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(Circulation. 1998;98:1735-1741.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Differential Regulation of Cardiac Angiotensin Converting Enzyme Binding Sites and AT₁ Receptor Density in the Failing Human Heart

Lawrence S. Zisman, MD; Koji Asano, MD, PhD; Darrin L. Dutcher, BS; Anthony Ferdensi, BS; Alastair D. Robertson, PhD; Matthew Jenkin, BS; Erik W. Bush, BS; Teresa Bohlmeyer, MD; M. Benjamin Perryman, PhD; ; Michael R. Bristow, MD, PhD

From the Department of Medicine, Division of Cardiology, University of Colorado Health Sciences Center, Denver.

Correspondence to Lawrence S. Zisman, MD, Center for Pulmonary Heart Disease, The Rush Heart Institute, 1725 W Harrison St, Suite 020, Chicago, IL 60612. E-mail lzisman{at}rush.edu

	Abstract

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Background—The regulation and interaction of ACE and the angiotensin II (Ang II) type I (AT₁) receptor in the failing human heart are not understood.

Methods and Results—Radioligand binding with ³H-ramiprilat was used to measure ACE protein in membrane preparations of hearts obtained from 36 subjects with idiopathic dilated cardiomyopathy (IDC), 8 subjects with primary pulmonary hypertension (PPH), and 32 organ donors with normal cardiac function (NF hearts). ¹²⁵I-Ang II formation was measured in a subset of hearts. Saralasin (¹²⁵I-{Sar¹,Ile⁸}-Ang II) was used to measure total Ang II receptor density. AT₁ and AT₂ receptor binding were determined with the AT₁ receptor antagonist losartan. Maximal ACE binding (B_max) was 578±47 fmol/mg in IDC left ventricle (LV), 713±97 fmol/mg in PPH LV, and 325±27 fmol/mg in NF LV (P<0.001, IDC or PPH versus NF). In IDC, PPH, and NF right ventricles (RV), ACE B_max was 737±78, 638±137, and 422±49 fmol/mg, respectively (P=0.02, IDC versus NF; P=0.08, PPH versus NF). ¹²⁵I-Ang II formation correlated with ACE binding sites (r=0.60, P=0.00005). There was selective downregulation of the AT₁ receptor subtype in failing PPH ventricles: 6.41±1.23 fmol/mg in PPH LV, 2.37±0.50 fmol/mg in PPH RV, 5.38±0.53 fmol/mg in NF LV, and 7.30±1.10 fmol/mg in NF RV (P=0.01, PPH RV versus PPH LV; P=0.0006, PPH RV versus NF RV).

Conclusions—ACE binding sites are increased in both failing IDC and nonfailing PPH ventricles. In PPH hearts, the AT₁ receptor is downregulated only in the failing RV.

Key Words: angiotensin • cardiomyopathy • pulmonary heart disease

	Introduction

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Systemic components of the renin-angiotensin system (RAS) are increased in patients with chronic heart failure, and treatment with oral ACE inhibitors attenuates progressive myocardial dysfunction and improves survival in this group of patients.^{1 2 3 4} In addition, the concept of an autocrine/paracrine RAS that locally regulates cardiac structure and function is supported by several lines of investigation.⁵ ACE catalyzes the formation of angiotensin (Ang) II from the prohormone Ang I. Ang II has multiple biological actions, including vasoconstriction, stimulation of myocyte and fibroblast cell growth, and facilitation of norepinephrine release from sympathetic neurons.⁶ These actions are mediated through the Ang II type I (AT₁) receptor. In the setting of selective AT₁ receptor antagonism or downregulation, Ang II may exert less-well-defined counterregulatory effects mediated by the AT₂ receptor.⁷

We and others^{8 9} have demonstrated that AT₁ receptor protein and mRNA abundance are selectively downregulated compared with AT₂ receptor protein and mRNA in the failing human heart. It is reasonable to argue that downregulation of the AT₁ receptor is the result of chronic stimulation by its agonist, Ang II, and that Ang II formation is increased because cardiac RAS components, including ACE, are upregulated in human heart failure. Previous studies have shown that ACE mRNA abundance is increased in failing compared with nonfailing (NF) human heart.¹⁰ However, it is not known if ACE protein density is correspondingly increased in cardiac tissue from failing human hearts. Furthermore, the cellular location of ACE production in the human heart is not known. Colocalization of ACE and AT₁ receptor mRNA would set the stage for an important local interaction of these 2 RAS components in the heart.

To investigate the relationships among ACE, Ang II–forming activity, and the AT₁ receptor, we quantified Ang II receptors and ACE binding sites and measured ACE activity in human hearts obtained from subjects with end-stage idiopathic dilated cardiomyopathy (IDC), subjects with primary pulmonary hypertension (PPH), and NF hearts obtained from organ donors. The major hypotheses for this study were that ACE protein is increased and AT₁ receptor density is selectively decreased in failing human heart ventricles.

	Methods

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Reagents
³H-ramiprilat (specific activity, 66.5 Ci/mmol) was a kind gift of Dr Klaus Wirth (Hoechst AG, Frankfurt, Germany). High-performance liquid chromatography (HPLC)–grade methanol and hydrochloric acid were obtained from Fisher Scientific. Enalaprilat and losartan were kindly provided by Merck Research Laboratories, West Point, Pa. The AT₂-selective antagonist PD123319 was a gift from Parke-Davis, Ann Arbor, Mich. ¹²⁵I-Ang I, ¹²⁵I-Ang II, ¹²⁵I-{ Sar¹,Ile⁸}-Ang II, -³²P-ATP, and ³H-cAMP were obtained from New England Nuclear. Superscript II reverse transcriptase (RT) was obtained from Life Technologies. The reagents for in situ polymerase chain reaction (PCR) were obtained from Perkin-Elmer. All other reagents were obtained from Sigma Chemical Co.

Patient Characteristics
Hearts with biventricular failure were obtained from 36 patients undergoing cardiac transplantation for treatment of end-stage IDC. Hearts with isolated advanced right ventricular (RV) failure were obtained from 8 patients undergoing combined heart-lung transplant for treatment of end-stage PPH. NF cardiac tissue was obtained from 32 organ donors whose hearts had documented normal left ventricular (LV) function by echocardiography but which were not placed for transplantation in the majority of cases because of donor/recipient size mismatch or ABO incompatibility.¹¹ Patient characteristics including hemodynamic data are summarized in the Table. Two of the PPH patients received prostacyclin as part of an acute drug study but were not taking prostacyclin chronically. In these 2 patients, the hemodynamics used were obtained before drug infusion. Ten patients with IDC were receiving dobutamine at the time of cardiac transplantation, and 14 were receiving parenteral furosemide at an average dose of 160 mg/d. Right heart catheterization data were obtained <12 months before cardiac transplantation. Pulmonary arterial pressure was significantly higher in the PPH group than in the IDC group (P<0.0001). Pulmonary capillary wedge pressure was significantly higher in the IDC group than in the PPH group (P<0.001).

View this table: [in this window] [in a new window]   Table 1. Patient Characteristics

Cardiac Membrane Preparation
Crude membrane preparations were prepared from hearts within 30 minutes of explantation as previously described^{11 12} and stored at -80°C until used for ACE and/or Ang II receptor binding. For ACE binding, the membrane preparation was solubilized at 4°C in an equal volume of resuspension buffer (0.01 mol/L HEPES, 0.3 mol/L KCl, 0.6% Triton-X, pH 7.5) for 4 hours and centrifuged at 25 000g for 1 hour. The supernatant was dialyzed extensively against 0.01 mol/L HEPES, pH 8.1, 0.3 mol/L KCl, 0.02% Triton-X, and 100 µmol/L ZnSO₄.

ACE Binding Assay
ACE binding sites were quantified according to the method of Vago et al¹³ with slight modifications. Solubilized/dialyzed membrane preparations were diluted 1:1 with ACE assay buffer (0.1 mol/L HEPES, 0.1 mol/L NaCl, 2 µmol/L ZnSO₄, 0.02% NaN₃, pH 8.1) and incubated for 1 hour with ³H-ramiprilat at 37°C with and without 10^-6 mol/L enalaprilat as a competitor to allow calculation of nonspecific binding. Seven-point saturation curve assays ranging from 0.15 to 10 nmol/L ³H-ramiprilat were performed in duplicate. The reaction was terminated by the addition of ice-cold assay buffer followed by three 5-mL washes over 25-mm glass filters subjected to vacuum. The filters were air-dried overnight and counted in a scintillation counter. Maximum binding sites (B_max) and the dissociation constant (K_d) were calculated by use of a nonlinear least squares fit of the specific binding curve.¹² Because each molecule of ACE has 2 active sites, it is necessary to divide the value obtained for ACE binding sites by a factor of 2 to calculate ACE protein concentration.¹⁴ The data, as presented, do not include this stoichiometric correction.

Ang II–Forming Activity Assay
Cardiac tissue Ang II–forming assays were performed as previously described with slight modifications.¹⁵ Dialyzed sample was diluted 1:2 in ACE assay buffer. One hundred microliters of this dilution was incubated in a total volume of 200 µL with 200 fmol of the ACE substrate ¹²⁵I-Ang I for 10 minutes at 37°C. To inhibit ACE in these preparations, parallel samples were made with the addition of l0 µmol/L enalaprilat and incubated for 20 minutes at 37°C. All samples were assayed in duplicate. Angiotensin peptides were separated by HPLC with a C18 Nucleosil column. Under the conditions of this assay, 85% of ¹²⁵I-Ang II formation is mediated by ACE.¹⁵

Ang II Receptor Binding Assay
Membrane preparations of human heart tissue (60 to 200 µg) were incubated with¹²⁵I-{Sar¹,Ile⁸}-Ang II (¹²⁵I-saralasin; specific activity, 2200 Ci/mmol) as previously described.⁸ Saralasin (1 mmol/L) was used to determine specific binding; competitive displacement with losartan (1 µmol/L) was used to quantify AT₁ and AT₂ receptor subtypes. Maximum radioligand binding sites (B_max) and K_d were determined from saturation binding curves with a nonlinear fitting computer program.¹²

ACE mRNA In Situ RT-PCR
In situ RT-PCR was performed as previously described with slight modifications for ACE mRNA.⁸ The primers for human ACE mRNA amplification were 5'-CGA ACT CCG CTC GCT CAG-3' (upstream) and 5'-GTG TTC CCA TCC CAG TCT CTG-3' (downstream). Briefly, in situ PCR glass slides were treated with pepsin, followed by DNase digestion and reverse transcription with an oligo dT primer and random primer mix. The PCR reaction was performed in the presence of the ACE primers digoxigenin-dUTP and dNTP under the following conditions: 1 cycle at 94°C for 2 minutes 30 seconds, 20 cycles at 94°C for 40 seconds, and 55°C for 1 minute 30 seconds. A levamisole solution was used for colorimetric detection.

Adenylate Cyclase Assays
To quantify membrane yield in failing vs NF hearts, basal and manganese-stimulated adenylate cyclase activity was determined in LV membrane preparations before solubilization, as previously described.¹¹

Statistical Analysis
Analyses referred to as "unpaired" used all data from LV and RV. Those analyses referred to as "paired" used data only from subjects with matching LV and RV data. Covariates examined were cause of heart failure, age, sex, LV ejection fraction, cardiac index, right atrial pressure, pulmonary capillary wedge pressure, mean pulmonary artery pressure, and prior treatment with an ACE inhibitor. Distributions of ACE binding were found to be nonnormal by the Shapiro-Wilk test, and analyses were performed with nonparametric tests. Distributions of total Ang II receptors and AT₁ and AT₂ subtypes were not significantly different from normal.

Nonparametric analyses used the Kruskal-Wallis or Wilcoxon rank sum tests as appropriate, with the Bonferroni correction. Paired analysis was by the Wilcoxon signed rank test. Identification of significant covariates was performed univariately for each etiology (NF, IDC, or PPH) with the Spearman correlation coefficient.

Parametric analyses used ANOVA and the Student-Newman-Keuls test or the unpaired t test as appropriate. Paired analysis was performed with the Student paired t test. Identification of significant covariates was done univariately by the Pearson correlation coefficient and multivariately by regression, with backward elimination to remove nonsignificant variables.

Software for analysis was Excel (Microsoft) and SAS (SAS Institute). All comparisons were 2-sided, with a significance level of 0.05. Unless otherwise noted, values are expressed as mean±SEM.

	Results

Top Abstract Introduction Methods Results Discussion References

 
ACE Binding Sites
Figure 1 demonstrates saturation binding curves and Scatchard plots from the LV and RV of a PPH heart. The binding of ³H-ramiprilat to solubilized membrane preparations of human heart ventricles was saturable, and Scatchard plot analysis was consistent with a single binding site. In addition, the assay was linear in the range of protein concentrations used, and specific binding reached steady state by 60 minutes (data not shown). Competition curves with unlabeled enalaprilat modeled to 1 active site (Figure 2). ACE B_max was 578±47 fmol/mg in IDC LV (n=36) and 713±97 fmol/mg in PPH LV (n=8) compared with 325±27 fmol/mg in NF LV (n=32) (P=0.0003, IDC versus NF and PPH versus NF). ACE B_max was 737±78 fmol/mg (n=10) in IDC RV membranes, 638±137 fmol/mg in PPH RV (n=8), and 422±49 fmol/mg in NF RV (n=13) (P=0.02, IDC versus NF; P=0.08, PPH versus NF; Figure 3). There was a significant correlation between LV and RV ACE B_max in the NF, IDC, and PPH hearts in which both LV and RV data were measured (Spearman r=0.82, P=0.0001; Figure 4). Paired analysis demonstrated that in NF hearts, ACE B_max was higher in the RV than the LV (NF LV ACE B_max=333±51 fmol/mg, NF RV ACE B_max=422±49 fmol/mg; P=0.02); however, this difference was lost in both the IDC and PPH hearts. In the subgroup of IDC patients taking ACE inhibitors (n=23), LV ACE B_max was 570.9±48.7 fmol/mg compared with 499.5±60.4 fmol/mg for the group of IDC patients who were not taking ACE inhibitors (n=11) (P=NS). The K_d for ³H-ramiprilat binding in the IDC hearts was 1.67±0.16 nmol/L compared with 1.63±0.18 nmol/L in the NF hearts examined (P=NS). In the subgroup of IDC patients treated with ACE inhibitors before transplantation, the K_d for ³H-ramiprilat binding was 1.93±0.19 nmol/L (n=23) compared with 1.15±0.26 nmol/L for the IDC subgroup not taking ACE inhibitors (n=11) (P=0.02). The 2 IDC hearts for which there was no documentation of prior ACE inhibitor treatment were not included in this subgroup analysis.

View larger version (19K): [in this window] [in a new window]   Figure 1. 3H-ramiprilat saturation binding curves from the LV (A) and RV (B) of a PPH heart. Nonspecific binding was evaluated in the presence of 1 µmol/L enalaprilat: (), total binding; (), specific binding; and (), nonspecific binding. Scatchard transformation of data from PPH LV and PPH RV are shown in C and D, respectively.

View larger version (13K): [in this window] [in a new window]   Figure 2. Competition curve from an NF human heart LV membrane preparation incubated with 3H-ramiprilat in the presence of increasing concentrations of enalaprilat.

View larger version (16K): [in this window] [in a new window]   Figure 3. Radioligand binding with 3H-ramiprilat in membrane preparations from IDC, PPH, and NF hearts (*P<0.001 versus NF LV; P=0.02 versus NF RV; §P=0.02 vs NF LV).

View larger version (16K): [in this window] [in a new window]   Figure 4. Correlation between LV and RV ACE Bmax in NF (), IDC (), and PPH () hearts in which both LV and RV data were measured (Spearman r=0.82, P=0.0001).

In the LV of PPH hearts, there was a positive correlation between cardiac index and ACE B_max (P=0.01; Figure 5). The correlation between RV ACE B_max and cardiac index in the PPH hearts did not reach statistical significance (P=0.09). In contrast, no relationship between cardiac index and ACE B_max was found in the IDC hearts. However, in the RV of IDC hearts, there was a positive correlation between pulmonary capillary wedge pressure and ACE B_max (P=0.03). Correlation coefficients for age, sex, LV ejection fraction, or mean pulmonary artery pressure with regard to ACE B_max or K_d were not significant. No significant effect of dobutamine or parenteral furosemide on ACE B_max was observed. No relationships were found among date of cardiac transplantation, date of membrane preparation, and ACE binding sites.

View larger version (13K): [in this window] [in a new window]   Figure 5. A, Correlation of cardiac index (CI) and LV ACE Bmax in PPH hearts () (n=8; r=0.84, P=0.01). B, No significant correlation between cardiac index and LV ACE Bmax in IDC hearts ().

ACE Activity
¹²⁵I-Ang II formation was measured in 24 IDC and 16 NF human heart LV membrane preparations. Regression analysis demonstrated a significant relationship between ACE activity with ¹²⁵I Ang I as substrate and ACE binding sites (n=40, r=0.60, r²=0.36, F=21.2, P=0.00005; Figure 6). Furthermore, ACE activity was higher in the failing than the NF hearts (101±8.0 versus 71.5±4.9 fmol of ¹²⁵I-Ang II formed per minute per milligram of protein, respectively; P<0.01). Analysis of the data from only the IDC hearts demonstrated a significant correlation between ACE B_max and ¹²⁵I-Ang II formation (n=24, r=0.54, P=0.007); however, there was no correlation between ACE binding sites and activity in the NF hearts. In LV membrane preparations from hearts without ACE-inhibitor exposure before explantation, ¹²⁵I-Ang II formation was 98.1±17.0 fmol · min^-1 · mg^-1 (n=8) compared with 102.4±9.0 fmol · min^-1 · mg^-1 in hearts explanted from patients who were taking ACE inhibitors before cardiac transplantation (n=16; P=NS). In the 16 IDC hearts with prior ACE-inhibitor exposure, there was a significant correlation between ACE B_max and ¹²⁵I-Ang II formation (r=0.54, P=0.03); the correlation between ACE B_max and ¹²⁵I-Ang II–forming activity in the IDC hearts without prior ACE-inhibitor exposure did not achieve statistical significance (r=0.59, P=0.12; n=8).

View larger version (16K): [in this window] [in a new window]   Figure 6. 125I-Ang II formation in 24 IDC () and 16 NF () human heart LV membrane preparations. ACE activity with 125I-Ang I as substrate and ACE binding sites were positively correlated (r=0.60, P=0.00005).

Ang II Receptor Binding
Total Ang II receptor density was 9.93±1.43 and 5.38±1.02 fmol/mg in PPH LV and RV, respectively (n=8), compared with 7.55±0.58 and 9.87±1.26 fmol/mg in NF LV (n=26) and RV (n=10), respectively (P=0.016 PPH RV versus PPH LV; P=0.03 PPH RV versus NF RV). The decrease in total Ang II receptor density was explained by selective downregulation of the AT₁ receptor subtype, which was 6.41±1.23 fmol/mg in PPH LV, 2.37±0.5 fmol/mg in PPH RV, 5.38±0.53 fmol/mg in NF LV, and 7.30±1.10 fmol/mg in NF RV (P=0.01 PPH RV versus PPH LV; P=0.0006 PPH RV versus NF RV; Figure 7). In IDC hearts, LV Ang II receptor density was 6.56±0.85 fmol/mg (n=17), and RV Ang II receptor density was 7.56±1.53 fmol/mg (n=9) (P=NS versus NF and PPH hearts). IDC LV AT₁ receptor density was 3.23±0.56 (n=17) (P<0.05 versus NF and PPH LVs), and IDC RV AT₁ receptor density was 2.58±0.46 fmol/mg (n=9) (P=0.0006 versus NF RV).

View larger version (16K): [in this window] [in a new window]   Figure 7. Total Ang II receptor (ATR) Bmax was lower in PPH RVs than in PPH LVs or NF RVs. Decrease in total Ang II receptor density was explained by selective downregulation of AT1 receptor subtype (*P<0.05 vs PPH LV or NF RV; P<0.05 vs PPH LV; §P<0.001 vs NF RV).

Gender effects on LV but not RV total Ang II receptor and AT₁ and AT₂ subtype receptor densities were revealed by univariate and multivariate analyses. Specifically, in IDC and NF LVs, by univariate analysis, total Ang II receptor density was higher in women than in men (P=0.03), but in PPH LVs, total Ang II receptor density was higher in men than in women (P=0.007). These opposite gender effects were borne out in the multivariate analysis, and both univariate analysis and stepwise regression indicated that PPH LV AT₁ receptor density was higher in men than in women (P=0.002). Univariate analysis also revealed that IDC LV AT₂ receptor density was higher in women than in men (P=0.007). A subgroup analysis of women younger than 50 years of age revealed that the AT₂ receptor concentration was higher than in women >50 years old (6.38±0.44 fmol/mg, n=4, versus 3.24±0.93 fmol/mg, n=4; P=0.01) and higher than in all men (P<0.001).

In the RV of NF hearts, both univariate and multivariate analyses demonstrated a positive correlation between age and AT₂ receptor density (P=0.0036 and P=0.002, respectively). Univariate analysis did not reveal any relationship between hemodynamic parameters and Ang II receptor density in the NF, IDC, or PPH groups.

Adenylate Cyclase Activity
Under basal conditions, adenylate cyclase activity in the LV membrane preparations of NF hearts was 2.57±0.32 compared with 2.37±0.31 cAMP pmol · min^-1 · mg^-1 in IDC hearts (P=NS). Manganese-stimulated activity was 284±28 cAMP pmol · min^-1 · mg^-1 in NF hearts compared with 267±13 cAMP pmol · min^-1 · mg^-1 in IDC hearts (P=NS).

ACE In Situ PCR
Liquid-phase RT-PCR was performed to verify the predicted 299-bp amplified product for human ACE. In situ PCR demonstrated ACE mRNA signal in microvascular endothelial cells as well as the subsarcolemmal surface of ventricular myocytes in tissue sections from 4 NF and 4 IDC hearts. Signal for ACE mRNA was also detected diffusely throughout the cytoplasm of cardiac myocytes. Tissue sections not treated with DNase served as positive controls and demonstrated PCR amplification of genomic DNA in nuclei. In the absence of RT, no PCR product was detected (Figure 8).

View larger version (113K): [in this window] [in a new window]   Figure 8. In situ PCR of ACE mRNA in tissue sections of LV myocardium from an explanted NF (top left) and failing (top right) human heart (magnification x150). ACE mRNA was localized to the microvasculature and cardiac myocytes. There was no difference in localization pattern in failing (n=4 IDC) versus NF hearts (n=4) examined. A negative control in which the RT was omitted is shown (bottom left), and a positive control in which the specimen was not treated with DNase is shown (bottom right).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have found for the first time that there is a highly significant increase in ACE protein concentration in failing compared with NF human hearts. This finding thus provides the first support for the argument that the previously reported increase in ACE steady-state mRNA abundance¹⁰ may translate to an increase in ACE protein expression in the failing human heart. Previous studies in the failing human heart have shown either no significant upregulation of ACE activity¹⁶ or a decrease in total Ang II–forming activity.¹⁷ The differences between these studies and the present study may be explained in part by the different methods used to prepare the cardiac tissue for assay¹⁸ or the smaller sample size and limited availability of NF heart tissue in previous studies.^{16 17}

In both failing and NF hearts, we found a good correlation between LV and RV ACE B_max. In the NF hearts, RV ACE B_max was higher than LV ACE B_max; however, in the IDC hearts, both LV and RV ACE B_max were increased (Figures 3 and 4). To determine whether an increase in ACE protein density could result in an increase in Ang II–forming activity, we measured ¹²⁵I-Ang II formation in the same preparations used to quantify ACE binding sites. We found a significant correlation between ACE B_max and ¹²⁵I-Ang II–forming activity. Furthermore, Ang II–forming activity was increased in the failing compared with the NF hearts.

The higher K_d for ³H-ramiprilat binding found in the IDC hearts with a history of ACE-inhibitor exposure compared with failing hearts without such exposure suggests that some residual ACE inhibitor was bound to ACE in these hearts. However, the actual percent of ACE binding sites occupied by inhibitor in the IDC hearts with prior ACE-inhibitor exposure and the efficacy of cardiac ACE inhibition in these hearts was not determined by the present study.

Contrary to our expectation, ACE binding sites were increased in the nonfailing PPH LV, with a trend toward an increase in the failing RV of PPH hearts. However, there was selective downregulation of the AT₁ receptor in failing PPH RVs compared with nonfailing LVs of PPH hearts or RVs of NF donor hearts.

An increase in ACE activity could result in higher local levels of Ang II, the endogenous agonist for the AT₁ receptor. Chronic stimulation of the AT₁ receptor could lead to its downregulation. For example, in vascular smooth muscle cells, Ang II decreases AT₁ receptor mRNA abundance.¹⁹ Our finding that an increase in ACE protein was not associated with downregulation of the AT₁ receptor in the nonfailing LV of PPH hearts suggests that additional factors may be required for the chamber-specific downregulation of the AT₁ receptor in the failing RV of PPH hearts.

To further understand the regulation of ACE protein in failing hearts, we examined the effect of hemodynamic variables on ACE B_max and K_d. Interestingly, in the PPH hearts, we found a positive correlation between ACE B_max and cardiac index (Figure 5). However, we found no relationship between LV ACE B_max and cardiac index in the IDC hearts.

Using the technique of in situ PCR, we detected ACE mRNA in both endothelial cells and cardiac myocytes in the human heart. The distribution of ACE mRNA was similar to that of the AT₁ receptor mRNA.⁸ Comparison of failing with NF myocardium did not reveal any significant differences in ACE mRNA distribution. Therefore, our in situ PCR data do not support the argument that dissociation of AT₁ receptor and ACE protein regulation is related to compartmentalization.

Potentially important effects of age and sex on total Ang II receptor and AT₁ or AT₂ subtype receptor densities were identified by univariate and multivariate analyses. These effects were different between the NF, IDC, and PPH groups. The higher LV total Ang II receptor density in the women with IDC appeared to be secondary to a relative increase in the AT₂ receptor subtype. This effect was greatest in women younger than 50 years of age, a finding that suggests a possible interaction between the premenopause hormonal milieu and the concentration of cardiac AT₂ receptors. In contrast, the higher LV total Ang II receptor density in male relative to female PPH hearts was due to an increase in AT₁ receptors. In the NF group, AT₂ receptor density was higher in the older donors. Information regarding the potential effects of age and sex on angiotensin receptors in human subjects or animal models is sparse.^{20 21} Our data suggest a complex interaction between age, sex, heart failure, and cardiac Ang II receptor concentration.

Limitations
There are several important limitations of this study. We demonstrated only a trend toward an increase in PPH RV ACE binding sites. We suspect that if further assays had been possible, a significant increase would have been found. Although ACE binding sites and Ang II–forming activity correlated in vitro, this study could not determine if the observed increase in ACE protein in failing ventricular myocardium resulted in higher local Ang II concentrations in vivo. Because our localization studies were restricted to mRNA, we cannot be certain that ACE protein was correspondingly expressed in human cardiac myocytes.^{22 23} Furthermore, although provocative, the positive correlation between ACE protein concentration and cardiac index in the PPH hearts does not establish a cause-and-effect relationship between these 2 parameters.

Conclusions
We have shown for the first time that the number of ACE binding sites is increased in ventricular membrane preparations of explanted hearts taken from patients with end-stage heart failure. We have also shown that ACE mRNA is localized to both endothelial cells and cardiac myocytes in the human heart in a pattern similar to that of the AT₁ receptor mRNA. Nevertheless, we found that ACE binding sites and AT₁ receptors are differentially regulated in the failing RV and nonfailing LV of PPH hearts. Future studies designed to address the mechanisms that might explain this observation will be needed.

	Acknowledgments

 
Dr Zisman was supported by an NIH clinical investigator development award (No. HL-03404).

	Footnotes

 
Published in abstract form (J Am Coll Cardiol. 1997;29:230A and J Am Coll Cardiol. 1997;29:244A–245A.).

Received February 14, 1998; revision received June 17, 1998; accepted June 22, 1998.

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