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Am J Physiol Regul Integr Comp Physiol 275: R1-R9, 1998;
0363-6119/98 $5.00
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Vol. 275, Issue 1, R1-R9, July 1998

Activation of angiotensinogen gene in cardiac myocytes by angiotensin II and mechanical stretch

Kouichi Tamura, Satoshi Umemura, Nobuo Nyui, Kiyoshi Hibi, Tomoaki Ishigami, Minoru Kihara, Yoshiyuki Toya, and Masao Ishii

Department of Internal Medicine II, Yokohama City University School of Medicine, Yokohama 236, Japan

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Circulating and cardiac renin-angiotensin systems (RAS) play important roles in the development of cardiac hypertrophy. Mechanical stretch of cardiac myocytes induces secretion of ANG II and evokes hypertrophic responses. Angiotensinogen is a unique substrate of the RAS. This study was performed to examine the regulation of the angiotensinogen gene in cardiac myocytes in response to ANG II and stretch. ANG II and stretch significantly increased the levels of angiotensinogen mRNA in cardiac myocytes. Actinomycin D completely inhibited ANG II- and stretch-mediated increases in angiotensinogen mRNA. Although CV-11974 abolished ANG II-mediated increases in mRNA level and promoter activity of the angiotensinogen gene, the inhibition of stretch-mediated activation by CV-11974 was significant but not complete. These results indicate that ANG II activates transcription of the angiotensinogen gene exclusively via ANG II type 1-receptor pathway and that stretch activates such transcription mainly via the same pathway in cardiac myocytes. Furthermore, factors other than ANG II may also be involved in stretch-mediated activation of the angiotensinogen gene in cardiac myocytes.

renin-angiotensin system; cardiac hypertrophy; angiotensin II receptor; mRNA expression; transcription

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE RENIN-ANGIOTENSIN SYSTEM (RAS) exerts a major influence on blood pressure as well as sodium and extracellular fluid balance through generation of ANG II, which has a variety of actions such as vasoconstrictor activity and stimulation of the production and release of aldosterone. The RAS has been strongly implicated in the development of several cardiovascular diseases, including hypertension, and accumulating evidence from biochemical and molecular studies of angiotensin suggests that distinct local RAS with different regulatory mechanisms from the classical plasma RAS may exist (12, 16). Local RAS may exist and function in the brain, heart, adrenal gland, kidney, blood vessel wall, and adipose tissue. Whether all components of local RAS are physiologically relevant is controversial, and the exact role of these systems remains elusive, but it is interesting to speculate that a local RAS may augment the effects of circulating ANG II on a particular tissue in specific physiological and pathophysiological processes. A number of studies indicate that the RAS plays a pivotal role in the development of cardiac hypertrophy (1, 26, 36). In addition, previous studies demonstrated the presence of a local cardiac RAS in normal adult hearts and showed that expression of angiotensinogen, renin, angiotensin-converting enzyme (ACE), and ANG II type 1 (AT1)-receptor genes is upregulated in the presence of pressure- or volume-overload cardiac hypertrophy (3, 4, 18, 43, 52). These results suggest that all of these components of RAS would be important for the pathogenesis of cardiac hypertrophy.

Among the components of the RAS, angiotensinogen is a unique substrate of renin in vivo. Expression of the angiotensinogen gene is regulated in a cell type-specific and differentiation-linked manner in cell culture systems (7, 13, 15, 28), and the 750-bp promoter element from the immediate 5'-flanking region is capable of directing most, but not all, tissue-specific and hormonal regulation of the angiotensinogen minigene in transgenic mice (11). Molecular variants of the human angiotensinogen gene are associated with essential hypertension (8, 22), and recent studies have shown that several mutations of the proximal promoter region of the human angiotensinogen gene that affect the binding activity of transcription factors are associated with essential hypertension (20, 21, 42). These results suggest that transcriptional regulation of the angiotensinogen gene is intimately involved in the pathogenesis of hypertension. We previously analyzed the transcriptional mechanism of the murine angiotensinogen gene using hepatic HepG2 cells and adipogenic differentiation-inducible 3T3-L1 cells and showed that the promoter region from -501 to +22 of the transcriptional initiation site is able to direct transcription of the gene in a hepatocyte-specific and adipogenic differentiation-dependent manner (45, 46). We also examined tissue-specific expression of the angiotensinogen gene during the development of hypertension using spontaneously hypertensive rats (SHR) and Wistar fatty hypertensive rats (WFR) and showed that expression of tissue angiotensinogen gene is regulated differently in hypertensive and normotensive rats (34, 47, 48). However, there were no significant differences in the levels of hepatic angiotensinogen mRNA between hypertensive and normotensive rats, and the levels of adipogenic angiotensinogen mRNA were lower in hypertensive rats than in normotensive rats (34, 47, 48). On the other hand, the expression of angiotensinogen mRNA is increased in hypertrophied hearts of SHR and WFR (47, 48), thereby suggesting that angiotensinogen plays a role in hypertension-induced hypertrophic responses in cardiac myocytes. Thus the aim of the present study was to investigate the regulation of angiotensinogen gene expression in rat cardiac myocytes exposed to ANG II or subjected to mechanical stretch.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Eagle's minimal essential medium (MEM) was purchased from Nissui Pharmaceutical (Tokyo, Japan), FBS from Gibco BRL (New York, NY), trypsin from Difco (Detroit, MI), and collagenase, ANG II, and saralasin from Sigma (St. Louis, MO). CV-11974 and PD-123319 were kind gifts of Takeda Chemical Industries (Osaka, Japan) and the Parke- Davis Pharmaceutical Research Division of Warner-Lambert (Ann Arbor, MI), respectively.

Cell culture of cardiac myocytes. Primary cultures of neonatal cardiac myocytes were prepared from the ventricles of 1- to 3-day-old Sprague-Dawley rats (Oriental Kobo, Tokyo, Japan), as previously described, basically according to the method of Nakamura et al. (31). Briefly, trypsinization and collagenization were performed, and cardiocytes were maintained at 37°C in humidified air with 5% CO2. To reduce the number of contaminating nonmuscle cells, dissociated cells were preplated on 100-mm culture dishes in MEM with 10% FBS for 1 h. The cardiac-myocyte-rich fraction was plated at a field density of 105/cm2 on plastic dishes or on laminin-coated (20 µg/ml) silicone dishes (33). The culture medium was changed 24 h after seeding to a serum-free chemically defined solution consisting of MEM, 10 mg/ml insulin, 5.5 mg/ml transferrin, and 6.7 mg/ml selenium. At this point, >90% of the cells displayed spontaneous contractile activity in the cardiac-myocyte-rich fraction. Uniaxial strain was applied by stretching the silicone dishes by 20%.

Isolation and Northern blot analysis of RNA. Total RNA was extracted from cardiac myocytes by the acid guanidinium thiocyanate-phenol-chloroform method (9), and Northern blot analysis was performed essentially as previously described (45). Briefly, total RNA (40 µg) was denatured with glyoxal and DMSO, electrophoresed on agarose gels, and transferred onto nylon membranes. The membranes were hybridized with 32P-labeled probes for angiotensinogen (35) or 18S ribosomal RNA (18S rRNA) (38). The radioactivities of the bands were measured with BAS2000 imaging plates and a Fujix Bio-Imaging Analyzer (Fuji Photo Film, Tokyo, Japan) (2), and expression of angiotensinogen mRNA was normalized to the signal generated by probing for the constitutively expressed 18S rRNA.

Plasmid construction. The promoterless plasmid pUCSV0CAT (chloramphenicol acetyltransferase) was used as a background reference and pUCSV3CAT was used as a positive control including the SV40 enhancer-promoter region (15). Plasmid Ag501 was constructed by insertion of an angiotensinogen promoter fragment (-501 to +22, relative to the major transcription start site) into the Bgl II/Hind III sites upstream of the CAT coding sequences of pUCSV0CAT as previously described (46).

DNA transfection and CAT assay. The angiotensinogen promoter-CAT chimeric construct Ag501 (5 µg) and a beta -galactosidase expression plasmid pCH110 (1 µg), which is used to normalize transfection efficiency, were transiently cotransfected into cardiac myocytes by the calcium phosphate precipitation method as previously described (15). Cells were incubated for 12 h, washed, and incubated for 12 h in fresh serum-free medium. Cells were then treated with ANG II or subjected to stretch for 12 h, and collected; cell extracts were prepared by freezing and thawing. The protein concentration was determined with BSA as a standard. The reaction mixture containing 40 µg of the cell extracts, was incubated, and the labeled chloramphenicol and acetylated derivatives were separated by ascending TLC as previously described (15). The chromatograms were exposed to the imaging plate of a Fujix Bio-Imaging Analyzer BAS2000. The conversion ratios of [14C]chloramphenicol were measured with the computer analyzer of the BAS2000. DNA transfection was performed four times for each construct. To correct for transfection efficiency, beta -galactosidase activity of cell extracts was measured. CAT activity was also normalized to the protein content of each extract.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of ANG II and mechanical stretch on angiotensinogen mRNA in cardiac myocytes. We first examined the time course of activation by ANG II of angiotensinogen gene expression in cardiac myocytes (100 nM; Fig. 1). The angiotensinogen mRNA was abundantly expressed in the liver but was very low in the lung. The expression of angiotensinogen mRNA was extremely low in nontreated and 1 h-treated cardiac myocytes and was difficult to detect. However, the analysis using the BAS2000 system allowed us to calculate the mRNA value. This system could discriminate the basal (nontreated control) angiotensinogen mRNA value from from the background value. The typical quantitation of the angiotensinogen mRNA values after exposure for 72 h were background 349.8 phosphostimulated luminescence units (PSL), basal (nontreated control) mRNA value 587.6 PSL (net value 237.8), and mRNA value with ANG II treatment for 1 h 647.7 PSL (net value 297.9). We repeated the quantitation of the angiotensinogen mRNA value and used the data from four independent experiments. The increase in angiotensinogen mRNA level was first detected 6 h after addition of ANG II, and the mRNA levels were still rising at 48 h (a 20.2 ± 2.1-fold increase compared with nontreated myocytes). We next examined the time course of mechanical stretch-induced activation of angiotensinogen mRNA in cardiac myocytes (20% stretch, Fig. 2). We found that stretching myocytes increased angiotensinogen mRNA accumulation as well. The increase was significant 6 h after stretching, and the maximal increase was observed after 24 h (a 19.7 ± 2.2-fold increase compared with nonstretched myocytes) and sustained for up to 48 h.


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  		Fig. 1.   Effects of ANG II on angiotensinogen mRNA levels. Cardiac myocytes incubated with serum-free medium for 12 h were treated with ANG II (100 nM) for indicated times, and angiotensinogen mRNA/18S rRNA levels were estimated. A: representative Northern blots of angiotensinogen mRNA (ATNG) and 18S rRNA (18S) from myocytes (total RNA 40 µg) treated with ANG II (exposed for 72 h for angiotensinogen and for 6 h for 18S rRNA). Total RNAs from rat liver (5 µg) and lung (20 µg) were also electrophoresed. B: relative angiotensinogen mRNA levels in cardiac myocytes exposed to ANG II. Angiotensinogen mRNA levels were measured with an Imaging Analyzer BAS2000 normalized to signal generated by probing for 18S rRNA gene expression and are expressed relative to those achieved with RNA from control myocytes (mean angiotensinogen mRNA level of control myocytes is expressed as 100%). Bars represent means ± SE of 4 independent experiments.


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  		Fig. 2.   Effects of stretching on angiotensinogen mRNA levels. Cardiac myocytes incubated with serum-free medium for 12 h were stretched (20%) for indicated times, and angiotensinogen mRNA/18S rRNA levels were estimated. A: representative Northern blots of angiotensinogen mRNA and 18S rRNA from myocytes (total RNA 40 µg) subjected to stretch (exposed for 72 h for angiotensinogen and for 6 h for 18S rRNA). Total RNAs from rat liver (5 µg) and lung (20 µg) were also electrophoresed. B: relative angiotensinogen mRNA levels in cardiac myocytes subjected to stretch. Data (n = 4) were analyzed as described in Fig. 1.

Effects of ANG II-receptor antagonists, transcriptional inhibitor, and protein synthesis inhibitor on ANG II- and stretch-mediated increases in angiotensinogen mRNA in cardiac myocytes. To determine the type of ANG II receptors involved in mediating the enhanced expression of angiotensinogen mRNA in response to ANG II, the effects of ANG II-receptor antagonists were investigated (Fig. 3A). Preincubation of cardiac myocytes for 30 min with CV-11974 (100 nM), an AT1-receptor-specific antagonist, before treatment with ANG II (100 nM) for 24 h virtually abolished the stimulatory effect of ANG II (93 ± 3% inhibition compared with ANG II-treated myocytes). In contrast, preincubation of cells with PD-123319 (100 nM), an ANG II type 2 (AT2)-receptor-specific antagonist, did not affect the response to ANG II. None of these ANG II-receptor antagonists alone had any influence on the expression of angiotensinogen mRNA (data not shown). To determine whether de novo RNA or protein synthesis was required for the ANG II-induced increase in angiotensinogen mRNA, cardiac myocytes were preincubated with actinomycin D (1 µg/ml) or cycloheximide (5 µg/ml) for 30 min before treatment with ANG II for 24 h (Fig. 3A). The RNA synthesis inhibitor actinomycin D abolished the ANG II-mediated increase in angiotensinogen mRNA (98 ± 9% inhibition compared with ANG II-treated myocytes), whereas the induction of angiotensinogen mRNA by ANG II was not significantly altered by the protein synthesis inhibitor cycloheximide in cardiac myocytes. Although it was possible that the lack of inhibition by PD-123319 or cycloheximide was due to an insufficient dose of the inhibitor, preincubation of cardiac myocytes with increasing doses of PD-123319 (1 and 10 mM) or cycloheximide (50 and 500 µg/ml) did not affect the response to ANG II (Fig. 3B).


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  		Fig. 3.   Effects of inhibitors on ANG II-induced expression of angiotensinogen mRNA. A: representative Northern blots of angiotensinogen mRNA and 18S rRNA from myocytes (total RNA 40 mg) treated with ANG II and inhibitors (exposed for 36 h for angiotensinogen and for 6 h for 18S rRNA; top), and relative angiotensinogen mRNA levels in cardiac myocytes exposed to ANG II with inhibitors (bottom). Cardiac myocytes incubated with serum-free medium were preincubated for 30 min with CV-11974 (lane 3, 100 nM), PD-123319 (lane 4, 100 nM), actinomycin D (lane 5, 1 µg/ml), or cycloheximide (lane 6, 5 µg/ml) and treated with ANG II (lanes 2-6, 100 nM) for 24 h, and angiotensinogen mRNA/18S rRNA levels were estimated. Data (n = 6) were analyzed as described in Fig. 1. B: representative Northern blots of angiotensinogen and 18S rRNA mRNAs from myocytes (total RNA 40 µg) treated with ANG II and inhibitors (exposed for 18 h for angiotensinogen and for 4 h for 18S rRNA). Cardiac myocytes incubated with serum-free medium were preincubated for 30 min with PD-123319 (lane 2, 1 mM; lane 3, 10 mM) or cycloheximide (lane 5, 50 µg/ml; lane 6, 500 µg/ml) and treated with ANG II (lanes 1-6, 100 nM) for 24 h.

Because previous studies reported that stretch-induced events mainly depend on endogenous ANG II secreted from stretched myocytes (41, 50, 51), we next examined the effects of ANG II-receptor antagonists on stretch (24 h)-induced expression of angiotensinogen mRNA (Fig. 4). It is notable that exposure to CV-11974 significantly but not completely inhibited the stretch-mediated activation of angiotensinogen mRNA expression in cardiac myocytes (58 ± 4% inhibition by 100 nM CV-11974 and 60 ± 5% inhibition by 10 mM CV-11974 compared with stretched myocytes), whereas exposure to PD-123319 (100 nM and 10 mM) had no effect on the stretch-mediated expression of angiotensinogen mRNA. On the other hand, actinomycin D (1 and 10 µg/ml) abolished the stretch-mediated increase in angiotensinogen mRNA (98 ± 3% inhibition by 1 µg/ml actinomycin D and 98 ± 4% inhibition by 10 µg/ml actinomycin D compared with stretched myocytes), whereas the induction of angiotensinogen mRNA by stretch was not significantly altered by cycloheximide (5 and 50 µg/ml).


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  		Fig. 4.   Effects of inhibitors on stretch-induced expression of angiotensinogen mRNA. Representative Northern blots of angiotensinogen mRNA and 18S rRNA from myocytes (total RNA 40 µg) stretched with inhibitors (exposed for 36 h for angiotensinogen and for 4 h for 18S rRNA; top), and relative angiotensinogen mRNA levels in stretched cardiac myocytes with inhibitors (bottom). Cardiac myocytes incubated with serum-free medium were preincubated for 30 min with CV-11974 (lane 3, 100 nM; lane 4, 10 mM), PD-123319 (lane 5, 100 nM; lane 6, 10 mM), actinomycin D (lane 7, 1 µg/ml; lane 8, 10 mg/ml), or cycloheximide (lane 9, 5 mg/ml; lane 10, 50 µg/ml) and stretched (lanes 2-10, 20%) for 24 h, and angiotensinogen mRNA/18S rRNA levels were estimated. Data (n = 4) were analyzed as described in Fig. 1.

Effects of ANG II and mechanical stretch on transcription of angiotensinogen gene in cardiac myocytes. Because previous studies showed that expression of the angiotensinogen gene is regulated at least partly at the level of transcription (13) and that the promoter region from -501 to +22 relative to the major transcription start site of the mouse angiotensinogen gene is able to direct cell type-specific and differentiation-dependent transcription (45, 46), we examined the effects of ANG II on the transcriptional activity of the angiotensinogen gene by transient transfection assay. As shown in Fig. 5, treatment of cardiac myocytes with ANG II (100 nM) for 12 h significantly increased CAT activity (4.5 ± 0.3-fold compared with untreated cells) directed by the promoter region from -501 to +22 of the angiotensinogen gene (Ag501). To identify which subtype of the ANG II receptor was involved in the activation of angiotensinogen promoter by ANG II, cells were preincubated with CV-11974 (10 nM and 100 nM) or PD-123319 (10 and 100 nM) for 30 min and were then treated with ANG II for 12 h. CV-11974 completely blocked the activation of angiotensinogen promoter induced by treatment with ANG II (112 ± 8% inhibition compared with ANG II-treated myocytes at 100 nM), whereas PD-123319 did not affect the increase in promoter activity.


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  		Fig. 5.   Effects of ANG II and inhibitors on angiotensinogen promoter activity. Plasmid Ag501 DNA was transfected in cardiac myocytes. Myocytes transfected and incubated with serum-free medium were preincubated for 30 min with CV-11974 (lane 5, 10 nM; lane 6, 100 nM) or PD-123319 (lane 7, 10 nM; lane 8, 100 nM) and treated with ANG II (lanes 4-8, 100 nM) for an additional 12 h. Promoter activity was estimated by chloramphenicol acetyltransferase (CAT) assay. A: representative results of CAT assay using cell extracts (40 µg) from myocytes treated with ANG II and inhibitors. Different forms of acetylated [14C]chloramphenicol (arrowheads) represent promoter activity. Promoterless plasmid pUCSV0CAT (SV0, lane 2) was used as a background reference, and pUCSV3CAT (SV3, lane 1) was used as a positive control including SV40 enhancer-promoter region. B: relative CAT activities of Ag501 in cardiac myocytes exposed to ANG II with inhibitors. CAT activities were measured with an Imaging Analyzer BAS2000 and are expressed relative to those achieved with cell extracts from control myocytes [mean CAT activity of control myocytes (lane 3) is expressed as 100%]. Bars represent means ± SE of 4 independent transfection experiments.

Because the above results suggest that ANG II activates the angiotensinogen promoter through an AT1-receptor pathway, we finally examined which subtype of the ANG II receptor was involved in the activation of angiotensinogen promoter in response to mechanical stretch (Fig. 6). Cardiac myocytes were preincubated with CV-11974 (100 nM), PD-123319 (100 nM), or saralasin (100 nM, an antagonist of both AT1 and AT2 receptors) for 30 min and were then stretched for 12 h. Similar to the effect of CV-11974 on the stretch-mediated induction of angiotensinogen mRNA, both CV-11974 and saralasin significantly but not completely inhibited the stretch-mediated activation of angiotensinogen promoter in cardiac myocytes (47 ± 5% inhibition by saralasin and 50 ± 5% inhibition by CV-11974 compared with stretched myocytes); however, PD-123319 had no significant effect on the stretch-mediated activation of angiotensinogen promoter.


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  		Fig. 6.   Effects of stretching and inhibitors on angiotensinogen promoter activity. Plasmid Ag501 DNA was transfected in cardiac myocytes. Myocytes transfected and incubated with serum-free medium were preincubated for 30 min with saralasin (lane 5, 100 nM), CV-11974 (lane 6, 100 nM), or PD-123319 (lane 7, 100 nM) and treated with ANG II (lanes 4-7, 100 nM) for an additional 12 h. Promoter activity was estimated by CAT assay. A: representative results of CAT assay using cell extracts (40 µg) from stretched myocytes with inhibitors. Different forms of acetylated [14C]chloramphenicol (arrowheads) represent promoter activity. Promoterless plasmid pUCSV0CAT (SV0, lane 2) was used as a background reference, and pUCSV3CAT (SV3, lane 1) was used as a positive control including SV40 enhancer-promoter region. B: relative CAT activities of Ag501 in cardiac myocytes stretched with inhibitors. Data (n = 4) were analyzed as described in Fig. 5.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mechanical stress exemplified by stretching cardiac myocytes stimulates the secretion of ANG II from myocytes, and the secreted ANG II activates protein kinase and produces myocyte hypertrophy through an AT1-receptor pathway (41, 50, 51). In the present study, we focused on the regulation of angiotensinogen gene expression in response to treatment with ANG II and stretching of myocytes and found that both ANG II and mechanical stress increased expression of the angiotensinogen gene in cardiac myocytes.

Accumulating evidence suggests that humoral or neural factors, or both, and hemodynamic overload contribute to the pathogenesis of cardiac hypertrophy (14, 29). A number of studies suggest that the circulating and cardiac RAS plays an important role in the development of cardiac hypertrophy. All components of the RAS (e.g., renin, angiotensinogen, ACE, and ANG II receptor) have been identified in the heart at both the mRNA and protein levels. Although the angiotensinogen mRNA level was extremely low in the ventricle of normal heart, previous studies showed that these levels increased significantly in hypertrophied left ventricles in a pressure-overload cardiac hypertrophy model and in hypertrophied hearts of genetically hypertensive rats (3, 47, 48). These findings suggest that cardiac angiotensinogen is activated in response to hypertrophic stimulation in vivo and plays an important role in the development of cardiac hypertrophy. An alternative explanation is that the increased angiotensinogen gene expression is a response to hypertrophy and is not involved in its development. The augmentation of angiotensinogen expression may also be an "epi" phenomenon that is merely associated with the development of hypertrophy but is not causally related.

By binding to the AT1 receptor, ANG II increases the activities of signal transduction pathways, the expression of immediate early genes, and the synthesis of proteins in cardiac myocytes (39). Mechanical stretch of cardiac myocytes also induces the secretion of ANG II and evokes these hypertrophic responses (41, 50, 51). After induction of immediate-early genes, the expression of several genes changes in response to pressure overload. In this study, ANG II and mechanical stretch were able to activate the expression of angiotensinogen mRNA in cardiac myocytes. Because ANG II- and stretch-induced upregulation of angiotensinogen mRNA was evident after 6 h of stimulation and increased expression of angiotensinogen is maintained during the chronic phase of hypertrophy in vivo (3, 47, 48), the cardiac angiotensinogen gene may be classified as a stable late marker of hypertrophy similar to the cardiac AT1 receptor (23, 44).

In the present study, actinomycin D completely inhibited the ANG II- and stretch-mediated increases in angiotensinogen mRNA. In addition, CV-11974 abolished the ANG II-induced increases in the levels of mRNA expression and promoter activity. On the other hand, suppression by CV-11974 of the stretch-mediated increases in the levels of angiotensinogen mRNA and transcriptional activity was significant but not complete in cardiac myocytes. These results indicate that ANG II activated angiotensinogen gene transcription exclusively via an AT1-receptor pathway and that stretch activated angiotensinogen gene transcription in cardiac myocytes mainly via an AT1-receptor pathway. The concentration of ANG II secreted into the media from cardiac myocytes exposed to mechanical stretch was reported to be between 400 and 500 pM (41). In addition, the concentration of the ANG II-receptor antagonists used in this study is enough to block the ANG II receptors because 100 nM CV-11974 was sufficient for the complete inhibition of the effects of 100 nM ANG II in both isolated rabbit aorta (32) and cardiac myocytes (19, 24), 100 nM saralasin and 100 nM CV-11974 abolished the maximal activation of extracellular signal-regulated kinases (ERK) induced by 100 nM ANG II (50), and 100 nM PD-123319 completely suppressed release of arachidonic acid from cardiac myocytes induced by 10 nM ANG II (27). Furthermore, the concentration of inhibitors of RNA synthesis and protein synthesis used in this study is enough to block the synthesis of RNA and protein, since 1 mg/ml actinomycin D and 1 mg/ml cycloheximide abolished the interleukin-1beta -induced expression of nitric oxide synthase gene in cardiac myocytes (49). Thus the concentration of CV-11974 used in this study should be able to completely block the AT1-mediated effect of ANG II secreted from cardiac myocytes in response to stretch. Therefore incomplete inhibition of the stretch-mediated increase in mRNA expression and promoter activity of the angiotensinogen gene suggests two possibilities. One possibilty is that factors other than ANG II would be involved in stretch-mediated transcription of the angiotensinogen gene in cardiac myocytes, as we and others have reported with respect to the stretch-mediated activation of ERK and other protein kinases in cardiac myocytes (33, 50). The other possibility is that ANG II is produced intracellularly and activates intracellular (e.g., nuclear) ANG II receptors (5). A recent study has shown that the intracellular ANG II effect is totally inhibited by the concomitant injection of CV-11974 but that extracellular CV-11974 does not influence the intracellular effects (17). It would therefore would be possible that stretch of cardiac myocytes produces ANG II intracellularly to the concentration enough to activate nuclear ANG II receptors which are insensitive to CV-11974 in the medium.

The results of Northern blot analysis indicated that ANG II, mechanical stretch, or both increased the levels of angiotensinogen mRNA conservatively by ~20-fold. However, the magnitude of changes in the CAT assay were approximately four- to fivefold. This would indicate that full inducibility is not contained within the promoter region from -501 to +22 of the mouse angiotensinogen gene used for CAT assay in this study. Previous studies have shown that the ANG II-responsive element of the c-fos gene, which is one of the first genes activated by myocyte stretch, is mapped to the serum response element (40). Although there is no such sequence motif in the 5'-flanking region of the murine angiotensinogen gene (7, 10), the promoter region of the rat angiotensinogen gene has an acute-phase response element (APRE, -557 to -531 of the transcriptional start site), and the APRE is able to function as an ANG II-inducible enhancer in cultured hepatocytes (6, 25). This APRE lies outside the limits of the promoter used in this study and may explain the discrepancy in the degree of induction between Northern blot analysis and CAT assay. Clearly, additional studies examining possible interactions between the upstream APRE and the proximal promoter region are needed to determine the role of angiotensinogen proximal promoter in ANG II- and stretch-mediated activation of the angiotensinogen gene in cardiac myocytes. In conclusion, our results indicate that treatment of cardiac myocytes with ANG II activates angiotensinogen gene transcription exclusively via an AT1-receptor pathway and that mechanical stretch of cardiac myocytes activates such transcription mainly via an AT1-receptor pathway. It is likely that the upstream APRE, specific proximal DNA elements, or both may be involved in ANG II- and stretch-mediated transcriptional activation of the angiotensinogen gene in cardiac myocytes. Experiments now in progress will define these promoter elements and will provide a molecular basis for the transcriptional machinery of the angotensinogen gene in response to hypertrophic stimuli. Our results also suggest one possible mechanism for the RAS positive-feedback loop in cardiac myocytes in the pathogenesis of cardiac hypertrophy and may provide a rationale for the use of AT1 antagonists in the management of cardiac hypertrophy associated with volume overload.

Perspectives

The local RAS is involved in the development of cardiovascular diseases including cardiac hypertrophy, and there seems to be tissue-specific regulation of the cardiac RAS component gene expression in cardiac hypertrophy. Data presented in this paper support the presence of cardiac hypertrophy-linked regulation of the angiotensinogen gene. A previous study showed that in vivo transfection of double-stranded oligonucleotides as "decoy" cis-elements to block the binding of nuclear factors to promoter regions of the angiotensinogen gene in the liver resulted in inhibition of gene trans-activation and a transient decrease in high blood pressure in SHR (30). Because the remodeling that occurs after cardiac ischemia has been shown to be prevented or attenuated by inhibition of the RAS (37), therapeutic methodology using antisense or decoy oligonucleotides, which specifically inhibit expression of the cardiac angiotensinogen gene in pathological states, may have a potential as a gene therapy.

    ACKNOWLEDGEMENTS

This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan, the Uehara Memorial Foundation, Ichiro Kanehara Foundation, and Yokohama Foundation for Advancement of Medical Science.

    FOOTNOTES

We acknowledge Takeda Chemical Industries (Osaka, Japan) and Parke-Davis (Ann Arbor, MI) for providing CV-11974 and PD-123319, respectively.

Address for reprint requests: K. Tamura, Dept. of Internal Medicine II, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-Ku, Yokohama 236, Japan.

Received 10 November 1997; accepted in final form 9 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Regul Integr Compar Physiol 275(1):R1-R9
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