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Upregulation of intracardiac adrenomedullin and its receptor system in rats with volume overload-induced cardiac hypertrophy
Regulatory Peptides 127 (2005) 239 – 244 www.elsevier.com/locate/regpep
Upregulation of intracardiac adrenomedullin and its receptor system in rats with volume overload-induced cardiac hypertrophy Fumiki Yoshiharaa,*, Toshio Nishikimib, Ichiro Okanoc, Jun Hinoc, Takeshi Horioa, Takeshi Tokudomec, Shin-ichi Sugac, Hiroaki Matsuokab, Kenji Kangawac, Yuhei Kawanoa a
Division of Hypertension and Nephrology, National Cardiovascular Center, Fujishirodai, Suita, Osaka 565-8565, Japan b Department of Cardiovascular Medicine, Dokkyo Medical School, Tochigi, 321-0293, Japan c Research Institute, National Cardiovascular Center, Suita, Osaka, 565-8565, Japan Received 16 June 2004; accepted 10 December 2004 Available online 15 January 2005
Abstract Specific adrenomedullin receptors have been identified as calcitonin receptor-like receptor (CRLR)/receptor activity-modifying proteins (RAMP2 and RAMP3) complexes. Although we have demonstrated that adrenomedullin is increased in volume overload-induced cardiac hypertrophy, it remains unknown whether the adrenomedullin receptor is altered or not. This study sought to investigate the significance of intracardiac adrenomedullin and its receptor system in volume overload-induced cardiac hypertrophy. Left ventricular adrenomedullin levels were higher in aortocaval shunt (ACS) rats than in controls (+58%). The left ventricular gene expressions of adrenomedullin, CRLR, RAMP2 and RAMP3 were increased (+27%, +76%, +108% and +131%, respectively) and the left ventricular collagen gene expressions were also increased (type I: +138%, type III: +87%). The left ventricular adrenomedullin level correlated with the gene expression of type III collagen (R=0.42). These results suggest that intracardiac adrenomedullin and its receptor system are upregulated and may participate in the regulation of cardiac remodeling in volume overload-induced cardiac hypertrophy. D 2005 Elsevier B.V. All rights reserved. Keywords: Adrenomedullin; Receptors; Volume overload; Cardiac hypertrophy
1. Introduction Cardiac adrenomedullin and its gene expression have been reported to be upregulated in rats with pressure and volume overload-induced cardiac hypertrophy [1–3] and with myocardial infarction [4] produced by coronary artery ligation. We previously reported that rat cardiac myocytes and nonmyocytes produce and secrete adrenomedullin and that its secretion from myocytes is increased by exposure to interleukin-1h [5], hypoxia and oxidative stress [6], and the secretion from nonmyocytes is increased by interleukin-1h and tumor necrosis factor-a [5]. Furthermore, adrenomedullin, which was secreted from both types of cardiac cells,
* Corresponding author. Tel.: +81 6 6833 5012; fax: +81 6 6872 7486. E-mail address: [email protected] (F. Yoshihara). 0167-0115/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.12.017
has been reported to play a role as an autocrine/paracrine modulator in the process of cardiac remodeling, mainly by suppressing mitogenesis and collagen synthesis in cardiac fibroblasts [7]. Adrenomedullin receptor complexes have recently been identified, namely the calcitonin receptor-like receptor (CRLR) and receptor-activity modifying proteins (RAMP2 or RAMP3) [8]. Previous reports have demonstrated that cardiac gene expressions of CRLR and RAMP2 are increased in rats with myocardial infarction [9] produced by left coronary artery ligation and that endothelin-1 modulates the gene expressions of CRLR, RAMP2 and RAMP3 in cultured rat cardiomyocytes [10]. However, the pathophysiological significance of cardiac adrenomedullin and cardiac specific adrenomedullin receptor in volume overload-induced cardiac hypertrophy have not been clarified. In the present study, we evaluated the pathophysiological significance of cardiac adrenomedullin and whether
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cardiac adrenomedullin specific receptors are involved in the left ventricular remodeling of volume overload-induced cardiac hypertrophy.
2. Methods This study was performed in accordance with the guidelines of the Animal Care Committee of the National Cardiovascular Center Research Institute. 2.1. Animals Seven- to eight-week-old male Wistar rats, weighing 250 to 300 g, were used for aortocaval shunt (ACS)-induced cardiac hypertrophy. ACS was produced in rats by a method previously described [11] and modified in our laboratory [2]. Control (C) rats underwent an identical operation, but no shunt was established (n=12). Approximately 30% of the ACS rats died. As a result, 20 AC shunt rats were studied. 2.2. Hemodynamic study Hemodynamic studies were performed at 5 weeks after the ACS operation as previously described [2], because the ACS operation has been reported to cause an increase of ventricular adrenomedullin mRNA expression and its peptide levels in a time dependent manner and an compensated heart failure state at 5 weeks after the operation [3,12]. The animals were anesthetized with pentobarbital sodium (40 mg/kg, i.p.) and blood (1 ml) was withdrawn through the femoral vein for plasma renin concentration (PRC) and atrial natriuretic peptide (ANP) measurements. After the hemodynamic measurements, the rats were then killed and their hearts were excised. Each heart was immediately separated into the right ventricle, left ventricle and septum as previously described [2]. Heart weights were also measured. 2.3. Radioimmunoassay (RIA) for ventricular adrenomedullin Left ventricular adrenomedullin levels at 5 weeks after the operation were measured in ACS and C rats. RIA for rat adrenomedullin was performed as described previously [13]. 2.4. Radioimmunoassay (RIA) for plasma ANP and PRC Plasma ANP and PRC were measured by the specific RIA as previously reported [14]. 2.5. cDNA probes and radiolabeling of probes An EcoRI/NaeI restriction fragment of rat adrenomedullin cDNA corresponding to nucleotides 153 to 436
was used as the rat adrenomedullin cDNA probe [2]. The rat CRLR, RAMP2, RAMP3, human collagen type I and type III cDNA probes were synthesized by polymerase chain reaction (PCR) using the primers CRLR sense, 5V-AGGACATGGACAAACTACAC-3V; CRLR antisense, 5V-GAATGAACTGGGACACCTTGC-3V; RAMP2 sense, 5V-AACACATGTCCTACCTTGCTG-3V; RAMP2 antisense, 5V-TCGCTGTCTTTACTCCTCCAC-3V; RAMP3 sense, 5V-AGCGACTGCACCTTCTTCCA-3V: RAMP3 antisense, 5V-GCCAGCCATAGCCACAGTCAG-3V: collagen type I sense, 5V-CAAGGTGTTGTGCGATGACG3V: collagen type I antisense, 5V-ATTCCTCCGGTTGATTTCTC-3V: collagen type III sense, 5V-ATCCGTTCTCTGCGATGACATAATA-3V: collagen type III antisense, 5V-GCCTGCGAGTCCTCCTACTGCTACT-3V [12,15]. Amplification of cDNA by these primers gave 301 bp (CRLR), 327 bp (RAMP2) and 386 bp (RAMP3) PCR products. These PCR products have 85.4% (CRLR), 82.1% (RAMP2) and 84.2% (RAMP3) nucleic identity with the corresponding human CRLR, RAMP2 and RAMP3, respectively. These probes were radiolabeled by random priming with [a-32P]dCTP (Amersham), and the labeled probes were purified by column chromatography (NICK column, Pharmacia Biotech, Sweden). 2.6. Northern blot analysis Total RNA (20 Ag per lane) for adrenomedullin, collagen type I and collagen type III mRNA evaluation and poly (A)+RNA (2.5 Ag per lane) for CRLR, RAMP2 and RAMP3 mRNA evaluation were denatured, electrophoresed and transferred to a nylon membrane. For hybridization with the cDNA probes, the conditions for hybridization and washing have been previously described
Table 1 Hemodynamics, humoral factors and heart weights in control and ACS rats n Heart rate (bpm) MAP (mm Hg) LVEDP (mm Hg) PRC (ng AngI/mL/h) ANP (pg/mL) RV/BW (mg/g) LV+SEP/BW (mg/g) LVAM conc. (fmol/mg)
C
ACS
12 409F39 129F7 4F3 1.5F0.2 226F39 0.53F0.06 1.64F0.15 0.31F0.22
20 405F23 116F10** 13F17** 1.7F0.6 651F194** 0.85F0.13** 2.30F0.26** 0.49F0.19*
C indicates sham-operated control; ACS, aortocaval shunt rat; MAP, mean arterial pressure; LVEDP, left ventricular end-diastolic pressure; PRC, plasma renin concentration; ANP, plasma ANP concentration; RV, right ventricular weight; BW, body weight; LV, left ventricular weight; SEP, septal weight; LVAM conc., left ventricular adrenomedullin concentration. Values are meanFS.D. * pb0.05. ** pb0.001 vs. C.
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[2,15]. Band intensity was estimated using a radio-image analyzer (BAS5000, Fuji) as previously reported [3]. To normalize these mRNA expression to the amounts of RNA loaded and the transfer efficiencies, the same membrane was rehybridized with an 18S probe for adrenomedullin mRNA and with GAPDH probe for its receptor component mRNAs.
3. Statistical analysis All values are presented as meanFS.D. Comparisons between the two groups were performed by the unpaired ttest. Differences were considered significant at a level of pb0.05. Correlation coefficients were calculated using linear regression analysis.
C
241
4. Results There were no significant differences in heart rate or plasma renin concentration between the two groups at 5 weeks after the ACS operation (Table 1). Mean arterial pressure was significantly lower in ACS than in C operation (Table 1). Left ventricular end-diastolic pressure, plasma ANP level, right ventricular weight and left ventricular and septal weights were significantly higher in ACS than in C at 5 weeks after the operation (Table 1). Left ventricular adrenomedullin concentration was also higher in ACS than in C (Table 1). The gene expressions of adrenomedullin, CRLR, RAMP2 and RAMP3 were also upregulated in the left ventricle in ACS compared with C (Fig. 1). Left ventricular adrenomedullin concentration correlated with the left ventricular end-diastolic pressure (R=0.43, pb0.05)
ACS p<0.05
AM
AM/18S mRNA
200
100
0
CRLR
p<0.0 CRLR/GAPDH mRNA
18S 200
100
0
RAMP2
RAMP2/GAPDH mRNA
p<0.05
200 100 0
GAPDH
RAMP3/GAPDH mRNA
RAMP3 p<0.05
400
200
0 C
ACS
Fig. 1. Representative Northern blots and relative abundance of adrenomedullin (AM), CRLR, RAMP2 and RAMP3 in the left ventricle in rats with ACS and sham-operated C rats at 5 weeks after operation. Values are meanFS.D.
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C
C
ACS
ACS Col III
Col I
18S
18S
p<0.001
p<0.001
300
200
200 100 100 0
0 C
ACS
C
ACS
Fig. 2. Representative Northern blots and relative abundance of collagen type I (Col I) and collagen type III (Col III) in the left ventricle in rats with ACS and sham-operated C rats at 5 weeks after operation. Values are meanFS.D.
and left ventricular and septal weights (R=0.46, pb0.01). The gene expressions of collagen type I and collagen type III were also upregulated in the left ventricle in ACS compared with C (Fig. 2). The gene expressions of both types of
collagen correlated with the left ventricular and septal weights (Fig. 3). Left ventricular adrenomedullin concentration correlated with the gene expression of collagen type III but not collagen type I (Fig. 3).
R=0.47 p<0.01
8
Col III mRNA level
Col I mRNA level
20 10
6 4 2 0 1.2
1.6
2
2.4
2.8
R=0.66 p<0.0001
16 12 8 4 0 1.2
LV+SEP/BW (mg/g)
2
2.4
2.8
LV+SEP/BW (mg/g) 20
Col III mRNA level
12
Col I mRNA level
1.6
N.S. 8
4
0 0
0.4
0.8
LVAM conc (fmol/mg)
1.2
16 12 8
R=0.42 p<0.05
4 0 0
0.4
0.8
1.2
LVAM conc (fmol/mg)
Fig. 3. Correlations between gene expression of collagen type I (Col I) and collagen type III (Col III) and the ratio of left ventricular and septal weight to body weight) LV+SEP/BW or left ventricular adrenomedullin concentration (LVAM conc) in rats with ACS and sham-operated C rats at 5 weeks after operation.
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5. Discussion In the present study, we examined adrenomedullin levels in the left ventricle in ACS rats and shamoperated C rats at 5 weeks after operation. The gene expressions of adrenomedullin and its receptor system in the left ventricle were also measured to determine whether adrenomedullin and its receptor system exist in rat hearts and whether this system is modulated by transcriptional regulation after the ACS operation. Furthermore, we evaluated the gene expressions of collagen type I and collage type III and examined the relationship between the left ventricular adrenomedullin concentration and the expression level of collagen mRNA. We demonstrated that (1) adrenomedullin and its receptor system gene expression exist in rat hearts; (2) an increased left ventricular adrenomedullin level may reflect an increased adrenomedullin production in the ventricular myocardium; and (3) an increased ventricular adrenomedullin may be in part involved in the regulation of ventricular remodeling in volume overload-induced cardiac hypertrophy. Adrenomedullin and its receptor system exist in the rat cardiac ventricular myocardium. The ventricular gene expressions of CRLR and RAMP2 and the density of adrenomedullin binding sites increased in rats with myocardial infarction [9,16] produced by coronary artery ligation. A recent study reported that endotheline-1 participates in the modulation of the gene expressions of CRLR, RAMP2 and RAMP3 in cultured rat cardiomyocytes [10]. We previously demonstrated an increase in ventricular adrenomedullin concentration, its gene expression and the adrenomedullin immunoreactive staining of myocytes in pressure and volume overloadinduced cardiac hypertrophy [1–3], suggesting that adrenomedullin production in the ventricular myocytes is increased in rats with mechanical stress-induced cardiac hypertrophy. However, our results showed that a relatively weak correlation between left ventricular adrenomedullin levels and left ventricular end-diastolic pressure and left ventricular weight. Angiotensin II [17], interleukin-1h [5] and tumor necrosis factor-a [5] have been also reported to stimulate adrenomedullin production in the heart, suggesting that these humoral factors may participate in the increased ventricular adrenomedullin production in the volume overload-induced cardiac hypertrophy. In the present study, the left ventricular gene expressions of CRLR, RAMP2 and RAMP3 increased in rats with volume overload-induced cardiac hypertrophy, suggesting that not only increased adrenomedullin, but also upregulation of its receptor system may be in part involved in the regulation of the development of cardiac hypertrophty. Renal adrenomedullin concentration is also increased in rats with ACS-induced heart failure [3]. Renal adrenomedullin may participate in the regulation of urinary sodium
243
excretion without the upregulation of the renal adrenomedullin receptor system. A previous report demonstrated that although ventricular CRLR and RAMP2 gene expressions were upregulated in rats with myocardial infarction produced by coronary artery ligation, renal CRLR and RAMP2 gene expression were unchanged [16]. Thus, these results suggested that there is a different transcriptional regulation for the adrenomedullin receptor system between the heart and kidney in rats with both volume overload-induced heart failure and myocardial infarction. Adrenomedullin has been reported to play a role as an autocrine/paracrine modulator in the process of cardiac remodeling, mainly by suppressing mitogenesis and collagen synthesis in rat cultured cardiac fibroblasts [7]. In addition, cardiac adrenomedullin and its mRNA expressions were upregulated in a rat experimental model of increased cardiac fibrosis such as myocardial infarction [16] and mechanical stress-induced cardiac hypertrophy [1–3]. Furthermore, in the present study, the ventricular adrenomedullin concentration correlated with the level of collagen type III mRNA expression. The myocardial connective tissue maintaining the functional integrity of the heart mainly consists of collagen type I (80%) and collagen type III (20%) [18]. Type I collagen represents a stiff fibrillar protein that provides substantial tensile strength to biological structures such as tendons and type III collagen forms a reticular supportive network. Both collagens are recognized as structures that are essential in the maintenance of myocardial structural integrity. Thus, our results suggested that ventricular adrenomedullin may be in part involved in the regulation of ventricular remodeling and that the upregulation of its receptor system may also participate in this regulation. However, the present study has limitations. Mishima et al. [10] had reported that adrenomedullin receptor mRNA expressed in the cultured rat cardiomyocytes. We also previously reported that adrenomedullin increased cyclic AMP, the second messenger of its peptide, in not only cardiomyocytes but also cardiac fibroblasts [19], suggesting that adrenomedullin specific receptor might exist cardiac myocytes and fibroblasts. On the other hand, the cellular origin of collagen matrix in the heart is cardiac fibroblast [18], suggesting that our detected significance of ventricular adrenomedullin and its receptor system may be limited in the area of ventricular interstitium. Further study is necessary to reveal the pathophysiological significance of ventricular adrenomedullin in the development of cardiac hypertrophy and left ventricular remodeling concerning each cell type. In conclusion, in the present study, we demonstrated that adrenomedullin and its receptor system exist in rat hearts. The present findings suggest that the increased left ventricular adrenomedullin may in part be involved in the regulation of left ventricular remodeling in rats with volume overload-induced cardiac hypertrophy.
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Acknowledgements This study was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan. We thank Yoko Saito for her technical assistance.
References [1] Morimoto A, Nishikimi T, Yoshihara F, Horio T, Nagaya N, Matsuo H, et al. Ventricular adrenomedullin levels correlate with the extent of cardiac hypertrophy in rats. Hypertension 1999;33:1146 – 52. [2] Nishikimi T, Horio T, Sasaki T, Yoshihara F, Takishita S, Miyata A, et al. Cardiac production and secretion of adrenomedullin are increased in heart failure. Hypertension 1997;30:1369 – 75. [3] Yoshihara F, Nishikimi T, Horio T, Yutani C, Nagaya N, Matsuo H, et al. Ventricular adrenomedullin concentration is a sensitive biochemical marker for volume and pressure overload in rats. Am J Physiol, Heart Circ Physiol 2000;278:H633 – 42. [4] Nagaya N, Nishikimi T, Yoshihara F, Horio T, Morimoto A, Kangawa K. Cardiac adrenomedullin gene expression and peptide accumulation after acute myocardial infarction in rats. Am J Physiol, Regul Integr Comp Physiol 2000;278:R1019 – 26. [5] Horio T, Nishikimi T, Yoshihara F, Nagaya N, Matsuo H, Takishita S, et al. Production and secretion of adrenomedullin in cultured rat cardiac myocytes and nonmyocytes: stimulation by interleukin-1beta and tumor necrosis factor-alpha. Endocrinology 1998;139:4576 – 80. [6] Yoshihara F, Horio T, Nishikimi T, Matsuo H, Kangawa K. Possible involvement of oxidative stress in hypoxia-induced adrenomedullin secretion in cultured rat cardiomyocytes. Eur J Pharmacol 2002;436: 1 – 6. [7] Horio T, Nishikimi T, Yoshihara F, Matsuo H, Takishita S, Kangawa K. Effects of adrenomedullin on cultured rat cardiac myocytes and fibroblasts. Eur J Pharmacol 1999;382:1 – 9. [8] McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998;393:333 – 9.
[9] Oie E, Vinge LE, Yndestad A, Sandberg C, Grogaard HK, Attramadal H. Induction of a myocardial adrenomedullin signaling system during ischemic heart failure in rats. Circulation 2000;101:415 – 22. [10] Mishima K, Kato J, Kuwasako K, Ito K, Imamura T, Kitamura K, et al. Effects of endothelin on adrenomedullin secretion and expression of adrenomedullin receptors in rat cardiomyocytes. Biochem Biophys Res Commun 2001;287:264 – 9. [11] Garcia R, Diebold S. Simple, rapid, and effective method of producing aortocaval shunts in the rat. Cardiovasc Res 1990;24:430 – 2. [12] Yoshihara F, Nishikimi T, Okano I, Horio T, Yutani C, Matsuo H, et al. Alterations of intrarenal adrenomedullin and its receptor system in heart failure rats. Hypertension 2001;37:216 – 22. [13] Sakata J, Shimokubo T, Kitamura K, Nishizono M, Iehiki Y, Kangawa K, et al. Distribution and characterization of immunoreactive rat adrenomedullin in tissue and plasma. FEBS Lett 1994;352:105 – 8. [14] Nishikimi T, Miura K, Minamino N, Takeuchi K, Takeda T. Role of endogenous atrial natriuretic peptide on systemic and renal hemodynamics in heart failure rats. Am J Physiol 1994;267:H182 – 6. [15] Yoshihara F, Nishikimi T, Sasako Y, Hino J, Kobayashi J, Minatoya K, et al. Plasma atrial natriuretic peptide (ANP) concentration inversely correlates with left atrial collagen volume fraction in patients with atrial fibrillation-plasma ANP as a possible biochemical marker to predict the outcome of the maze procedure. J Am Coll Cardiol 2002;39:288 – 94. [16] Totsune K, Takahashi K, Mackenzie HS, Murakami O, Arihara Z, Sone M, et al. Increased gene expression of adrenomedullin and adrenomedullin-receptor complexes, receptor-activity modifying protein (RAMP)2 and calcitonin-receptor-like receptor (CRLR) in the hearts of rats with congestive heart failure. Clin Sci (Colch) 2000; 99:541 – 6. [17] Tsuruda T, Kato J, Kitamura K, Imamura T, Koiwaya Y, Kangawa K, et al. Enhanced adrenomedulin production by mechanical stretching in cultured rat cardiomyocytes. Hypertension 2000;35:1210 – 4. [18] Weber TK. Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol 1989;13:1637 – 52. [19] Nishikimi T, Horio T, Yoshihara F, Nagaya N, Matsuo H, Kangawa K. Effect of adrenomedullin on cAMP and cGMP levels in rat cardiac myocytes and nonmyocytes. Eur J Pharmacol 1998;353:337 – 44.
Upregulation of intracardiac adrenomedullin and its receptor system in rats with volume overload-induced cardiac hypertrophy Fumiki Yoshiharaa,*, Toshio Nishikimib, Ichiro Okanoc, Jun Hinoc, Takeshi Horioa, Takeshi Tokudomec, Shin-ichi Sugac, Hiroaki Matsuokab, Kenji Kangawac, Yuhei Kawanoa a
Division of Hypertension and Nephrology, National Cardiovascular Center, Fujishirodai, Suita, Osaka 565-8565, Japan b Department of Cardiovascular Medicine, Dokkyo Medical School, Tochigi, 321-0293, Japan c Research Institute, National Cardiovascular Center, Suita, Osaka, 565-8565, Japan Received 16 June 2004; accepted 10 December 2004 Available online 15 January 2005
Abstract Specific adrenomedullin receptors have been identified as calcitonin receptor-like receptor (CRLR)/receptor activity-modifying proteins (RAMP2 and RAMP3) complexes. Although we have demonstrated that adrenomedullin is increased in volume overload-induced cardiac hypertrophy, it remains unknown whether the adrenomedullin receptor is altered or not. This study sought to investigate the significance of intracardiac adrenomedullin and its receptor system in volume overload-induced cardiac hypertrophy. Left ventricular adrenomedullin levels were higher in aortocaval shunt (ACS) rats than in controls (+58%). The left ventricular gene expressions of adrenomedullin, CRLR, RAMP2 and RAMP3 were increased (+27%, +76%, +108% and +131%, respectively) and the left ventricular collagen gene expressions were also increased (type I: +138%, type III: +87%). The left ventricular adrenomedullin level correlated with the gene expression of type III collagen (R=0.42). These results suggest that intracardiac adrenomedullin and its receptor system are upregulated and may participate in the regulation of cardiac remodeling in volume overload-induced cardiac hypertrophy. D 2005 Elsevier B.V. All rights reserved. Keywords: Adrenomedullin; Receptors; Volume overload; Cardiac hypertrophy
1. Introduction Cardiac adrenomedullin and its gene expression have been reported to be upregulated in rats with pressure and volume overload-induced cardiac hypertrophy [1–3] and with myocardial infarction [4] produced by coronary artery ligation. We previously reported that rat cardiac myocytes and nonmyocytes produce and secrete adrenomedullin and that its secretion from myocytes is increased by exposure to interleukin-1h [5], hypoxia and oxidative stress [6], and the secretion from nonmyocytes is increased by interleukin-1h and tumor necrosis factor-a [5]. Furthermore, adrenomedullin, which was secreted from both types of cardiac cells,
* Corresponding author. Tel.: +81 6 6833 5012; fax: +81 6 6872 7486. E-mail address: [email protected] (F. Yoshihara). 0167-0115/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.12.017
has been reported to play a role as an autocrine/paracrine modulator in the process of cardiac remodeling, mainly by suppressing mitogenesis and collagen synthesis in cardiac fibroblasts [7]. Adrenomedullin receptor complexes have recently been identified, namely the calcitonin receptor-like receptor (CRLR) and receptor-activity modifying proteins (RAMP2 or RAMP3) [8]. Previous reports have demonstrated that cardiac gene expressions of CRLR and RAMP2 are increased in rats with myocardial infarction [9] produced by left coronary artery ligation and that endothelin-1 modulates the gene expressions of CRLR, RAMP2 and RAMP3 in cultured rat cardiomyocytes [10]. However, the pathophysiological significance of cardiac adrenomedullin and cardiac specific adrenomedullin receptor in volume overload-induced cardiac hypertrophy have not been clarified. In the present study, we evaluated the pathophysiological significance of cardiac adrenomedullin and whether
240
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cardiac adrenomedullin specific receptors are involved in the left ventricular remodeling of volume overload-induced cardiac hypertrophy.
2. Methods This study was performed in accordance with the guidelines of the Animal Care Committee of the National Cardiovascular Center Research Institute. 2.1. Animals Seven- to eight-week-old male Wistar rats, weighing 250 to 300 g, were used for aortocaval shunt (ACS)-induced cardiac hypertrophy. ACS was produced in rats by a method previously described [11] and modified in our laboratory [2]. Control (C) rats underwent an identical operation, but no shunt was established (n=12). Approximately 30% of the ACS rats died. As a result, 20 AC shunt rats were studied. 2.2. Hemodynamic study Hemodynamic studies were performed at 5 weeks after the ACS operation as previously described [2], because the ACS operation has been reported to cause an increase of ventricular adrenomedullin mRNA expression and its peptide levels in a time dependent manner and an compensated heart failure state at 5 weeks after the operation [3,12]. The animals were anesthetized with pentobarbital sodium (40 mg/kg, i.p.) and blood (1 ml) was withdrawn through the femoral vein for plasma renin concentration (PRC) and atrial natriuretic peptide (ANP) measurements. After the hemodynamic measurements, the rats were then killed and their hearts were excised. Each heart was immediately separated into the right ventricle, left ventricle and septum as previously described [2]. Heart weights were also measured. 2.3. Radioimmunoassay (RIA) for ventricular adrenomedullin Left ventricular adrenomedullin levels at 5 weeks after the operation were measured in ACS and C rats. RIA for rat adrenomedullin was performed as described previously [13]. 2.4. Radioimmunoassay (RIA) for plasma ANP and PRC Plasma ANP and PRC were measured by the specific RIA as previously reported [14]. 2.5. cDNA probes and radiolabeling of probes An EcoRI/NaeI restriction fragment of rat adrenomedullin cDNA corresponding to nucleotides 153 to 436
was used as the rat adrenomedullin cDNA probe [2]. The rat CRLR, RAMP2, RAMP3, human collagen type I and type III cDNA probes were synthesized by polymerase chain reaction (PCR) using the primers CRLR sense, 5V-AGGACATGGACAAACTACAC-3V; CRLR antisense, 5V-GAATGAACTGGGACACCTTGC-3V; RAMP2 sense, 5V-AACACATGTCCTACCTTGCTG-3V; RAMP2 antisense, 5V-TCGCTGTCTTTACTCCTCCAC-3V; RAMP3 sense, 5V-AGCGACTGCACCTTCTTCCA-3V: RAMP3 antisense, 5V-GCCAGCCATAGCCACAGTCAG-3V: collagen type I sense, 5V-CAAGGTGTTGTGCGATGACG3V: collagen type I antisense, 5V-ATTCCTCCGGTTGATTTCTC-3V: collagen type III sense, 5V-ATCCGTTCTCTGCGATGACATAATA-3V: collagen type III antisense, 5V-GCCTGCGAGTCCTCCTACTGCTACT-3V [12,15]. Amplification of cDNA by these primers gave 301 bp (CRLR), 327 bp (RAMP2) and 386 bp (RAMP3) PCR products. These PCR products have 85.4% (CRLR), 82.1% (RAMP2) and 84.2% (RAMP3) nucleic identity with the corresponding human CRLR, RAMP2 and RAMP3, respectively. These probes were radiolabeled by random priming with [a-32P]dCTP (Amersham), and the labeled probes were purified by column chromatography (NICK column, Pharmacia Biotech, Sweden). 2.6. Northern blot analysis Total RNA (20 Ag per lane) for adrenomedullin, collagen type I and collagen type III mRNA evaluation and poly (A)+RNA (2.5 Ag per lane) for CRLR, RAMP2 and RAMP3 mRNA evaluation were denatured, electrophoresed and transferred to a nylon membrane. For hybridization with the cDNA probes, the conditions for hybridization and washing have been previously described
Table 1 Hemodynamics, humoral factors and heart weights in control and ACS rats n Heart rate (bpm) MAP (mm Hg) LVEDP (mm Hg) PRC (ng AngI/mL/h) ANP (pg/mL) RV/BW (mg/g) LV+SEP/BW (mg/g) LVAM conc. (fmol/mg)
C
ACS
12 409F39 129F7 4F3 1.5F0.2 226F39 0.53F0.06 1.64F0.15 0.31F0.22
20 405F23 116F10** 13F17** 1.7F0.6 651F194** 0.85F0.13** 2.30F0.26** 0.49F0.19*
C indicates sham-operated control; ACS, aortocaval shunt rat; MAP, mean arterial pressure; LVEDP, left ventricular end-diastolic pressure; PRC, plasma renin concentration; ANP, plasma ANP concentration; RV, right ventricular weight; BW, body weight; LV, left ventricular weight; SEP, septal weight; LVAM conc., left ventricular adrenomedullin concentration. Values are meanFS.D. * pb0.05. ** pb0.001 vs. C.
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[2,15]. Band intensity was estimated using a radio-image analyzer (BAS5000, Fuji) as previously reported [3]. To normalize these mRNA expression to the amounts of RNA loaded and the transfer efficiencies, the same membrane was rehybridized with an 18S probe for adrenomedullin mRNA and with GAPDH probe for its receptor component mRNAs.
3. Statistical analysis All values are presented as meanFS.D. Comparisons between the two groups were performed by the unpaired ttest. Differences were considered significant at a level of pb0.05. Correlation coefficients were calculated using linear regression analysis.
C
241
4. Results There were no significant differences in heart rate or plasma renin concentration between the two groups at 5 weeks after the ACS operation (Table 1). Mean arterial pressure was significantly lower in ACS than in C operation (Table 1). Left ventricular end-diastolic pressure, plasma ANP level, right ventricular weight and left ventricular and septal weights were significantly higher in ACS than in C at 5 weeks after the operation (Table 1). Left ventricular adrenomedullin concentration was also higher in ACS than in C (Table 1). The gene expressions of adrenomedullin, CRLR, RAMP2 and RAMP3 were also upregulated in the left ventricle in ACS compared with C (Fig. 1). Left ventricular adrenomedullin concentration correlated with the left ventricular end-diastolic pressure (R=0.43, pb0.05)
ACS p<0.05
AM
AM/18S mRNA
200
100
0
CRLR
p<0.0 CRLR/GAPDH mRNA
18S 200
100
0
RAMP2
RAMP2/GAPDH mRNA
p<0.05
200 100 0
GAPDH
RAMP3/GAPDH mRNA
RAMP3 p<0.05
400
200
0 C
ACS
Fig. 1. Representative Northern blots and relative abundance of adrenomedullin (AM), CRLR, RAMP2 and RAMP3 in the left ventricle in rats with ACS and sham-operated C rats at 5 weeks after operation. Values are meanFS.D.
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C
C
ACS
ACS Col III
Col I
18S
18S
p<0.001
p<0.001
300
200
200 100 100 0
0 C
ACS
C
ACS
Fig. 2. Representative Northern blots and relative abundance of collagen type I (Col I) and collagen type III (Col III) in the left ventricle in rats with ACS and sham-operated C rats at 5 weeks after operation. Values are meanFS.D.
and left ventricular and septal weights (R=0.46, pb0.01). The gene expressions of collagen type I and collagen type III were also upregulated in the left ventricle in ACS compared with C (Fig. 2). The gene expressions of both types of
collagen correlated with the left ventricular and septal weights (Fig. 3). Left ventricular adrenomedullin concentration correlated with the gene expression of collagen type III but not collagen type I (Fig. 3).
R=0.47 p<0.01
8
Col III mRNA level
Col I mRNA level
20 10
6 4 2 0 1.2
1.6
2
2.4
2.8
R=0.66 p<0.0001
16 12 8 4 0 1.2
LV+SEP/BW (mg/g)
2
2.4
2.8
LV+SEP/BW (mg/g) 20
Col III mRNA level
12
Col I mRNA level
1.6
N.S. 8
4
0 0
0.4
0.8
LVAM conc (fmol/mg)
1.2
16 12 8
R=0.42 p<0.05
4 0 0
0.4
0.8
1.2
LVAM conc (fmol/mg)
Fig. 3. Correlations between gene expression of collagen type I (Col I) and collagen type III (Col III) and the ratio of left ventricular and septal weight to body weight) LV+SEP/BW or left ventricular adrenomedullin concentration (LVAM conc) in rats with ACS and sham-operated C rats at 5 weeks after operation.
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5. Discussion In the present study, we examined adrenomedullin levels in the left ventricle in ACS rats and shamoperated C rats at 5 weeks after operation. The gene expressions of adrenomedullin and its receptor system in the left ventricle were also measured to determine whether adrenomedullin and its receptor system exist in rat hearts and whether this system is modulated by transcriptional regulation after the ACS operation. Furthermore, we evaluated the gene expressions of collagen type I and collage type III and examined the relationship between the left ventricular adrenomedullin concentration and the expression level of collagen mRNA. We demonstrated that (1) adrenomedullin and its receptor system gene expression exist in rat hearts; (2) an increased left ventricular adrenomedullin level may reflect an increased adrenomedullin production in the ventricular myocardium; and (3) an increased ventricular adrenomedullin may be in part involved in the regulation of ventricular remodeling in volume overload-induced cardiac hypertrophy. Adrenomedullin and its receptor system exist in the rat cardiac ventricular myocardium. The ventricular gene expressions of CRLR and RAMP2 and the density of adrenomedullin binding sites increased in rats with myocardial infarction [9,16] produced by coronary artery ligation. A recent study reported that endotheline-1 participates in the modulation of the gene expressions of CRLR, RAMP2 and RAMP3 in cultured rat cardiomyocytes [10]. We previously demonstrated an increase in ventricular adrenomedullin concentration, its gene expression and the adrenomedullin immunoreactive staining of myocytes in pressure and volume overloadinduced cardiac hypertrophy [1–3], suggesting that adrenomedullin production in the ventricular myocytes is increased in rats with mechanical stress-induced cardiac hypertrophy. However, our results showed that a relatively weak correlation between left ventricular adrenomedullin levels and left ventricular end-diastolic pressure and left ventricular weight. Angiotensin II [17], interleukin-1h [5] and tumor necrosis factor-a [5] have been also reported to stimulate adrenomedullin production in the heart, suggesting that these humoral factors may participate in the increased ventricular adrenomedullin production in the volume overload-induced cardiac hypertrophy. In the present study, the left ventricular gene expressions of CRLR, RAMP2 and RAMP3 increased in rats with volume overload-induced cardiac hypertrophy, suggesting that not only increased adrenomedullin, but also upregulation of its receptor system may be in part involved in the regulation of the development of cardiac hypertrophty. Renal adrenomedullin concentration is also increased in rats with ACS-induced heart failure [3]. Renal adrenomedullin may participate in the regulation of urinary sodium
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excretion without the upregulation of the renal adrenomedullin receptor system. A previous report demonstrated that although ventricular CRLR and RAMP2 gene expressions were upregulated in rats with myocardial infarction produced by coronary artery ligation, renal CRLR and RAMP2 gene expression were unchanged [16]. Thus, these results suggested that there is a different transcriptional regulation for the adrenomedullin receptor system between the heart and kidney in rats with both volume overload-induced heart failure and myocardial infarction. Adrenomedullin has been reported to play a role as an autocrine/paracrine modulator in the process of cardiac remodeling, mainly by suppressing mitogenesis and collagen synthesis in rat cultured cardiac fibroblasts [7]. In addition, cardiac adrenomedullin and its mRNA expressions were upregulated in a rat experimental model of increased cardiac fibrosis such as myocardial infarction [16] and mechanical stress-induced cardiac hypertrophy [1–3]. Furthermore, in the present study, the ventricular adrenomedullin concentration correlated with the level of collagen type III mRNA expression. The myocardial connective tissue maintaining the functional integrity of the heart mainly consists of collagen type I (80%) and collagen type III (20%) [18]. Type I collagen represents a stiff fibrillar protein that provides substantial tensile strength to biological structures such as tendons and type III collagen forms a reticular supportive network. Both collagens are recognized as structures that are essential in the maintenance of myocardial structural integrity. Thus, our results suggested that ventricular adrenomedullin may be in part involved in the regulation of ventricular remodeling and that the upregulation of its receptor system may also participate in this regulation. However, the present study has limitations. Mishima et al. [10] had reported that adrenomedullin receptor mRNA expressed in the cultured rat cardiomyocytes. We also previously reported that adrenomedullin increased cyclic AMP, the second messenger of its peptide, in not only cardiomyocytes but also cardiac fibroblasts [19], suggesting that adrenomedullin specific receptor might exist cardiac myocytes and fibroblasts. On the other hand, the cellular origin of collagen matrix in the heart is cardiac fibroblast [18], suggesting that our detected significance of ventricular adrenomedullin and its receptor system may be limited in the area of ventricular interstitium. Further study is necessary to reveal the pathophysiological significance of ventricular adrenomedullin in the development of cardiac hypertrophy and left ventricular remodeling concerning each cell type. In conclusion, in the present study, we demonstrated that adrenomedullin and its receptor system exist in rat hearts. The present findings suggest that the increased left ventricular adrenomedullin may in part be involved in the regulation of left ventricular remodeling in rats with volume overload-induced cardiac hypertrophy.
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Acknowledgements This study was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan. We thank Yoko Saito for her technical assistance.
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