This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Asano, K. Articles by Bristow, M. R. Search for Related Content PubMed PubMed Citation Articles by Asano, K. Articles by Bristow, M. R.

(Circulation. 1997;95:1193-1200.)
© 1997 American Heart Association, Inc.

Articles

Selective Downregulation of the Angiotensin II AT₁-Receptor Subtype in Failing Human Ventricular Myocardium

Koji Asano, MD, PhD; Darrin L. Dutcher, BS; J. David Port, PhD; Wayne A. Minobe, BS; Kelli D. Tremmel, BS; Robert L. Roden, MS; Teresa J. Bohlmeyer, MD; Erik W. Bush, BS; Matthew J. Jenkin, BS; William T. Abraham, MD; Mary V. Raynolds, PhD; Lawrence S. Zisman, MD; M. Benjamin Perryman, PhD; Michael R. Bristow, MD, PhD

the Division of Cardiology, The Temple Hoyne Buell Heart Center Research Laboratories, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colo.

Correspondence to Michael R. Bristow, MD, PhD, Division of Cardiology, B-139, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Denver, CO 80262. E-mail bristow_m{at}defiance.hsc.colorado.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background The regulation of angiotensin II receptors and the two major subtypes (AT₁ and AT₂) in chronically failing human ventricular myocardium has not been previously examined.

Methods and Results Angiotensin II receptors were measured by saturation binding of ¹²⁵I-[Sar¹,Ile⁸]angiotensin II in crude membranes from nonfailing (n=19) and failing human left ventricles with idiopathic dilated cardiomyopathy (IDC; n=31) or ischemic cardiomyopathy (ISC; n=21) and membranes from a limited number of right ventricles in each category. The AT₁ and AT₂ fractions were determined by use of an AT₁-selective antagonist, losartan. ß-Adrenergic receptors were also measured by binding of ¹²⁵I-iodocyanopindolol with the ß₁ and ß₂ fractions determined by use of a ß₁-selective antagonist, CGP20712A. AT₁ but not AT₂ density was significantly decreased in the combined (IDC+ISC) failing left ventricles (nonfailing: AT₁ 4.66±0.48, AT₂ 2.73±0.39; failing: AT₁ 3.20±0.29, AT₂ 2.70±0.33 fmol/mg protein; mean±SE). The decrease in AT₁ density was greater in the IDC than in the ISC left ventricles (IDC: 2.73±0.40, P<.01; ISC: 3.89±0.39 fmol/mg protein, P=NS versus nonfailing). ß₁ but not ß₂ density was decreased in the failing left ventricles. AT₁ density was correlated with ß₁ density in all left ventricles (r=.43). AT₁ density was also decreased in IDC right ventricles. In situ reverse transcription–polymerase chain reaction in sections of nonfailing and failing ventricles indicated that AT₁ mRNA was present in both myocytes and nonmyocytes.

Conclusions AT₁ receptors are selectively downregulated in failing human ventricles, similar to the selective downregulation of ß₁ receptors. The relative lack of AT₁ downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.

Key Words: receptors • polymerase chain reaction • angiotensin • radioisotopes

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The chronically failing human heart exhibits evidence of neurohormonal activation.^{1 2 3 4} In chronic heart failure, cardiac adrenergic activation and the resultant signal-transduction abnormalities have been well documented.^{1 2 3} Induction of the cardiac renin-angiotensin system in failing myocardium has also been described.^{4 5} However, unlike ß-adrenergic receptor regulation, there is limited and conflicting information on the status of Ang-II receptors in chronically failing human heart,^{6 7} with one study⁶ describing no change in total Ang-II receptor density in failing ventricles and another⁷ describing a decrease in the total Ang-II receptor density in failing atria and possibly in ventricles. Moreover, there are no reports on the behavior of the two major Ang-II receptor subtypes (AT₁ and AT₂)^{8 9} in failing human ventricles.

Activation of the cardiac renin-angiotensin and adrenergic systems may be interrelated.^{10 11 12} Therefore, we hypothesized that one or both Ang-II and ß-adrenergic receptor subtypes would be downregulated due to coactivation/induction of the renin-angiotensin and adrenergic systems in chronically failing human myocardium. To test this hypothesis, we measured Ang-II receptors with the fractions of AT₁ and AT₂ subtypes in membrane fractions from both NF and failing human ventricles. We also measured ß-adrenergic receptors to determine the relationship of these two signal-transduction pathways in the failing heart. Finally, to examine the cellular expression of AT₁ receptors, we performed in situ RT-PCR in sections of NF and failing ventricles.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Tissue Procurement
All human hearts were provided by the Utah and Colorado cardiac transplant programs. Failing LVs were harvested at the time of cardiac transplantation from 52 patients with end-stage heart failure (NYHA class III or IV) due to either IDC (n=31) or ISC (n=21). NF LVs were obtained from 19 organ donors with no history of cardiac disease who were excluded from organ donation because of age, body size, or blood type incompatibilities. None of the above subjects were treated with catecholamines, phosphodiesterase inhibitors, or other positive inotropic agents except for low-dose dopamine given to 13 of 19 donors for maintenance of peripheral vascular tone and renal blood flow. Eighteen of 19 organ donors had normal left ventricular function (echocardiographically determined shortening fractions >0.25 before cardiac explantation).

A high-yield crude membrane fraction was prepared within 30 minutes of excision as previously described.^{1 3 13 14} A 5- to 6-g aliquot of left ventricular free wall was dissected free of epicardial and endocardial connective tissue and finely minced in a 10-fold volume (wt/vol) of ice-cold 10 mmol/L Tris and 1 mmol/L EGTA buffer (pH 8.0). From the ISC ventricles, the noninfarcted area with no visible fibrosis was dissected. The tissue was homogenized with a Polytron (Brinkmann) by use of three 5-second bursts at full speed. After extraction of the contractile proteins in 0.5 mol/L of KCl, the homogenate was centrifuged for 15 minutes at 50 000g, 4°C. The pellet was washed with 75 mmol/L ice-cold Tris and 10 mmol/L MgCl₂ buffer (pH 7.8) and centrifuged as above. After another washing and centrifugation, the final pellet was resuspended in a 10-fold volume of ice-cold 250 mmol/L sucrose, 50 mmol/L Tris, and 1 mmol/L EGTA buffer and stored at –80°C.

Radioligand Binding Assays
Ang-II receptors were measured by binding of ¹²⁵I-saralasin (specific activity, 2200 Ci/mmol; New England Nuclear). Membranes (60 to 200 µg) were incubated with increasing concentrations of this radioligand (0.05 to 1.3 nmol/L) in a buffer containing 150 mmol/L NaCl, 20 mmol/L Tris, 1 mmol/L ascorbate, and 0.07% BSA (pH 7.5) for 60 minutes at 30°C. 1,10-Phenanthroline (1 mmol/L) was included in the buffer to inhibit peptide degradation.¹⁵ Total assay volume was 225 µL. Specific binding was determined by the displacement of bound ¹²⁵I-saralasin by 1 µmol/L of unlabeled saralasin. To quantify the AT₁ and AT₂ subtype fractions, the proportion of ¹²⁵I-saralasin binding displaceable by 1 µmol/L losartan (a gift from DuPont-Merck, Wilmington, Del), an AT₁-receptor–specific antagonist,^{7 9 16} was determined. This concentration was chosen on the basis of the competitions for ¹²⁵I-saralasin and unlabeled saralasin, losartan, or PD123319 (an AT₂-selective antagonist,^{7 9} a gift from Parke-Davis, Ann Arbor, Mich), which demonstrated the ability of 1 µmol/L losartan to displace >95% of specific ¹²⁵I-saralasin binding from AT₁ receptors. The incubation was terminated by the addition of 5 mL of ice-cold incubation buffer without BSA, followed by vacuum filtration over glass fiber filters (type A/E, Gelman Sciences Inc) presoaked in 0.2% BSA solution. The filters were rapidly washed with an additional 20 mL of buffer. Filter-bound radioactivity was measured by counting (model 1470 WIZARD, Wallac).

ß-Adrenergic receptors were measured by binding of ¹²⁵I-ICYP (specific activity, 2200 Ci/mmol; New England Nuclear) as previously described.^{3 13 14} To quantify the ß₁- and ß₂-receptor fractions, the proportion of specific ¹²⁵I-ICYP binding displaceable by 0.2 µmol/L CGP20712A (a gift from Ciba-Geigy, Summit, NJ), a selective ß₁-receptor antagonist,¹⁷ was determined. This concentration was chosen from the competition for ¹²⁵I-ICYP and increasing concentration of CGP20712A as a concentration that would displace >95% and <5% of bound ¹²⁵I-ICYP from ß₁-receptors and ß₂-receptors, respectively.³ This method yields ß₁- and ß₂-receptor fraction data that are highly correlated (r=.95) with determinations from full ¹²⁵I-ICYP versus CGP20712A competition curves. Protein concentration was determined with the Lowry method as modified by Peterson¹⁸ with BSA used as a standard.

All assays were run in duplicate or triplicate. B_max and dissociation constant (K_d) were determined from saturation binding curves using a nonlinear-fitting computer program.¹⁹ Subtype densities of the respective receptor systems were calculated by multiplying their B_max by each subtype's fractions.

Membrane Markers
To examine membrane yield, we measured manganese (10 mmol/L MnCl₂)-stimulated adenylyl cyclase activity in crude membranes as previously described.^{3 14}

In Situ RT-PCR
To examine the localization of AT₁-receptor mRNA expression in human ventricular myocardium, we performed in situ RT-PCR as previously described²⁰ with the use of the GeneAmp In Situ PCR System 1000 (Perkin Elmer).

Tissue Preparation
Four-micrometer-thick sections of formalin-fixed, paraffin-embedded human heart tissue were placed on amino alkysilane-coated in situ PCR glass slides (three sections per slide). The tissue was deparaffinized with xylene and 100% ethanol. Protease digestion was performed with the use of pepsin at 2 mg/mL (Sigma Chemical Co) in 0.01N HCl for 90 minutes, which was inactivated with DEPC-treated water. The slides were then washed for 1 minute in 100% ethanol and air-dried.

DNase Digestion
Fifty microliters of the following reaction mixture was placed over two of three tissue sections: 5 µL of 10x DNase buffer (1 mol/L NaOAc, 50 mmol/L MgSO₄, pH 5.0, sterile), 5 µL RNase-free DNase (10 U/µL, 1 U/µL final concentration, Boehringer-Mannheim), and 40 µL DEPC water. The section without DNase served as the positive control. The slides were incubated overnight at 37°C with the GeneAmp In Situ PCR System 1000, washed for 1 minute in DEPC water and 1 minute in 100% ethanol, and air-dried.

Reverse Transcription
Fifty microliters of the following reaction mixture was placed over two of three sections of each slide: 5 µL each of dATP, dCTP, dGTP, and dTTP (final concentration, 1 mmol/L); 2.5 µL 3' AT₁ primer (20 µmol/L AT₁RV1a-3'-5'-GTGGTCTTGCTTTGTCTTGTTG-3'; final concentration, 1 µmol/L); 2.5 µL RNAsin (70 U, Promega); 2.5 µL reverse transcriptase (Superscript II, Gibco-BRL); 10 µL MgCl₂ (25 mmol/L, final concentration, 5 mmol/L); and 2.5 µL DEPC water. The section without reverse transcriptase served as the negative control. The slides were incubated at 42°C for 30 minutes, washed in xylene for 5 minutes and in 100% ethanol for 5 minutes, and air-dried.

PCR
The following master mix was prepared for a final volume of 50 µL per section: 32 µL water, 5 µL 10x PCR buffer II (GeneAmp In Situ PCR Core Kit, Perkin-Elmer), 1 µL each of dATP, dCTP, dTTP, and dGTP (final concentration, 200 µmol/L); 0.5 µL digoxigenin-dUTP (final concentration, 10 µmol/L; Boehringer-Mannheim); 2.5 µL each of 3' and 5' primers (20 µmol/L stock AT₁RV1a-3'-5'-GTGGTCTTGCTTTGTCTTGTTG-3', AT₁RV1a-5'-5'-AAAATGAGCACGCTTTCCTACC-3'; final concentration, 1 µmol/L); and 3 µL MgCl₂ (25 mmol/L stock; final concentration, 1.5 mmol/L). The master mix tube was heated to 70°C before addition of Taq polymerase per the Hot Start protocol. One-half microliter of AmpliTaq DNA polymerase IS was added to the master mix tube for every section. Fifty microliters of the reaction mixture was added to each tissue section, and the slides were immediately placed in the thermocycler, which had been programmed to 70°C incubation. The following program was begun: 1 cycle at 94°C for 90 seconds20 cycles at 94°C for 40 seconds, 55°C for 90 seconds4°C soak. After completion of the program, the slides were washed in xylene for 3 minutes and in 100% ethanol for 3 minutes.

Direct Colorimetric Detection
The slides were washed for 1 minute in buffer I (100 mmol/L Tris-HCl, 150 mmol/L NaCl; pH 7.5), then incubated for 30 minutes in buffer I containing 2% normal sheep serum and 0.3% Triton X-100. One hundred microliters of the following reaction mixture was placed over each section: anti-digoxigenin antibody conjugate (Genius detection kit, Boehringer-Mannheim) diluted 1:50 with buffer I containing 1% normal sheep serum and 0.3% Triton X. The slides were incubated in a humid chamber for 30 minutes, then washed with shaking for 10 minutes in buffer I and for 10 minutes in buffer II (100 mmol/L Tris-HCl, 100 mmol/L NaCl, 50 mmol/L MgCl₂; pH 9.5). Five hundred microliters of the color solution (45 µL nitroblue tetrazolium solution, 35 µL X-phosphate solution [Genius detection kit], and 2.4 mg levamisole) was placed over each section, and the slides were incubated in a dark, humid chamber for 5 to 10 minutes. When color development was apparent to the naked eye, the reaction was stopped by washing in buffer III (10 mmol/L Tris-HCl, 1 mmol/L EDTA; pH 8.0). The slides were dehydrated in a series of ethanol dilutions, washed in xylene, and coverslipped with a water-based mounting medium.

Statistics
Data are expressed as mean±SEM. Comparisons were made with one-way ANOVA with Scheffe's multiple comparison test, contingency table analysis, linear regression, or Student's unpaired t test as appropriate. Differences were considered statistically significant when P<.05 in a two-tailed distribution.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Clinical Information
Although age was not different between the NF (41.9±3.9 years) and the combined (IDC+ISC) failing group (42.6±1.9 years), the age of the ISC group was higher than the other two groups (Table 1). The ISC group also had a higher percentage of male subjects. Ventricular function appeared to be slightly less impaired in the ISC group than in the IDC group, with lower pulmonary capillary wedge and right atrial pressures.

View this table: [in this window] [in a new window]   Table 1. Clinical Information

Characterization of Ang-II Receptor Binding
Fig 1 shows a representative binding curve with a Scatchard transformation, and Fig 2 shows a representative set of competition curves for ¹²⁵I-saralasin and increasing concentrations of unlabeled saralasin, Ang-II, the AT₁ antagonist losartan, or the AT₂ antagonist PD123319 in membranes from an NF LV. The Scatchard plot in Fig 1 indicates a homogeneous population of saturable binding sites. The data in Fig 2 indicate that the antagonist saralasin identifies the same population of binding sites as the agonist Ang-II and, on the basis of the degree of displacement of losartan or PD123319, that AT₁ receptors are the predominant subtype in NF LVs. In data not shown, the specific binding of ¹²⁵I-saralasin reached steady state by 40 minutes and was linear with respect to added membrane protein between concentrations of 250 and 1400 µg/mL.

View larger version (21K): [in this window] [in a new window]   Figure 1. A, Binding curves of 125I-saralasin in a crude membrane fraction prepared from an NF human LV. , Total binding; , nonspecific binding determined with 1 µmol/L of unlabeled [Sar1, Ile8]-Ang-II (saralasin); and , derived specific binding. Kd was 0.20 nmol/L and Bmax was 5.85 fmol/mg protein in this preparation. B, Scatchard transformation of the same data.

View larger version (18K): [in this window] [in a new window]   Figure 2. 125I-saralasin binding (0.8 nmol/L) in a crude membrane fraction prepared from an NF human LV; competition with unlabeled [Sar1, Ile8]-Ang-II (saralasin) (), unlabeled Ang-II (), losartan (), or PD123319 ().

Ang-II Receptors in NF and Failing Ventricular Myocardium
Comparisons Between NF and Combined Failing LVs
Fig 3A compares total Ang-II receptors (B_max) and subtype densities in the NF, IDC, ISC, and combined failing (IDC+ISC) groups. The B_max tended to be decreased (P=.07) and the AT₁ density was significantly decreased (P<.02) in the combined failing group, with no significant difference in AT₂ density between the two groups (NF [n=19]: B_max 7.39±0.57, AT₁ 4.66±0.48, and AT₂ 2.73±0.39; failing [n=52]: B_max 5.90±0.44, AT₁ 3.20±0.29, and AT₂ 2.70±0.33 fmol/mg). The K_d was not significantly different between the two groups (NF: 0.40±0.04; failing: 0.35±0.03 nmol/L). ACEI treatment did not significantly affect either B_max, AT₁, or AT₂ density within the failing group (with ACEI [n=28]: B_max 5.72±0.46, AT₁ 3.21±0.38, and AT₂ 2.51±0.41; without ACEI [n=24]: B_max 6.10±0.79, AT₁ 3.18±0.47, and AT₂ 2.92±0.53 fmol/mg).

View larger version (38K): [in this window] [in a new window]   Figure 3. Summary of subtype densities (A) and fractions (B) of Ang-II receptors in NF and failing human LVs. Bmax indicates total Ang-II receptor density; combined F, combined (IDC+ISC) failing group.

Comparisons Between IDC and ISC LVs
When the IDC and ISC groups were separately compared with the NF group (Fig 3A), the AT₁ density was significantly (by ANOVA) decreased in the IDC group but not in the ISC group, with no significant difference in AT₂ density among the three groups (IDC [n=31]: AT₁ 2.73±0.40, AT₂ 2.97±0.46; ISC [n=21]: AT₁ 3.89±0.39, AT₂ 2.30±0.45 fmol/mg). As a result, in the IDC group, the fractions of AT₁ and AT₂ (Fig 3B) were altered compared with the other two groups ([ANOVA P=.03] NF: AT₁ 62.4±4.0%, AT₂ 37.6±4.0%; IDC: AT₁ 48.6±5.0%, AT₂ 51.4±5.0%; ISC: AT₁ 65.4±4.7%, AT₂ 34.6±4.7%). The K_d was not significantly different among the three groups (IDC: 0.33±0.04; ISC: 0.39±0.04 nmol/L). Furthermore, ACEI treatment did not significantly affect either B_max, AT₁, or AT₂ density within the IDC group or within the ISC group (data not shown). Because patients in the ISC group were older than patients in the other two groups, we analyzed the relationship between AT₁ density and age. There was no significant positive correlation between AT₁ density and age in either the NF (r=-.37, n=19, P=.12), the IDC (r=.17, n=31, P=.38), or the ISC groups (r=-.05, n=21, P=.84).

Because the ISC group was composed of only male subjects, comparisons were also made among male subjects in the three groups. This comparison again revealed that the AT₁ density was significantly decreased in the IDC but not in the ISC group, with no significant change in AT₂ density in either group (NF [n=8]: AT₁ 5.36±0.88, AT₂ 1.65±0.32; IDC [n=22]: AT₁ 2.35±0.44 [P<.01 versus NF], AT₂ 1.94±0.34; ISC [n=21]: AT₁ 3.89±0.39, AT₂ 2.30±0.45 fmol/mg).

Because the hemodynamic profile in the ISC group tended to be less impaired than in the IDC group (Table 1), comparisons were made within the ISC subgroups on the basis of the degree of myocardial dysfunction. The ISC subjects who had ejection fraction 20% plus either a cardiac index <2.2 or pulmonary capillary wedge pressure 20 mm Hg were defined as the "worse-function" subgroup, and the remaining subjects were assigned to the "better-function" subgroup. As shown in Table 2, LVs from the worse-function subgroup tended to have lower AT₁ density than those from the better-function subgroup.

View this table: [in this window] [in a new window]   Table 2. Comparisons of Receptor Subtype Densities Within ISC LVs

Additional experiments were performed in ultracentrifuged (two sets of 158 000g, 60-minute spins at 4°C) subcellular fractions prepared from the supernatants obtained during the preparation of crude membrane (see "Methods"). In this light vesicle membrane preparation, the AT₁ density appeared to be selectively decreased in the IDC group (NF [n=3]: B_max 7.45±1.45, AT₁ 4.14±1.43, and AT₂ 3.31±0.69 versus IDC [n=3]: B_max 4.73±0.79, AT₁ 1.68±0.14, and AT₂ 3.05±0.92 fmol/mg).

Ang-II Receptors in NF and Failing RVs
In a limited number of subjects in the current study population, AT₁ but not AT₂ density was significantly decreased in IDC but not ISC RVs (NF [n=4]: AT₁ 6.94±0.85, AT₂ 3.32±0.78; IDC [n=10]: AT₁ 3.09±0.66 [P<.05 versus NF], AT₂ 4.99±1.44; ISC [n=3]: AT₁ 5.42±0.29, AT₂ 2.21±0.52 fmol/mg).

ß-Adrenergic Receptors in NF and Failing LVs
Fig 4A compares total ß-receptor density (B_max) and subtype densities in LVs of the three groups. The B_max and the ß₁-receptor density were significantly (P<.001) decreased in the combined failing group, with no significant difference in ß₂-receptor density (NF: B_max 77.5±3.6, ß₁ 58.8±3.7, and ß₂ 18.8±1.6; failing: B_max 59.5±2.4, ß₁ 40.5±2.0, and ß₂ 18.9±0.9 fmol/mg). The K_d was not significantly different between the two groups (NF: 15.1±2.3; failing: 18.5±1.8 pmol/L). When the IDC and ISC groups were separately compared with the NF group, the B_max and the ß₁-receptor density were significantly (by ANOVA) decreased in the IDC group but not the ISC group, with no significant difference in ß₂-receptor density among the three groups (IDC [n=31]: B_max 53.2±2.4, ß₁ 35.2±2.0, and ß₂ 17.9±1.3; ISC [n=21]: B_max 68.7±4.1, ß₁ 48.4±3.2, and ß₂ 20.3±1.2 fmol/mg). As a result, the ß₁- and ß₂-receptor fractions (Fig 4B) were significantly (P<.01) different among the three groups (NF: ß₁ 75.2±2.0%, ß₂ 24.8±2.0%; IDC: ß₁ 65.7±2.2%, ß₂ 34.3±2.2%; ISC: ß₁ 70.4±1.1%, ß₂ 29.6±1.1%). There was no significant positive correlation between ß₁-receptor density and age in either the NF (r=-.42, n=19, P=.08), IDC (r=-.18, n=31, P=.32), or ISC groups (r=-.23, n=21, P=.33). Comparisons among male subjects showed that the ß₁-receptor density remained significantly decreased in the IDC but not the ISC group, with no significant change in ß₂-receptor density in either group (NF [n=8]: ß₁ 55.6±5.5, ß₂ 17.9±2.9; IDC [n=22]: ß₁ 33.3±2.5 [P<.01 versus NF], ß₂ 17.9±1.7; ISC [n=21]: ß₁ 48.4±3.2, ß₂ 20.3±1.2 fmol/mg). Furthermore, comparisons within the ISC group showed that LVs from the worse-function subgroup appeared to have slightly lower ß₁-receptor density and slightly lower ß₂-receptor density than those from the better-function subgroup (Table 2).

View larger version (39K): [in this window] [in a new window]   Figure 4. Summary of subtype densities (A) and fractions (B) of ß-adrenergic receptors in NF and failing human LVs. Bmax indicates total ß-receptor density; Beta1, ß1-receptor subtype; Beta2, ß2-receptor subtype; and combined F, combined (IDC+ISC) failing group.

There was a significant positive correlation between AT₁ and ß₁-receptor densities in a combined group of all LVs (Fig 5). Correlations within each of the three groups were as follows: NF, r=.22 (P=.36); IDC, r=.28 (P=.13); and ISC, r=.39 (P=.08), respectively.

View larger version (21K): [in this window] [in a new window]   Figure 5. Relation between the density of the Ang-II receptor type 1 subtype (AT1) and that of ß1-adrenergic receptors (Beta1) in crude membranes prepared from NF and failing LVs with IDC (n=71).

Membrane Marker Measurements
There was no significant difference in Mn²⁺-stimulated adenylyl cyclase activity (pmol·min^-1·mg protein^-1) between the NF and the IDC LV membranes (NF [n=15]: basal 3.8±1.4, stimulated 333±63; IDC [n=27]: basal 4.1±0.8, stimulated 378±30; P=NS).

In Situ RT-PCR
In situ RT-PCR was performed in two NF LVs, three IDC LVs, three ISC LVs, and three ISC RVs in the present study population. Hematoxylin-and-eosin–stained sections (data not shown) demonstrated no obvious pathological change in the NF LVs. In the IDC LVs, myocyte hypertrophy with marked nuclear pleomorphism and focal mild interstitial fibrosis was observed. In the ISC ventricles, extensive interstitial fibrosis with moderate myocyte hypertrophy was observed.

Fig 6 shows representative examples of in situ PCR performed in one NF LV, one IDC LV, one ISC RV, and one ISC LV. When in situ PCR was performed without the addition of reverse transcriptase, sections from all ventricles exhibited no staining (negative control, Fig 6A). When DNase was omitted before the RT-PCR reactions, sections from all ventricles exhibited intense nuclear staining in myocytes and interstitial cells, with little or no signal identified in the cytoplasm (positive control, Fig 6B). As shown in Fig 6C (NF LV), 6D (IDC LV), 6E (ISC RV), and 6F (ISC LV), in situ RT-PCR with AT₁-receptor primers demonstrated the signal in both the myocytes and the interstitium. Diffuse myocyte cytoplasmic staining was observed with myocyte cell membranes strongly highlighted in both NF and failing ventricles. In the interstitium, the staining in vessel walls was particularly prominent. In some cells, nuclear RNA was stained due to detection of hnRNA. Quantitative comparisons cannot be made between different tissue samples because the in situ PCR technique was performed at different times.

View larger version (683K): [in this window] [in a new window]   Figure 6. In situ RT-PCR with AT1-receptor primers in NF and failing human ventricles. A, Negative control (without reverse transcription) (magnification x50). B, Positive control (without DNase) (magnification x50). C, NF LV: diffuse myocyte cytoplasmic staining, stronger myocyte cell membrane staining, and intense interstitial vessel wall staining were noted (magnification x100). D, IDC LV: myocyte cytoplasmic and cell membrane staining with intense staining of interstitial vessel walls (magnification x100). E, ISC RV and (F) ISC LV: diffuse myocyte cytoplasmic staining with more intense staining of myocyte cell membrane and interstitial vessel walls (magnification x100).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Previous reports on the regulation of Ang-II receptors in normal and diseased human hearts are in conflict.^{6 7} The present study considered the possibility that cardiac Ang-II–receptor regulation may exhibit disease-specific effects (IDC versus ISC) and that differences in age/sex distribution of subjects and in assay conditions may affect the findings. A high-yield crude membrane fraction was prepared because receptor density can be greatly affected by membrane yield when receptor density is low. ¹²⁵I-saralasin was used because this radioligand is known to identify a single class of Ang-II receptors with high affinity.^{16 21} Although the existence of other Ang-II–receptor subtypes (non-AT₁/AT₂) has been suspected in some tissue,⁹ the sum of the ¹²⁵I-saralasin binding displaceable by 1 µmol/L losartan and 1 µmol/L PD123319 was comparable to the binding displaceable by unlabeled saralasin or Ang-II (Fig 2), indicating that only AT₁ and AT₂ subtypes were detected by our assay conditions.

In the present study, receptor subtype analysis revealed a selective decrease in AT₁-receptor density in failing human ventricles. Mn²⁺-stimulated adenylyl cyclase activity, a membrane marker,^{3 14} was not different in membranes from the NF and IDC LVs in which the decrease in AT₁ was more pronounced. Therefore, the decrease in AT₁-receptor density in IDC LVs is not explained by a general loss of membrane proteins. In addition, decreased AT₁-receptor density in membranes from the IDC LVs did not seem to be due to receptor sequestration into a light vesicle fraction in which the AT₁ receptors also appeared to be selectively decreased.

Regitz-Zagrosek et al⁷ recently reported that the AT₂ subtype predominated in membranes from NF human right atrium and that AT₁ and AT₂ subtypes were downregulated to a comparable degree in failing right atrium. Although direct comparisons cannot be made between the two studies because Regitz-Zagrosek et al did not report data from NF ventricles, there may be chamber-specific regulation in cardiac Ang-II–receptor subtypes. Alternatively, membrane protein yield may account for the discrepancy. Because we did not use low-speed centrifugation before high-speed centrifugation to prepare membrane fractions as did Regitz-Zagrosek et al, our membrane yield was likely to be substantially higher than theirs inasmuch as an initial low-speed centrifugation markedly decreases the yield of membrane-associated ß-receptors.¹

The significant positive correlation between AT₁ and ß₁-receptor densities in all LVs suggests a possibility that the downregulation of the two receptor systems is biologically related. Local chamber-specific activation of the adrenergic system in failing human heart has been demonstrated previously.³ In addition, we and others have recently shown that ACE activity²² and mRNA abundance^{4 22} are increased in the failing human heart with IDC. The finding that ACEI treatment did not significantly affect Ang-II–receptor regulation in failing LVs is not necessarily evidence against activation of the cardiac renin-angiotensin system because a component of Ang-II generation in human cardiac tissue may be ACE independent,²³ and oral ACEI treatment may not completely inhibit cardiac ACE activity.²⁴ Because increased levels of first-messenger agonists are the most likely explanation for downregulation of AT₁ and ß₁-receptors, our present findings suggest an interaction between coactivation/induction of the renin-angiotensin and adrenergic systems in the failing human heart.

The exact mechanisms responsible for the selective downregulation of AT₁ receptors are not clear. Only the AT₁ subtype is reportedly guanine-nucleotide sensitive,²⁵ which may be a condition required for downregulation inasmuch as the AT₁ subtype may have a higher agonist-binding affinity. Differences may also exist in the molecular mechanisms that regulate gene expression of the AT₁ and AT₂ subtypes.²⁶ Haywood et al²⁷ recently demonstrated that AT₁- but not AT₂-receptor mRNA abundance is also selectively downregulated in failing human ventricles, providing a molecular explanation for the selective decrease in AT₁-receptor density.

The decrease in AT₁ density was less in ISC LVs (17% compared with 41% in IDC LVs) and was not statistically significant compared with NF LVs. This pattern is similar to ß₁-receptor downregulation in the present study and in our previous report.²⁸ The difference in degree of AT₁ downregulation between the IDC and ISC LVs did not appear to be related to differences in sex distribution or age. Subjects with ISC had slightly better global ventricular function (Table 1). In addition, the ISC LVs with worse ventricular function tended to have lower AT₁-receptor density than those with better function (Table 2). Therefore, a difference in global ventricular function may account for the lesser AT₁ downregulation in the ISC group.

We demonstrated the presence of AT₁ mRNA in ventricular myocytes and interstitial cells in both NF and failing ventricles, indicating AT₁-receptor gene expression in cardiac myocytes and nonmyocytes as previously reported.^{6 7 26} The cell specificity of AT₁-receptor gene regulation in failing ventricles will need to be elucidated by future studies.

In conclusion, the AT₁-subtype receptor is selectively downregulated in failing human ventricles. AT₁ receptors are known to mediate important Ang-II effects in the myocardium, ie, inotropic,¹⁶ hypertrophic,²⁹ and cardiotoxic effects.³⁰ Our data suggest that in the end-stage failing human heart, the AT₁-receptor pathway is being exposed to increased concentrations of Ang-II, even with ACEI treatment. This argues for improved methods of inhibiting the cardiac renin-angiotensin system in diseased hearts.^{5 31} Finally, AT₂ receptors constitute a relatively large proportion of Ang-II receptors in failing human ventricles. If this subtype is ultimately shown to mediate adverse biological effects of Ang-II, such as apoptosis,^{32 33} this receptor pathway would warrant specific therapeutic targeting in the failing human heart.

	Selected Abbreviations and Acronyms

 

125I-ICYP = (-)-[125I]iodocyanopindolol 125I-saralasin = 125I-[Sar1,Ile8]angiotensin II ACEI = angiotensin-converting enzyme inhibitor Ang-II = angiotensin II AT1 = angiotensin II receptor type 1 subtype AT2 = angiotensin II receptor type 2 subtype Bmax = maximum radioligand binding sites DEPC = diethyl pyrocarbonate IDC = idiopathic dilated cardiomyopathy ISC = ischemic cardiomyopathy LV = left ventricle NF = nonfailing PCR = polymerase chain reaction RT-PCR = reverse transcription–polymerase chain reaction RV = right ventricle

	Acknowledgments

 
Dr Asano was a recipient of a fellowship from the Uehara Memorial Foundation, Tokyo, Japan. The present study was supported in part by NIH grants HL-48013 and HL-13108, and a grant from Merck and Co. We greatly appreciate the DuPont-Merck Pharmaceutical Co for supplying losartan, Parke-Davis for supplying PD123319, and Ciba-Geigy for supplying CGP20712A. We thank Patti Larrabee, Mary Beth Hagan, and Patrice Mealey for help in collecting patient information. We also thank Frank Stewart, Susan Veach, and Becky Olson for help in manuscript preparation.

Received September 16, 1996; revision received November 13, 1996; accepted November 25, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Bristow MR, Ginsburg R, Minobe W, Cubicciotti RS, Sageman WS, Lurie K, Billingham ME, Harrison DC, Stinson EB. Decreased catecholamine sensitivity and ß-adrenergic receptor density in failing human hearts. N Engl J Med. 1982;307:205-211.[Abstract]
   
2. Bristow MR. Changes in myocardial and vascular receptors in heart failure. J Am Coll Cardiol. 1993;22(suppl A):61A-71A.
   
3. Bristow MR, Minobe W, Rasmussen R, Larrabee P, Skerl L, Klein JW, Anderson FL, Murray J, Mestroni L, Karwande SV, Fowler M, Ginsburg R. ß-Adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms. J Clin Invest. 1992;89:803-815.
   
4. Studer R, Reinecke H, Muller B, Holtz J, Just H, Drexler H. Increased angiotensin-1 converting enzyme gene expression in the failing human heart: quantification by competitive RNA polymerase chain reaction. J Clin Invest. 1994;94:301-310.
   
5. Hirsch AT, Talsness CE, Schunkert H, Paul M, Dzau VJ. Tissue-specific activation of cardiac angiotensin converting enzyme in experimental heart failure. Circ Res. 1991;69:475-482.[Abstract/Free Full Text]
   
6. Urata H, Healy B, Stewart RW, Bumpus M, Husain A. Angiotensin II receptors in normal and failing human hearts. J Clin Endocrinol Metab. 1989;69:54-66.[Abstract]
   
7. Regitz-Zagrosek V, Friedel N, Heymann A, Bauer P, Neuß M, Rolfs A, Steffen C, Hildebrandt A, Hetzer R, Flecke E. Regulation, chamber localization, and subtype distribution of angiotensin II receptors in human hearts. Circulation. 1995;91:1461-1471.[Abstract/Free Full Text]
   
8. Bumpus FM, Catt KJ, Chiu AT, DeGasparo M, Goodfriend T, Husain A, Peach MJ, Taylor DG, Timmermans PBMWM. Nomenclature for angiotensin receptors. Hypertension. 1991;17:720-723.[Free Full Text]
   
9. Timmermans PBMWM, Wong PC, Chieu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JAM, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev. 1993;45:205-251.[Medline] [Order article via Infotrieve]
   
10. Xiang JZ, Linz W, Becker H, Ganten D, Lang RE, Scholkens B, Unger T. Effects of converting enzyme inhibitors ramipril and enalapril on peptide action and sympathetic neurotransmission in the isolated heart. Eur J Pharmacol. 1985;113:215-223.[Medline] [Order article via Infotrieve]
    
11. Bristow MR, Abraham WT. Anti-adrenergic effects of angiotensin converting enzyme inhibitors. Eur Heart J. 1995;16(suppl K):37-41.
    
12. Nagano M, Higaki J, Nakamura F, Higashimori K, Nagano N, Mikami H, Ogihara T. Role of cardiac angiotensin II in isoproterenol-induced left ventricular hypertrophy. Hypertension. 1992;19:708-712.[Abstract/Free Full Text]
    
13. Bristow MR, Ginsburg R, Umans V, Fowler M, Minobe W, Rasmussen R, Zera P, Menlove R, Shar P, Jamieson S, Stinson EB. ß₁- and ß₂-adrenergic receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective ß₁-receptor downregulation in heart failure. Circ Res. 1986;59:297-309.[Abstract/Free Full Text]
    
14. White M, Roden R, Minobe W, Khan MF, Larrabee P, Wollmering M, Port JD, Anderson F, Campbell D, Feldman AM, Bristow MR. Age-related changes in ß-adrenergic neuroeffector systems in the human heart. Circulation. 1994;90:1225-1238.[Abstract/Free Full Text]
    
15. Koziarz P, Moore GJ. Inhibition of enzymatic degradation of angiotensin II in membrane binding assays: utility of 1,10-phenanthroline. Biochem Cell Biol. 1990;68:218-220.[Medline] [Order article via Infotrieve]
    
16. Scott AL, Chang RSL, Lotti VJ, Siegl PKS. Cardiac angiotensin II receptors: effects of selective angiotensin II receptor antagonists, DUP753 and PD121981, in rabbit heart. J Pharmacol Exp Ther. 1992;261:931-935.[Abstract/Free Full Text]
    
17. Dooley DJ, Bittiger H, Reymann NC. CGP20712A: a useful tool for quantitating ß₁- and ß₂-adrenoceptors. Eur J Pharmacol. 1986;130:137-139.[Medline] [Order article via Infotrieve]
    
18. Peterson GL. A simplification of the protein assay method of Lowry et al, which is more generally applicable. Anal Biochem. 1977;83:346-356.[Medline] [Order article via Infotrieve]
    
19. Munson PJ, Rodbard D. LIGAND: a versatile computerized approach for characterization of ligand-binding system. Anal Biochem. 1980;107:220-239.[Medline] [Order article via Infotrieve]
    
20. Nuovo G. RT in situ PCR with direct incorporation of digoxigenin-11-dUTP: protocol and applications. Biochemica. 1994 (Winter);11:4-6.
    
21. De Lean A, Ong H, Gutkowska J, Schiller PW, McNicoll N. Evidence for agonist-induced interaction of angiotensin II receptor with a guanine nucleotide-binding protein in bovine adrenal zona glomerulosa. Mol Pharmacol. 1984;26:498-508.[Abstract]
    
22. Zisman L, Bush EW, Taft CS, Bristow MR, Perryman MB, Raynolds MV. Increase in angiotensin converting enzyme gene expression and activity in the failing human ventricle. Circulation. 1994;90(part 2):I-578. Abstract.
    
23. Urata H, Healy B, Stewart RW, Bumpus M, Husain A. Angiotensin II-forming pathways in normal and failing human hearts. Circ Res. 1990;66:883-890.[Abstract/Free Full Text]
    
24. Cushman DW, Wang FL, Fung WC, Harvey CM, DeForrest JM. Differentiation of angiotensin-converting enzyme (ACE) inhibitors by their selective inhibition of ACE in physiologically important target organs. Am J Hypertens. 1989;2:294-306.[Medline] [Order article via Infotrieve]
    
25. Sechi LA, Griffin CA, Grady EF, Kalinyak JE, Schambelan M. Characterization of angiotensin II receptor subtypes in rat heart. Circ Res. 1992;71:1482-1489.[Abstract/Free Full Text]
    
26. Matsubara H, Kanasaki M, Murasawa S, Tsukaguchi Y, Nio Y, Inada M. Differential gene expression and regulation of angiotensin II receptor subtypes in rat cardiac fibroblasts and cardiomyocytes in culture. J Clin Invest. 1994;93:1592-1601.
    
27. Haywood GA, Gullestad L, Katsuya T, Hutchinson HH, Pratt RE, Horiuchi M, Fowler MB. AT₁ and AT₂ angiotensin receptor gene expression in human heart failure. Circulation. 1997;95:1201-1206.[Abstract/Free Full Text]
    
28. Bristow MR, Anderson FL, Port JD, Skerl L, Hershberger RE, Larrabee P, O'Connel JB, Renlud DG, Volkman K, Murray J, Feldman AM. Differences in ß-adrenergic neuroeffector mechanisms in ischemic versus idiopathic dilated cardiomyopathy. Circulation. 1991;84:1024-1039.[Abstract/Free Full Text]
    
29. Dostal DE, Baker KM. Angiotensin II stimulation of left ventricular hypertrophy in adult rat heart: mediation by the AT₁ receptor. Am J Hypertens. 1992;5:276-280.[Medline] [Order article via Infotrieve]
    
30. Kabour A, Henegar JR, Janicki JS. Angiotensin II (AII)-induced myocyte necrosis: role of the AII receptor. J Cardiovasc Pharmacol. 1994;23:547-553.[Medline] [Order article via Infotrieve]
    
31. Baker KM, Booz GW, Dostal DE. Cardiac actions of angiotensin II: role of an intracardiac renin-angiotensin system. Annu Rev Physiol. 1992;54:227-241.[Medline] [Order article via Infotrieve]
    
32. Tanaka M, Ohnishi J, Ozawa Y, Sugimoto M, Usuki S, Naruse M, Murakami K, Miyazaki H. Characterization of angiotensin II receptor type 2 during differentiation and apoptosis of rat ovarian cultured granulosa cells. Biochem Biophys Res Commun. 1995;207:593-598.[Medline] [Order article via Infotrieve]
    
33. Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci U S A. 1996;93:156-160.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				J. van der Velden, N. A. Narolska, R. R. Lamberts, N. M. Boontje, A. Borbely, R. Zaremba, J. G.F. Bronzwaer, Z. Papp, K. Jaquet, W. J. Paulus, et al. Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium Cardiovasc Res, March 1, 2006; 69(4): 876 - 887. [Abstract] [Full Text] [PDF]

				A. Modesti, I. Bertolozzi, T. Gamberi, M. Marchetta, C. Lumachi, M. Coppo, F. Moroni, T. Toscano, G. Lucchese, G. F. Gensini, et al. Hyperglycemia Activates JAK2 Signaling Pathway in Human Failing Myocytes via Angiotensin II-Mediated Oxidative Stress Diabetes, February 1, 2005; 54(2): 394 - 401. [Abstract] [Full Text] [PDF]

				M. Nakayama, X. Yan, R. L. Price, T. K. Borg, K. Ito, A. Sanbe, J. Robbins, and B. H. Lorell Chronic ventricular myocyte-specific overexpression of angiotensin II type 2 receptor results in intrinsic myocyte contractile dysfunction Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H317 - H327. [Abstract] [Full Text] [PDF]

				P. van der Harst, M. Volbeda, A. A. Voors, H. Buikema, S. Wassmann, M. Bohm, G. Nickenig, and W. H. van Gilst Vascular Response to Angiotensin II Predicts Long-Term Prognosis in Patients Undergoing Coronary Artery Bypass Grafting Hypertension, December 1, 2004; 44(6): 930 - 934. [Abstract] [Full Text] [PDF]

				A. Goette and U. Lendeckel Expression of angiotensin II receptors in human left and right atrial tissue in atrial fibrillation with and without underlying mitral valve disease J. Am. Coll. Cardiol., June 16, 2004; 43(12): 2363 - 2363. [Full Text] [PDF]

				M. Yousufuddin, S. Haji, R. C. Starling, E. M. Tuzcu, N. B. Ratliff, D. J. Cook, A. Abdo, Y. Saad, S. S. Karnik, D. Wang, et al. Cardiac angiotensin II receptors as predictors of transplant coronary artery disease following heart transplantation Eur. Heart J., March 1, 2004; 25(5): 377 - 385. [Abstract] [Full Text] [PDF]

				R. Latini, S. Masson, I. Anand, M. Salio, A. Hester, D. Judd, S. Barlera, A. P Maggioni, G. Tognoni, J. N Cohn, et al. The comparative prognostic value of plasma neurohormones at baseline in patients with heart failure enrolled in Val-HeFT Eur. Heart J., February 2, 2004; 25(4): 292 - 299. [Abstract] [Full Text] [PDF]

				A. Boldt, U. Wetzel, J. Weigl, J. Garbade, J. Lauschke, G. Hindricks, H. Kottkamp, J. F. Gummert, and S. Dhein Expression of angiotensin II receptors in human left and right atrial tissue in atrial fibrillation with and without underlying mitral valve disease J. Am. Coll. Cardiol., November 19, 2003; 42(10): 1785 - 1792. [Abstract] [Full Text] [PDF]

				X. Yan, R. L. Price, M. Nakayama, K. Ito, A. J. T. Schuldt, W. J. Manning, A. Sanbe, T. K. Borg, J. Robbins, and B. H. Lorell Ventricular-specific expression of angiotensin II type 2 receptors causes dilated cardiomyopathy and heart failure in transgenic mice Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H2179 - H2187. [Abstract] [Full Text] [PDF]

				L. S. Zisman, R. S. Keller, B. Weaver, Q. Lin, R. Speth, M. R. Bristow, and C. C. Canver Increased Angiotensin-(1-7)-Forming Activity in Failing Human Heart Ventricles: Evidence for Upregulation of the Angiotensin-Converting Enzyme Homologue ACE2 Circulation, October 7, 2003; 108(14): 1707 - 1712. [Abstract] [Full Text] [PDF]

				R. M. Carey and H. M. Siragy Newly Recognized Components of the Renin-Angiotensin System: Potential Roles in Cardiovascular and Renal Regulation Endocr. Rev., June 1, 2003; 24(3): 261 - 271. [Abstract] [Full Text] [PDF]

				J van der Velden, Z Papp, R Zaremba, N.M Boontje, J.W de Jong, V.J Owen, P.B.J Burton, P Goldmann, K Jaquet, and G.J.M Stienen Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins Cardiovasc Res, January 1, 2003; 57(1): 37 - 47. [Abstract] [Full Text] [PDF]

				A. Goette, U. Lendeckel, and H. U Klein Signal transduction systems and atrial fibrillation Cardiovasc Res, May 1, 2002; 54(2): 247 - 258. [Abstract] [Full Text] [PDF]

</

				D. Schultz, X. Su, C.-C. Wei, S. P. Bishop, P. Powell, G. H. Hankes, A. R. Dillon, P. Rynders, F. G. Spinale, G. Walcott, et al. Downregulation of ANG II receptor is associated with compensated pressure-overload hypertrophy in the young dog Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H749 - H756. [Abstract] [Full Text] [PDF]