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Cardiovascular Research 2001 51(1):131-139; doi:10.1016/S0008-6363(01)00267-X
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Combined angiotensin converting enzyme inhibition and angiotensin AT1 receptor blockade up-regulates myocardial AT2 receptors in remodeled myocardium post-infarction

Sunghou Leea, Christopher M. Kramerb, Sunil Mankadc, Sung-eun Yood and Kathryn Sandberga,*

aDepartments of Medicine and Physiology and Biophysics, Georgetown University Medical Center, 394 Building D, 4000 Reservoir Road, NW, Washington, DC 20007, USA
bDepartments of Medicine and Radiology, University of Virginia Health System, Richmond, VA, USA
cDepartment of Medicine, Allegheny General Hospital, Allegheny, NY, USA
dKorea Research Institute of Chemical Technology, Taejon, South Korea

* Corresponding author. Tel.: +1-202-687-4179; fax: +1-202-687-7278 sandberg{at}gusun.georgetown.edu

Received 25 August 2000; accepted 21 February 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: In an ovine model of left ventricular (LV) remodeling after transmural anteroapical myocardial infarction (MI), we have previously demonstrated that the combination of angiotensin converting enzyme (ACE) inhibition and AT1 receptor blockade is more effective at limiting LV remodeling than either therapy alone. We hypothesized that the beneficial effect of combined therapy is due in part to upregulation of AT2 receptor levels. Methods: Two days after transmural anteroapical MI by coronary ligation, 16 sheep were randomized to losartan (50 mg/day), ramipril (10 mg/day), ramipril+losartan (combined therapy), or no therapy. At 8 weeks after MI, radioligand receptor assay were deployed with homogenates from regional LV tissues. Results: We found that AT receptors in normal sheep myocardium are predominantly of the AT2 receptor subtype. Binding studies of remodeled myocardium 8 weeks later showed that the apparent maximum binding (Bmax) was increased from 23 to 48 fmol/mg protein only in animals with combined therapy. The AT2/AT1 proportion was increased significantly in animals with combined therapy compared to infarcted controls (18.0 vs. 5.17). Conclusions: These results indicate that AT2 receptor expression increased significantly during LV remodeling with combined therapy but not with either therapy alone. In combination with prior work demonstrating the effectiveness of combined therapy in limiting LV remodeling, this study is consistent with the hypothesis that AT2 receptors play a cardioprotective role in LV remodeling after MI.

KEYWORDS ACE inhibitors; Angiotensin; Antihypertensive/diuretic agents; Infarction; Remodeling; Receptors; Renin angiotensin system


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The octapeptide hormone angiotensin II (Ang II) plays a major role in the pathogenesis of hypertension [1,2] and in the structural alterations of the heart and kidney such as left ventricular (LV) hypertrophy, remodeling after myocardial infarction (MI) and nephrosclerosis [3–5]. Two main AT receptor subtypes (AT1 and AT2) have been characterized, which can be distinguished on the basis of their different affinities for synthetic nonpeptide ligands.

The expression of these AT receptor subtypes is tissue-specific; some tissues express only one subtype while others express both [6]. However, the AT1 receptor is the predominant receptor subtype found in the mammalian adult. Furthermore, most of the classic Ang II-mediated vasoconstrictive actions are mediated by AT1 receptors, whereas less information is available regarding the physiological role of the AT2 receptor and its signal-transduction pathway [7,8]. In addition to tissue specificity, there is species variability as well. While AT receptor binding sites have been identified in the heart of many species [9], the ratio of the two receptor populations (AT1/AT2) varies enormously between species. In the rat ventricle membrane preparation, the AT1 receptor is the major subtype expressed [10]. In contrast, the expression pattern of AT receptors in the human heart is quite different from that in the rat, and human adult hearts express substantial amounts of the AT2 receptor [7]. The densities of AT1 and AT2 expression in normal human atria were 5–10 fmol/mg protein and in the LV were 3.8–17.3 fmol/mg protein, representing AT2 as the dominant receptor subtype, amounting to about 70% [11,12].

Accumulating evidence suggests that AT2 receptors play a role in tissue injury in the cardiovascular system [13]. In the hypertrophied rat heart, the ratio of AT2 to AT1 receptor densities is increased compared to noninfarcted normal controls [14]. Furthermore, the density of myocardial AT2 receptors is increased in infarcted regions 1 day after MI. AT2 receptor expression is further increased 7 days after infarction in both the infarcted and noninfarcted regions [15]. In humans, AT2 receptor expression is increased in the right atrial biopsies from patients with coronary artery disease [16] and in the right and left ventricles from patients with end-stage heart failure [16–18]. In failing human heart, the relative ratio of AT2 receptor expression to AT1 receptors is also reported to be higher than in ventricular myocardium from normal heart [18,19]. These results suggest that the AT2 receptor plays a role in cardiovascular remodeling and the pathogenesis of myocardial hypertrophy and fibrosis.

We have previously used an ovine model of reproducible transmural anteroapical MI [20] to study post-MI LV remodeling and have demonstrated regional differences in function [21], sympathetic innervation [22], and cellular hypertrophy [23] within noninfarcted myocardium. We have shown that the combination of ACE inhibition and AT1 receptor blockade is more effective at limiting LV remodeling than either therapy alone [24], although the mechanisms of the combined effect remain poorly understood. We hypothesized that alterations in AT1 and AT2 receptor numbers may relate to the beneficial effects of combined therapy. In this paper, we describe a method for measuring AT receptor expression in sheep heart as well as characterize the AT receptor subtype population in the heart after MI in tissue at the site of infarct and in areas adjacent and remote to the site of infarct using a LV membrane fraction. We also examine the regulation of AT receptor expression in this model during ACE inhibition and AT1 receptor blockade alone and in combination.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
125I-[Sar1, Ile8]Ang II (2176 Ci/mmol) was purchased from Peptide Radioiodination Center (Pullman, WA). Ang II (human) and [Sar1, Val5, Ala8]Ang II (Saralasin) were purchased from Sigma Chemical (St. Louis, MO) and PD123319 was purchased from Research Biochemical International (Natick, MA). The non-peptide antagonists were kindly provided as follows: SK1080 (S. Yoo, Korea Research Institute of Chemical Technology); L163017 (W.J. Greenlee, Merck Sharp & Dohme, Rahway, NJ); and losartan (P.C. Wong, Du Pont Merck, Wilmington, DE).

2.2 Animals
In 16 female Q fever-negative Dorsett sheep, a left thoracotomy was performed with ligation of the left anterior descending coronary artery and its second diagonal branch to create a moderate-sized transmural anteroapical infarction as previously described [20,21,23]. The infarct size was 26±3% of the endocardial surface area [20,21]. In addition, three normal sheep (noninfarcted controls) were included for the AT receptor characterization. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). On day 2 post-MI, animals were randomized to no therapy (control, n=6), ramipril 10 mg p.o. qd (RT, n=3), losartan 25 mg p.o. bid (LT, n=4), and combined therapy with ramipril 10 mg p.o. qd and losartan 25 mg p.o. bid (CT, n=3). At 8 weeks after MI, the heart was excised and the sharply defined infarct borders were identified. Tissue within 2 cm of the transmural infarct was termed adjacent and tissue beyond 2 cm was termed remote, as previously defined [22,25]. Then, 5–20 g pieces of infarcted, adjacent, and remote myocardial LV tissue were immediately resected and flash frozen in liquid nitrogen for shipment to Georgetown University for AT receptor analysis.

2.3 Tissue preparation
All tissue preparations were performed at 4°C. Sheep heart LV tissues from various myocardial regions including infarct, adjacent and remote tissue sections were thawed on ice and diluted in 10 volumes (w/v) of ice-cold buffer A [10 mM Tris buffer (pH 7.2) containing 0.32 M sucrose, 2 mM EDTA, and 3 mM MgCl2]. After mincing and rinsing the tissue with buffer A, the tissue was homogenized with a polytron homogenizer for 30 s on setting 7 (PT-10, Brinkmann Instruments, Inc.). The homogenate was centrifuged at 500xg for 10 min and the supernatant was decanted through a standard testing sieve #80 (VWR scientific, West Chester, PA). The decanted supernatant was centrifuged at 10 000xg for 10 min. The final supernatant was centrifuged at 102 000xg for 60 min. The resultant pellet was washed with buffer B [10 mM Tris (pH 7.2) containing 3 mM MgCl2 and 1 mM EGTA] by adding 30 ml to the pellet and recentrifuging for 60 min at 102 000xg. After re-suspending the final pellet in buffer C [10 mM Tris (pH 7.2) containing 3 mM MgCl2], the protein content was determined by the Lowry method [26] using the Bio-Rad DC protein assay. The membrane protein was adjusted to 1.0–1.5 mg/ml in buffer C and supplemented with a final concentration of 0.2% bovine serum albumin (BSA). Membranes were stored in aliquots at –80°C. In initial experiments, we determined that AT receptor binding levels did not change under our experimental conditions when the membrane fractions were stored at –80°C compared to freshly prepared membranes (data not shown).

2.4 Radioligand binding assays
For the saturation binding analysis, membranes (100–200 µg) were incubated with increasing concentrations of 125I-[Sar1, Ile8]Ang II (0.05–4 nM) in buffer C supplemented with 0.2% BSA for 3 h at 37°C in a 250 µl assay volume. Specific binding of 125I-[Sar1, Ile8]Ang II was defined as the total binding minus the nonspecific. Nonspecific binding was determined experimentally in the presence of 1 µM unlabeled saralasin.

In radioligand competition experiments, membranes (100–200 µg) were incubated with increasing concentrations of drugs in buffer C supplemented with 0.2% BSA and 0.3 nM of 125I-[Sar1, Ile8]Ang II. All test compounds were prepared at 2.5 mM in dimethyl sulfoxide (DMSO) and serially diluted to various concentrations (10–5–10–13 M). Under these conditions, the added amount of DMSO was less than 5% and showed no significant interference with the binding reactions. The proportions of AT1 and AT2 in each tissue preparation were measured with SK1080 [27] and PD123319, respectively, by calculating the specific binding at the maximum dose response derived from non-linear curve fitting.

The membrane preparations made from normal sheep LV were preincubated with 10 µM PD123319, which is a concentration sufficient to saturate all the AT2 receptors without affecting the AT1 subtype. Conversely, competitive binding studies were performed to confirm and identify the presence of the AT1 receptor subtype by performing competition binding studies in the presence of 10 µM SK1080. All binding reactions were terminated by the addition of 4 ml ice cold buffer B and rapid filtration through glass fiber filters (GF/C Whatman, presoaked with assay buffer) with a Brandel cell harvester system (Brandel M-24R). The filters were washed with an additional 12 ml of ice-cold buffer B and the membrane bound radioactivity trapped on the filters was measured in a Packard Cobra II {gamma}-counter. Every binding experiment was performed twice in duplicate for each animal sample.

2.5 Data analysis
Data from binding experiments were analyzed by nonlinear regression analysis, by using PRISM computer software (GraphPad Software Inc, San Diego, CA). The dissociation constant (Kd) and the maximum number of specific binding sites (Bmax) were calculated. The ability of antagonists to inhibit specific 125I-[Sar1, Ile8]Ang II binding was estimated by IC50 values, which are the molar concentrations of unlabeled drugs necessary to reduce specific binding by 50%. The Ki value was calculated from the equation Ki=IC50/(1+L/Kd), where L equals the concentration of 125I-[Sar1, Ile8]Ang II [28]. Results were expressed as the mean±standard error of the mean (S.E.M.). The sample size estimation was determined by power analysis based on preliminary studies [29]. Statistical significance was evaluated with the two-way ANOVA tests according to tissue region and treatment followed by one-way ANOVA with Student–Newman–Keuls multiple comparison methods. Our data showed normal distribution and used parametric analysis. P<0.05 was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Ang II receptor characterization in normal sheep heart
To investigate AT receptor expression in the sheep myocardium, we prepared membranes from LV tissues by differential centrifugation as described in Section 2. Specific binding of 125I-[Sar1,Ile8]Ang II increased in a linear manner between 10 and 250 µg membrane protein. The signal to noise was approximately 1 at 50 µg and 3 at 100 µg, using 300 000 cpm of 125I-[Sar1,Ile8]Ang II in a 250 µl assay volume. The time course of 125I-[Sar1,Ile8]Ang II binding reached steady state by 3 h at 37°C. Hence, all further experiments were performed with 0.1–0.2 mg membrane protein and for a 3 h incubation period at 37°C.

A representative saturation binding curve is shown in Fig. 1, along with Scatchard transformation of the data (Fig. 1, inset). The Scatchard revealed a linear distribution, which suggests the ligand interacts with a single population of sites with a dissociation constant (Kd) of 0.35±0.05 (mean±S.E.M.) and an apparent maximum binding (Bmax) of 23.3±5.6 fmol/mg protein, under these experimental conditions.


Figure 1
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  		Fig. 1 Saturation binding curves in ovine LV. Left ventricular membranes were incubated with increasing concentrations of 125I-[Sar1, Ile8]Ang II (0.05–4 nM). Inset, Scatchard transformation of the binding data. Each point represents the mean of specific binding from two separate experiments carried out in duplicate.

 
In competition binding studies, the balanced affinity antagonist, L163017, caused a concentration-dependent decrease of specifically bound 125I-[Sar1, Ile8]Ang II in noninfarcted normal sheep LV homogenates. About 100% of the bound 125I-[Sar1, Ile8]Ang II was inhibited at a maximum concentration of L163017 (Ki=24.6±3.4 nM). However, the AT1 antagonists, SK1080 and losartan, showed only 23% inhibition, while the AT2 antagonist, PD123319, exhibited approximately 84% inhibition at maximum concentration. These results demonstrate the existence of both of AT1 and AT2 receptors in ovine LV tissue and also that the AT2 receptor is the predominant receptor subtype. Subsequently, we preincubated (see Section 2) membrane preparations with 10 µM PD123319 to assess the small AT1 receptor population (23%). The non-peptide AT1 antagonist, SK1080, revealed a typical dose–response curve (Ki=28.7 nM) with a Hill coefficient of –0.873.

Fig. 2 shows a representative set of radioligand competition curves for 125I-[Sar1, Ile8]Ang II with increasing concentrations of competitors including: AT1 antagonists, SK1080 and Losartan; the AT2 antagonist PD123319; and, the AT1/AT2 balanced affinity ligand, L163017. Interestingly, SK1080 and losartan were poor competitors of 125I-[Sar1, Ile8]Ang II binding, while PD123319 was able to inhibit binding by more than 80% at maximum dose (10 µM).


Figure 2
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  		Fig. 2 Radioligand competition curves in ovine LV. Left ventricular membranes were incubated with 125I-[Sar1, Ile8]Ang II and the following competitors: the AT1 selective antagonists, SK1,080 ({blacktriangleup}) and losartan ({blacktriangledown}); the AT2 selective antagonist, PD123319 (bullet); and the AT1/AT2 balanced affinity antagonist, L163,017 ({blacksquare}).

 
3.2 Comparisons between noninfarcted normal animals and infarcted control animals
Table 1 summarizes the total AT receptor numbers (Bmax) and dissociation constants (Kd) in noninfarcted normal and infarcted control groups. The Kd and Bmax values were not changed (Kd: 0.12–0.35 nM; Bmax: 23.3–29.2 fmol/mg protein) and no significant difference in the AT1 and AT2 receptor proportion was observed between the two groups (noninfarcted normal: AT1, 23.1±3.2% and AT2, 83.8±0.82%; infarcted control-infarct: AT1, 19.2±1.6% and AT2, 84.5±2.4%; infarcted control-adjacent: AT1, 20.2±1.9% and AT2, 84.7±2.4%; infarcted control-remote: AT1, 15.6±1.7% and AT2, 91.6±5.6%). The AT receptor population was not significantly different between regions within the infarcted control group. However, as shown in Table 2, the affinity constant (Ki) for the balanced affinity antagonist L163017 was significantly increased (P<0.001) in infant tissues (80.4±0.65 nM) from infarcted control animals compared to myocardial tissue from the noninfarcted normal group (24.6±3.4 nM) and compared to other regions in the infarcted control groups (adjacent: 25.6±2.2 nM; remote: 23.9±2.8 nM).


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  		Table 1 Effect of ramipril and losartan on AT receptor binding parameters in ovine LV after myocardial infarct

 

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  		Table 2 Effect of ramipril and losartan on AT receptor affinities and subtype distribution in ovine LV after myocardial infarct

 
3.3 Comparisons between infarcted control and treated infarcted animals
When the ramipril (RT), losartan (LT), and combined therapy (CT) treatment groups of infarcted animals were separately compared to the infarcted control group, the receptor density (Bmax) was significantly increased (P<0.05) only in the tissues from the CT group but not in the RT and LT groups (Fig. 3A). Furthermore, the Kd was significantly increased (P<0.001) in the CT group but not in the RT and LT groups (Fig. 3B). Adjacent (P<0.05) and remote (P<0.05) tissues from the CT group also showed significant increases in the Kd value. The proximity to the infarct site did not significantly affect either Kd or Bmax within the treatment groups (Table 1).


Figure 3
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  		Fig. 3 Effect of ramipril and losartan on AT receptor binding parameters in ovine LV after MI. (A) AT receptor densities (Bmax) and (B) affinity constants (Kd) in LV membranes from infarcted control sheep or infarcted animals treated with ramipril (RT), losartan (LT) or the combination (CT). Data from infarct tissues from each group were shown. *P<0.001 and #P<0.05.

 
Competition of the labeled antagonist 125I-[Sar1, Ile8]Ang II with the balanced affinity antagonist, L163017, in tissues from the different treatment groups is shown in Table 2. The affinity constant (Ki) for L163017, was significantly increased in infarct tissues from RT (P<0.005), LT (P<0.01), and CT (P<0.001) groups compared to the tissues in the infarcted control group. Only the infarct tissues from the control, RT, and CT groups showed a significant increase in the Ki values compared to the Ki values in various regions of the LV group. In the LT group, no significant differences in affinity constants were observed between regions and the Ki values were similar to the Ki from normal LV tissues.

Fig. 4 compares the receptor subtype proportions in tissues from noninfarcted normal and in infarcted tissues from control, RT, LT, and CT groups. The AT1 proportion was significantly decreased in the infarcted CT group (infarct: AT1, 8.75±2.7%; adjacent: AT1, 3.52±1.7%; remote: AT1, 2.33±1.5%) compared to the infarcted control group (infarct: AT1, 19.2±1.6%; adjacent: AT1, 20.2±1.9%; remote: AT1, 15.6±1.7%), and the AT2 proportion was significantly increased in the infarcted CT group (infarct: AT2, 92.1±0.60%; adjacent: AT2, 93.8±1.7%; remote: AT2, 101±2.6%) compared to the infarcted control group (infarct: AT2, 84.5±2.4%; adjacent: AT2, 84.7±2.4%; remote: AT2, 91.6±5.6%). The difference in the receptor subtype proportions in the infarcted RT group (infarct: AT1, 27.7±6.9% and AT2, 76.7±3.7%; adjacent: AT1, 23.3±5.5% and AT2, 78.0±3.8%; remote: AT1, 22.6±4.9% and AT2, 80.6±1.2%) and in the infarcted LT group (infarct: AT1, 17.2±4.3% and AT2, 84.6±2.8%; adjacent: AT1, 21.8±2.9% and AT2, 85.1±4.1%; remote: AT1, 20.1±10% and AT2, 72.7±3.5%) was not significant. The ratio of AT2/AT1 increased significantly in all tissues from the infarcted CT group (infarct: 12.5±2.2; adjacent: 25.8±13; remote: 15.6±3.6) compared to the tissues from the infarcted control, RT and LT groups (Table 2).


Figure 4
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  		Fig. 4 Effect of ramipril and losartan on AT receptor subtype distribution in ovine LV membranes after MI in infarcted control and infarcted animals treated with ramipril (RT), losartan (LT), and ramipril+losartan (CT). *P<0.001 (AT1, AT2 and AT2/AT1 ratio). Data from normal LV and infarct tissues from each group were shown.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
There is increasing evidence for the role of the RAS and AT receptors in post-infarction remodeling, in cardiac hypertrophy and in heart failure [7,8]. Several clinical trials have established that ACE inhibitors blunt post-infarction LV remodeling and limit mortality after anterior MI and MI with clinical heart failure [30–32]. The role of AT1 receptor blockade remains to be elucidated in this clinical setting [33]. We have demonstrated the efficacy of combined therapy with ACE inhibitors and AT1 receptor blockade over either therapy alone [24] in a well-characterized ovine model of LV remodeling after anteroapical MI. In the present study, we investigated the regulation of AT receptor expression after ACE inhibition, AT1 receptor blockade and combined therapy in this model.

Competition binding studies with AT1 and AT2 selective antagonists and with AT1/AT2 balanced affinity ligands revealed that the ovine myocardial AT receptor population consists of 23.1±3.2% AT1 and 83.8±0.82% AT2 (Fig. 2). These results indicate that the AT2 receptor is the predominant subtype in ovine LV, as it is in human LV [18,34,35]. The AT receptor population in sheep ventricle membrane preparation is thus distinctly different from the receptor population in ventricle membrane preparations of rat, rabbit and monkey, in which 90, 64 and 71% of the AT receptor population is AT1, respectively [10]. Accordingly, studies of AT receptor regulation in the ovine model are likely to be more clinically relevant than rodent, rabbit or monkey models.

In rats, cardiac remodeling following experimental MI induces changes in AT receptor expression. Using competitive PCR and radioligand binding assays, Nio et al. [15] demonstrated that MI caused a 2–4 fold increase in AT1a and AT2 receptor subtypes, with no changes in AT1b receptor expression in both infarcted and noninfarcted areas of the LV. However, we found that MI did not induce significant changes in AT receptor expression in sheep LV tissues compared to noninfarcted normal animals. These differences between rodent and ovine species might reflect the molecular differences in regulation of different myocardial AT receptor populations between these species.

Sheep treated with the combination of AT1 receptor blockers and ACE inhibitors exhibited significant differences in AT receptor number. In particular, co-treatment with AT1 receptor blockers and ACE inhibitors increased the Bmax from 26.8±4.6 to 60.6±7.4 fmol/mg protein in infarct LV tissue compared to the infarcted LV control group (Fig. 3). The increased AT receptor numbers from combined therapy were due to increased AT2 receptor expression (Fig. 4; Table 2).

Recently, combined therapy of candesartan and enalapril was found to provide more beneficial effects for preventing LV remodeling in patients with congestive heart failure in the Randomized Evaluation of Strategies for Left Ventricular Dysfunction (RESOLVD) Pilot Study compared to either treatment alone [36]. In this study of 768 patients with congestive heart failure, an ejection fraction (EF) less than 40% and poor exercise tolerance, EF increased more with the combination therapy (2.5%) than with either treatment alone (1.5% for both single treatments). Furthermore, EF increased more and the end-diastolic and end-systolic volumes increased less with combination therapy than with either treatment alone. In another study, the addition of valsartan to standard therapies including ACE inhibitors in patients with congestive heart failure did not reduce mortality but did reduce hospitalizations and improved quality of life and was associated with an increased EF [37]. In normotensive male volunteers with mild sodium depletion, the combined administration of an ACE inhibitor and an AT1 antagonist had a major additive effect on preventing the plasma renin to rise as well as reducing mean blood pressure [38].

Suppression of bradykinin breakdown by ACE inhibition is likely involved in the protective effects of combined therapy since this nonapeptide (produced by the kallikrein cascade) has been implicated in playing a direct role in myocardial remodeling and functional recovery from myocardial ischemia [39]. ACE inhibition prevents the rapid degradation of bradykinin and thereby potentiates the beneficial effects of this peptide in myocardial ischemia. It is also possible that a chymase-like system [40] is present in the ovine heart that generates Ang II that is not sensitive to ACE inhibition. In a porcine infarct model, ACE inhibition reduced plasma Ang II concentrations yet local Ang II concentrations actually increased [41]. This study also emphasizes the differences between regulatory control mechanisms of the systemic and tissue-specific renin angiotensin systems. In the human heart, ACE contributes only a small amount to total Ang I conversion [42,43]. In this regard, high doses of ACE inhibitors were not able to decrease plasma Ang II levels over 24 h to the level observed a few hours after the initial dosing in normal volunteer studies [44,45]. An increase in circulating and tissue levels of Ang II could also occur through interruption of the Ang II-mediated negative feedback pathway on renin release as is observed in humans [46]. Therefore, increasing the occupation of the AT2 receptor populations via increased Ang II levels and/or blockade of AT1 receptors could account for the up-regulation of AT2 receptor expression in the sheep LV.

The AT2 receptor has been shown to activate the kinin–nitric oxide–cGMP system in cardiovascular and renal systems, resulting in AT2-mediated cardioprotection through vasodilation, and pressure natriuresis. Furthermore, cardiac specific over expression of the AT2 gene using the {alpha}-myosin heavy chain promoter resulted in decreased sensitivity to AT1-mediated pressor and chronotropic actions [47]. Recently, Tsutsumi et al. [48] suggested that AT2 receptors in aortic vascular smooth muscle cells promote vasodilation by stimulating the production of bradykinin, which subsequently stimulates the nitric oxide–cGMP system in a paracrine manner. Thus, a possible contributory mechanism to the beneficial effects of combined ACE inhibition and AT1 blockade in our ovine model may be due to enhanced bradykinin levels within the myocardium by the combination of AT2 receptor up-regulation and inhibition of bradykinin breakdown by ACE inhibition.

The doses of ramipril and losartan used have been demonstrated to block the RAS individually in this model [24,25]. Previous work demonstrated that this ramipril dose (10 mg/day) inhibited circulating ACE activity and reliably attenuated LV remodeling in this ovine model. In addition, this ramipril dose per body mass is higher than the ramipril dose that significantly limited mortality in the AIRE trial in humans with clinical congestive heart failure after MI [32]. Moreover, this same ramipril dose was shown to be highly effective at reducing the number of cardiovascular events in the HOPE study in high-risk patients [49]. The dose of losartan used in these studies was shown to markedly blunt the pressor response (<50%) to a 10 min intravenous infusion of Ang II at a dose that consistently increased mean arterial pressure by 30% [24]. Furthermore, this losartan dose maintained the inhibitory effect against repeated challenges of Ang II up to 11 h after the last dosing. It is therefore likely that combined therapy up-regulated AT2 receptors and reduced LV remodeling because of the combination of distinct inhibitory mechanisms rather than simply a lack of complete and long-lasting blockade of the RAS. However, it is important to note that the relationship between the beneficial effects of combination therapy and the up-regulation of AT2 receptors is associative rather than causal. In this regard, it is possible, that combination therapy is beneficial to left ventricular remodeling because of mechanisms unrelated to the AT2 receptor including protective actions by bradykinin and inhibition of Ang II-calcium signaling and MAP kinase pathways via AT1 receptor blockade. Moreover, although our study indicates that combination therapy increased the AT2/AT1 ratio under conditions in which either therapy alone did not, it is possible that larger numbers of animals would reveal a difference between the control and the ramipril-treated and/or losartan-treated groups.

In summary, we found that the predominant AT receptor subtype in ovine myocardium is the AT2 receptor, which is similar to the human heart and in distinct contrast to myocardial receptor population in rodents, rabbits and monkeys. Thus, the ovine model is likely to be a clinically relevant and valuable model in which to study the role of AT receptors and pharmaceutical inhibitors of the RAS in Ang II remodeling. Our present study also demonstrates that AT1 receptor blockade combined with ACE inhibition after transmural anteroapical MI, resulted in up-regulation of AT2 receptors in LV tissues leading to a significantly increased AT2/AT1 ratio in all myocardial regions. Therefore, up-regulation of the AT2 receptor subtype and the significantly increased AT2/AT1 ratio after ACE inhibition and AT1 blockade may be an important contributing mechanism in the beneficial effects of combined therapy on LV remodeling. The association of AT2 receptor up-regulation demonstrated in the present study and the limitation of LV remodeling previously shown with combined therapy is consistent with the hypothesis that AT2 receptors are cardioprotective.

Time for primary review 28 days.


   Acknowledgements
 
This study was supported in part by NIH grant HL57980 (CMK); a grant-in-aid from the American Heart Association, National Office, #96014830 (CMK); a Losartan Medical School Grant, Merck & Co., Inc. (SM); a NIH grant HL57502 (KS) and an American Heart Association Established Investigator Award (KS).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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