaneously hypertensive rats. In both sham operated and CHF rats, the myocardium was diffusely immunostained with AM2/IMD. The fibrotic infarcted layer was not immunostained with AM2/IMD but was surrounded by positively immunostained myocardial layers. These findings suggest that the expression of AM2/IMD is enhanced in the failing heart, and AM2/IMD has a certain pathophysiological role in heart failure.

Key Words: Adrenomedullin • Adrenomedullin 2/Intermedin • Heart failure • RT-PCR • Immunohistochemistry

Received February 7, 2008; Revised April 29, 2008; Accepted June 30, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Adrenomedullin 2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family, which includes calcitonin, CGRP, amylin and adrenomedullin (AM) [1-4]. The calcitonin/CGRP family of peptides, which are widely distributed in various peripheral tissues as well as in the central nervous system, induce multiple biological effects including potent vasodilation (CGRP and AM), reduction in nutrient intake (amylin) and decreased bone resorption (calcitonin) [5]. Two research groups recently discovered the peptide almost simultaneously by searching the genome database, and named it intermedin (IMD) [1] and adrenomedullin 2 (AM2) [2], respectively. Human AM2/IMD and rat AM2/IMD consist of 47 amino acids. Rat AM2/IMD has 34% similarity with rat AM. Reverse-transcriptase polymerase chain reaction (RT-PCR) has shown that AM2/IMD mRNA is widely distributed in various tissues of mice [2]. Immunohistochemical investigations in mice and human have revealed that AM2/IMD immunoreactivity was detected in the heart and kidney [3,6].

CGRP, AM and AM2/IMD activate the complex of calcitonin receptor-like receptor (CRLR) and one of the three types of receptor activity-modifying proteins (RAMPs) to transfer their signals [1,2,7]. AM2/IMD interacts nonselectively with all three CRLR/RAMP complexes, whereas CGRP preferentially interacts with CRLR/RAMP1, and AM with CRLR/RAMP2 or CRLR/RAMP3 [1]. AM2/IMD stimulates cAMP production and has a potent vasodilator action like AM and CGRP [1,2]. Intravenous injection of AM2/IMD decreased arterial blood pressure [1,3,8], and this effect was partially blocked with CGRP and AM receptor antagonists [1]. AM2/IMD was reported to have a positive inotropic action on murine cardiomyocytes [9]. In addition, AM2/IMD reduced pulmonary vascular resistance via nitric oxide dependent mechanism in a rat model with increased pulmonary vasoconstrictor tone [10]. Moreover, intrarenal infusion of AM2/IMD caused diuresis and natriuresis without significant decrease in systemic blood pressure in rats [11]. These previous reports suggest that AM2/IMD is a possible novel modulator of systemic circulation and blood pressure homeostasis, and plays an important role in the pathophysiology of cardiovascular diseases like AM.

The importance of AM as an organ protective peptide has been extensively studied, and AM is now a therapeutic target in cardiovascular and renal diseases. Plasma concentrations of AM are elevated in many cardiovascular disorders, including heart failure, renal dysfunction and diabetes mellitus, and AM acts as an autocrine or paracrine factor to prevent organ damage [12-14]. The pathophysiological roles of AM2/IMD in congestive heart failure (CHF) and hypertension, however, have not previously been investigated. In this study, we therefore examined the expression of AM2/IMD, AM and receptor complex components (CRLR, RAMP1, RAMP2 and RAMP3) in the hearts and kidneys of rats with CHF induced by coronary ligation. We also studied the expression of AM2/IMD in the hearts of spontaneously hypertensive rats (SHR).


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animals
The investigation conforms 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 1985). Animal experiments were approved by the Animal Care Committee of Tohoku University Graduate School of Medicine.

Eight week old male Wistar-Kyoto rats (WKY) (Charles River Japan Inc., Tsukuba, Japan) underwent either coronary ligation or sham operation under sodium pentobarbital anaesthesia (50 mg/kg), as previously described [15,16]. In short, in the coronary ligation group (CHF rats, n=35), a left thoracotomy was performed, the heart was exteriorized, and the left coronary artery was ligated between the pulmonary trunk and the left auricle. The heart then returned to the thoracic cavity, and the thorax was closed. Sham operated (SO) rats (n=6) were treated similarly, but without the suture around the coronary artery.

Rats received standard rat chow and water ad libitum for the 8 weeks after surgery. All rats underwent blood pressure measurement and echocardiography one day before organ collection under anaesthesia. Blood samples were obtained during the organ collection, and plasma brain natriuretic peptide (BNP) levels were measured by SRL, Inc. (Tokyo, Japan).

Rats which met the following criteria were defined as the CHF group: rats which survived eight weeks after coronary ligation, with infarct size over 30%, and echocardiographic data and plasma BNP concentrations out of the range of mean±2-standard deviation of the SO rats. Using these criteria, we excluded 27 of 35 rats which underwent the coronary ligation (eleven died, fourteen had infarct size less than 30% and normal echocardiographic data, and two had only slightly elevated plasma BNP levels). The remaining eight rats were included in the CHF group. The rats used in this study were also used in a previous study by the same authors [16].

The cardiac atria, right ventricles, non-infarcted and the infarcted part of the left ventricles and the kidneys were harvested, snap-frozen in liquid nitrogen and maintained at –80 °C until RNA extraction. In CHF rats, the thin fibrotic infarcted region was carefully dissected from the viable non-ischaemic myocardium, and used as the infarcted tissue sample. Tissues for histological examination were excised, fixed with 10% neutral buffered formalin, and embedded into paraffin. The central part of the ventricle was microscopically examined using the section stained with haematoxylin-eosin (HE) to measure the infarct area. The infarct area was manually measured using Scion Image for Windows (Scion Corporation, USA).

In another experiment, twelve week old male WKY rats (n=6) and SHR (Charles River Japan Inc., Tsukuba, Japan) (n=6) were killed under anaesthesia, and the cardiac atria and ventricles were harvested. Before anaesthesia, tail systolic blood pressure was measured, and significantly elevated blood pressure was observed in the SHR (202.8±7.1 mmHg) compared with the WKY rats (156.2±6.6 mmHg).

2.2. Reverse-transcriptase polymerase chain reaction (RT-PCR)
Total RNA was extracted by the guanidinium isothiocyanate/cesium chloride method and were reverse transcribed with 400 units of Moloney murine leukaemia virus reverse transcriptase (PrimeScript, TaKaRa, Otsu, Japan) using an oligo(dT) primer. Expression levels of AM2/IMD, AM, CRLR, RAMP1, RAMP2, RAMP3 and ribosomal protein L32 (RPL32) mRNAs were determined using competitive, quantitative RT-PCR methods, as previously described [16-19]. In our preliminary study, expression levels of RPL32 mRNA (mol/1 µg of total RNA) were the most stable among the several housekeeping genes tested including glyceraldehydes-3-phosphate dehydrogenase and β-actin. RPL32 was therefore selected as an internal control.

Primers used for RT-PCR analysis are shown in Table 1. To determine the equivalent concentration point, the competitive reference standard (CRS-) DNA for AM2/IMD, AM, CRLR, RAMP1, RAMP2, RAMP3 and RPL32 was prepared, and a constant amount of wild-type cDNA and increasing amounts of CRS-DNA were added to each PCR tube, as reported previously [16-19]. AM2/IMD, AM, CRLR, RAMP1, RAMP2 and RAMP3 mRNA concentrations were normalized by RPL32 mRNA expression levels.


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  		Table 1 The sequence of the primers for polymerase chain reaction (PCR)

 
The obtained PCR products were purified by agarose gel, sequenced by an autosequencer (Model 3100, Applied Biosystems), and confirmed 100% identity with the respective nucleotide sequence registered in the NCBI Data Bases.

2.3. Immunohistochemistry
Rat heart tissues were fixed in 10% neutral buffered formalin and embedded into paraffin for immunohistochemistry. Immunohistochemistry of AM2/IMD was performed by the ABC method using the Vector ABC kit (Vector Laboratories, Burlingame, CA, USA), as previously reported [6,20]. Briefly, 1.5 µm sections were deparaffinized and incubated in methanol containing 0.3% H2O2 for 30 min and then with normal goat serum (1:20) to block non-specific staining. Sections were intensely washed in 0.01 mol/l phosphate buffered saline (pH 7.4) between the procedures. The sections were then incubated in antiserum against AM2/IMD (1:2000) for 20 h at 4 °C. Sections were incubated in biotinylated secondary antibody to rabbit IgG (1:400) for 30 min at room temperature and subsequently incubated with peroxidase-conjugated avidin for 30 min using the Vector ABC kit. These sections were visualized by immersion in 3,3'-diaminobenzidine solution (0.01 mol/l 3,3'-diaminobenzidine in 0.05 mol/l Tris-HCl buffer (pH 7.6) and 0.006% H2O2). In negative controls, normal rabbit serum (at a dilution of 1:2000) was used instead of the respective antiserum.

The antiserum against AM2/IMD (No. 0403-721) was raised in a rabbit by injecting human AM2/IMD (17-47) conjugated with bovine serum albumin [6]. The characteristics of this antiserum have been reported previously [6]. Furthermore, the specificity of the AM2/IMD antiserum was examined by the absorption test. The diluted antiserum (1:2000) was incubated with rat AM2/IMD, rat AM, human AM2/IMD or human AM (all these peptides were obtained from Peptide Institute, Minoh, Japan) at concentrations of 10 and 50 nmol peptide/ml of the diluted antiserum for 20 h at 4 °C prior to use.

2.4. Statistics
Data are given as mean±SEM. mRNA concentration were analyzed by unpaired Student's t-test or one-way analysis of variance (ANOVA) and Scheffe's post hoc test for the multiple comparison of differences among the groups. Statistical significance was accepted at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Characteristic data for SO rats and CHF rats
The histological examinations using HE staining confirmed an infarction size of about 35% of the left ventricle in the eight rats with CHF. Furthermore, significantly elevated levels of BNP (about 2.0-fold), heart weight (about 1.3-fold), lung weight (about 1.7-fold), systolic left ventricular inner diameter (about 1.6-fold) and diastolic left ventricular inner diameter (about 1.2-fold) and significantly decreased mean arterial blood pressure (about 86% of SO rats), ejection fraction (about 56% of SO rats) and fractional shortening (about 46% of SO rats) were confirmed in the CHF rats, compared with SO rats (Table 2).


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  		Table 2 Characteristic data for sham operated (SO) rats and congestive heart failure (CHF) rats

 
3.2. mRNA expression
In the CHF rats, AM2/IMD mRNA levels were significantly increased by about 2.8-fold in the atrium (P<0.01), 1.7-fold in the right ventricle (P=0.012), 1.7-fold in the non-infarcted part of the left ventricle (P=0.024) and 2.5-fold in the infarcted part of the left ventricle (P<0.01), when compared with SO rats (Fig. 1A). AM2/IMD mRNA expression in the infarcted part of the left ventricle was higher than that in the non-infarcted part of the left ventricle of the CHF rats (P<0.01) (Fig. 1A). AM2/IMD mRNA levels in the kidney were markedly higher than those in the heart, but there was no significant difference in AM2/IMD mRNA expression levels in the kidney between CHF rats and SO rats (P=0.789).


Figure 01
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  		Fig. 1 Expression levels of (A) adrenomedullin 2/intermedin (AM2/IMD) mRNA, (B) adrenomedullin (AM) mRNA, (C) calcitonin-receptor-like receptor (CRLR) mRNA, (D) receptor-activity modifying receptor 1 (RAMP1) mRNA, (E) RAMP2 mRNA and (F) RAMP3 mRNA in the atrium, the right ventricle (RV), the left ventricle (LV) and the kidney. These tissues were obtained from sham operated (SO) rats and congestive heart failure (CHF) rats. Non-I: non-infarcted part of LV obtained from CHF rats. INF: infarcted part of LV obtained from CHF rats. *P<0.05 vs. SO, **P<0.01 vs. SO, {dagger}P<0.05 vs. Non-I, {ddagger}P<0.01 vs. Non-I.

 
AM mRNA was much more abundantly expressed in the heart than AM2/IMD mRNA. However, the increase of the expression levels of AM mRNA in CHF was not so marked as that of AM2/IMD mRNA. In the CHF rats, AM mRNA levels were significantly increased by about 1.4-fold in the atrium (P<0.05), 1.3-fold in the right ventricle (P<0.01), 1.6-fold in the non-infarcted part of the left ventricle (P<0.05) and 1.9-fold in the infarcted part of the left ventricle (P<0.01), when compared with SO rats (Fig. 1B).

CRLR mRNA levels in CHF rats were significantly increased about 1.4-fold in the atrium (P<0.05), and 1.5-fold in the infarcted part of the left ventricle (P<0.05), when compared with SO rats (Fig. 1C). RAMP1 mRNA levels in CHF rats were significantly increased about 1.9-fold in the atrium (P<0.01), 1.4-fold in the right ventricle (P<0.05) and 2.6-fold in the infarcted part of the left ventricle (P<0.01), when compared with SO rats (Fig. 1D). RAMP2 mRNA levels in CHF rats were significantly increased about 1.3-fold in the atrium and 1.8-fold in the infarcted part of the left ventricle, when compared with SO rats (Fig. 1E). RAMP3 mRNA levels in CHF rats were significantly increased about 1.6-fold only in the infarcted part of the left ventricle, when compared with SO rats (Fig. 1F). In the kidney, there was no significant difference in expression levels of AM, CRLR, RAMP1, RAMP2 and RAMP3 mRNA between CHF rats and SO rats.

There was no significant difference in the expression levels of AM2/IMD mRNA between WKY rats and SHR in either the atrium (WKY: 10.7±1.8 µmol/mole RPL32, SHR: 10.5±1.7 µmol/mole RPL32, P=0.918) or the ventricle (WKY: 17.7±2.9 µmol/mole RPL32, SHR: 19.9±3.3 µmol/mole RPL32, P=0.618).

3.3. Immunohistochemistry
Immunohistochemistry of AM2/IMD in rat hearts showed positive immunostaining in normal rat heart (Fig. 2A). The antiserum absorbed with synthetic rat AM2/IMD (Fig. 2B), or human AM2/IMD (data not shown) significantly attenuated positive immunostaining in normal rat heart whereas the antiserum absorbed with synthetic rat AM or human AM (data not shown) did not affect the immunostaining. Normal rabbit serum showed no positive immunostaining (Fig. 2C).


Figure 02
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  		Fig. 2 The absorption test of adrenomedullin 2/intermedin (AM2/IMD) antiserum in rat heart. A: positive control immunostained with AM2/IMD antiserum. B: absorption test using AM2/IMD antiserum preabsorbed with synthetic rat AM2/IMD. C: negative control using normal rabbit serum. Bar=50 µm.

 
The myocardium was diffusely immunostained with AM2/IMD in SO rats (Fig. 3A, C, E, I) and CHF rats (Fig. 3B, D, F, G, H, J). On the other hand, the subendocardial area and the subepicardial area were barely immunostained with AM2/IMD, and these areas with low AM/IMD immunostaining were widened in CHF (Fig. 3B, D, F) compared with SO rats (Fig. 3A, C, E). In the infarcted region, the fibrotic layer was barely immunostained but was surrounded by positively immunostained myocardial layers (Fig. 3G, H). The vascular smooth muscle cells in the blood vessels of the CHF rats were positively immunostained (Fig. 3J), whereas those of the SO rat were barely immunostained (Fig. 3I).


Figure 03
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  		Fig. 3 Immunohistochemistry of adrenomedullin 2/intermedin (AM2/IMD) in rat heart. A and B: the right ventricle of sham operated (SO) rats (A) and congestive heart failure (CHF) rats (B). C and D: the interventricular septum of SO rats (C) and CHF rats (D). E and F: the left ventricle of SO rats (E) and CHF rats (F). G and H: infarcted part of left ventricle of CHF rats. I and J: the blood vessels of SO rats (I) and CHF rats (J). Endo: endocardium site, Epi: epicardium site. Bar=100 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The present study shows for the first time increased gene expression of AM2/IMD in the hearts of CHF rats. Although increased gene expression of AM2/IMD has been reported in isoproterenol-induced hypertrophic myocardium in rats [8], there has been no previous report on AM2/IMD expression in the heart and kidney tissues in a CHF model. We have also confirmed the increased cardiac expression of AM and receptor complex components (CRLR, RAMP1, RAMP2 and RAMP3), which is consistent with previous reports [18,21-23]. The increase in AM2/IMD mRNA expression in the failing heart was more marked than that of AM mRNA, whereas the expression pattern of AM2/IMD mRNA in the failing heart was similar to that of AM mRNA. Increased expression of AM2/IMD, AM and receptor complex components in the failing heart seems to be an adaptive response to compensate for failed cardiac function as an autocrine or paracrine factor.

It is known that haemodynamic stress, hypoxia and inflammatory cytokines all induce AM expression. Thus the direct haemodynamic consequences of heart failure, the accompanying myocardial ischaemia or increased levels of cytokines in the cardiac tissues following infarction, might elevate the expression levels of AM mRNA in the heart. In contrast, there have been no reports on the induction of AM2/IMD expression by such stimuli or stresses. We therefore evaluated AM2/IMD mRNA levels in the hearts of WKY rats and SHR to study the effect of pressure overload on AM2/IMD expression, but found no significant change in AM2/IMD mRNA levels in the hearts of the two types of rats. Pressure overload is therefore unlikely to explain the increased expression of AM2/IMD mRNA in the cardiomyocytes in the failing heart.

Our immunohistochemical studies showed immunostaining of AM2/IMD in the myocardium and the vascular smooth muscle cells of the failing heart. The positive immunostaining of AM2/IMD in the vasculature of the rat heart is consistent with previous findings in mice [3] and in humans [6]. Increased expression of AM2/IMD mRNA in the ventricle of CHF rats was shown by RT-PCR, but the subpericardial and subendocardial areas were barely immunostained in our CHF rats. AM2/IMD peptide in these subpericardial and subendocardial areas may be secreted by stresses accompanied by heart failure, and the negative AM2/IMD immunostaining in these areas may reflect the depletion of this peptide from the cytoplasm of the cardiomyocytes. Using immunohistochemistry, Hagner et al. showed that CRLR and RAMPs are immunostained in the entire vasculature and that CRLR is expressed mainly in the endothelial layer [24]. Therefore, AM2/IMD secreted from the myocardium in the failing heart may act as a cardiovascular regulator via the activation of CRLR/RAMPs complexes in vascular endothelial cells.

Increased AM2/IMD mRNA levels were shown in the infarcted part of the left ventricle by quantitative RT-PCR. Immunohistochemical analysis suggested that this increase was due to the upregulation of AM2/IMD expression in the myocardium surrounding the fibrotic layer. However, it is possible that the increase may be due to increased levels of AM2/IMD in the vascular smooth muscle cells or an increased number of vascular vessels in the infarcted part of the left ventricle.

There were no significant changes in the mRNA expression levels of AM2/IMD, AM and receptor complex components in the kidney of CHF rats. It is therefore likely that the renal haemodynamic changes induced by heart failure had no significant influence on the expression of the AM system in the kidney including AM2/IMD.

The pathophysiological role of the upregulated AM2/IMD in the failing heart seems to be related to its cardio-protective effects, like AM and CGRP. There is accumulating evidence, which shows that AM and CGRP are potent endogenous cardio- and reno-protective substances. Exogenous administration of AM and CGRP peptides or their gene delivery is a new preventive and therapeutic strategy for cardiovascular diseases such as hypertension, myocardial ischaemia, heart failure and renal failure [13,14,25]. The cardio-protective effects of AM and CGRP are thought to be mediated by CRLR/RAMPs complexes [26]. Yang and co-workers recently demonstrated the cardio-protective effects of AM2/IMD in rat hearts, both in vitro and in vivo [27,28]. These cardio-protective effects of AM2/IMD are similar to those of CGRP and AM, which share the same receptors consisting of CRLR/RAMPs complexes and the cAMP signalling pathway [1,25]. Thus, AM2/IMD could have cardio-protective effects by cross-reacting on the CGRP receptor and the AM receptor, and could be a new target for the prevention and treatment of cardiovascular diseases.

Furthermore, there have been some reports suggesting that AM2/IMD might act on receptor systems other than CRLR/RAMPs, particularly in the hypothalamus and/or pituitary [29]. Contrary to previous findings that AM2/IMD elevated intracellular cAMP levels [1,3] and that CGRP stimulated growth hormone secretion in rats and humans [30], Taylor et al. revealed that AM2/IMD inhibited growth hormone release in Sprague-Dawley rats [29]. There were no reports about the existence of such unidentified AM2/IMD unique receptors in peripheral tissues. Further studies are therefore required to clarify whether the cardio-protective effects of AM2/IMD are mediated by CRLR/RAMPs complexes or by unidentified AM2/IMD unique receptors.

Production and secretion of certain vasoactive peptides and cytokines, such as atrial natriuretic peptide, brain natriuretic peptide, AM, angiotensin II, tumour necrosis factor-{alpha} and urotensin II, are known to be increased in the failing heart [16]. The present study has shown that expression of AM2/IMD was also elevated in the heart of CHF rats. These findings have raised the possibility that AM2/IMD plays a certain pathophysiological role in myocardial infarction and the heart failure syndrome.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
The authors are grateful to Ms. Kumi Kikuchi for her technical assistance, and to the Biomedical Research Core of Tohoku University Graduate School of Medicine for the use of their equipment. This study was supported in part by Grants-in-aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan, and by the Tohoku University 21st Center of Excellence Program "Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation" (CRESCENDO).


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

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