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Circulation. 1996;93:2059-2067

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(Circulation. 1996;93:2059-2067.)
© 1996 American Heart Association, Inc.

Articles

Evidence for Load-Dependent and Load-Independent Determinants of Cardiac Natriuretic Peptide Production

Tsuneo Ogawa, MD, PhD; Wolfgang Linz, PhD; Michelle Stevenson, BSc; Benoit G. Bruneau, BSc; Mercedes L. Kuroski de Bold, PhD; Jia Hua Chen, MD, PhD; Hoda Eid, PhD; Bernward A. Schölkens, MD; Adolfo J. de Bold, PhD

From the University of Ottawa Heart Institute at the Ottawa Civic Hospital and the Departments of Pathology (A.J.d.B., M.L.K.d.B., H.E.), Physiology (B.G.B., A.J.d.B.), and Biochemistry (H.E.), University of Ottawa, Ottawa, Ontario, Canada, and the Department of Pharmacology, Hoechst AG (W.L., B.A.S.), Frankfurt/Main, Germany.

Correspondence to Adolfo J. de Bold, PhD, University of Ottawa Heart Institute Research Centre at the Ottawa Civic Hospital, 1053 Carling Ave, Ottawa, Ontario K1Y 4E9, Canada.


*    Abstract
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 *Abstract
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 down arrowResults
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Background In hypertension with cardiac hypertrophy, the specific contributions to increased production of the cardiac natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) by load and the hypertrophic process are not known. In the present work we determine ANF and BNP synthesis and secretion in the aortic-banded rat treated with dosage schedules of the ACE inhibitor ramipril that result in the prevention or regression of both hypertension and hypertrophy (high dosage) or in the prevention or regression of hypertrophy alone with persistent hypertension (low dosage). Myosin heavy chain (MHC) isoform switch was studied as an indicator of ventricular cardiocyte hypertrophy as well as the levels of collagen III mRNA as a measure of changes in extracellular matrix.

Methods and Results Ramipril was administered for 6 weeks just after suprarenal aortic banding, or rats were banded for 6 weeks, after which ramipril was administered during the following 6 weeks. Banding caused an increase in blood pressure, left ventricular weight–to–body weight ratio, plasma and ventricular NP, ventricular NP mRNA, collagen III, and ß-MHC mRNA. Ramipril at 1 mg/kg normalized all these parameters while ramipril at 10 µg/kg normalized left ventricular weight–to–body weight ratio but not blood pressure. Plasma and ventricular NP content and mRNA levels were partially normalized by ramipril (10 µg/kg). Ramipril (10 µg/kg) prevented increased collagen III mRNA levels but did not affect ß-MHC mRNA levels.

Conclusions (1) NP production and secretion in aortic-banded rats are independently related to increased blood pressure and hypertrophy. (2) A load-dependent component is more important than a load-independent component in regulating left ventricular NP production. (3) ANF production is more sensitive than BNP production to the load-independent component. (4) Low-dose ramipril treatment reverses hypertrophy and the increased collagen III expression but does not reverse the increased ß-MHC isoform expression, suggesting that these are independently regulated processes. (5) Aortic banding and ACE inhibition do not affect atrial NP production and content.


Key Words: natriuretic peptides • myosin • collagen • aorta


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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The cardiac hormones ANF and BNP have very diverse biological properties but generally oppose the actions of the renin-angiotensin-aldosterone system.1 2 In the healthy adult, these NPs are predominantly stored in the atria, and their rate of secretion varies in response to changes in hemodynamic and neuroendocrine balance.3

On the basis of observations made on the types of response of the endocrine heart in different experimental situations, we have proposed3 that NP gene expression and NP release differ, depending on whether the stimulus is acute, subacute, or chronic. Acute stimuli such as acute volume expansion in vivo or atrial muscle stretch in vitro are met by the endocrine heart with an increase in the rate of release of ANF from stores without an apparent effect on synthesis and with a significant increase in plasma levels of ANF but not BNP.4 5 After 3 days of mineralocorticoid administration (a stimulus referred to as subacute), we observed an increase in both atrial ANF and BNP mRNA steady state levels but, as seen in acute stimulation, ANF but not BNP plasma levels were significantly elevated.6 Chronic mineralocorticoid plus salt administration leads to an increase in synthesis and plasma levels of ANF and BNP and to an increase in circulating levels of both these hormones.7 This is accompanied by cardiac hypertrophy and activation of the cardiac fetal gene program in the hypertrophied ventricle in which reexpression of ANF and BNP is a hallmark change not only of mineralocorticoid-induced hypertrophy but of ventricular hypertrophy in general.3 8

We have recently reported7 that in DOCA-salt–treated hypertensive rats with LV hypertrophy, NP production, MHC isoform expression, and anatomic hypertrophy may be dissociated from each other. However, it was not clear from these investigations whether the observed changes in NP production were primarily related to hypertrophy or to hypertension. Investigations using ACE inhibitors have shown that an effective treatment leads to the normalization of ANF gene expression and plasma levels of this hormone in hypertension associated with cardiac hypertrophy.9 10 11 12 13 14 15 16 It has also been observed in clinical studies that elevated plasma ANF and BNP levels in essential hypertension17 and heart failure are decreased after treatment with ACE inhibitors.18 19 These findings, however, offer no insight as to whether it is the normalization of blood pressure or the regression of hypertrophy that is responsible for the normalization of NP gene expression and circulating levels.

The finding that treatment of rats made hypertensive by aortic banding with high doses of ramipril (1 mg/kg) prevents and regresses both hypertension and hypertrophy but low doses of ramipril (10 µg/kg) prevent and regress only hypertrophy but not hypertension20 21 offers an opportunity to determine separately the contribution of cardiac hypertrophy and hemodynamic load to changes in cardiac NP production and secretion.

In the present work, we studied NP production and secretion after aortic banding during prevention or regression of hypertrophy with or without attendant hypertension. In addition, we determined whether other markers of cardiac hypertrophy (MHC isoform switch and collagen III mRNA levels) followed a pattern similar to that observed for ANF and BNP.


*    Methods
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 *Methods
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Experimental Protocols
Adult male Sprague-Dawley rats weighing 270 to 280 g were fasted for 12 hours before surgery. Treatment of animals was conducted following institutional guidelines. After anesthesia (induced by halothane–nitrous oxide–oxygen), the abdomen was opened by a cut parallel to the linea alba. The abdominal aorta above the kidney was exposed, and a cannula was placed longitudinally to the aorta and the two tied together. The cannula was pulled out, leaving the aorta constricted to the outer diameter of the cannula. Tetracycline was applied to the open area, and the abdomen was closed by clipping. Sham-operated animals were subjected to the same procedure without aortic banding. Rats then were separated into five groups as follows: (1) control, (2) sham-operated, (3) aortic-banded, (4) aortic-banded with high-dose ramipril (1 mg/kg), and (5) aortic-banded with low-dose ramipril (10 µg/kg). Two protocols were followed: (1) Ramipril was administered by daily oral gavage for 6 weeks to rats immediately after the aortic banding operation (prevention experiment). (2) Ramipril was started 6 weeks after the aortic banding and was continued for 6 weeks (regression experiment). Ramipril dosage was adjusted weekly according to body weight. At the end of the treatment period, the animals were instrumented for measurement of carotid blood pressure. Blood was collected in chilled tubes containing EDTA and immediately centrifuged at 4°C. After centrifugation, the plasma was stored at -80°C until used for RIA. After blood collection, the heart was excised, rapidly weighed, and dissected in cold saline into right and left atrium and right and left ventricle with their respective septa as part of the left chambers. After the tissue weight was measured, the tissue was quickly wrapped in aluminum foil and snap-frozen in liquid nitrogen.

Extraction of Plasma and Tissue Samples
Plasma samples were acidified by adding 100 µL/mL of 1 mol/L HCl and passed through Sep-Pak C18 cartridges (Millipore) that were prewetted with 5 mL of 80% ACN in 0.1% TFA and 10 mL of 0.1% TFA. The cartridges with the absorbed peptides were washed with 20 mL of 0.1% TFA and eluted with 3 mL of 60% ACN in 0.1% TFA. Tissue samples were homogenized in 10 vol of an extracting mixture consisting of 0.1N HCl, 1.0 mol/L acetic acid, and 1% NaCl and centrifuged at 10 000g for 30 minutes at 4°C. The supernatants were then extracted with the use of Sep-Pak C18 cartridges as described above for plasma except that elution was with 80% ACN in 0.1% TFA. The eluates from tissue or plasma were freeze-dried and processed for RIA as previously described.22

Assay Methods
PRA was measured using an RIA kit (Du Pont). Plasma and cardiac tissue concentrations of immunoreactive ANF and BNP were determined by RIA as previously described with anti-rat ANF99-126 and anti-rat BNP64-95 sera, respectively, from Peninsula Laboratories.22 The ANF and BNP antisera showed <0.01% cross-reactivity with BNP and ANF peptides, respectively.

Total RNA Extraction and Northern Blot Analysis
Atrial and ventricular tissue samples from individual rats (n=4 or 5, each chamber, each group) were extracted using Trizol (GIBCO BRL). Total RNA from the atrium (10 µg) and ventricle (20 µg) were electrophoretically separated in an agarose-formaldehyde gel followed by blotting to nylon membranes (Hybond N+, Amersham) overnight. Membranes were prehybridized in 2.5x Denhardt's solution, 5x SSC, 50% formamide, 25 mmol/L KH2PO4, pH 6.4, 0.2% SDS, and 0.2 mg/mL herring sssDNA for 3 hours at 42°C for cDNA probes, or prehybridized in 5x Denhardt's solution, 6x SSC, 50 mmol/L NaH2PO4, 0.5% SDS, and 0.2 mg/mL herring sssDNA for 3 hours at 5°C below the calculated Tm for oligonucleotide probes. Hybridization was then carried out for 16 hours at the same temperature and the same solution as the prehybridization condition except for the presence of the radiolabeled probes. Five cDNA probes and two oligonucleotide probes were used. The cDNA probes used are as follows: (1) a 900-bp EcoRI/HindIII fragment containing the full-length rat ANF cDNA,23 (2) a 595-bp SalI fragment containing full-length rat BNP cDNA,24 (3) a 5-kb EcoRI/SalI fragment of the mouse 28S rRNA cDNA probe, (4) a 2-kb BamHI/BglII fragment of the mouse PGK gene cDNA,25 and (5) rat {alpha}1-III collagen cDNA containing 1300 bp of the 3' noncoding and coding regions.26 The two oligonucleotide probes were 39 and 24 base fragments specific for unique regions in the 3' untranslated regions of the rat {alpha}-MHC and ß-MHC genes.27 28 The {alpha} sequence was 5'-GGGATAGCAACAGCGAGGCTCTTTCTGCTGGACAGGTTA-3' (Tm=60°C), and the ß sequence was 5'-CTCCAGGTCTCAGGGCTTCACAGG-3' (Tm=52°C). The cDNAs were labeled with 5'-[{alpha}32P]dCTP (3000 Ci/mmol; Amersham) using the Megaprime DNA labeling system (Amersham). The oligonucleotides were labeled with [{gamma}-32P]ATP (3000 Ci/mmol, Amersham) using a 5'–end-labeling kit (Amersham). At the end of hybridization, the membranes were washed twice at 42°C with 2x SSC and 1% SDS and twice at 55°C with 1x SSC and 0.1% SDS for the cDNA probes or were washed once at 30°C with 5x SSC and 0.1% SDS and twice at the same temperature as the hybridization with 1x SSC and 0.1% SDS for the oligonucleotide probes. Before additional probing, bound counts were completely stripped from the membranes by washing twice in 10 mmol/L sodium citrate, pH 6.8, 0.25% SDS for 10 minutes at 100°C. Autoradiographs were scanned with an Ultrascan XL laser densitometer (LKB Produckter) and LKB 2400 Gelscan XL software package. The scanning values of ANF, BNP, collagen-III, and {alpha}-MHC and ß-MHC mRNAs were normalized to 28S ribosomal RNA or PGK mRNA as internal controls to correct for differences in the amount of RNA applied and transfer efficiency.

Statistical Analysis
All results are expressed as mean±SEM. A level of P<.05 was considered significant. ANOVA was performed to determine statistical differences among multiple groups. When significance was obtained by ANOVA, Fisher's least squares difference post hoc analysis was used to determine pairwise differences.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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Mean Blood Pressure and Body and Heart Weights
Mean blood pressure at the end of the experimental period in both the prevention and regression experiments of the aortic-banded untreated rats and of low-dose ramipril–treated rats was significantly higher than that of the other groups, but heart rates were similar among all groups in both experiments (Table 1Down). Rats treated with high-dose ramipril had mean blood pressures comparable to that of control animals. LV weight/BW and RV weight/BW ratios of the aortic-banded untreated rats were significantly higher than those of any other group in the regression experiment. In the prevention experiment, only the LV-BW ratio of the aortic-banded rats was significantly higher than that of other groups (Table 1Down). Left atrial weight/BW and right atrial weight/BW ratios were not significantly different among all groups in both experiments (data not shown). Both low-dose and high-dose ramipril–treated rats had LV and RV/BW ratios not significantly different from control animals.


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  		Table 1. Hemodynamic Data and Tissue Weight

Plasma Renin Activity
In both the prevention and regression experiments, high-dose ramipril treatment was accompanied by significantly higher PRA compared with any of the other experimental groups. PRA in the prevention, low-dose ramipril protocol was similar to those of control, sham-operated, and untreated aortic-banded rats. In the regression experiment, however, PRA of the low-dose ramipril–treated rats was significantly lower than that of rats treated with high-dose ramipril but significantly higher than those of control, sham-operated, and aortic-banded rats (Table 2Down).


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  		Table 2. Plasma Renin Activity

irANF and irBNP Plasma Levels
In both the prevention and regression protocols, plasma irANF and irBNP in aortic-banded, untreated rats were significantly higher than those of controls and sham-operated rats (Fig 1Down). By the end of the treatment with high-dose ramipril, NP plasma levels were similar to those of controls and sham-operated animals and significantly lower than those of aortic-banded rats. In low-dose ramipril–treated animals, irANF and irBNP levels were significantly lower than those in aortic-banded rats. The plasma irBNP levels were significantly higher than those of high-dose ramipril rats. Plasma irANF levels in animals treated with low-dose ramipril had a tendency to be higher than those in animals treated with high-dose ramipril.



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  		Figure 1. Plasma irANF (open columns) and irBNP (solid columns) in the prevention and regression experiments in control, sham-operated, aortic-banded rats and in rats treated with high-dose ramipril [Band+R (high)] or with low-dose ramipril [Band+R (low)]. n=7 to 10. **P<.01 vs control and sham-operated; {dagger}P<.05, {dagger}{dagger}P<.01 vs aortic-banded; {ddagger}{ddagger}P<.01 vs aortic-banded with high-dose ramipril (1 mg/kg).

Cardiac Tissue Concentrations of irANF and irBNP
In both the prevention and regression protocols, ventricular NP content in aortic-banded, untreated rats was significantly higher than that of control and sham-operated rats (Fig 2Down). In animals treated with high-dose ramipril, the ventricular NP content was generally similar to that in sham-operated and control rats and significantly lower than that in aortic-banded rats. In animals treated with low-dose ramipril, the findings were similar to those for high-dose ramipril, although the changes were not as pronounced. In general, the decrease in irBNP content with either high- or low-dose ramipril was not as pronounced as those found for irANF content. Atrial irANF and irBNP content was similar in all groups (data not shown).



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  		Figure 2. Cardiac ventricular irANF (open columns) and irBNP (solid columns) content in the prevention and regression experiments in control, sham-operated, aortic-banded rats and in rats treated with high-dose ramipril [Band+R (high)] or with low-dose ramipril [Band+R (low)]. n=4 to 5. *P<.05; **P<.01 vs control and sham-operated; {dagger}P<.05, {dagger}{dagger}P<.01 vs aortic-banded; {ddagger}{ddagger}P<.01 vs aortic-banded with high-dose ramipril (1 mg/kg).

ANF and BNP mRNA Levels
The relative abundance of mRNAs for ANF and BNP in atria and ventricles of all of the different groups in both the prevention and regression experiments closely paralleled the changes described for irANF and irBNP content. (Figs 3Down and 4Down).



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  		Figure 3. Collage of representative Northern blot analysis of LV total RNA in the regression experiment. For each group, a single lane of a single membrane used for successive hybridizations with 32P-labeled probes is shown. Probes hybridized to single bands of the expected size for each mRNA: ANF (0.9 kb), BNP (0.9 kb), collagen III (5.3 kb), {alpha}-MHC (7.2 kb), ß-MHC (7.2 kb), and PGK (1.9 kb).



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  		Figure 4. Relative abundance of ANF (open columns) and BNP (solid columns) transcripts in cardiac ventricles in the prevention and regression experiments in control, sham-operated, aortic-banded rats and in rats treated with high-dose ramipril [Band+R (high)] or with low-dose ramipril [Band+R (low)]. n=3 to 5. *P<.05; **P<.01 vs control and sham-operated; {dagger}{dagger}P<.01 vs aortic-banded; {ddagger}P<.05, {ddagger}{ddagger}P<.01 vs aortic-banded with high-dose ramipril (1 mg/kg).

Collagen III mRNA Levels
In the regression experiment, the relative LV collagen III mRNA content of aortic-banded rats was significantly elevated compared with any other group. This elevation was reverted by both doses of ramipril to the levels found for control and sham-operated animals. In all groups of the prevention experiment as well as in the right ventricle of the regression experiment, collagen III mRNA content remained similar among all groups (Fig 5Down). Atrial levels remained unchanged in all groups (data not shown).



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  		Figure 5. Relative abundance of collagen III transcripts in cardiac ventricles in the prevention and regression experiments in control, sham-operated, aortic-banded rats and in rats treated with high-dose ramipril [Band+R (high)] or with low-dose ramipril [Band+R (low)]. n=3 to 5. *P<.05 vs control and sham-operated; {dagger}{dagger}P<.01 vs aortic-banded.

{alpha}-MHC and ß-MHC mRNA Levels
Northern blot analysis with the rat {alpha}-MHC or ß-MHC oligonucleotide probes identified single 7.2-kb mRNA species in the atria ({alpha}-MHC) and ventricles ({alpha}-MHC and ß-MHC). In both the prevention and regression experiments, atrial and RV {alpha}-MHC remained unchanged in all groups (Fig 6Down; also data not shown). LV {alpha}-MHC mRNA levels of the aortic-banded rats in both experiments had a tendency to be decreased compared with control and sham-operated animals, but the difference did not reach statistical significance. Treatment with low-dose ramipril in the regression experiment induced a significant decrease in LV {alpha}-MHC mRNA levels compared with control, sham-operated, and high-dose ramipril treatment. Ventricular ß-MHC mRNA levels of the aortic-banded rats were significantly higher than those of control and sham-operated rats (Fig 6Down). Treatment with high-dose ramipril returned ß-MHC mRNA levels to levels comparable to those in control and sham-operated animals in the prevention and regression experiments. However, treatment with low-dose ramipril did not reverse the increase in ventricular ß-MHC mRNA levels found after aortic banding.



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  		Figure 6. Relative abundance of {alpha}-MHC (open columns) and ß-MHC (solid columns) transcripts in cardiac ventricles in the prevention and regression experiments in control, sham-operated, aortic-banded rats and in rats treated with high-dose ramipril [Band+R (high)] or with low-dose ramipril [Band+R (low)]. n=3 to 5. *P<.05, **P<.01 vs control and sham-operated; {dagger}P<.05; {dagger}{dagger}P<.01 vs aortic-banded; {ddagger}P<.05, {ddagger}{ddagger}P<.01 vs aortic-banded with high-dose ramipril (1 mg/kg); §no statistical calculation because of the small number of samples.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Increased ventricular expression of the genes encoding for the NPs ANF and BNP is a hallmark change of cardiac hypertrophy. Successful management of hypertension associated with ventricular hypertrophy by use of ACE inhibitors results in a decrease in blood pressure, regression of the ventricular hypertrophy, and reduction of ventricular ANF mRNA and peptide content as well as plasma ANF levels in different experimental models associated with increased cardiac hemodynamic load such as the spontaneously hypertensive rat,9 11 12 13 after myocardial infarction,15 and rats with heart failure.16 Whereas the temporal association of increased ventricular NP gene expression and hypertrophy is well established, the details of this association remain unknown; nor is it clear whether the normalization of ventricular NP gene expression observed after ACE inhibition is related to the normalization of the hemodynamic load or to regression of the hypertrophy per se. In the present work, we aimed at elucidating the relationship between cardiac NP production and cardiac hypertrophy and hemodynamic load by taking advantage of the fact that a low dosage schedule (10 µg/kg per day) treatment with the ACE inhibitor ramipril prevents or regresses LV hypertrophy in aortic-banded rats without decreasing blood pressure.20 21 29 Further, we compared the results obtained with those from animals treated with a high-dosage schedule of ramipril (1 mg/kg per day) that allows for the prevention or regression of both high blood pressure and ventricular hypertrophy.

In the present studies, we confirm that in both the prevention and regression experiments, high-dose ramipril decreased anatomic hypertrophy (LV weight/BW ratio) and blood pressure, whereas low-dose ramipril decreased hypertrophy without affecting blood pressure.20 21 29 Our results are also in agreement with clinical findings by Lièvre et al,30 who reported that in human essential hypertension with LV hypertrophy, a nonantihypertensive dose of ramipril (1.25 mg/d) induced regression of LV hypertrophy independent of changes in blood pressure.

The comparison of LV NP production between aortic-banded animals and those treated with either low-dose or high-dose ramipril provides a useful insight into the possible influence of pressure overload and cardiac hypertrophy on NP gene expression. It is apparent that regression of the hypertrophy and normalization of blood pressure in both the regression and prevention protocols reduces LV NP production to levels comparable to normal or sham-operated controls. This might occur independent of the hemodynamic load, as suggested by the study of Rockman et al,31 who demonstrated in aortic-banded mice treated with losartan that the prevention of ANF mRNA elevation and LV hypertrophy occurred without a change in the transstenotic pressure gradient. However, low-dose ramipril treatment unveils what appears to be a discrete contribution by the hypertensive stimulus to the activation of NP genes in ventricular muscle. That is, reversal or prevention of hypertrophy results in decreased expression of NP in the left ventricle. However, in neither case is the increased NP expression completely reverted, which suggests that the persisting hypertension constitutes a continuing hemodynamic stimulus of enough magnitude to maintain significantly higher levels of expression of ventricular ANF and BNP independent from hypertrophy.

The neuroendocrine events leading to cardiac hypertrophy and chronically enhanced NP gene expression are not completely understood. In rats treated with DOCA-salt for 5 weeks, we have recently demonstrated7 that anatomic and biochemical indices of cardiac hypertrophy may occur simultaneously with activation of atrial and ventricular gene expression but that causality is not necessarily involved. For example, activation of NP production may occur without MHC switch or anatomic hypertrophy of the atria, nor are ANF and BNP gene expression coordinately regulated. Nevertheless, increased expression of NP genes is a hallmark change of LV hypertrophy in vivo.3 7 8 32 In vitro, the expression of ANF and ß-MHC and protein synthesis are increased after stretch of rat neonatal ventricular cardiocytes in culture.33 It appears that Ang II is released from ventricular cardiocytes, thus inducing ANF gene expression, which is completely blocked by blocking the type 1 Ang II receptor.34 These results suggest that stretch-induced ANF gene expression is mediated by the cardiac RAS. It is important to point out that atrial cardiocytes do not appear to have the ability to produce Ang II in a regulated manner,34 thus suggesting that the neuroendocrine control of the endocrine heart is not entirely under the control of the cardiac RAS. Indeed, the present investigations show that ramipril at either high or low doses does not affect atrial NP production as measured by cardiac content or steady-state mRNA levels.

The differential effects of ramipril at high or low doses in preventing or regressing hypertension and hypertrophy or hypertrophy alone seem to be interpreted best in light of the effect of this ACE inhibitor on the circulating and local RAS at high doses or, at low doses, on the local RAS alone, which works in a paracrine or autocrine manner independent from the circulating RAS.35 36 37 38 In support of this view, it has been previously established in suprarenal aortic-banded rats and in the spontaneously hypertensive rat that high-dose ramipril (1 mg/kg) decreases plasma ACE activity but low-dose ramipril (10 µg/kg) does not.20 21 39 40 Ramipril at 10 mg/kg, as well as the Ang II type-1 receptor antagonist losartan, can decrease blood pressure and cardiac ACE activity together with LV hypertrophy.41 In a previous investigation with the same model and in the spontaneously hypertensive rat, it has been shown that tissue ACE activity was suppressed by both doses of ramipril used here.21 40 42 Other than ramipril, lisonopril, captopril, and CS622 are reported to decrease cardiac hypertrophy associated with decreases of plasma or cardiac ANF.12 15 16 Because ramipril has a relatively high lipophilicity, ramipril at the relatively low dose of 10 µg/kg effectively inhibits Ang II production.43

The possible relationship between the cardiac RAS and NP production obtained in the present studies should be made taking into consideration that the antihypertrophic effect of ramipril is abolished by a specific B2-bradykinin receptor antagonist.39 Therefore, ramipril not only decreases Ang II formation but also increases bradykinin, which may induce antihypertrophic effects through pathways involving nitric oxide and prostacyclins. However, it is not known whether the B2-bradykinin receptor antagonist also counteracts the effects of ramipril on NP production and secretion; therefore, the contribution of the potentiation of bradykinin in our study is unknown. Nevertheless, the possibility that the antihypertrophic effect of ramipril may be mediated directly at least in part by inhibition of Ang II formation may not be ruled out completely, given that losartan and ramipril reduce LV hypertrophy and ANF mRNA levels to the same extent.41 However, it has been shown in spontaneously hypertensive stroke-prone rats and in suprarenal aortic-banded rats that bradykinin receptor blockade does not affect the antihypertensive or the antihypertrophic effect of ramipril.44 45

In view of the fact that inhibition of the cardiac RAS by low-dose ramipril does not completely inhibit NP production by the left ventricle, alternative mediators of the hemodynamic effects of elevated blood pressure must be sought. An important mediator candidate might be ET-1, which can induce the expression and release of ANF and BNP in cultured ventricular cardiocytes.46 47 48 Blockade of ET-1 receptor prevents ventricular hypertrophy and increases ANF gene expression in aortic-banded rats.49 ET-1 is released from endothelial and mesothelial cells, which lie immediately adjacent to cardiocytes,50 51 so there is a possibility that hemodynamic change can directly affect ET-1 release from endothelial cells in the ventricle and that ET-1 so released may stimulate NP production and secretion and the expression of {alpha}- and ß-MHC.52 ET-1 is also synthesized in and secreted from cardiocytes,53 and its expression in cardiocytes is increased by pressure overload.54 However, this increase may be due to actions of Ang II.53

In pressure overload models such as aortic banding in which compensatory LV hypertrophy may eventually be insufficient to compensate for the elevation of afterload, the LV end-diastolic pressure increases, causing remodeling of the pulmonary capillary bed, resulting in an elevation of pulmonary and RV pressures. Probably due to this mechanism, RV hypertrophy was seen in the regression but not in the prevention experiment, reflecting the fact that the pressure overload lasted twice as long in the regression experiment than in the prevention experiment. With both protocols, RV NP content in banded rats was elevated compared with that of control and sham-operated rats. Treatment with either high- or low-dose ramipril returned NP content back to control values. The increase in NP in the right ventricle without evidence of anatomic hypertrophy is reminiscent of findings previously reported with DOCA-salt rats.7 The significant increase in RV NP content but not in RV NP mRNA levels may be due to increased translation, compared with transcription.

Changes in ANF tissue content and plasma levels were mirrored by BNP in the present experiments in all groups except in the low-dose ramipril groups, in which the normalization of ANF content was more pronounced than the normalization of BNP content. This indicates that a load-dependent component may be involved in regulating both ANF and BNP production and secretion, whereas a load-independent component related to hypertrophy predominantly affects ANF. Supporting this view, the ACE inhibitor alacepril was shown to decrease plasma ANF only 1 hour after administration of the drug, whereas it took more than 6 hours for plasma BNP to decrease.18 Altogether, these results suggest that ANF and BNP production and secretion are partially independently regulated.

The findings of the present investigation show that atrial NP content and specific steady-state mRNA levels remained similar in all groups. These results may reflect a lesser hemodynamic stimulus than that reported in rats made hypertensive by treatment with DOCA-salt for 5 weeks7 or in the two kidney–one clip rat model of renovascular hypertension.14 In the DOCA-salt–treated animals, we observed left atrial hypertrophy with elevation of ANF mRNA content. The peptide content, however, was decreased.7 This appears to reflect a common reactive pattern for the atria because it also occurred in a volume overload model in which elevated atrial pressure stimulated both atrial NP production and secretion, leading to depletion of peptide stores.6 In our experiments, increased secretion from the atria is still possible either in response to atrial stretch or neurohormonal factors, and because NP content in the atrium is much greater than that in the ventricles, even a slight increase in NP secreted from the atria would significantly affect NP plasma levels.

Cardiac collagen, a major component of the cardiac extracellular matrix, is mainly composed of type I and III collagens.55 Both increase similarly in hypertension, compromising cardiac function by increasing diastolic stiffness or by causing arrhythmia.56 57 In the regression experiment, LV collagen III mRNA levels in banded rats were significantly elevated, and they returned to control levels in both high- and low-dose ramipril–treated animals. This result parallels the anatomic LV hypertrophy observed, suggesting that the increase of cardiac extracellular matrix is related to anatomic hypertrophy and that ramipril can revert the increased collagen deposition. However, in the left ventricle of the prevention experiment and in the right ventricle of the regression experiment, collagen III mRNA levels in banded rats were not elevated, even though these tissues were hypertrophied by anatomic criteria.

In the rat, LV hypertrophy caused by pressure overload is accompanied by MHC isoform switch from {alpha} to ß,8 although MHC isoform switch does not always accompany ventricular hypertrophy.7 In the present experiments, low-dose ramipril did not affect the change in MHC expression, indicating that increased hemodynamic load is likely to be the major determinant mediating the observed changes in MHC isoform gene expression. The pressure stress against the LV wall may directly induce MHC isoform switch without inducing hypertrophy. This concept is supported by the fact that in aortic-banded rats, LV MHC switch at the mRNA level occurs only 1 day after banding, but hypertrophy occurred after 35 days.58 However, this result is different from our result in DOCA-salt rats in which LV hypertrophy and NP mRNA elevation occurred without MHC isoform switch after 1 week of treatment.7 This is probably because in volume-overload hypertension as seen in the early phase of DOCA-salt treatment, the stress against the LV wall may be too weak to induce MHC isoform switch, whereas in pressure overload, this stress may be stronger, inducing MHC isoform switch. The results above suggest that MHC isoform switch is regulated independent from hypertrophy and NP production and seems to be influenced predominantly by the ventricular load.

Summary
We have shown that (1) the normalization of NP production or secretion in aortic-banded rats after ACE inhibition with ramipril is related to blood pressure decrease and the regression of hypertrophy independently, (2) a load-dependent component is more important than a load-independent component in regulating LV NP production, (3) ANF production is more sensitive than BNP production to the load-independent component, (4) low-dose ramipril reverses anatomic hypertrophy and increased collagen III expression but does not reverse the increased ß-MHC isoform expression, suggesting that these are independently regulated processes, and (5) aortic banding and ACE inhibition do not affect atrial NP production and content as measured by tissue NP content and specific mRNA steady-state levels.


*    Selected Abbreviations and Acronyms
 
ACN = acetonitrile
ANF = atrial natriuretic factor
Ang = angiotensin
BNP = brain natriuretic peptide
BW = body weight
DOCA = deoxycorticosterone acetate
ET = endothelin
ir = immunoreactive
LV = left ventricular
MHC = myosin heavy chain
NP = natriuretic peptide
PGK = phosphoglycerate kinase
PRA = plasma renin activity
RAS = renin-angiotensin system
RIA = radioimmunoassay
RV = right ventricular
TFA = trifluoroacetic acid


*    Acknowledgments
 
This work was supported by the Heart and Stroke Foundation of Ontario. We thank Dr E. Vuorio (University of Turku, Finland) for providing rat pro-{alpha}1(III) collagen plasmid.

Received August 24, 1995; revision received October 23, 1995; accepted November 3, 1995.


*    References
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