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(Circulation. 1997;96:3053-3062.)
© 1997 American Heart Association, Inc.

Articles

Endothelin-1 Is Involved in Stretch-Induced Early Activation of B-Type Natriuretic Peptide Gene Expression in Atrial but Not in Ventricular Myocytes

Acute Effects of Mixed ET_A/ET_B and AT₁ Receptor Antagonists In Vivo and In Vitro

Jarkko Magga, MD; Olli Vuolteenaho, MD, PhD; Minna Marttila, MD; ; Heikki Ruskoaho, MD, PhD

From the Department of Pharmacology and Toxicology and the Department of Physiology (O.V.), Biocenter Oulu, University of Oulu, Finland.

Correspondence to Heikki Ruskoaho, MD, Department of Pharmacology and Toxicology, University of Oulu, Kajaanintie 52 D, FIN-90220 Oulu, Finland. E-mail heikki.ruskoaho{at}oulu.fi

	Abstract

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Background The precise role of paracrine and autocrine factors in mechanical load–induced activation of cardiac gene expression is unknown. Here we report the effects of endothelin-1 (ET-1) and angiotensin II (Ang II) receptor antagonism on acute pressure overload–induced activation of cardiac B-type natriuretic peptide (BNP) gene expression in spontaneously hypertensive rats (SHRs) in vivo and on mechanical stretch–induced increase in atrial BNP gene expression in vitro.

Methods and Results Acute pressure overload produced in conscious SHRs by infusion of arginine⁸-vasopressin (0.05 µg · kg^-1 · min^-1) for 2 hours resulted in an increase in BNP mRNA levels in the left ventricle as well as in the atrium. Bolus injections of bosentan (mixed ET_A/ET_B receptor antagonist, 10 mg/kg IV) but not losartan (AT₁ receptor antagonist, 10 mg/kg IV) blocked the increase of the BNP mRNA levels produced by pressure overload in the left atria, whereas the elevation of BNP mRNA levels was similar (a 1.9-fold increase) in the left ventricles of vehicle-, losartan-, and bosentan-infused SHRs. In an isolated perfused rat heart preparation, infusion of bosentan (1 µmol/L) for 2 hours inhibited the mechanical stretch–induced increase in BNP mRNA levels in the right atria, whereas an AT₁ receptor antagonist, CV-11974 (10 nmol/L), had no effect.

Conclusions The findings of the present study demonstrate that Ang II and ET-1 are not obligatorily required for stretch to trigger the increased BNP gene expression in ventricular myocytes in vivo. In contrast, mechanical load on the atrial myocytes did initiate an ET-1–dependent expression of BNP gene showing that endogenous ET-1 production differentially regulates BNP gene expression in atrial and ventricular myocytes.

Key Words: pressure • natriuretic peptides • endothelin • angiotensin • hypertrophy

	Introduction

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The heart adapts to cardiac overload by the hypertrophy of individual muscle cells and differential expression of several cardiac-specific genes.^{1 2} The early genetic response (within 2 hours) to mechanical loading in cardiac myocytes includes transcription of immediate early response genes such as c-fos, c-myc, c-jun, and EGR-1. Upregulation or subtype switch of the contractile protein gene expression such as ß-myosin heavy chain, skeletal muscle -actin, and myosin light chain-2 genes follows later during ventricular hypertrophy.^{1 2} Hypertrophy also results in induction of noncontractile protein genes such as ANP and BNP,^{3 4 5} which are the known members of the mammalian cardiac natriuretic peptide system. In vivo induction of ANP gene expression is seen within a few days after initiation of increased cardiac overload,³ whereas pressure load induces rapid activation of BNP gene expression in the heart of normal and hypertensive rats.⁶ The acute increase in both atrial and ventricular BNP mRNA levels has been shown to be parallel to the pressure changes in each chamber and to occur within 1 hour,⁶ thus mimicking the rapid induction of proto-oncogenes in response to hemodynamic stress.^{1 2} Because proto-oncogenes are also expressed in nonmuscular cells, BNP can be used as a myocyte-specific marker to identify mechanisms that couple acute mechanical overload to alterations in cardiac gene expression in normal and failing hearts.

Although cardiac overload is known to alter the expression of several cardiac-specific genes, it has not yet been established whether wall stretch acts directly or via local paracrine and autocrine factors liberated in response to hemodynamic load. In particular, the local renin-angiotensin system may play an important role in the adaptation of the heart to pressure and volume overload.^{7 8} Increased myocardial angiotensinogen mRNA levels, ACE activity, rate of Ang II production, and AT₁ receptor mRNA levels are found in pressure-overload cardiac hypertrophy in rats.^{9 10 11} ACE inhibitors and AT₁ receptor antagonists produce significant regression of left ventricular hypertrophy in patients with hypertension and in animal models, including SHRs.^{12 13 14} Stretching of cardiac myocytes in vitro causes release of Ang II in the short term (10 to 30 minutes) and increases the expression of the angiotensinogen gene in the long term.¹⁵ Because Ang II also stimulates cardiac protein synthesis,^{16 17} Ang II acting through the AT₁ receptors may act as a mediator of stretch-induced adaptive response in cardiac myocytes. Another candidate is ET-1, because ET-1 is released rapidly when cultured endothelial cells are stretched.^{18 19} Production of ET-1 has been shown to increase in the hypertrophied rat heart as a result of pressure overload,²⁰ further suggesting that ET-1 may be involved in the mechanical load–induced activation of cardiac gene expression. However, the potential roles of endogenous ET-1 or Ang II in the regulation of early activation of cardiac gene expression in response to mechanical load in vivo are unknown.

The aim of the present study was to examine whether ET-1 or Ang II is playing a causal role in the activation of cardiac gene expression induced by acute pressure overload in cardiac myocytes by using the BNP gene as a molecular marker for increased load. Therefore, we determined in conscious SHRs the effects of the mixed ET_A/ET_B receptor antagonist bosentan and the AT₁ receptor antagonist losartan on the induction of cardiac BNP gene expression produced by infusion of AVP for 2 hours. The actions of ET-1 and Ang II receptor antagonism on atrial and ventricular BNP mRNA and irBNP levels under basal conditions (without acute pressure overload) in conscious SHRs were also analyzed. In addition, we used perfused rat hearts to study the involvement of ET-1 and Ang II in the mechanical stretch–induced atrial BNP gene expression. The findings of the present study show that Ang II is not obligatorily required for stretch to trigger the induction of cardiac BNP gene expression and that endogenous ET-1 production differentially regulates stretch-induced BNP gene expression in atrial and ventricular myocytes.

	Methods

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Materials
Bosentan was generously supplied by Dr Martine Clozel (F. Hoffmann-La Roche Ltd), losartan by Dr Ronald D. Smith (DuPont Merck Pharmaceutical Co), and CV-11974 by Dr Hajime Toguchi (Takeda Chemical Industries Ltd). Chemicals used in this study were AVP (Peninsula Laboratories Europe), formaldehyde and guanidine isothiocyanate (Fluka Chemie AG), CsCl (Serva Feinchemica GmbH & Co), heparin (Leiras), and radioiodine (Amersham). Unless otherwise stated, all other chemicals were from Sigma Chemical Co.

Animals
Male SHRs of the Okamoto-Aoki strain and male 2-month-old Sprague-Dawley rats from the Center for Experimental Animals at the University of Oulu, Finland, were used. SHRs were studied at 13 to 20 months of age, and the strain was originally obtained from Mollegaards Avslaboratorium, Skensved, Denmark. The choice of rat strains for this study was based on our previous studies, in which we found that infusion of AVP rapidly stimulates BNP gene expression in the hearts of normal and hypertensive SHRs in vivo⁶ and that mechanical stretch in vitro in Sprague-Dawley rats produces an acute induction of BNP gene expression.²¹ Thus, by choosing these rat strains, we were able to study the role of ET-1 and Ang II under identical, previously documented experimental conditions. The rats were housed in plastic cages in a room with a controlled 40% humidity and a temperature of 22°C. A 6 AM on/6 PM off environmental light cycle was maintained. The experimental design was approved by the Animal Experimentation Committee of the University of Oulu, Finland.

Chronically Instrumented Rats
The rats were instrumented as described previously.^{22 23} The experiments were started by measurement of MAP, heart rate, and RAP in the conscious, freely moving animals for 25 minutes before 1.0 mL of blood was withdrawn from the arterial catheter for measurement of plasma irANP and irBNP. An equal volume of blood from a donor rat was then infused. The baseline hemodynamic measurements were done 5 minutes later, when MAP, heart rate, and RAP were stabilized near the control values. Then, bosentan (10 mg/kg), losartan (10 mg/kg), or vehicle (0.9% NaCl) was injected as an intravenous bolus. Injection volume was 0.1 mL/100 g body wt. Next, AVP (0.05 µg · kg^-1 · min^-1 IV) or vehicle (0.9% NaCl IV) was infused for 2 hours via an infusion pump (B. Braun Perfusor ED, Braun Melsungen). The infusion rate was 37.5 µL/min. Arterial blood samples were taken 1 and 2 hours after the start of administration of vehicle or AVP. Blood samples were taken into precooled tubes containing 1.5 mg EDTA/mL blood on ice and immediately centrifuged (2000g, 10 minutes, at +4°C). The plasma was stored at -20°C until assayed by RIA.²³ Tissues were prepared at the end of drug and vehicle infusions as described previously.^{6 22} All cardiac tissue samples were blotted dry, weighed, immersed in the liquid nitrogen, and stored at -70°C until assayed.

Isolated Perfused Heart Preparation
The isolated perfused rat heart preparation was similar to that described previously.^{21 24} The hearts were stimulated (11 V, 0.5 ms) with a Grass stimulator (model S88, Grass Instruments) to increase heart rate up to the level of 300 bpm. The hearts were paced via the pulmonary artery cannula and the cannula in the inferior vena cava. During the equilibration period (40 minutes), the hearts were perfused with a peristaltic pump (Minipuls 3, model 312, Gilson) at a constant flow rate of 5 mL/min. After a 10-minute control period, the right atria were stretched for 2 hours by elevation of the level of the pulmonary artery cannula tip. RAP was kept constant at the desired level by adjustment of the level of the pulmonary artery cannula tip.²¹ Drugs were infused continuously throughout the stretch period via an infusion pump (Secan PSA 55, Sky Electronics SA). The intracardial concentrations of bosentan and CV-11974 were 1 µmol/L and 10 nmol/L, respectively. The right auricles were carefully removed immediately after perfusion, weighed, immersed in liquid nitrogen, and stored at -70°C until assayed.

Isolation and Analysis of Cytoplasmic RNA
RNA was isolated from atria and ventricles by the guanidine thiocyanate–CsCl method.²⁵ For the RNA Northern blot and dot blot analyses, 3.0-µg samples of the RNA from atria and 22-µg samples from ventricles were transferred to the Schleicher & Schuell BAS 85 nitrocellulose membrane. A 390-bp fragment of rat BNP cDNA probe²⁶ (a generous gift from Dr Kazuwa Nakao, Kyoto University School of Medicine, Kyoto, Japan), full-length rat ANP cDNA probe²⁷ (a generous gift from Dr Peter L. Davies, Queen's University, Kingston, Canada), and full-length cDNA probe complementary to GAPDH²⁸ were labeled, and the membranes were hybridized and washed as described previously.⁶ The hybridization signal of ANP mRNA and BNP mRNA was normalized to that of GAPDH mRNA for each sample.

RIA of irBNP and irANP
For the BNP RIA, the atrial and ventricular guanidine thiocyanate extracts were diluted 100- and 50-fold, respectively. Tissue and plasma samples were extracted by Sep-Pak C₁₈ cartridges.^{6 29} Eluates were lyophilized and redissolved in RIA buffer. For the ANP RIA, the atrial and ventricular guanidine thiocyanate extracts were diluted 5x10⁴- and 400-fold, respectively. The incubation of the tissue and plasma extracts and precipitation of the immunocomplexes were done as previously described.^{6 21} The sensitivities of the BNP and ANP assays were 2 fmol/tube and 1 fmol/tube, respectively. Fifty percent displacements of the respective standard curve occurred at 16 and 25 fmol/tube. The intra-assay and interassay variations were <10% and <15%, respectively. Serial dilutions of perfusate and tissue extracts showed parallelism with the standards. The ANP antiserum recognized ANP and proANP with equal avidity but did not cross-react with BNP or CNP (<.01%). The BNP antiserum did not recognize ANP or CNP (<.01%). Tissue BNP and ANP are expressed as a concentration per milligram wet weight.

Statistical Analysis
The results are expressed as mean±SEM. For the comparison of statistical significance between two groups, Student's t test was used. The hemodynamic variables and plasma irANP and irBNP levels were analyzed with one-way ANOVA followed by the Student-Newman-Keuls post hoc test. Repeated-measures ANOVA was used for multivariate analysis. Differences at the 95% level were considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of Bosentan and Losartan on Hemodynamic Variables in Conscious SHRs
We reported earlier that infusion of AVP (0.05 µg · kg^-1 · min^-1) rapidly stimulates BNP gene expression in the hearts of normal and hypertensive rats.⁶ During AVP infusion, an 2-fold increase in ventricular BNP mRNA levels was already evident after 1 hour, and peak levels of BNP mRNA associated with an increase in the irBNP concentration in the left ventricle were reached at 4 hours. Left atrial BNP mRNA levels increased significantly in response to 1-hour infusions, and values peaked (about 3.5-fold increase) in 1-year-old SHRs at 2 hours.⁶ Furthermore, the differences in ventricular BNP mRNA levels in AVP-infused animals at 2 and 4 hours were small (2.2-fold versus 2.6-fold increase). On the basis of these findings, AVP was infused in conscious SHRs for 2 hours in the present study. The age range of animals used varied between 13 and 20 months (Table 1). MAP, heart rate, and RAP were measured continuously throughout the experiments in conscious animals, and plasma concentrations of irBNP and irANP were measured before as well as 1 and 2 hours after the infusions were begun. There were no significant differences in baseline hemodynamic variables and plasma peptide concentrations between the vehicle-, AVP-, bosentan-, and losartan-infused groups, except that the plasma irBNP level of the bosentan plus AVP group and the irANP level of the bosentan group were lower than those of the respective vehicle-infused groups (Table 1).

View this table: [in this window] [in a new window]   Table 1. Body and Cardiac Weights, Basal Hemodynamic Variables, and Baseline Plasma Levels of Immunoreactive Natriuretic Peptides in Spontaneously Hypertensive Rats

Both losartan and bosentan were administered at a concentration of 10 mg/kg IV as a bolus injection. Previously, it was reported that losartan at a dose of 10 mg/kg IV causes an inhibition of the Ang II pressor response for at least 24 hours in conscious SHRs³⁰ and that bosentan at a dose of 10 mg/kg IV inhibits cardiovascular responses of ET-1 in pithed rats.³¹ We recently showed that in conscious rats, bosentan at a concentration of 10 mg/kg completely blocked any increase in MAP produced by big ET-1 and losartan at a concentration of 10 mg/kg completely blocked any increase in MAP produced by Ang II infusion.²³ In the present study, bolus administration of losartan led to a significant decrease in MAP within 2 hours (20.6±7.5 mm Hg, F=2.8, P<.05) (Fig 1). There was also a tendency for MAP to decrease after bosentan infusion, but this change was not statistically significant (20.0±7.3 mm Hg, F=1.9, P=NS). Administration of receptor antagonists had no effect on heart rate and RAP, and during blood sampling in the vehicle-infused group, hemodynamic variables remained stable (Fig 1).

View larger version (27K): [in this window] [in a new window]   Figure 1. Hemodynamic variables in bosentan- and losartan-infused conscious SHRs. After control period (arrow), AVP (0.05 µg · kg-1 · min-1), bosentan (10 mg/kg), losartan (10 mg/kg), or vehicle was infused intravenously for 2 hours. indicates controls; , bosentan or losartan; , AVP; and , AVP plus bosentan or losartan. Results are mean±SEM. For number of experiments in each group, see Table 1.

During AVP infusion, MAP reached maximum values within 10 minutes and remained elevated throughout the experimental period (Fig 1). Intravenous infusion of AVP increased MAP similarly both in the bosentan-treated (AVP plus bosentan versus AVP, F=0.4, P=NS) and in the losartan-infused (AVP plus losartan versus AVP, F=1.5, P=NS) rats (Fig 1). Expressed in absolute values, MAP increased in response to AVP infusion in the vehicle-injected SHRs maximally by 43.3± 4.9 mm Hg and in the bosentan-treated SHRs by 43.8±5.7 mm Hg. The corresponding increase in MAP produced by AVP infusion in vehicle-injected SHRs was 53.8±5.5 mm Hg and in the losartan-treated SHRs, 55.0±5.3 mm Hg. In addition, heart rate decreased similarly in the bosentan-pretreated (AVP plus bosentan, 31%, versus AVP alone, 24%) and losartan-pretreated (AVP plus losartan, 29%, versus AVP, 24%) animals during AVP infusion (Fig 1). There were no statistically significant differences in changes of RAP between vehicle and drug infusions (Fig 1). Thus, because changes in all hemodynamic variables in response to AVP in bosentan- and losartan-pretreated rats were similar to those seen in vehicle-infused rats, this experimental model in intact animals allowed us to characterize the role of ET-1 and Ang II in mechanical stretch–induced changes in cardiac natriuretic gene expression at the identical degree of cardiac load.

Effect of Bosentan on Cardiac and Plasma Levels of the Natriuretic Peptides in SHRs
Northern blot analysis with both rat BNP and ANP cDNA probes identified a single 0.9-kb mRNA species in the ventricles and atria (Fig 2). As shown in Fig 3, administration of bosentan resulted in a 31% decrease (P<.05) in BNP mRNA levels in the epicardium of the left ventricle and a 47% decrease (P<.05 to.01) in irBNP concentrations in both left ventricular endocardial and epicardial layers. The elevation of BNP mRNA levels in response to acute pressure load produced by AVP was similar (a 1.9-fold increase) in the epicardial and endocardial layers of the left ventricle in vehicle- and bosentan-infused SHRs, whereas no changes in left ventricular irBNP concentrations were found (Fig 3). In contrast to the significant changes of BNP gene expression, ventricular ANP mRNA and irANP levels after administration of bosentan or during AVP infusion remained unchanged, except that a slight decrease (28%) in ANP mRNA levels of the left ventricular endocardial layer in bosentan-infused animals was noted (Fig 3, upper right).

View larger version (72K): [in this window] [in a new window]   Figure 2. Northern blot analysis showing effects of bosentan (A and B) and losartan (C and D) on cardiac ANP mRNA and BNP mRNA levels. At end of vehicle or drug infusions, total RNA was prepared from epicardium (epi) and endocardium (endo) of left ventricles and from right and left atria of SHRs. Northern blot analysis with rat ANP and BNP cDNA probes identified a single 0.9-kb mRNA species in left ventricle and atrium. Hybridization signal for GAPDH is also shown.

View larger version (46K): [in this window] [in a new window]   Figure 3. Effects of intravenous administration of bosentan, AVP, and their combination on left endocardial (endo) and epicardial (epi) BNP mRNA, irBNP, ANP mRNA, and irANP levels in conscious SHRs at 2 hours. mRNA results are expressed as ratio of ANP mRNA or BNP mRNA to GAPDH, as determined by dot blot analysis. Open bars indicate control; hatched bars, bosentan; crosshatched bars, AVP; and solid bars, bosentan plus AVP. Data are mean±SEM. For number of experiments in each group, see Table 1. *P<.05, **P<.01, ***P<.001 vs control (Student's t test).

As shown in Fig 4, AVP infusion also resulted in an early activation of BNP gene expression (a 1.5-fold increase in the BNP mRNA levels) in both left and right atria of conscious SHRs. The increase in left but not right atrial BNP mRNA levels produced by acute pressure overload was blocked by bosentan administration (Fig 4). In addition, bolus injection of bosentan alone significantly (47%, P<.05) decreased baseline left atrial BNP mRNA levels, whereas it had no effect on right atrial BNP mRNA levels. Bosentan, AVP, or the combined treatment of SHRs with bosentan and AVP did not significantly affect atrial irBNP and irANP concentrations. Right atrial ANP mRNA levels after administration of bosentan or during AVP infusion also remained unchanged. Conversely, left atrial ANP mRNA levels were lower (14% to 22%) in all drug-treated animals compared with vehicle-infused control animals (Fig 4). The reason for this finding is not clear, but it is of interest to note that the degree of left ventricular hypertrophy was also smaller (7% to 10%) in these groups than in the control group (see Table 1).

View larger version (38K): [in this window] [in a new window]   Figure 4. Effects of intravenous administration of bosentan, AVP, and their combination on left and right atrial BNP mRNA, irBNP, ANP mRNA, and irANP levels in conscious SHRs at 2 hours. mRNA results are expressed as ratio of ANP mRNA or BNP mRNA to GAPDH, as determined by dot blot analysis. Bars as in Fig 3. Data are mean±SEM. For number of experiments in each group, see Table 1. *P<.05, **P<.01 vs control (Student's t test).

When AVP was infused for 2 hours, significant increases in both plasma irBNP (F=11.4, P<.001) and irANP (F=12.1, P<.001) concentrations in conscious SHRs were seen (Fig 5). Plasma irBNP concentration reached maximal values at the end of AVP infusion, whereas plasma irANP values peaked in the AVP-infused SHRs at 60 minutes. After administration of bosentan, the increases in plasma concentration of natriuretic peptides in response to AVP did not differ significantly from the vehicle-infused group (irBNP: AVP plus bosentan versus AVP, F=1.6, P=NS; irANP: AVP plus bosentan versus AVP, F=0.7, P=NS) (Fig 5). Injections of bosentan alone had no effect on baseline plasma irBNP and irANP levels, and during blood sampling in vehicle-infused animals, the plasma concentrations of natriuretic peptides remained constant (Fig 5).

View larger version (54K): [in this window] [in a new window]   Figure 5. Effects of bosentan and losartan on plasma irBNP and irANP concentrations in conscious SHRs. AVP (0.05 µg · kg-1 · min-1), bosentan (10 mg/kg), losartan (10 mg/kg), or vehicle was infused intravenously for 2 hours. Blood samples were taken before and 60 and 120 minutes after start of infusions. Open bars indicate control; hatched bars, bosentan or losartan; crosshatched bars, AVP; and striped bars, AVP plus bosentan or losartan. Results are mean±SEM. For number of experiments in each group, see Table 1. *P<.05 (each group was analyzed with one-way ANOVA followed by Student-Newman-Keuls t test).

Effect of Losartan Cardiac and Plasma Levels of the Natriuretic Peptides in SHRs
In the losartan-injected conscious SHRs, a 34% decrease (P<.05) in baseline BNP mRNA levels in the endocardium of the left ventricle was seen (Fig 6). Administration of AVP produced a 1.5-fold increase in BNP mRNA levels in the endocardium of the left ventricle and a 1.9-fold increase in left ventricular epicardial BNP mRNA levels, whereas no changes in left ventricular irBNP concentrations were found (Fig 7). The increase in BNP mRNA levels in the left ventricle in response to acute pressure load produced by AVP did not differ between vehicle- and losartan-treated SHRs (Fig 6). Losartan, AVP, or their combination did not significantly affect ventricular ANP gene expression in SHRs, except that endocardial ANP mRNA levels were lower in animals treated with losartan and AVP than in the vehicle-infused group (Fig 6).

View larger version (44K): [in this window] [in a new window]   Figure 6. Effects of intravenous administration of losartan, AVP, and their combination on left endocardial and epicardial BNP mRNA, irBNP, ANP mRNA, and irANP levels in conscious SHRs at 2 hours. Abbreviations as in Fig 3. mRNA results are expressed as ratio of ANP mRNA or BNP mRNA to GAPDH, as determined by dot blot analysis. Open bars indicate control; hatched bars, losartan; crosshatched bars, AVP; and solid bars, losartan plus AVP. Data are mean±SEM. For number of experiments in each group, see Table 1. *P<.05, **P<.01 vs control (Student's t test).

View larger version (36K): [in this window] [in a new window]   Figure 7. Effects of administration of bosentan (1 µmol/L) and CV-11974 (10 nmol/L) for 2 hours on right atrial BNP mRNA, irBNP, ANP mRNA, and irANP levels in perfused Sprague-Dawley rat heart preparation. mRNA results are expressed as ratio of ANP mRNA or BNP mRNA to GAPDH, as determined by dot blot analysis. Open bars indicate control; solid bars, stretch (5 mm Hg). Data are mean±SEM. For number of experiments in each group, see Table 3. **P<.05 vs control (Student's t test).

Losartan had no effect on the AVP-stimulated increase of BNP mRNA levels in the left atria (AVP plus losartan, 2.5-fold increase versus AVP, 2.2-fold increase, P=NS) and in the right atria (AVP plus losartan, 1.4-fold increase versus AVP, 1.3-fold increase, P=NS). Furthermore, bolus administration of losartan alone (without acute pressure load) did not alter atrial ANP and BNP mRNA levels (data not shown). Left atrial irBNP and irANP concentrations were slightly lower in the group treated with both losartan and AVP than in the vehicle-infused group; otherwise, atrial ANP concentrations remained unchanged (data not shown). As shown in Fig 5, losartan injections had no effect on baseline plasma irBNP and irANP concentrations in conscious SHRs. AVP infusion for 2 hours resulted in significant increases in plasma concentrations of both irBNP (F=3.0, P<.05) and irANP (F=29.5, P<.001). The increase in plasma natriuretic peptide concentrations in the SHR strain induced by AVP was similar in vehicle- and losartan-infused animals (irBNP: losartan plus AVP versus AVP, F=0.4, P=NS; irANP: losartan plus AVP versus AVP, F=2.1, P=NS) (Fig 5).

Effect of Bosentan and CV-11974 on Atrial Levels of Natriuretic Peptides in Isolated Perfused Rat Heart Preparation
To further characterize the role of ET-1 and Ang II in the mechanical stretch–induced BNP gene activation, we studied the effects of bosentan (1 µmol/L) and AT₁ receptor antagonist (CV-11974, 10 nmol/L) on right atrial stretch-stimulated increases in BNP mRNA levels in vitro. Previously, it has been reported that bosentan at this concentration decreases the vasoconstrictor effect of ET-1 (0.4 nmol/L) in perfused rat heart³² and that CV-11974 inhibits the submaximal dose of Ang II (10^-8 mol/L)–induced contraction of rabbit aorta by 50% at a concentration of 0.3 nmol/L.³³ The right atria were stretched for 2 hours by increasing RAP 5 mm Hg above the baseline RAP, as described previously.²¹ Bosentan and CV-11974 infusions were started simultaneously with and infused continuously during right atrial stretch. Perfusion pressure, heart rate, RAP, and contractility were measured continuously throughout the experiments, and the baseline hemodynamic parameters did not differ between vehicle and drug-infused groups (Table 2).

View this table: [in this window] [in a new window]   Table 2. Hemodynamic Variables in the Isolated Perfused Sprague-Dawley Rat Heart

As shown in Fig 7, right atrial stretch for 2 hours produced a 2.0-fold increase in right atrial BNP mRNA levels but had no effect on irBNP concentration. Infusion of bosentan alone (without stretch) had no effect on BNP gene expression, but it significantly decreased the mechanical stretch–induced increase in BNP mRNA levels in the atria (P<.05, Fig 7). In contrast, infusion of the AT₁ receptor antagonist CV-11974 had no statistically significant effect on the stretch-stimulated increase in atrial BNP mRNA levels. When bosentan was infused alone, a 27% increase (P<.05) in the right atrial irBNP concentration was seen, whereas atrial irBNP concentrations remained unchanged when bosentan was infused continuously during stretch. Mechanical stretch, drug infusions, or their combination had no effect on atrial ANP mRNA and irANP levels (Fig 7).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
These data show that endogenous production of ET-1 plays a role in stretch-induced activation of atrial BNP gene expression, whereas Ang II and ET-1 are not required for pressure overload–induced increase of BNP mRNA levels in the left ventricle. Induction of BNP gene expression serves as a characteristic myocyte-specific response to load, because the expression of BNP mRNA is rapidly increased at the onset of cardiac overload,^{6 21} its protein is increased in the ventricular myocytes within 4 hours of pressure overload,⁶ and its expression remains elevated in chronic pressure and volume overload.^{26 34 35 36 37} In this study, acute pressure overload was produced by infusion of AVP for 2 hours in intact animals. AVP caused rapid increase in MAP followed by an increase in the BNP mRNA levels in both atria and ventricles. Because in conscious animals, the different stimuli themselves invoke a complex array of hemodynamic, neural, and hormonal responses, we carefully documented changes in the degree of cardiac load between vehicle- and drug-infused groups. The results indicate that injections of bosentan or losartan did not significantly alter the hemodynamic response evoked by AVP infusion, thus allowing us to examine the direct action of load versus a requirement for Ang II and ET-1 to mediate stretch-induced initiation of early cardiocyte response associated with the onset of cardiac hypertrophy.

Several studies have used cardiocytes in culture to examine the potential role of Ang II in the mechanical stretch–induced hypertrophic response. Neonatal rat cardiocytes subjected to passive stretch increased protein synthesis within 24 hours and the expression of the immediate early gene c-fos within 30 minutes. Both of these responses were blocked by AT₁ receptor antagonists.¹⁵ CV-11974 (an AT₁ receptor–selective antagonist) and saralasin (an antagonist of both AT₁ and AT₂ receptors) also significantly suppressed activation of MAP kinases and MAP kinase kinase activators stimulated by stretching of myocytes,^{14 38} but the AT₂ receptor–selective antagonist PD123319 did not show any inhibitory effects on these events.³⁸ Moreover, cyclical stretch-induced angiotensinogen mRNA expression has been reported to be suppressed by the losartan treatment in a neonatal rat cardiocyte culture system.³⁹ Because mechanical stretch also caused direct secretion of Ang II from the cytoplasmic granules of ventricular myocytes,^{15 40} these results have led to the conclusion that mechanical stretch–induced myocyte hypertrophy is dependent on Ang II acting through the AT₁ receptor. Our present results in intact animals disagree with these reports in cultured cardiocytes. We noted that the pressure overload–induced increase in BNP mRNA levels was not altered by the selective AT₁-receptor antagonist losartan in conscious SHRs. Thus, in vivo Ang II is not required for early events of transducing mechanical load signal into BNP gene expression. In agreement with the present results in vivo, a recent study using passively loaded adult cardiocytes within 1 day after isolation showed that load-accelerated protein synthesis and increased Na⁺/Ca²⁺-exchanger mRNA expression were not altered by saralasin treatment.⁴¹

The lack of requirement for Ang II in the transduction pathway between the load on the ventricular myocytes and the early induction of cardiac gene expression demonstrated for first time in vivo in this study suggests two possibilities. Mechanical stretch may directly induce cardiac BNP gene expression, or autocrine/paracrine mechanisms other than Ang II are activated by pressure overload and evoke rapid induction of BNP gene expression. An important mediator could be ET-1, which has potent effects on cell growth and induces hypertrophy of cultured cardiac myocytes.^{42 43} ET-1 has also been suggested to mediate the effects of hypertrophic stimuli such as Ang II in vitro⁴⁴ and norepinephrine in vivo.⁴⁵ ET-1 is also synthesized in and secreted from cardiocytes,⁴⁴ and it can induce the expression of ANP and BNP^{46 47} in cultured cardiomyocytes as well as in isolated atrium preparations.⁴⁸ Pressure overload due to aortic constriction^{49 50} or pulmonary hypertension⁵¹ and norepinephrine-induced ventricular hypertrophy⁴⁵ have been shown to be associated with large increases in ventricular expression of ET-1 mRNA, further suggesting that endogenous cardiac production of ET-1 may play a functional role in mechanical load–induced cardiac gene expression. In the present study, we examined the effects of the mixed ET_A/ET_B receptor antagonist bosentan on pressure overload–induced early activation of BNP gene expression. The results show that the production of ET-1 in vivo is not playing a causal role in the induction of ventricular BNP gene expression. However, although ET-1 does not appear to act as an initiating factor during the very early phase of pressure overload, ET-1 may be involved in a more sustained phase of the hypertrophic response. For example, Ito et al⁴⁹ showed that ventricular expression of ANP mRNA due to aortic constriction was partially blocked at 1 week by subcutaneous administration of the ET_A receptor antagonist BQ 123. Another ET_A receptor antagonist, FR139317, was shown to block the hypertrophic response to the pressure increase within 21 days.⁵² Taken together, our data in the pressure overload model in intact animals do not support the hypothesis that local Ang II and ET-1 production act as triggering factors to an early increase in ventricular gene expression.

Another key finding of the present study was that the transduction of mechanical load signal into changes in BNP mRNA levels was dependent on ET-1 in the atria but not in the left ventricle. The lack of effect of acute administration of combined ET_A/ET_B receptor antagonism on ventricular BNP mRNA levels compared with the attenuation of expression in the atria suggests that alternative pathways are involved in the regulation of atrial and ventricular BNP gene expression. Indeed, BNP is secreted from ventricular cells promptly after synthesis via a constitutive pathway, whereas it is stored in the secretory granules and released via a regulated pathway in the atrium.^{3 4 5 47 53} Thus, our data suggest that endogenous ET-1 plays a more important role in the latter system and a lesser or absent role in the former. A further explanation for the difference between the effect of bosentan on BNP gene expression is that atrial cells may be more sensitive to ET-1 than ventricular cardiocytes. In support of this hypothesis, stimulation of cardiocytes by ET-1 (10 nmol/L) has been shown to double the secretion rate of BNP in atrial cardiocytes but not in ventricular cardiocytes.⁵⁴ Because bosentan is a combined antagonist, it is not possible to conclude whether the effect seen in the present study on atrial BNP gene expression is exerted though the ET_A or ET_B receptors, and further studies with selective ET receptor antagonists are needed to address this issue.

We also analyzed changes in ventricular BNP gene expression under basal conditions (without acute pressure overload) and found that antagonism of AT₁ receptors by losartan resulted in decreased left ventricular endocardial BNP mRNA levels within 2 hours. Similarly, infusion of bosentan for 2 hours decreased BNP mRNA and irBNP levels in the left ventricle of SHRs. Because transcription for BNP is known to be increased in ventricles of SHRs compared with normotensive WKY rats^{6 26 55} and production of Ang II and ET-1 increases in the hypertrophic myocardium because of pressure overload,^{9 10 45 49 50 51} these results suggest that receptor antagonists may act directly on ventricular myocardium to attenuate the effects of endogenous ET-1 and Ang II. However, losartan and also bosentan decreased blood pressure (20 mm Hg), which could have been responsible for the decrease in ventricular BNP gene expression as well. Recently, a load-dependent component has been reported to be more important than a load-independent component in regulating left ventricular natriuretic peptide production in the established phase of hypertension in aortic-banded rats.⁵⁶

Interestingly, the increase in left but not right atrial BNP mRNA levels produced by AVP infusion in conscious SHRs was blocked by bosentan administration. In our previous study,⁶ right atrial BNP mRNA levels and RAP remained generally unchanged during AVP infusion in SHRs, whereas an increase in RAP with elevation of right atrial BNP mRNA levels was observed in the WKY strain. In the present study, the induction of right atrial BNP gene expression did not parallel changes in RAP, ie, right atrial BNP mRNA levels increased significantly in response to AVP infusion, but pressure remained unchanged in the right atria. One possible explanation for this finding is that right atrial BNP gene expression may not be a specific marker for pressure overload in this model but may, in addition, reflect the direct influence of AVP on the right atrial myocytes. In support of this hypothesis, a vasopressin receptor has recently been detected in the heart.⁵⁷ Nevertheless, further studies with other hypertensive agents, such as phenylephrine,⁶ are needed to resolve this question.

Compared with the rapid induction of BNP gene expression in response to acute increase in MAP in conscious SHRs, alterations in ANP gene expression during AVP infusion were insignificant. Administration of losartan and bosentan for 2 hours alone or in combination with AVP had no or only a small effect on cardiac ANP mRNA and irANP levels, suggesting that ET-1 and Ang II do not significantly contribute to the short-term regulation of ANP gene expression in vivo. Because ET-1 and Ang II have been show to modulate stretch-activated ANP secretion^{58 59} and they are potent secretagogues for ANP secretion,^{3 60} ET-1 and Ang II could play an important role in mechanical load–induced natriuretic peptide secretion as well. In the present study, antagonism of AT₁ and ET_A/ET_B receptors by losartan and bosentan, respectively, had no effect on baseline plasma irANP and irBNP levels. Furthermore, plasma irANP and irBNP responses to acute pressure overload were not altered in ET_A/ET_B and AT₁ receptor antagonist–treated animals, suggesting that endogenous ET-1 and Ang II do not play a major role in pressure overload–induced cardiac hormone release. These data agree with the previous studies showing that myocyte stretch is the predominant stimulus controlling the release of ANP^{24 61} and BNP^{6 21 28} from the heart. In agreement with previous studies,^{6 26} BNP appeared to be secreted from cardiac cells promptly after its synthesis, because tissue irBNP levels remained unchanged despite the increase in BNP mRNA levels in both atria and ventricles in response to pressure overload.

Findings reported here have a number of important implications for further studies mapping the potential role of autocrine/paracrine factors in load-induced cardiac gene expression. First, the observation that ET-1 is involved in the mechanical stretch–induced activation of the BNP gene expression in the atria but not in the ventricle demonstrates that generalization of the results from ventricular myocytes to atrial cells should be done cautiously. Second, the results from isolated neonatal myocytes may not be applicable to the whole heart, because in vivo pressure overload–induced activation of the BNP gene expression was independent of ET-1 and Ang II in the ventricles, whereas several studies using cultured neonatal cells show that Ang II is required for load-stimulated protein synthesis and cardiac gene expression in vitro. Third, the results on the role of autocrine and paracrine factors seem to depend on the marker (for example, c-fos versus natriuretic peptides) used to characterize the hypertrophic response. Finally, the previous findings that the induction of c-fos expression after loading of neonatal ventricular myocytes can be blocked by AT₁ receptor antagonists but not that of BNP gene expression in vivo, observed in the present study, may also be interpreted to suggest that separate pathways are involved in stimulating natriuretic peptide and proto-oncogene transcription.

In summary, we used intact animals to map the role of two paracrine/autocrine factors, ET-1 and Ang II, in the induction of cardiac BNP gene expression produced by acute pressure overload. We showed for the first time that antagonism of ET_A/ET_B and AT₁ receptors in vivo had no effect on mechanical load–induced early activation of BNP gene expression in the ventricles. In contrast, ET-1 played a causal role in the activation of transcription for BNP by stretch in atrial tissue in vivo and in vitro. Therefore, endogenous ET-1 liberated in response to stretch appears to differentially regulate the early activation of BNP gene expression in atrial and ventricular myocytes.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II ANP = atrial natriuretic peptide AVP = arginine8-vasopressin BNP = B-type natriuretic peptide CNP = C-type natriuretic peptide ET-1 = endothelin-1 irANP = immunoreactive ANP irBNP = immunoreactive BNP MAP = mean arterial pressure RAP = right atrial pressure RIA = radioimmunoassay SHR = spontaneously hypertensive rat WKY = Wistar-Kyoto rat

	Acknowledgments

 
This study was supported by the Medical Research Council of the Academy of Finland, Sigrid Juselius Foundation, Finnish Heart Research Foundation, Ida Montin Foundation, Emil Aaltonen Foundation, Aarne Koskelo Foundation, and the Foundation of Oulu University. We thank Tuula Lumijärvi, Sirpa Rutanen, Tuula Räisänen, and Marja-Leena Vainikka for expert technical assistance.

	Footnotes

 
Guest editor for this article was John C. Burnett, Jr, MD, Mayo Clinic, Rochester, Minn.

Received October 16, 1996; revision received May 19, 1997; accepted May 28, 1997.

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