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(Circulation Research. 1996;78:302-311.)
© 1996 American Heart Association, Inc.

Articles

Endothelin-1 and Angiotensin II Receptors in Cells From Rat Hypertrophied Heart

Receptor Regulation and Intracellular Ca²⁺ Modulation

Jeannette Fareh, Rhian M. Touyz, Ernesto L. Schiffrin, Gaetan Thibault

From the MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal (Canada).

	Abstract

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Abstract This study investigates the cellular localization and regulation of endothelin-1 (ET-1) and angiotensin II (Ang II) receptors and the effects of ET-1 and Ang II on [Ca²⁺]_i in cardiac hypertrophy due to volume overload in the rat. Radioligand binding assays and [Ca²⁺]_i measurements by fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from the heart of rats with a 4-week aortocaval shunt. In the hypertrophied myocardium, ET-1 and Ang II concentrations were unchanged in ventricles. Ventricular ET-1 receptors had a cell-specific distribution: >90% of ET receptors in cardiomyocytes are of the ET_A subtype, whereas fibroblasts had a nearly equal proportion of the ET_A and ET_B subtypes. ET-1 receptor densities, affinities, and ET-1–induced [Ca²⁺]_i were not significantly different from control in both ventricular cell types from hypertrophied myocardium. Ang II specific binding was very low on isolated ventricular cardiomyocytes, suggesting few receptors in control conditions. However, [Ca²⁺]_i responses induced by Ang II at concentrations >10^-8 mol/L were detectable and were significantly higher in hypertrophied cardiomyocytes. Ang II receptor density (exclusively AT₁) on fibroblasts was significantly reduced (42 970±3330 versus 73 870±7940 sites per cell for control cells, P<.01), but AT₁ receptor affinity was unchanged after volume overload. The maximum increase in [Ca²⁺]_i evoked by 10^-6 to 10^-4 mol/L Ang II was significantly lower in fibroblasts from overloaded hearts. In conclusion, ET-1 receptor proportion is cell specific, with cardiomyocytes possessing predominantly the ET_A subtype and fibroblasts possessing both ET_A and ET_B receptors. Plasma and cardiac ET-1 concentrations and ET-1 receptor regulation on both ventricular cell types are not altered in cardiac volume overload, suggesting that cardiac ET-1 may not play a significant role in this model. Cardiac hypertrophy induced a significant downregulation of AT₁ receptors on fibroblasts, whereas total binding and [Ca²⁺]_i sensitivity to Ang II were significantly enhanced in hypertrophied cardiomyocytes. This suggests that cardiac Ang II may be involved in the pathophysiology of the cardiac hypertrophy of volume overload.

Key Words: cardiac hypertrophy • adult cardiomyocytes • ET-1 • intracellular Ca²⁺ • angiotensin II

	Introduction

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Chronic volume or pressure overload induces a series of cellular and molecular events leading to an adaptive cardiac hypertrophy. The hypertrophic response is characterized by increased cardiomyocyte cell volume, proliferation of nonmyocyte cells (mainly fibroblasts), activation of the fetal gene expression (myosin light chain 2, skeletal -actin, ß-myosin heavy chain, ANP, and BNP) and proto-oncogenes,¹ and altered [Ca²⁺]_i regulation.²

ET-1 and Ang II are two potent vasoactive peptides that have an extensive tissue distribution, including the heart. ET-1 and Ang II exert similar effects on myocardial growth, heart rate, and cardiac contractility. More precisely, ET-1^{3 4 5 6} and Ang II^{3 7} induce the hypertrophic phenotype in cultured cardiomyocytes. ET-1 and Ang II seem to interact in trophic cardiomyocyte events.⁴ Several lines of evidence based on cardiac expression of preproET-1 gene,^{4 8 9} as well as renin, angiotensinogen, and angiotensin-converting enzyme genes,^{10 11} suggest the existence of a local ET production and a local renin-angiotensin system in the myocardium. Therefore, intracardiac ET-1 and Ang II, acting probably in an autocrine/paracrine fashion, may exert hypertrophic effects on the heart that are independent of blood pressure regulation.

Recent studies performed on various tissues, including rat heart, have revealed the presence of ET-1 and its receptors in the heart.^{12 13 14 15 16} Two receptor subtypes (ET_A and ET_B) are well identified.^{17 18} The ET_A subtype binds with high-affinity ET-1 and ET-2 but not ET-3 and may be selectively blocked by the ET antagonist BQ123. The ET_B subtype presents an equal affinity for the three isopeptides. However, little information on the cellular distribution and regulation of cardiac ET-1 receptor subtypes is available.^{16 19 20} Various cardiac effects, such as trophic effects,²¹ collagen deposition in hypertrophied hearts,²² and induction of ANP secretion,²⁰ are mediated via ET_A subtype receptors.

Previous studies have reported the existence of at least two subtypes of Ang II cell surface receptors (AT₁ and AT₂) in various peripheral tissues as well as in the brain. The receptor antagonist DuP 753 (losartan) blocks selectively the AT₁ receptor subtype, whereas PD 123177 or CGP 42112A are selective for the AT₂ receptor subtype. In the rat myocardium, Ang II receptors have been identified, but the AT₁ and AT₂ subtype proportion remains controversial,^{23 24 25 26} probably because most previous investigations were performed on the whole heart, whereas cardiac tissue is composed of several cell types, including cardiomyocytes, fibroblasts, endothelial cells, vascular smooth muscle cells, and neurons. To our knowledge, few studies involving Ang II receptors and their distribution on cardiac cells have been undertaken.^{7 24 27} It has been reported in vivo²⁸ and in vitro⁷ that hypertrophic effects of Ang II are mainly mediated via the AT₁ subtype.

Intracellular mechanisms underlying ET-1 and Ang II actions include phospholipase C stimulation, resulting in the production of water-soluble inositol phosphates and diacylglycerol, which induce an increase in intracellular Ca²⁺ and protein kinase C activation, respectively. Inotropic and chronotropic effects of ET-1 and Ang II on cardiomyocytes have been shown to be mediated mainly by phospholipase C and intracellular Ca²⁺ modulation.^{29 30} However, the exact intracellular signaling pathway underlying ET-1– and Ang II–induced hypertrophic responses remains to be elucidated.

The aims of the present study were (1) to determine the role of local cardiac ET-1 and Ang II in stable rat cardiac hypertrophy due to chronic volume overload (aortocaval shunt model of 4 weeks), (2) to characterize ET-1 and Ang II receptors on adult rat cardiomyocytes and fibroblasts in control conditions, (3) to investigate the regulation of cardiac ET-1 and Ang II cellular receptor subtypes in stable cardiac hypertrophy, and (4) to establish the effects of ET-1 and Ang II on [Ca²⁺]_i in ventricular cardiomyocytes and fibroblasts from hypertrophied hearts.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peptides and Reagents
ET-1, S6c, and [Sar¹,Ile⁸]Ang II were purchased from Peninsula Laboratories and Bachem California. BQ 123 was obtained from Peptide International Inc. DuP 753 and PD 123319 were generous gifts from the Du Pont Merck Pharmaceutical Co and Parke-Davis, respectively. All chemical products for cell isolation and culture were obtained from Sigma Chemical Co, Life Technology, and Fisher Scientific Co.

Animals and Aortocaval Shunt Surgery
Ninety male Sprague-Dawley rats (200 to 220 g, Charles River, St Constant, Quebec, Canada) were housed in a room with controlled temperature under a light/dark cycle of 12 hours. They were fed with standard rat chow and tap water ad libitum. The present experiment was in accordance with the guiding principles of the council of the Canadian Council on Animal Care. The surgical procedure to induce cardiac hypertrophy has been previously well described.³¹ Abdominal aortocaval shunt was performed with an 18-gauge disposable needle above the aortic bifurcation. Sham-operated rats (control group) were subjected to the same surgical procedure with no abdominal aortic and inferior vena caval puncture. Four weeks after chronic volume overload, rats were killed.

Adult Cardiomyocyte Isolation
Animals were first injected intraperitoneally with 500 U of heparin sulfate (Hepalean, Organon Canada Ltd) and anesthetized with pentobarbital sodium (60 mg/kg IP). The hearts were then rapidly removed. The Ca²⁺-tolerant cardiomyocytes were isolated by cardiac retrograde aortic perfusion (Langendorff method) as described previously by Eid et al.³² Briefly, hearts were rinsed (4 mL/min) for 5 minutes in KH solution containing (mmol/L) NaCl 118, KCl 4.7, CaCl₂ 1.25, MgSO₄-7H₂O 1.2, KH₂PO₄ 1.2, NaHCO₃ 25, and dextrose 11 at 37°C. Ca²⁺-free KH was then used for 5 minutes to stop spontaneous cardiac contraction. Hearts were next perfused for 20 minutes by 0.05% CLS2 (Worthington Biochemical Corp) and 0.03% hyaluronidase (Sigma) in KH buffer (solution A). Ventricles were then separated from atria and vessels. Ventricles were finely minced and incubated in solution A containing trypsin (0.2 mg/mL) and DNAse I (0.2 mg/mL) for 20 minutes at 37°C with agitation (120 cycles per minute). The cell suspension was filtered through a nylon mesh and centrifuged for 2 minutes at 1000g. Cells were diluted in 10 mL of washed solution (1:1 medium 199/KH) and were allowed to sediment at 1g for 20 minutes at room temperature. This procedure was repeated three times. Cells were then gently layered on 10 mL of 6% BSA solution to separate heavy cells (cardiomyocytes) from light cells (noncardiomyocytes and debris). Freshly isolated cells were gently diluted in sterile culture medium 199, pH 7.4, with 10% FBS. The culture medium (medium 199) contained 0.2% BSA, insulin (10^-7 mol/L), creatine (5 mmol/L), L-carnitine (2 mmol/L), taurine (5 mmol/L), penicillin (100 IU/mL), and streptomycin (100 µg/mL). For intracellular free Ca²⁺ determinations, ventricular cells were seeded onto round glass coverslips in culture dishes (7000 cells per 2 cm²), which had been coated previously with laminin for 1 hour at room temperature (3 µg/2 cm², Collaborative Research Inc). After 1 hour at 37°C (in a humidified incubator at 5% CO₂/95% air), the medium was changed to remove damaged cells (globular-shaped cells) and debris. In this way, we obtained 90% Ca²⁺-tolerant cardiomyocytes (rod-shaped cells) or 2 to 2.5x10⁶ cells per heart, which corresponds to >95% cardiomyocyte purity. Serum-free medium was added overnight, and the intracellular Ca²⁺ measurements were performed the following day. For radioligand binding assays, ventricular isolated cardiomyocytes (60% to 70% of rod-shaped cells) were resuspended in medium 199 (pH 7.4). Binding receptor studies were performed the same day on freshly suspended cells (15 000 cells per 100 µL) at room temperature. Preliminary studies showed that binding analysis performed immediately (on isolated cells) or 24 hours after cell isolation (on cultured cells) gave the same binding level (data not shown).

Immediately after the isolation procedure, adult cardiomyocytes from control and hypertrophied hearts were seeded onto round glass coverslips in culture dishes, and ventricular cells were observed on an inverted microscope and photographed to measure cell length and width (40 to 50 cells per preparation). Adult cardiomyocytes were considered to have a cylindrical shape to evaluate cell volume in cubic micrometers. Geometric dimensions (length and width) estimated are in accordance with a previous study performed with a computerized image analysis system.²⁴

Primary Culture of Adult Ventricular Fibroblasts
Animals were injected with 500 U of heparin sulfate and pentobarbital. After cardiac dissection, ventricles were removed from atria and large vessels and washed in sterile PBS solution containing 0.05 mol/L sodium phosphate and 0.9 g/dL NaCl. They were finely minced and digested in 15 mL DMEM containing 0.1% trypsin and 100 U/mL CLS2 (Worthington Biochemical Corp) at 37°C with agitation (150 cycles per minute) for 15 minutes.²⁷ After sedimentation at 1g, the remaining tissue was digested for 15 minutes. This procedure was repeated four times. After the fifth digestion, isolated cells were pooled and centrifuged for 3 minutes at 2000g. The pellet was resuspended in DMEM plus 10% FBS. The cell preparation was diluted in 150 mL DMEM/10% FBS and seeded in 24-well plates for binding assays and in six-well plates (onto glass coverslips) for intracellular Ca²⁺ assays. Cells were incubated for 2 hours at 37°C in a 10% CO₂/90% air humidified incubator. After the preplating step, nonadherent cells were removed, and fresh serum medium was added. The remaining cells (mostly fibroblasts) were grown until confluence (4 to 5 days, 2x10⁵ cells per 2 cm²). In a preliminary investigation, we found that primary cultured fibroblasts maintained their cell phenotype after 5 days in culture according to an immunostaining approach and that ET-1 and Ang II binding measurements were stable for a period of 2 to 7 days in culture (data not shown). Twenty-four hours before radioligand binding and intracellular Ca²⁺ assays, culture medium was replaced by serum-free medium. Before binding studies, attached cells were washed twice with DMEM (pH 7.2).

Indirect Immunofluorescence
Immunocytochemical staining was performed to estimate cell purity in cardiomyocyte and fibroblast preparations. Cultured cells were washed twice with PBS and fixed for 10 minutes at room temperature in 4% formaldehyde in PBS. Fixed cells were rinsed in 70% ethanol and then incubated for 10 minutes in cold acetone. The chamber slides were stored at 4°C in 70% ethanol until assay. After two PBS washings, the chamber slides were incubated for 30 minutes at room temperature with 1.5% normal goat serum to eliminate nonspecific sites. Cells were then incubated overnight at 4°C with either monoclonal antibodies directed against -sarcomeric actin, desmin, or vimentin (Sigma) or polyclonal antibody directed against human von Willebrand factor (factor VIII, Sigma). Anti-actin and -desmin antibodies are used to identify cardiomyocytes and smooth muscle cells, whereas anti-vimentin antibodies served to identify cardiac fibroblasts. Contaminating cells such as endothelial cells react with anti–von Willebrand factor antibody. After two washes with PBS, cells were incubated for 45 minutes at room temperature in the dark with the secondary antibody, ie, goat anti-mouse IgG or IgM fluorescein solution (1:100) or goat anti-rabbit IgG fluorescein solution (1:100). The chamber slides were rinsed twice with PBS and then rinsed with distilled water. Control reactions were performed by omitting the first specific antibody. Cells were then mounted with 90% glycerol in PBS. Immunoreactions were observed with an epifluorescence microscope.

Receptor Binding Assays
All binding reactions (in duplicate) were performed in the respective serum-free culture medium at room temperature for 90 minutes. ET-1 and [Sar¹,Ile⁸]Ang II were radiolabeled (¹²⁵I) by the lactoperoxidase method and purified by high-performance liquid chromatography.³³ For ET-1 receptors, the competitive binding reaction was determined in the presence of increasing concentrations (from 10^-12 to 10^-6 mol/L) of ET-1, BQ 123 (an ET_A-selective antagonist) or S6c (an ET_B-selective agonist), and 40 to 60 pmol/L iodinated ET-1 (1500 Ci/mmol). Volume of reaction was 0.25 and 0.5 mL for isolated cardiomyocytes (15 000 cells) and cultured fibroblasts (2x10⁵ cells), respectively. In saturation experiments, cells were incubated with increasing concentrations of ¹²⁵I-ET-1 (10 to 700 pmol/L), and nonspecific binding was determined with the same concentrations of iodinated ET-1 in the presence of 10^-6 mol/L unlabeled ET-1. Similarly, increasing concentrations (from 10^-12 to 10^-6 mol/L) of [Sar¹,Ile⁸]Ang II, DuP 753 (an AT₁-selective antagonist) or PD 123319 (an AT₂-specific ligand), and 100 to 120 pmol/L ¹²⁵I-[Sar¹,Ile⁸]Ang II (2200 Ci/mmol) were used for Ang II receptor characterization. Saturation experiments were performed by incubating ventricular cells with increasing concentrations of ¹²⁵I-[Sar¹,Ile⁸]Ang II (20 pmol/L to 2 nmol/L). Nonspecific binding was determined using unlabeled [Sar¹,Ile⁸]Ang II at 10^-6 mol/L.

For isolated cardiomyocyte binding experiments, the reaction was stopped with 3.5 mL of 50 mmol/L Tris-HCl (pH 7.2) and 0.15 mol/L NaCl. A rapid filtration through glass filters (Schleicher & Schuell) with a cell harvester (Brandel) was performed. Filters were rinsed three times with the same solution. Before filtration, filters were soaked in 5% dry skim milk (Carnation) to reduce nonspecific ET binding. After the binding reaction, attached fibroblasts were washed twice with 0.5 mL of culture medium (DMEM), and cells were digested by 0.5 mL of NaOH (1N). Radioactivity on filters or on digested cells was counted in a gamma counter with 80% efficiency (LKB Wallac). Binding data were analyzed using EBDA-LIGAND program software of McPherson³⁴ (Biosoft). Receptor densities are reported as numbers of sites per cell for isolated cardiomyocytes and primary cultured fibroblasts.

Radioimmunoassays: ANP-(1–98), ANP-(99–126), BNP, ET-1, Ang II, and Plasma Renin Activity
Plasma and cardiac peptide concentrations and PRA were measured by radioimmunoassay. Blood was collected in ice-chilled tubes containing EDTA and pepstatin, except for PRA determination, for which blood was collected with EDTA only. Blood samples were centrifuged at 1500g for 10 minutes at 4°C and stored at -20°C until assay. Plasma ANP-(1–98),³⁵ Ang II,³⁶ and ET-1³⁷ as well as PRA³⁸ determinations were assessed as previously described. Cardiac tissues were dissected (atria separated from ventricles) and rapidly frozen in liquid N₂. Ventricles and atria were homogenized with a Polytron tissue homogenizer in 4 mL of 1N HCl, 0.1% trifluoroacetic acid, 1% formic acid, and 1% NaCl solution for Ang II and ET-1 determinations and in boiling acetic acid (1 mol/L) for ANP-(99–126) and BNP. Samples were centrifuged at 30 000g for 15 minutes, and supernatants were extracted on C₁₈ Sep-Paks (Millipore Corp).

Measurements of [Ca²⁺]_i
Measurements of [Ca²⁺]_i were performed using fura 2 methodology as described previously.³⁹ Briefly, adult cardiomyocytes and fibroblasts were loaded with 4 µmol/L fura 2-AM for 30 minutes at 37°C in a humidified incubator with 95% air/5% CO₂ and washed three times with modified Hanks' buffer containing (mmol/L) NaCl 137, NaHCO₃ 4.2, NaHPO₄ 3, KCl 5.4, KH₂PO₄ 0.4, CaCl₂ 1.3, MgCl₂ 0.5, MgSO₄ 0.8, glucose 10, and HEPES 5 (pH 7.4). Fluorescence experiments were performed using the Axiovert 135 inverted microscope and Attofluor digital fluorescence system (Zeiss). Fluorescence measurements were assessed using double excitatory wavelengths (343 and 380 nm) and a single emission wavelength (510 nm).⁴⁰ After an equilibration period, cultured cells were exposed to single concentrations of ET-1 or Ang II (10^-12 to 10^-5 mol/L) at room temperature. [Ca²⁺]_i determinations were performed on cardiac cells from control and hypertrophied myocardium. In the resting and stimulated state, ventricular cardiomyocytes (Ca²⁺-tolerant rod-shaped cells) are characterized by spontaneous Ca²⁺ release from sarcoplasmic reticulum and contractile activity, corresponding to oscillatory intracellular Ca²⁺ waves. Ventricular fibroblasts, on the other hand, do not present any spontaneous contractile waves.³⁹

Statistical Analysis
All data are reported as mean±SEM. Peptide determinations were performed on 10 to 15 animals per group, and all binding and [Ca²⁺]_i experiments were performed on 5 animals per group. Statistical significance was evaluated using the unpaired Student's t test or Mann-Whitney test where appropriate. Concentration-response curves were fitted by nonlinear regression. The concentration in moles per liter giving 50% of the maximal [Ca²⁺]_i response (EC₅₀) was determined to calculate pD₂ as -log EC₅₀. [Ca²⁺]_i results are compared by ANOVA for repeated measures or by Student's t test where appropriate. The significance level was set at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Body and Tissue Weights
No significant differences were found between the body weight of shunted and sham-operated rats (Table 1). Absolute and relative total heart weights were significantly increased in 4-week–shunted rats (P<.001). A significant increase was found in the relative atrial and ventricular weights after volume overload (Table 1).

View this table: [in this window] [in a new window]   Table 1. Effects of a 4-Week Volume Overload on Body and Heart Weights

Plasma Peptide Concentrations and PRA
Ventricular hypertrophy induced by a 4-week aortocaval shunt was accompanied by a large increase in plasma ANP-(1–98) (1445±118 versus 600±25 pmol/L for sham-operated rats, P<.001) as well as a moderate augmentation in PRA (2.1±0.2 versus 1.6±0.1 ng/mL per hour for sham-operated rats, P<.05). Only aortocaval-shunted rats with such a high plasma ANP-(1–98) level were included in the present experiment, in agreement with previous studies.³¹ Plasma concentrations of ET-1 (2.87±0.18 versus 2.78±0.09 pmol/L for sham-operated rats) and Ang II (6.78±1.0 versus 7.21±1.2 ng/L for sham-operated rats) were not significantly different from control values after chronic volume overload.

Cardiac Peptide Concentrations
Ventricular ANP-(99–126) and BNP concentrations were significantly increased (P<.01) in hearts from shunted rats. No significant change was found in atrial ANP-(99–126) and BNP concentrations or in ventricular ET-1 and Ang II concentrations after chronic volume overload (Table 2).

View this table: [in this window] [in a new window]   Table 2. Peptide Cardiac Concentrations After Chronic Aortocaval Shunt

Cardiac Cell Characterization
Immunochemical staining of cell cultures showed that adult cardiomyocyte and cardiac fibroblast preparations presented a cell purity of >95%. In cardiac fibroblast cultures, positive staining obtained with anti-actin, anti-desmin, or anti–von Willebrand factor antibodies corresponded to <5%. Intact adult cardiomyocytes identified as rod-shaped cells (Ca²⁺-tolerant cells) reacted negatively to anti-vimentin and anti–von Willebrand antibodies.

Chronic volume overload induced a significant increase in cardiomyocyte length (141±6 versus 107±4 µm for control cells, P<.001) and no significant change in cell width (25±1.3 versus 22±1.4 µm for control cells). Thus, cell volume was increased after cardiac volume overload (7.2±0.8x10⁴ versus 4.2±0.5x10⁴ µm³ for control cells, P<.005), demonstrating that adult cardiomyocytes were significantly hypertrophied in volume-overloaded rats.

ET-1 Receptor Binding Assay
Saturation and competition curves were performed on isolated cardiomyocytes and cultured fibroblasts to determine binding properties (affinity and receptor density) and the subtype characteristics (ratio of ET_A to ET_B) of cardiac ET-1 receptors. Saturation of ET-1 receptors was obtained with between 0.25 to 0.35 nmol/L of iodinated ET-1 on cardiomyocytes and primary cultured fibroblasts (Fig 1). Scatchard analysis demonstrated a single class of binding sites on both ventricular cell types. The apparent dissociation constant (K_d) was estimated at 0.13±0.02 nmol/L on both ventricular cardiomyocytes (Fig 1A) and fibroblasts (Fig 1B). The maximum binding densities (B_max) were calculated at 167 890±25 800 sites per cell (83.6±13 fmol/mg protein) and 20 890±2030 sites per cell (21.5±2 fmol/mg protein) on cardiomyocytes (Fig 1A) and fibroblasts (Fig 1B), respectively. Competition analyses were performed using ET-1, BQ 123 (the ET_A-selective antagonist), and S6c (the ET_B-selective agonist). As shown in Fig 2A and Table 3, BQ 123 displaced >90% and S6c displaced <10% of the tracer in isolated cardiomyocytes, whereas in cultured fibroblasts, equal displacement was obtained with BQ 123 and S6c (Fig 2B). These data indicated that 90% of ET-1 receptors were of the ET_A subtype and 10% were of the ET_B subtype on cardiomyocytes, whereas on ventricular fibroblasts ET_A and ET_B subtypes were present in equal proportions.

View larger version (22K): [in this window] [in a new window]   Figure 1. Saturation binding of 125I-ET-1 and Scatchard transformation (inset) performed on adult ventricular cardiomyocytes (A) and fibroblasts (B). Bmax corresponds to maximum binding sites; Kd, to the dissociation constant. Binding analyses were performed on 15 000 cardiomyocytes or 2x105 cardiac fibroblasts. Five separate cell preparations from hearts from 5 different animals for each group were assessed. Results are mean±SEM.

View larger version (21K): [in this window] [in a new window]   Figure 2. Representative curves of competitive displacement of 125-I-ET-1 on adult ventricular cardiomyocytes (A) and fibroblasts (B) produced by increasing concentrations (from 10-12 to 10-6 mol/L) of unlabeled ET-1, BQ123 (ETA antagonist), or S6c (ETB agonist). B/Bo (percentage) represents the bound-to-free ratio. Binding studies were performed on five separate cell preparations from hearts from 5 different animals for each group.

View this table: [in this window] [in a new window]   Table 3. ET Isopeptide Binding Parameters (Kd and Bmax) of Adult Ventricular Cells After Chronic Volume Overload

As summarized in Table 3, stable cardiac hypertrophy due to volume overload did not significantly modify the maximum density of binding sites and the affinity of ET-1 receptors on adult cardiomyocytes and fibroblasts.

Ang II Receptor Binding Assay
Specific binding was low on rat adult cardiomyocytes, even after increasing cell numbers (10 000 to 100 000 cells) and concentrations of ¹²⁵I-[Sar¹,Ile⁸]Ang II (0.06 to 0.60 nmol/L), suggesting that at the most a very low number of Ang II receptors are present on adult cardiomyocytes. When 70 000 cells were incubated with 0.12 nmol/L of ¹²⁵I-[Sar¹,Ile⁸]Ang II for 90 minutes, a total binding of 1% and nonspecific binding of 0.75% were obtained. Ang II saturation and competition curves could not be performed on isolated ventricular cardiomyocytes. Saturation experiments with cultured ventricular fibroblasts showed that ¹²⁵I-[Sar¹,Ile⁸]Ang II bound with high affinity to a single class of Ang II receptors with a K_d of 0.42±0.01 nmol/L and a B_max of 66 620±2140 sites per cell (68.7±2 fmol/mg protein). Saturation was obtained between 1.5 to 2 nmol/L of iodinated [Sar¹,Ile⁸]Ang II (Fig 3). The characteristics of Ang II receptors were determined using [Sar¹,Ile⁸]Ang II, DuP 753 (specific AT₁ antagonist), and PD 123319 (specific AT₂ ligand). [Sar¹,Ile⁸]Ang II and DuP 753 completely displaced the tracer, which indicated that Ang II receptors on ventricular fibroblasts were exclusively of the AT₁ subtype.

View larger version (26K): [in this window] [in a new window]   Figure 3. Saturation binding of 125I-[Sar1,Ile8]Ang II and Scatchard transformation (inset) performed on ventricular fibroblasts. Binding analyses were assessed on five separate cell preparations from hearts from 5 different animals. Results are mean±SEM.

After cardiac volume overload, AT₁ receptor density on fibroblasts was significantly decreased, without change in affinity (Table 4). In parallel, Ang II total binding was higher on adult hypertrophied cardiomyocytes (70 000 cells) than on control cells in the same experimental conditions (1.4% versus 1% for control cells), suggesting an increase in Ang II receptor number.

View this table: [in this window] [in a new window]   Table 4. Effects of Chronic Volume Overload on Ang II Isopeptide Parameters (Kd and Bmax) of Primary Cultured Ventricular Fibroblasts

ET-1– and Ang II–Induced [Ca²⁺]_i in Control Cells
For adult cardiomyocytes, [Ca²⁺]_i measurements correspond to diastolic (average of the lowest point of each tracing over a 30-second period) and systolic (average of the maximum points corresponding to the diastolic points) values. To establish whether the ET-1 and Ang II receptors identified on ventricular cardiomyocytes and fibroblasts are functional, we studied the capacity of these vasoactive peptides to stimulate [Ca²⁺]_i. The concentration-response curve of effects of ET-1 (Fig 4) and Ang II (Fig 5) on [Ca²⁺]_i in control and hypertrophied cells was investigated. ET-1 increased [Ca²⁺]_i in a concentration-dependent manner in adult cardiomyocytes and fibroblasts (Fig 4), whereas Ang II induced concentration-dependent effects only on cardiac fibroblasts (Fig 5B). Various Ang II concentrations (10^-10 to 10^-7 mol/L) failed to modify [Ca²⁺]_i in normal adult cardiomyocytes, and systolic [Ca²⁺]_i was significantly increased only at 10^-6 to 10^-5 mol/L Ang II (Fig 5A).

View larger version (22K): [in this window] [in a new window]   Figure 4. Line graph of concentration-response curves for ET-1 (10-12 to 10-4 mol/L) in adult ventricular cardiomyocytes (A) and fibroblasts (B) from control (sham) and overloaded (shunt) hearts. Cardiomyocyte [Ca2+]i corresponds to the systolic value. pD2 values are 7.7±0.3 for cardiomyocytes from hypertrophied hearts vs 6.8±0.5 for cardiomyocytes from control hearts and 7.6±0.3 for fibroblasts from hypertrophied myocardium vs 6.8±0.5 for fibroblasts from control hearts. Values are the average from five separate cell preparations from hearts from 5 different animals for each group and are mean±SEM.

View larger version (21K): [in this window] [in a new window]   Figure 5. Line graph of concentration-response curves for Ang II (10-12 to 10-4 mol/L) in adult cardiomyocytes (A) and fibroblasts (B) from control (sham) and hypertrophied (shunt) myocardium. Cardiomyocyte [Ca2+]i corresponds to the systolic value. pD2 values are 8.5±0.2 for cardiomyocytes from overloaded hearts vs 7.0±0.2 for cardiomyocytes for control hearts (P<.05) and 7.1±0.4 for fibroblasts from hypertrophied hearts vs 6.5±0.3 for fibroblasts from control hearts. Values are the average from five separate cell preparations from hearts from 5 different animals for each group and are mean±SEM. *P<.05 vs control cells.

ET-1– and Ang II–Induced [Ca²⁺]_i in Hypertrophied Cells
The concentration-response curve of ET-1 was not significantly modified in either cell type from overload hearts (Fig 4). Adult cardiomyocytes from shunted rats (pD_2, 7.7±0.3 versus 6.8±0.5 for control cardiomyocytes) and cardiac fibroblasts from shunted rats (pD_2, 7.6±0.3 versus 6.8±0.5 for control fibroblasts) did not exhibit change in their [Ca²⁺]_i sensitivity to ET-1. Fig 5A shows the concentration-response curve to Ang II of [Ca²⁺]_i in cardiomyocytes from control and hypertrophied myocardium. The main finding was a displacement to the left of the concentration-response curve of Ang II in hypertrophied cardiomyocytes, with a significant increase in [Ca²⁺]_i sensitivity to Ang II (pD₂, 8.5±0.2 versus 7.2±0.2 for control cells; P<.05). For adult fibroblasts from hypertrophied hearts, [Ca²⁺]_i sensitivity to Ang II was not modified (pD₂, 7.1±0.4 versus 6.5±0.3 for control cells), although [Ca²⁺]_i increase was significantly lower at 10^-6 mol/L Ang II (Fig 5B). Thus, the maximum [Ca²⁺]_i response was significantly reduced in fibroblasts from hypertrophied myocardium compared with control cells (at 10^-5 to 10^-4 mol/L Ang II), in agreement with the reduced Ang II receptor number on fibroblasts from overloaded hearts.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that (1) ET-1 and Ang II receptors are present and functional with a cell-specific distribution in adult rat cardiomyocytes and fibroblasts; (2) ET-1 receptor densities and ET-1–induced [Ca²⁺]_i are not altered in adult cardiomyocytes and fibroblasts from hypertrophied hearts; (3) cardiac hypertrophy induces a significant downregulation of Ang II receptors (which are exclusively of the AT₁ subtype) on adult fibroblasts, with no alteration in [Ca²⁺]_i sensitivity to Ang II, but reduces efficacy of the [Ca²⁺]_i response; and (4) [Ca²⁺]_i sensitivity to Ang II and Ang II total binding are significantly higher in hypertrophied cardiomyocytes, suggesting upregulation of Ang II receptors.

Cultured adult rat cardiomyocytes⁴¹ and cardiac fibroblasts²⁷ are good cell models for studying molecular and cellular changes in control and experimental conditions, such as cardiac hypertrophy. As we reported here, on the basis of morphological and biochemical characteristics, the cell preparation used was of a high level of purity. ET receptors are present on the cell surface of rat adult cardiomyocytes and fibroblasts and increase [Ca²⁺]_i in a concentration-dependent manner. We reported for the first time that adult cardiomyocytes possess both ET_A and ET_B receptor subtypes, with a predominance of the ET_A subtype. To date, most studies have demonstrated ET receptors in the intact adult rat heart^{12 13 42 43 44} but only a few have reported the proportion of receptor subtypes in the myocardium.¹⁶ Recently, the existence of a third ET receptor subtype (ET_C), which is highly ET-3 selective, has been reported in cardiac tissue and brain.¹⁷ However, in current binding investigations, adult ventricular cardiomyocyte and fibroblast cultures did not reveal the existence of the ET_C receptor on the cell surface. The predominance of ET_A receptors (>90%) on adult cardiomyocytes is in agreement with our previous studies on neonatal cardiomyocytes²⁰ and on the entire spontaneously hypertensive rat heart.¹⁶ These results suggest that most ET-1 actions on the heart, such as proto-oncogene expression events⁴⁵ and regulation of ANP/BNP secretion,²⁰ are mediated in cardiomyocytes via the ET_A subtype. In contrast, ET receptors of both subtypes are nearly equally represented on cardiac fibroblasts, in accordance with a previous report.¹⁹ This suggests that both ET_A and ET_B subtypes mediate ET-1 effects in fibroblasts from adult hearts, such as production and deposition of extracellular matrix.^{46 47} ET-1 has stimulatory effects on collagen synthesis (types I and III) and inhibitory effects on collagenase activity in cardiac fibroblasts.⁴⁷ Also, ET-1 activates collagen deposition, resulting in enhanced myocardial stiffness as observed in ventricular hypertrophy.⁴⁷ Various studies have demonstrated that local cardiac ET-1 synthesis is enhanced at the early^{8 21 44 49} and later⁹ stages of cardiac pressure overload but not in cardiac volume overload.⁴⁹ Recently, Arai et al⁴⁴ reported an elevated density of ET-1 receptors in rat ventricular tissue after 8 days of pressure overload, suggesting the involvement of cardiac ET-1 in the development of cardiac hypertrophy. However, that study, based on binding to membrane preparations, did not take into consideration that nonmyocyte cells possess high ET-1 receptor density. In the present study, plasma and cardiac ET-1 levels as well as ET receptor regulation on cardiomyocytes and fibroblasts are not modified in stable cardiac hypertrophy due to volume overload. Similarly, the ET-1 intracellular signaling pathway ([Ca²⁺]_i) is not altered in either cardiac cell type. Our present data suggest that cardiac ET-1 may not be involved in the maintenance of the characteristics of the hypertrophic phenotype, such as cardiomyocyte size increase, nonmyocyte cell proliferation, fibrosis, and molecular changes. Together with previous studies, the present cardiac ET assessment suggests that ET-1 could act in the developmental rather than in the established phase of cardiac hypertrophy^{8 16 21 44} and may be more important in the mechanisms of cardiac pressure overload than of cardiac volume overload.⁸ This approach, based on cardiac peptide concentrations, cell receptor regulation, and intracellular signaling pathways, demonstrates for the first time that the cardiac ET-1 system may not be altered in cardiac volume overload in the rat.

Various studies provide evidence that the cardiac renin-angiotensin system is activated in cardiac hypertrophy. Cardiac volume overload induced an increase in renin activity and expression in left ventricles.⁵⁰ In contrast, in the present study, Ang II concentrations in plasma and ventricles are not modified, but PRA is significantly increased in volume-overloaded rats. Data on cardiac Ang II levels should be interpreted with caution, because these tissue levels correspond to circulating and locally produced Ang II. Several previous studies report conflicting results regarding Ang II receptor distribution in the rat myocardium.^{23 24 25 26} Reasons for these conflicting reports may be attributable to the fact that most investigations were performed on the entire heart, which is composed of several cellular compartments, including myocytes and vascular and interstitial tissues, and that cardiac Ang II receptors are less abundant in myocardial tissue (mainly in cardiomyocyte cells) than in adrenal, kidney, liver, or vascular tissue.^{11 51} Consequently, previous data involving cardiac Ang II receptors in control and experimental conditions should be regarded cautiously. As mentioned, data involving Ang II receptor regulation in cardiac hypertrophy are controversial: cardiac pressure overload was reported to induce either upregulation of Ang II receptors in intact hearts²⁵ or ventricular cardiomyocytes²⁴ or downregulation in the intact heart.²⁶ In our experiments in control cardiomyocytes, total Ang II binding is very low, and Ang II failed to increase [Ca²⁺]_i at physiological concentrations. Only 10^-6 to 10^-5 mol/L Ang II enhances the intracellular signal, which suggests that Ang II cell surface receptors are present but in very low number. The absence of measurable Ang II binding sites in adult cardiomyocytes could be explained in part by the type of the binding site studied. Recent investigations have reported the existence of Ang IV receptors in adult cardiomyocytes⁵² and in cardiac fibroblasts of mammalian heart⁵³ clearly distinct from the well-known AT₁ and AT₂ receptor subtypes. Although the role of Ang IV in the myocardium remains to be elucidated, Ang IV receptors mediated stimulation of DNA and RNA synthesis in cardiac fibroblasts,⁵³ suggesting the implication of Ang IV in cardiac hypertrophy. It will be interesting to investigate the cellular localization and the regulation of Ang IV receptors in cardiac hypertrophy. Moreover, ventricular Ang II receptors seem to be developmentally regulated, because previous studies have reported an elevated number of Ang II binding sites in the neonatal period,^{54 55} suggesting that cardiac Ang II receptors may play a role in early cardiac development.¹¹ In cardiac adaptation, Ang II–induced [Ca²⁺]_i is significantly higher in hypertrophied cardiomyocytes, with a significant increase in [Ca²⁺]_i sensitivity to Ang II and in total Ang II binding, which suggests upregulation of Ang II receptors and/or a tighter coupling of the receptors. Increase in the density of cardiomyocyte Ang II receptors is accompanied by a significant cell volume increase (+71%). Cardiomyocyte hypertrophy is associated with the impairment of mechanical function, leading to an alteration of cardiac performance.²⁴ Cardiac volume overload seems to induce an upregulation of Ang II receptor and an enhanced intracellular signal transduction in hypertrophied cardiomyocytes, indicating that myocardial hypertrophy may be facilitated by Ang II receptor and effector pathway activation in cardiomyocytes. Our data are in agreement with recent studies, which reported an increase in [Ca²⁺]_i sensitivity to Ang II⁵⁶ and upregulation of Ang II receptors and higher Ang II–induced phosphoinositide turnover in hypertrophied cardiomyocytes,²⁴ suggesting that Ang II may indeed have a role in the maintenance of stable cardiac hypertrophy.

Binding analyses demonstrated in the present study that Ang II cell surface receptors are exclusively of the AT₁ subtype in cardiac fibroblasts, as previously reported.^{7 27 57} Consequently, the low Ang II receptor number previously reported in the whole heart of adult rat^{25 26} seems to be mainly localized in adult cardiac fibroblasts, which represent 90% of nonmyocyte cells. In the present study, activation of AT₁ receptors results in an increase [Ca²⁺]_i in a concentration-dependent manner. Cardiac AT₁ cell surface receptors have been reported to mediate proliferative growth of nonmyocyte cells^{7 57} and myocardial fibrosis.²² To our knowledge, no data have been reported on the regulation of Ang II receptors and the modulation of the intracellular signaling pathways in cardiac fibroblasts from hypertrophied hearts. We demonstrate in the present study that cardiac volume overload induced a downregulation of AT₁ receptor density on cardiac fibroblasts (42%). These binding results are in agreement with the lowered Ang II–induced [Ca²⁺]_i changes observed with high Ang II concentrations in fibroblasts from hypertrophied hearts compared with those in control fibroblasts. Ang II has been reported to stimulate collagen synthesis and inhibit collagenase activity in cardiac fibroblasts,⁴⁸ as well as to enhance fibroblast proliferation.⁷ Consequently, chronic renin-angiotensin system activation is associated with enhanced fibrosis and hyperplasia in ventricles. Chronic volume overload induces an elongation of cardiomyocytes, which is responsible for an eccentric cardiac hypertrophy. However, myocardial fibrosis was reported only in cardiac pressure overload (concentric remodeling) and not in cardiac volume overload.⁴⁷ Indeed, cardiac volume overload induced by aortocaval shunt in rats²² or in dogs⁵⁸ induced a decreased collagen deposition in left ventricles, suggesting that increased diastolic wall stress leads to the inhibition of fibrosis. From the present study, it may be speculated that AT₁ receptor downregulation in cardiac fibroblasts, associated with a significant decrease in the intracellular signaling pathway in response to high Ang II concentrations, may attenuate the effects of chronic renin-angiotensin system activation and prevent ventricular fibrosis in cardiac volume overload.^{22 47 58}

In conclusion, the present study demonstrates that ET-1 and Ang II receptors are present on the cell surface of adult cardiomyocytes and fibroblasts and modulate [Ca²⁺]_i in a concentration-dependent manner. ET-1 receptors have a cell-specific distribution, with the ET_A subtype predominating on cardiomyocytes, whereas on cardiac fibroblasts, both subtypes are equally represented. This suggests a cell-specific function of cardiac ET-1 receptors. Plasma and cardiac ET-1 levels and ET-1 receptor regulation on both ventricular cell types are not modified in cardiac volume overload, indicating that ET-1 may not play a significant role in the maintenance of cardiac hypertrophy due to volume overload. Furthermore, cardiac hypertrophy induces a significant downregulation of AT₁ receptors on adult fibroblasts, with no alteration in [Ca²⁺]_i sensitivity to Ang II, whereas total binding and [Ca²⁺]_i sensitivity to Ang II are enhanced in hypertrophied cardiomyocytes. Cardiac volume overload induces a complex cellular response, whereby cardiac Ang II may be involved in the maintenance of myocardial hypertrophy (cell enlargement) and inhibition of cardiac fibrosis defined in eccentric cardiac remodeling. Results from the present study provide evidence that cardiac ET-1 and Ang II may be differentially involved in the cardiac adaptive mechanisms associated with volume overload.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II ANP = atrial natriuretic peptide AT = Ang II receptor subtype BNP = brain natriuretic peptide CLS2 = collagenase DuP 753 = losartan potassium ET = endothelin KH = Krebs-Henseleit bicarbonate (pH 7.4) PD 123319 = 1-(4-amino-3-methylphenyl)methyl-5-diphenylacetyl-4,5,6,7-tetrahydro-1-H-imidazole (4,5-c)pyridine-6-carboxylic acid PRA = plasma renin activity S6c = sarafotoxin 6c

	Acknowledgments

 
This study was supported by a group grant to the Multidisciplinary Research Group on Hypertension from the Medical Research Council of Canada (MRC) and by a grant to Dr Thibault from the Heart and Stroke Foundation of Canada. Drs Fareh and Touyz are recipients of fellowships from the Canadian Hypertension Society/MRC and the MRC, respectively, and Dr Fareh is also a recipient of "Programme Lavoisier 1994" award of the "Ministère des Affaires Etrangères de France." The scientific advice of Dr Hoda Eid (University of Ottawa Heart Institute, Canada) on adult cardiomyocyte isolation was appreciated. The authors thank Suzanne Diebold, Chantal Arguin, and Micheline Vachon for their technical assistance.

	Footnotes

 
Reprint requests to Gaetan Thibault, PhD, Clinical Research Institute of Montreal, 110, Pine Ave West, Montreal, Quebec H2W 1R7, Canada. E-mail thibaug@ircm.umontreal.ca.

Received June 23, 1995; accepted November 6, 1995.

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