Selective Downregulation of the Angiotensin II AT1-Receptor Subtype in Failing Human Ventricular Myocardium
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. β-Adrenergic receptors were also measured by binding of 125I-iodocyanopindolol with the β1 and β2 fractions determined by use of a β1-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). β1 but not β2 density was decreased in the failing left ventricles. AT1 density was correlated with β1 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 β1 receptors. The relative lack of AT1 downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.
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 β-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 β-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 β-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.
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 γ counting (model 1470 WIZARD, Wallac).
β-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 β1- and β2-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 β1-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 β1-receptors and β2-receptors, respectively.3 This method yields β1- and β2-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. Bmax and dissociation constant (Kd) 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 Bmax by each subtype's fractions.
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).
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.
Fifty microliters of the following reaction mixture was placed over two of three tissue sections: 5 μL of 10× 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.
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.
The following master mix was prepared for a final volume of 50 μL per section: 32 μL water, 5 μL 10× 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 seconds→20 cycles at 94°C for 40 seconds, 55°C for 90 seconds→4°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.
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.
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.
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.
Ang-II Receptors in NF and Failing Ventricular Myocardium
Comparisons Between NF and Combined Failing LVs
Fig 3A⇓ compares total Ang-II receptors (Bmax) and subtype densities in the NF, IDC, ISC, and combined failing (IDC+ISC) groups. The Bmax 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]: Bmax 7.39±0.57, AT1 4.66±0.48, and AT2 2.73±0.39; failing [n=52]: Bmax 5.90±0.44, AT1 3.20±0.29, and AT2 2.70±0.33 fmol/mg). The Kd 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 Bmax, AT1, or AT2 density within the failing group (with ACEI [n=28]: Bmax 5.72±0.46, AT1 3.21±0.38, and AT2 2.51±0.41; without ACEI [n=24]: Bmax 6.10±0.79, AT1 3.18±0.47, and AT2 2.92±0.53 fmol/mg).
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 Kd 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 Bmax, 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.
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]: Bmax 7.45±1.45, AT1 4.14±1.43, and AT2 3.31±0.69 versus IDC [n=3]: Bmax 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).
β-Adrenergic Receptors in NF and Failing LVs
Fig 4A⇓ compares total β-receptor density (Bmax) and subtype densities in LVs of the three groups. The Bmax and the β1-receptor density were significantly (P<.001) decreased in the combined failing group, with no significant difference in β2-receptor density (NF: Bmax 77.5±3.6, β1 58.8±3.7, and β2 18.8±1.6; failing: Bmax 59.5±2.4, β1 40.5±2.0, and β2 18.9±0.9 fmol/mg). The Kd 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 Bmax and the β1-receptor density were significantly (by ANOVA) decreased in the IDC group but not the ISC group, with no significant difference in β2-receptor density among the three groups (IDC [n=31]: Bmax 53.2±2.4, β1 35.2±2.0, and β2 17.9±1.3; ISC [n=21]: Bmax 68.7±4.1, β1 48.4±3.2, and β2 20.3±1.2 fmol/mg). As a result, the β1- and β2-receptor fractions (Fig 4B⇓) were significantly (P<.01) different among the three groups (NF: β1 75.2±2.0%, β2 24.8±2.0%; IDC: β1 65.7±2.2%, β2 34.3±2.2%; ISC: β1 70.4±1.1%, β2 29.6±1.1%). There was no significant positive correlation between β1-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 β1-receptor density remained significantly decreased in the IDC but not the ISC group, with no significant change in β2-receptor density in either group (NF [n=8]: β1 55.6±5.5, β2 17.9±2.9; IDC [n=22]: β1 33.3±2.5 [P<.01 versus NF], β2 17.9±1.7; ISC [n=21]: β1 48.4±3.2, β2 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 β1-receptor density and slightly lower β2-receptor density than those from the better-function subgroup (Table 2⇑).
There was a significant positive correlation between AT1 and β1-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.
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.
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 β-receptors.1
The significant positive correlation between AT1 and β1-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 abundance4 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 β1-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 β1-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
|ACEI||=||angiotensin-converting enzyme inhibitor|
|AT1||=||angiotensin II receptor type 1 subtype|
|AT2||=||angiotensin II receptor type 2 subtype|
|Bmax||=||maximum radioligand binding sites|
|IDC||=||idiopathic dilated cardiomyopathy|
|PCR||=||polymerase chain reaction|
|RT-PCR||=||reverse transcription–polymerase chain reaction|
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.
- Copyright © 1997 by American Heart Association
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