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(Hypertension. 1997;30:1253-1259.)
© 1997 American Heart Association, Inc.

Articles

Cardiac Type-1 Angiotensin II Receptor Status in Deoxycorticosterone Acetate–Salt Hypertension in Rats

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

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

Correspondence to Gaetan Thibault, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, Quebec, Canada H2W 1R7. Email thibaug{at}ircm.umontreal.ca

	Abstract

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Abstract The regulation of angiotensin II (Ang II) receptors and Ang II–induced modulation of intracellular Ca²⁺ concentration in cardiac cells from hearts of experimentally induced hypertensive deoxycorticosterone acetate (DOCA)–salt and control unilaterally nephrectomized (Uni-Nx) Sprague-Dawley rats was assessed. Ang II receptor density and intracellular Ca²⁺ concentration measurements were examined in adult ventricular myocytes and fibroblasts by radioligand binding assay and digital imaging using fura 2 methodology, respectively. Four-week DOCA-salt treatment induced hypertension associated with cardiac hypertrophy. Ang II binding studies demonstrated that adult ventricular myocytes and fibroblasts possess mainly the AT₁ subtype receptor. Moreover, DOCA-salt hypertension was associated with a 1.8-fold increase in Ang II–specific binding compared with myocytes from Uni-Nx control rats. Intracellular Ca²⁺ responses induced by increasing Ang II concentrations (10^-12 to 10^-4 mol/L) were significantly enhanced in cardiomyocytes from DOCA-salt rats. The effects of Ang II on intracellular Ca²⁺ spike frequency were unaltered in cardiomyocytes from DOCA-salt–hypertensive rats. The density of AT₁ subtype receptors was not modified in ventricular fibroblasts after DOCA-salt treatment. Ang II increased intracellular Ca²⁺ concentration similarly in ventricular fibroblasts from normal and hypertensive rats. In conclusion, DOCA-salt hypertension is characterized by an increased AT₁ receptor density and intracellular calcium responses in ventricular myocytes, whereas in ventricular fibroblasts the AT₁ receptor status is unaltered. These findings report for the first time the cardiac cell–specific implication of Ang II and the intracellular calcium signaling pathway stimulated by the AT₁ receptor in cardiac hypertrophy in DOCA-salt–hypertensive rats.

Key Words: hypertrophy • myocytes • fibroblasts • calcium

	Introduction

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Cardiac remodeling, defined as enhanced cell growth, myocardial fibrosis, and cardiac contractile dysfunction, is the pathological consequence of chronic pressure overload observed in hypertension. Morphological and contractile abnormalities of the myocardium lead ultimately to the development of congestive heart failure. A role of the cardiac renin-angiotensin system in the development of cardiac hypertrophy has been suggested.^{1 2 3} Ang II enhances in vitro protein synthesis⁴ and myocyte cell growth and induces hyperplasia of nonmyocyte cells.^{5 6} The precise role of Ang II in vivo in the initiation and maintenance of hypertensive cardiac hypertrophy, however, remains unclear. Ang II receptor antagonists or angiotensin-converting enzyme inhibitors can regress cardiac hypertrophy and improve survival,⁷ but they may have no effect on blood pressure and concentric cardiac hypertrophy in the DOCA-salt pressure overload model.⁸

Cardiovascular actions of Ang II are mediated by two distinct cell surface receptor subtypes, AT₁ and AT₂. The AT₁ receptor has been cloned in the rat, and two isoforms, AT_1a and AT_1b, have been characterized. In the heart, some studies have shown by radioautography or binding assays on total ventricular membranes that both subtypes are consistently present in equal proportion and are distributed throughout cardiac structures, with a predominance in the conduction system and the autonomic nervous system.^{9 10 11} However, other studies documented the presence of only AT₁ receptors,^{12 13} and this was confirmed in isolated ventricular myocytes^{14 15 16} and cultured cardiac fibroblasts.^{16 17 18} In addition, only AT_1a receptors could be detected in the adult myocardium.^{10 19} Upregulation of the AT₁ receptor was observed in myocardial infarction and in cardiac hypertrophy associated with hypertension, in spontaneously hypertensive rats, in two-kidney, one clip hypertension, and after aldosterone infusion.^{10 12 13 14} More specifically, this upregulation was associated with myofibroblasts of the fibrous scar in the myocardial infarction model.²⁰ We recently demonstrated that Ang II receptor regulation in the overloaded myocardium of the rat was also cardiac cell–specific,^{15 16} which suggests a complex differential role of Ang II in myocyte and nonmyocyte cell adaptation. However, cardiac hypertrophy associated with chronic infusion of Ang II was accompanied by a downregulation of the AT₁ receptor.¹²

The presence of AT₁ receptors in the heart implies a local action for Ang II. Apart from the well-known renin-angiotensin system, which generates Ang II from components in the circulation, local cardiac production of Ang II has been postulated. There is evidence that Ang II can be generated in cardiac tissues and that generation is augmented in myocardial infarction.²¹ Angiotensin-converting enzyme is detected in the myocardium and is present in high concentrations in cardiac valves and coronary vessels.²² Its expression is considerably increased together with AT₁ receptor density in infarcted areas of myocardium and in areas with fibrosis, and it is clearly associated with the localization of myofibroblasts.^{20 23} The case for cardiac renin is still debated. As reviewed by van Lutteroti et al²⁴ and based on mRNA analysis or on enzymatic activity, it is still unclear whether cardiac tissues can synthesize prorenin or process it to active renin. Uptake of renin to explain its presence in cardiac tissues remains a possibility.²⁵

It has been suggested that intracellular calcium and protein kinase C mediate the effects of Ang II on chronotropy, inotropy, and myocardial growth.²⁶ Moreover, abnormal protein kinase C activation²⁷ and impairment of intracellular calcium handling, leading to cardiac arrhythmias,^{28 29} may be intracellular effectors of cardiac hypertrophy and heart failure. However, the intracellular pathways of Ang II–induced mechanisms underlying cardiac hypertrophy such as contractile dysfunction, myocyte enlargement, nonmyocyte proliferation, and fibrosis remain to be elucidated.

The DOCA-salt rat model is one of several animal models used in studies of cardiac hypertrophy related to chronic hypertension. This hypertensive model is characterized by enhanced sympathetic activity, severe concentric remodeling of the heart, and perivascular and interstitial fibrosis.³⁰ To elucidate whether cardiac Ang II is involved in this model, we assessed Ang II receptor status in adult ventricular myocytes and fibroblasts. We investigated Ang II receptor density and modulation of Ang II–induced [Ca²⁺]_i in isolated cells from hearts of DOCA-salt and unilaterally nephrectomized Sprague-Dawley rats.

	Methods

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Animal Experiments
Male Sprague-Dawley rats (150 to 180 g, Charles River, St Constant, Quebec, Canada) were used. Experiments were carried out in accordance with the guidelines of the Canadian Council on Animal Care. The rats of the experimental group were anesthetized with sodium pentobarbital (50 mg/kg IP), unilaterally nephrectomized, and implanted subcutaneously with silicone rubber containing 200 mg of DOCA (Sigma Chemical Co) per rat.³¹ Animals were given 0.171 mol/L saline solution in tap water ad libitum (DOCA-salt group). Control rats were also unilaterally nephrectomized, but DOCA and saline treatments were not administered (Uni-Nx control rats). Systolic blood pressure was measured weekly by the tail-cuff method, and animals were studied 4 to 5 weeks after DOCA-salt treatment. At this stage, animals were 11 to 12 weeks old.

Preparation and Isolation of Adult Myocytes
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 heart was then rapidly removed. Calcium-tolerant myocytes were isolated by cardiac retrograde aortic perfusion (Langendorff method) as previously described.^{15 32} Freshly isolated cells were gently diluted in sterile culture M199 medium, pH 7.4, with 10% fetal bovine serum. The culture medium (M199) was supplemented with 0.2% BSA, 10^-7 mol/L insulin, 5 mmol/L creatine, 2 mmol/L L-carnitine, 5 mmol/L taurine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Ventricular cells were seeded onto round glass coverslips in culture dishes (7000 cells/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. We obtained 90% calcium-tolerant myocytes (rod-shaped cells) or 2.5 to 3.0x10⁶ cells per heart, which corresponds to >95% myocyte purity. Serum-free medium was added overnight, and the [Ca²⁺]_i measurements were performed the following day.

Preparation and Primary Culture of Adult Ventricular Fibroblasts
Animals were injected with heparin sulfate and pentobarbital. After cardiac dissection, ventricles were dissected from atria and large vessels and washed in sterile 0.05 mol/L PBS. They were finely minced and digested in 15 mL of Dulbecco's modified Eagle's medium containing 0.1 g/dL trypsin and 100 U/mL collagenase (CLS2, Worthington Biochemical Corp) at 37°C with agitation (150 cycles per minute) for 15 minutes as previously described.^{15 32} 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 confluency (4 to 5 days, 2x10⁵ cells/2 cm²). The identity of the cells was confirmed by immunohistochemistry with antisera against protein markers. The cells were positive for vimentin but negative for desmin and von Willbrand factor as previously reported.¹⁵ However, less than 5% of cultured fibroblasts from control heart were stained with anti--smooth muscle actin, whereas between 10% and 20% of the cells from DOCA-salt–treated rats were positive, indicating that some fibroblasts were phenotypically modified into myofibroblasts.³³ Twenty-four hours before [Ca²⁺]_i assays, culture medium was replaced by serum-free medium. Our previous studies, showing modulation of Ang II receptors on fibroblasts depending on the model used,^{16 34} suggest that fibroblasts maintain their phenotype after 5 days in culture, although it cannot be excluded that these cells may have dedifferentiated to some degree.

Ang II Receptor Binding Assays on Cardiac Cells
All binding reactions (in duplicate) were performed in the respective serum-free culture medium at room temperature for 90 minutes. [Sar¹,Ile⁸]-Ang II was radiolabeled with ¹²⁵I by the lactoperoxidase method and purified by high-pressure liquid chromatography.³⁵ As we demonstrated previously,¹⁵ Ang II–specific binding was very low in rat adult ventricular myocytes, suggesting the presence of few receptors under control conditions. Because Ang II competition curves could not be performed, total and nonspecific Ang II binding was obtained in adult myocytes (60 000 cells) with 0.12 nmol/L ¹²⁵I-[Sar¹,Ile⁸]-Ang II in the absence or presence of 10^-6 mol/L competing unlabeled agents ([Sar¹,Ile⁸]-Ang II or losartan [DuP 753, an AT₁-selective antagonist]), respectively. As previously described,¹⁵ the competitive binding assay on ventricular fibroblasts was performed in the presence of increasing concentrations (10^-12 to 10^-6 mol/L) of [Sar¹,Ile⁸]-Ang II, losartan, or PD 123319 (an AT₂-specific ligand) and 100 to 120 pmol/L ¹²⁵I-[Sar¹,Ile⁸]-Ang II (2200 Ci/mmol). The volume of reaction was 1.0 and 0.5 mL for myocytes (60 000 cells) and cultured fibroblasts (2x10⁵ cells/well), respectively. The Ang II binding reaction on isolated myocytes was stopped with 3.5 mL of 50 mmol/L Tris-HCl (pH 7.2) and 0.15 mol/L NaCl. After a rapid filtration through glass filters (Schleider & Schuell) with a cell harvester (Brandel), filters were rinsed three times with the same solution. After the binding reaction, attached fibroblasts were washed twice with 0.5 mL of culture medium (Dulbecco's modified Eagle's medium) and cells were digested by 0.5 mL of 1 mol/L NaOH. Radioactivity on filters or on digested cells was counted in a gamma counter with 80% efficiency (LKB Wallac). Binding data were analyzed using the EBDA-LIGAND program software of McPherson (Biosoft).³⁶

Measurement of [Ca²⁺]_i in Cardiac Cells
Measurement of [Ca²⁺]_i was performed using fura 2 methodology.³² Adult myocytes 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 (in mmol/L): 137 NaCl, 4.2 NaHCO₃, 3 NaHPO₄, 5.4 KCl, 0.4 KH₂PO₄, 1.3 CaCl₂, 0.5 MgCl₂, 0.8 MgSO₄, 10 glucose, 5 HEPES (pH 7.4). Fluorescence measurements were assessed using double-excitatory wavelengths (343 and 380 nm) and a single-emission wavelength (520 nm).^{15 32} [Ca²⁺]_i was measured in isolated cells by fluorescent digital imaging. [Ca²⁺]_i was calculated according to the formula of Grynkiewicz et al,³⁷ in which the dissociation constant for fura 2–Ca²⁺ (K_d) was taken to be 224 nmol/L. Fluorescence experiments were performed using the Axiovert 135 inverted microscope and Attofluor Digital Fluorescence system (Zeiss). After an equilibration period, cultured cells were exposed to single concentrations of Ang II (10^-12 to 10^-4 mol/L) at room temperature. [Ca²⁺]_i determinations were performed on cardiac cells from control and hypertrophied hearts (50 to 75 cells). In resting and stimulated state, calcium-tolerant myocytes are characterized by spontaneous calcium spikes, corresponding to calcium release and contraction.³² Consequently, cardiomyocyte [Ca²⁺]_i measurements are reported as diastolic and systolic [Ca²⁺]_i values (in nmol/L). Diastolic [Ca²⁺]_i was determined as the average of the lowest point of each tracing over a 30-second period, and systolic [Ca²⁺]_i was taken as the average of the maximum points. The frequency of [Ca²⁺]_i spikes was defined over a 60-second time interval (spikes per minute). Ventricular fibroblasts do not present any spontaneous calcium or contractile waves in the resting and stimulated states, as previously reported.^{15 32}

Statistics
Values are expressed as mean±SEM. Ang II binding assays and [Ca²⁺]_i measurements were performed on isolated ventricular myocytes and cultured fibroblasts from 5 to 6 animals per group. Unpaired Student's t test or two-way ANOVA was used to show statistical significance between DOCA-salt and Uni-Nx rats. [Ca²⁺]_i measurements were compared by ANOVA or by Student's t test where appropriate. The significance level was set at P<.05.

	Results

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Systolic Blood Pressure and Body and Heart Weight
Systolic blood pressure and heart weight rose significantly in unilaterally nephrectomized DOCA-salt–treated rats (Table 1). Heart weight–to–body weight ratio was markedly increased in DOCA-salt–hypertensive compared with Uni-Nx rats, indicating the presence of cardiac hypertrophy. Correction of heart weight by tibia length rather than by body weight (which was lower in DOCA-salt rats and could be affected by different factors) also indicated that cardiac hypertrophy was present.

View this table: [in this window] [in a new window]   Table 1. Characteristics of DOCA-Salt– Hypertensive Rats

Cellular Ang II Receptor Regulation
Table 2 illustrates the effects of DOCA-salt hypertension on total and nonspecific binding of Ang II in adult ventricular myocytes. In control and experimental conditions, Ang II binding was statistically similar in the presence of either 10^-6 mol/L [Sar¹,Ile⁸]-Ang II or 10^-6 mol/L losartan (DuP 753, an AT₁-selective antagonist), whereas 10^-6 mol/L PD 123319 failed to affect Ang II binding to cardiomyocytes (data not shown). These binding data suggest that Ang II receptors were mainly of the AT₁ subtype and that the AT₂ subtype was undetectable on adult myocytes. DOCA-salt hypertension was associated with a 1.8-fold increase (P<.01) in Ang II–specific binding to ventricular myocytes. Because specific binding levels were similar with [Sar¹,Ile⁸]-Ang II and losartan in cells from DOCA-salt rats, the Ang II receptor increase in myocytes from DOCA-salt rats probably corresponded to the AT₁ subtype (P<.001) (Table 2).

View this table: [in this window] [in a new window]   Table 2. Effects of DOCA-Salt Hypertension on Ang II Binding to Adult Ventricular Myocytes

In ventricular fibroblasts, as calculated from the displacement curves obtained with increasing concentrations of [Sar¹,Ile⁸]-Ang II, losartan, or PD 123319, Ang II receptor density (mainly AT₁ subtype) was unaltered in DOCA-salt hypertension (52780±4550 versus 66360±3960 sites per cell for [Sar¹,Ile⁸]-Ang II binding on control cells and 54540±4570 versus 69730±5770 sites per cell for losartan binding on control cells). Similarly, Ang II receptor affinity did not differ between ventricular fibroblasts from DOCA-salt and Uni-Nx rats. Binding analysis showed the following K_d values for [Sar¹,Ile⁸]-Ang II and losartan in DOCA-salt versus Uni-Nx cells: 225±19 versus 218±39 pmol/L for [Sar¹,Ile⁸]-Ang II and 5.01±0.3 versus 6.3±0.7 nmol/L for losartan.

Basal and Ang II–Induced Intracellular Calcium Responses
Under basal conditions, systolic [Ca²⁺]_i in myocytes from hypertensive rats was higher compared with control cells (115±1.3 versus 103±3.0 nmol/L for control cells, P<.05), whereas diastolic [Ca²⁺]_i did not differ (70±2.4 versus 73±2.8 nmol/L for control cells). Similarly, basal [Ca²⁺]_i in fibroblasts was significantly greater in DOCA-salt hypertension (115±1.3 versus 82±1.1 nmol/L for control cells, P<.01).

The effects of increasing concentrations of Ang II on [Ca²⁺]_i (systolic values) in control and hypertrophied cardiac myocytes are illustrated in Fig 1. In control rats, only 10^-8 to 10^-4 mol/L Ang II concentrations elicited a weak increase in systolic [Ca²⁺]_i in ventricular myocytes, possibly because of the very low Ang II receptor density in adult cardiac myocytes, as previously reported.^{14 15} However, in myocytes from hypertrophied myocardium from DOCA-salt rats, the Ang II–induced [Ca²⁺]_i response was significantly greater (P<.01) at lower Ang II concentrations compared with control cells (10^-12 to 10^-4 mol/L) (Fig 1), resulting in a significant displacement of the Ang II concentration-response curve. As illustrated in Fig 2, in control rats Ang II increased [Ca²⁺]_i spike frequency in a concentration-dependent manner. Basal (26.2±2.0 versus 26.0±1.1 spikes per minute for control myocytes) and Ang II–induced [Ca²⁺]_i spike frequency, although showing a trend to increase, failed to achieve statistically significant differences in myocytes from hypertensive rats compared with control cells (Fig 2).

View larger version (19K): [in this window] [in a new window]   Figure 1. Ang II–induced [Ca2+]i transients in adult ventricular myocytes from DOCA-salt–treated and control rats. [Ca2+]i values correspond to the systolic [Ca2+]i values, defined as the average of the maximum points of each tracing over a 30-second period. The concentration-dependent curve was assessed in myocytes from hearts of control Uni-Nx and DOCA-salt rats. [Ca2+]i values, expressed in nanomoles per liter, are the average of separate cell preparations (50 to 75 cells) from the hearts of 6 different animals for each group. Data are mean±SEM. *P<.05, **P<.01 vs Uni-Nx.

View larger version (17K): [in this window] [in a new window]   Figure 2. [Ca2+]i spike frequency in ventricular cardiomyocytes from DOCA-salt and control rats. Increasing Ang II concentrations (10-12 to 10-4 mol/L) were used to obtain concentration-response curves to Ang II of [Ca2+]i spike frequency, defined over a 60-second time interval (spikes per minute). Measurements were performed on 50 to 75 cardiomyocytes from myocardium of 6 different animals for each group. Values are mean±SEM. No statistical differences were found between the two curves.

To identify the Ang II receptor subtypes responsible for [Ca²⁺]_i responses in adult fibroblasts, Ang II (10^-8 mol/L)–induced responses were assessed in cells in the absence or presence of 10^-6 mol/L [Sar¹,Ile⁸]-Ang II (nonspecific Ang II receptor antagonist), 10^-6 mol/L losartan (selective AT₁ receptor antagonist), or 10^-6 mol/L PD 123319 (selective AT₂ receptor antagonist). Because findings obtained in cells from Uni-Nx and DOCA-salt rats were similar, only results in fibroblasts from DOCA-salt fibroblasts rats are reported in Table 3. Ang II effects on [Ca²⁺]_i were completely abolished by 10^-6 mol/L [Sar¹,Ile⁸]-Ang II (P<.01) or by 10^-6 mol/L losartan (P<.01). The AT₂ antagonist PD 123319 (10^-6 mol/L) did not modify the Ang II effects on [Ca²⁺]_i. These data clearly demonstrate that in fibroblasts Ang II increases [Ca²⁺]_i via the AT₁ subtype receptor.

View this table: [in this window] [in a new window]   Table 3. Identification of the Ang II Receptor Subtype Mediating [Ca2+]i Responses in Adult Ventricular Fibroblasts

In fibroblasts from Uni-Nx and DOCA-salt rats, Ang II (10^-12 to 10^-4 mol/L) increased [Ca²⁺]_i in a concentration-dependent manner, as demonstrated in Fig 3. Moreover, Ang II–induced [Ca²⁺]_i responses were similar in fibroblasts from DOCA-salt and Uni-Nx rats, and only concentrations greater than 10^-6 mol/L (nonphysiological concentrations) elicited significantly higher [Ca²⁺]_i responses in fibroblasts from DOCA-salt rats compared with control cells (P<.05) (Fig 3).

View larger version (19K): [in this window] [in a new window]   Figure 3. Ang II–induced [Ca2+]i responses in ventricular fibroblasts from DOCA-salt and control rats. Increasing Ang II concentrations (10-12 to 10-4 mol/L) were used to obtain concentration-response curves of Ang II on [Ca2+]i in fibroblasts from hearts of Uni-Nx and DOCA-salt rats. Measurements were performed on 50 to 75 fibroblasts from the hearts of 6 different animals for each group. Values are mean±SEM. *P<.05, **P<.01 vs Uni-Nx.

	Discussion

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In the present study, we demonstrate for the first time that AT₁ receptor levels and Ang II–induced [Ca²⁺]_i responses in hypertrophied myocytes are enhanced in the hearts of DOCA-salt hypertensive rats, whereas Ang II receptor density and its intracellular signaling pathway did not differ in nonmyocyte cells. Additionally, we report that myocyte and nonmyocyte cells are characterized by intracellular calcium overload in this model of hypertensive cardiac hypertrophy. Local Ang II receptor activation and the intracellular calcium pathway related to the AT₁ subtype receptor may be involved in myocyte enlargement¹⁴ and the alterations of sarcoplasmic reticulum calcium handling²⁹ observed in pressure overload–induced cardiac hypertrophy.

Several studies have documented the hypertrophic process as individual myocyte enlargement and hyperplasia/hypertrophy of nonmyocyte cells such as fibroblasts and vascular smooth muscle cells. Interestingly, although myocytes and fibroblasts are closely associated structurally in myocardial tissue, myocyte and nonmyocyte cell growth mechanisms are clearly independent in volume and pressure overload cardiac hypertrophy.³⁸ The present report shows that basal [Ca²⁺]_i was significantly increased in myocytes (systolic Ca²⁺ values) and in nonmyocyte cells in the cardiac hypertrophy present in DOCA-salt–hypertensive rats. These findings are in agreement with previous investigations³⁹ but differ from others.⁴⁰ Discrepancies in intracellular calcium responses could be explained in part by the different degree of cardiac overload investigated.²⁹ Recently, we showed that basal [Ca²⁺]_i was higher in rat cardiac cells in chronic volume overload hypertrophy³⁴ and in genetic hypertension,¹⁶ suggesting that increased intracellular calcium overload may be independent of the mechanisms involved in the pathogenesis of hypertension. Rather, calcium elevation in the heart may be related to development of cardiac hypertrophy. Impairment of calcium homeostasis in cardiac overload in hypertension can be attributed to dysfunction of Ca²⁺ uptake by the sarcoplasmic reticulum, Ca²⁺-ATPase depression, and alterations in Ca²⁺ channel density.²⁹ However, the exact mechanisms involved in the impairment of calcium handling in cardiac hypertrophy and heart failure need to be clarified.

There is evidence of the existence of a local cardiac angiotensin system in normal and hypertrophied myocardium because expression of angiotensinogen and angiotensin-converting enzyme genes has been demonstrated in the heart.^{21 22 23 41} The intracardiac angiotensin system may be activated in various animal models of pressure^{21 22 42} and volume overload.⁴¹ In addition, regression of cardiac hypertrophy has been reported with angiotensin-converting enzyme inhibitors.^{7 43} Previous studies were performed on various experimental models such as aortic banding, myocardial infarction, aorto-caval shunt, or genetic models (spontaneously hypertensive rats). To our knowledge, few data are available on the potential role of Ang II in mineralocorticoid-induced hypertension in rats. Kim et al⁸ reported that an AT₁ receptor antagonist (TCV-116) and an angiotensin-converting enzyme inhibitor (enalapril) failed to reduce blood pressure and cardiac hypertrophy but improved renal lesions in DOCA-salt rats. These findings, associated with overexpression of endothelin-1 in the endothelium of coronary vessels,³¹ regression of cardiac hypertrophy following endothelin antagonist (bosentan or FR139317) infusion in some studies^{44 45} but only in small arteries in others,⁴⁶ and the autocrine/paracrine interaction of endothelin-1 with Ang II,^{47 48} indicate the complexity of the potential role of Ang II in the development of cardiac hypertrophy in this model. In the present study, pressure overload caused by chronic DOCA-salt hypertension induced a 1.8-fold increase in AT₁ receptor subtype binding in hypertrophied myocytes. In a similar model (aldosterone infusion), Sun and Weber¹² observed an increased density of AT₁ receptor throughout the myocardium. These results provide evidence for a potential role of cardiac Ang II in an endothelin-dependent hypertensive model. We speculate that Ang II and endothelin-1, which has been shown to be overexpressed in the endothelium of coronary arteries of DOCA-salt rats,³¹ could closely interact in the induction of cardiac hypertrophy in this model. We previously reported that physiological Ang II concentrations (10^-12 to 10^-8 mol/L) failed to increase [Ca²⁺]_i in normal myocytes,^{15 16} demonstrating the low level of Ang II receptors in normal myocytes.^{14 15} However, in myocytes from DOCA-salt rats, Ang II–induced intracellular signal transduction was markedly enhanced at both low and high Ang II concentrations. The present findings support our previous studies in volume overload¹⁵ and genetic hypertensive cardiac hypertrophy.¹⁶ They are in agreement with results of Meggs et al,¹⁴ who previously reported Ang II receptor upregulation associated with enhanced Ang II–induced phosphoinositide turnover in hypertrophied myocytes. Data on cardiac Ang II receptor regulation in the hypertrophied heart are controversial, especially because myocardial tissue, and particularly adult myocytes, possesses few Ang II receptors^{14 15} compared with nonmyocardial tissue.⁴⁹ Indeed, previous reports demonstrated that pressure or volume cardiac overload induced not only upregulation of Ang II receptors in whole hearts¹⁰ or ventricular myocytes,^{14 15 16} but also downregulation in the intact myocardium.¹¹ Discrepancies regarding cardiac Ang II receptor status in failing hearts could be explained in part by differences in the experimental approach.³⁸ Some studies examined intact myocardium, and others, such as the present one, studied cardiomyocytes and fibroblasts separately.

Nonmyocyte cells comprising mainly fibroblasts correspond to two-thirds of cardiac cells and produce cardiac extracellular matrix (collagen types I and III, fibronectin) and collagenase. In hypertension, there is exaggerated accumulation of collagen and fibroblast proliferation, which is responsible for increased myocardial stiffness.⁵⁰ However, cardiac fibrosis seems to be associated more with pressure-overloaded myocardium, where hormones such as Ang II or aldosterone play a role,^{51 52} than in volume-overload models in which hemodynamic factors are primarily involved.⁵³ Recently, we¹⁵ and others^{17 18} have characterized functional Ang II receptors on cultured adult fibroblasts (mainly AT₁ subtype). In the present study, we showed that Ang II receptor density and Ang II–induced [Ca²⁺]_i at physiological concentrations are unaltered in adult fibroblasts from the myocardium of DOCA-salt rats. Enhanced [Ca²⁺]_i responses were observed, but only in the presence of supraphysiological Ang II concentrations. It can be speculated that other altered intracellular mechanisms such as receptor coupling, phospholipase C, or Ca²⁺ channels may affect Ca²⁺ metabolism and modulate Ang II responses. Because mineralocorticoid-induced hypertension is characterized by ventricular interstitial and perivascular collagen deposition,^{52 53} and because Ang II has been reported to act on collagen function and metabolism,^{18 54 55} nonmyocyte Ang II receptors and the associated intracellular calcium pathway related to AT₁ receptor may play an important role in either the initiation or the maintenance of growth and in the function of cardiac fibroblasts in the cardiac hypertrophy present in DOCA-salt–hypertensive rats.

In conclusion, we report for the first time that cardiac hypertrophy in DOCA-salt–hypertensive Sprague-Dawley rats is characterized by cytosolic free calcium overload in myocyte and nonmyocyte cells. There is upregulation of AT₁ receptors and enhanced responsiveness of [Ca²⁺]_i transients to Ang II in hypertrophied myocytes, whereas AT₁ receptor status (binding and signal transduction) was unaltered in fibroblasts. These findings indicate that in response to chronic pressure overload associated with DOCA-salt hypertension, cardiac AT₁ receptors and the AT₁ signal transduction pathway are regulated in a differential manner in myocyte and nonmyocyte cells. Ang II, through the enhanced function of AT₁ receptors in myocytes, may contribute to the initiation or maintenance of hypertensive cardiac hypertrophy in DOCA-salt rats.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II [Ca2+]i = intracellular free Ca2+ concentration DOCA = deoxycorticosterone acetate Uni-Nx = unilaterally nephrectomized

	Acknowledgments

 
This work was supported by a group grant to the Multidisciplinary Research Group on Hypertension from the Medical Research Council of Canada and grants from the Fondation des Maladies du Coeur du Québec. Dr Fareh is the recipient of a Heart and Stroke Foundation-Merck-Frosst Canada Inc postdoctoral fellowship. We thank André Turgeon for technical assistance.

Received September 19, 1996; first decision October 21, 1996; accepted May 12, 1997.

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