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(Hypertension. 1996;28:797-805.)
© 1996 American Heart Association, Inc.

Articles

Intracellular Ca²⁺ Modulation by Angiotensin II and Endothelin-1 in Cardiomyocytes and Fibroblasts From Hypertrophied Hearts of Spontaneously Hypertensive Rats

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

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

	Abstract

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The vasoactive peptides angiotensin II (Ang II) and endothelin-1 (ET-1) have been implicated in cardiac hypertrophy. This study investigates Ang II and ET-1 effects on intracellular free calcium concentration and the receptor subtype through which agonist-induced calcium responses are mediated in isolated cardiomyocytes and fibroblasts from hypertrophied hearts of spontaneously hypertensive rats (SHR). We measured intracellular free calcium concentration by fura 2 methodology and determined receptor status by radioligand binding assays. Ang II (10^-12 to 10^-7 mol/L) had no effect on cardiomyocyte calcium levels in control Wistar-Kyoto rats but significantly increased (P<.01) intracellular free calcium concentration in a dose-dependent manner in cardiomyocytes from SHR. Ang II total and specific binding were increased (P<.05) in SHR cardiomyocytes. Calcium responses elicited by 10^-7 to 10^-5 mol/L Ang II were significantly reduced (P<.01) in SHR fibroblasts despite no significant change in Ang II receptor density. The angiotensin type 1 receptor blocker losartan (1 µmol/L) blocked Ang II–stimulated calcium transients, whereas the angiotensin type 2 receptor blocker PD 123319 had no effect. ET-1– and sarafotoxin S6c–induced calcium responses in cardiomyocytes and fibroblasts were not different between hypertensive and control groups. In conclusion, Ang II and ET-1 elicit distinct and differential responses in a cell-specific manner in cardiomyocytes and fibroblasts from hypertrophied hearts of SHR. Whereas Ang II–mediated effects, which are elicited via angiotensin type 1 receptors, are detectable in cardiomyocytes from SHR, responses to Ang II are blunted in fibroblasts from SHR, and ET-1–related actions are similar in cells from both rat groups. Stimulation of cardiomyocytes by Ang II in hypertrophied hearts associated with pressure overload in genetic hypertension suggests that Ang II could modulate the function of cardiomyocytes of SHR but not those of Wistar-Kyoto rats, whereas cardiac actions of ET-1 do not change with the development of hypertension.

Key Words: rats, inbred SHR • hypertrophy • angiotensin II • endothelin • intracellular • receptors, angiotensin

	Introduction

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Cardiac cells are targets for a variety of growth factors and vasoactive peptides. Among these, Ang II and ET-1 play an important role in cardiac growth and differentiation^{1 2} and in the regulation of myocardial contraction.^{3 4} In addition to these physiological actions, Ang II and ET-1 have been associated with pathological conditions such as myocardial ischemia, arrhythmias, and ventricular hypertrophy.^{5 6 7} Locally produced cardiac peptides have been implicated in exerting a hypertrophic effect on the heart in an autocrine/paracrine fashion.^{1 8}

The multiple direct cardiac actions of Ang II and ET-1 are initiated by their binding to specific cell membrane receptors. High-affinity Ang II binding sites are found in neonatal and adult cardiomyocytes and fibroblasts of many species and in the conduction system of rat and guinea pig hearts.^{9 10 11} Two Ang II receptor subtypes, AT₁-R and AT₂-R, have been identified by ligand binding studies with nonpeptide Ang II antagonists.¹² AT₁-R are selectively blocked by DuP 753 (losartan), and AT₂-R are selectively blocked by PD 123177 or CGP 42112A.¹² Two subvariants of the AT₁-R, AT_1A and AT_1B, have been characterized by DNA sequencing.¹³ In the rat heart, Ang II receptor expression is increased during the neonatal period and decreases with maturation.¹⁴ Recently, we demonstrated that neonatal rats possess AT₁-R and that atrial cardiomyocytes express both AT_1A-R and AT_1B-R genes but ventricular cardiomyocytes express only AT_1B-R genes.¹⁵ In cardiomyocytes from adult rats, AT₁-R are expressed in very low numbers, whereas in fibroblasts, receptors are detectable and ample calcium transients may be recorded in response to Ang II.¹⁶ In cardiomyocytes from volume-overload hypertrophied hearts, Ang II receptor density is increased, whereas in cardiac fibroblasts, Ang II receptors are simultaneously downregulated.¹⁶ The cardiac hypertrophy of SHR and two-kidney, one clip renovascular hypertensive rats also exhibits alterations in Ang II receptor status, with ventricular AT_1A-R mRNA levels and Ang II receptor densities increased compared with normotensive controls.^{14 17} Further evidence for a role of Ang II in cardiac hypertrophy is the effect of Ang II receptor antagonists and angiotensin-converting enzyme inhibitors, which prevent myocardial hypertrophy in SHR.^{18 19}

ET-1 also influences cardiac function by modulating chronotropic and inotropic responses of the heart, by inducing coronary vasoconstriction, and by potentiating atrial natriuretic peptide release by myocytes.^{20 21} ET-1 effects are mediated by two receptor subtypes, ET_A, which is characterized by a higher affinity toward ET-1 compared with ET-3, and ET_B, which has an equal affinity for the three endothelin peptides ET-1, ET-2, and ET-3.²² Endothelin receptors are present in rat neonatal and adult myocardium and have been demonstrated in both cardiomyocytes and fibroblasts.^{16 21 23} The proportion of ET_A-R to ET_B-R is approximately 85% to 15% in cardiomyocytes and is almost equal in fibroblasts.^{16 24} Like Ang II, ET-1 has also been postulated to contribute to cardiac hypertrophy. In cultured ventricular cardiac cells, ET-1 stimulates proto-oncogene expression and increases protein synthesis,²⁵ and in pressure-overload cardiac hypertrophy, ET-1 and its binding sites are upregulated.^{26 27}

In cardiac fibroblasts, as in other tissues, Ang II and ET-1 mediate their intracellular effects through receptor-mediated stimulation of the breakdown of inositol phospholipids, resulting in the generation of inositol trisphosphate and 1,2-diacylglycerol.^{28 29} Diacylglycerol stimulates membrane-bound, phospholipid-dependent, Ca²⁺-dependent protein kinase C, whereas inositol trisphosphate releases Ca²⁺ from sarcoplasmic reticular stores, leading to elevation of [Ca²⁺]_i.²⁸ In cardiomyocytes, the major mechanism for [Ca²⁺]_i elevation is through Ca²⁺-induced Ca²⁺ release. [Ca²⁺]_i is a major determinant of myocardial excitation-contraction coupling, and uncontrolled changes in [Ca²⁺]_i may result in pathological sequelae.²⁸ In myocytes from hypertrophied guinea pig hearts, basal [Ca²⁺]_i is reduced,³⁰ whereas in renovascular hypertensive rats, basal [Ca²⁺]_i is increased, sensitivity to Ang II is enhanced, and the Ca²⁺ transient is significantly prolonged.^{17 31} In volume-overload cardiac hypertrophy, we recently demonstrated that ET-1–mediated [Ca²⁺]_i transients are unchanged, whereas [Ca²⁺]_i responses to Ang II are increased.¹⁶ These [Ca²⁺]_i changes may be associated with spontaneous Ca²⁺ oscillations and with the generation of arrhythmias in cardiac hypertrophy.

Despite extensive data in the literature regarding the pathophysiological role of Ang II and ET-1 in experimentally induced cardiac hypertrophy,^{16 17 18 19 25 26 27} relatively little is known about the effect of these peptides in cardiac hypertrophy associated with spontaneous hypertension. The SHR is a model of genetic hypertension that in many respects resembles the course of human hypertensive cardiovascular disease,³² and the vascular smooth muscle cells of these rats have elevated basal [Ca²⁺]_i and display enhanced responsiveness to Ang II³³ ; therefore, we investigated whether intracellular Ca²⁺ mobilization and peptide actions are altered in SHR hypertrophied myocytes.

Our objectives in the present study were to assess [Ca²⁺]_i responses to Ang II and ET-1 in isolated cardiomyocytes and fibroblasts from SHR hearts and to determine the receptor subtypes through which Ang II and ET-1 mediate their [Ca²⁺]_i effects. We studied SHR at 17 weeks of age because at this age, hypertension is well established and cardiac hypertrophy has developed.

	Methods

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Reagents and Peptides
Ang II, [Sar¹,Ile⁸]-Ang II, ET-1, and sarafotoxin S6c were obtained from Peninsula Laboratories. DuP 753 and PD 123319 were gifts from DuPont-Merck Pharmaceutical Co and Parke-Davis, respectively. Fura 2-acetoxymethyl ester (fura 2-AM) and pluronic F-127 were from Molecular Probes. All other chemicals were obtained from Sigma Chemical Co, Fisher Scientific Co, and Life Technologies Inc.

Animals
Animal experiments were performed following the recommendations of the Canadian Council for Animal Care and were approved by the Animal Care Committee of the Clinical Research Institute of Montreal. Eleven adult (16-week-old) male SHR and 10 WKY (Taconic Farms, Germantown, NY) were maintained on a standard rat chow, housed under standardized conditions of constant temperature (22°C), and exposed to a 12-hour light/dark cycle for 1 week before use. Systolic pressure was measured in conscious, restrained, warmed rats by the tail-cuff method with a PCB photoelectric pulse sensor and was recorded on a model 7 polygraph fitted with a 7-P8 preamplifier (all from Grass Medical Instruments).

Isolation of Cardiomyocytes
Each rat was used for a separate cell preparation. On each occasion, cells were prepared from one SHR and its normotensive counterpart. Rats were injected with heparin sulfate (500 U IP) (Hepalcan, Organon Canada Ltd) and anesthetized with 60 mg/kg pentobarbital sodium IP. The hearts were removed rapidly. Ca²⁺-tolerant cardiomyocytes were isolated by the Langendorff method (cardiac retrograde aortic perfusion) as previously described by Eid et al³⁴ and already reported by our group.^{16 35} Briefly, hearts were rinsed (4 mL/min) for 5 minutes in Krebs-Henseleit (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 solution was then used to stop spontaneous cardiac contraction. Hearts were perfused for 20 minutes with 0.05% collagenase (CLS2, Worthington Biochemical Corp) and 0.03% hyaluronidase in KH buffer, after which ventricles were separated from atria. Ventricles were minced and incubated in KH buffer 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 at 1000g. Cells were diluted and allowed to sediment in washed solution (medium 199/KH, 1:1). Cells were then layered on 10 mL of 6% bovine serum albumin solution to separate cardiomyocytes (heavy cells) from noncardiomyocytes (light cells). Freshly isolated cells were diluted in culture medium 199 that contained 0.2% bovine serum albumin, 10^-7 mol/L insulin, 5 mmol/L creatine, 2 mmol/L L-carnitine, 5 mmol/L taurine, 100 IU/mL penicillin, and 100 µg/mL streptomycin with 10% fetal bovine serum.

For [Ca²⁺]_i measurements, cells were prepared as previously described.^{16 36} Briefly, cells were seeded onto laminin-coated round glass coverslips (25-mm diameter) in culture dishes (7000 cells per 2 cm²). After 1 hour in a humidified incubator (5% CO₂/95% air) at 37°C, the medium was changed to remove globular-shaped cells (damaged cells) and debris. Using this method, we obtained more than 90% Ca²⁺-tolerant rod-shaped cardiomyocytes. Serum-free medium was added overnight, and [Ca²⁺]_i was determined the following day. For radioligand binding assays, ventricular cardiomyocytes 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).

Preparation of Cultured Ventricular Fibroblasts
Rats were injected with heparin sulfate and pentobarbital as described above. Once the heart had been isolated, the ventricles were removed and washed in sterile saline solution. They were minced and digested in 15 mL Dulbecco's modified Eagle's medium (DMEM) (containing 0.1% trypsin and 100 U/mL collagenase CLS2) with agitation (150 cycles per minute) for 15 minutes. Cells were repeatedly sedimented and digested according to previously described methods.¹⁶ Isolated cells were pooled and centrifuged (3 minutes at 2000g) and the pellet resuspended in DMEM plus 10% fetal bovine serum. The cell preparation was diluted in 150 mL DMEM/10% fetal bovine serum and seeded in six-well plates (onto glass coverslips) for [Ca²⁺]_i determinations and in 24-well plates for binding assays. Plated cells were incubated for 2 hours at 37°C (10% CO₂/90% air humidified incubator). The nonadherent cells were then removed, and fresh serum medium was added. The fibroblasts were grown until confluence (5 days after isolation). In preliminary studies, we found that primary cultured fibroblasts maintained their cell phenotype after 5 days in culture according to an immunostaining method and that Ang II and ET-1 binding assays were stable for 2 to 7 days in culture. Twenty-four hours before [Ca²⁺]_i measurements and radioligand binding studies, culture medium was replaced by serum-free medium.

Measurement of [Ca²⁺]_i
[Ca²⁺]_i was determined with the use of the fluorescent probe fura 2-AM according to previously described methods.³⁵ Briefly, cardiomyocytes and fibroblasts were loaded with 4 µmol/L fura 2-AM (dissolved in dimethyl sulfoxide with 0.02% pluronic) for 30 minutes at 37°C in a humidified incubator with 95% air/5% CO₂. Cells were then 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 was determined with the Axiovert 135 inverted microscope and Attofluor digital fluorescence system (Zeiss) using dual excitatory wavelengths of 343 and 380 nm and a single-emission wavelength of 520 nm.³⁶ Both in situ and in vitro calibrations were made according to our previously described methods.³⁵ [Ca²⁺]_i was determined with the equation of Grynkiewicz et al³⁶ : [Ca²⁺]_i=K_dxß(R-R_min)/(R_max-R), where K_d is the dissociation constant for fura 2–Ca²⁺ and taken to be 224 nmol/L,^{36 37 38} ß is the ratio of fluorescence at 380 nm and zero Ca²⁺ (F_380min) and saturating Ca²⁺ (F_380max) conditions, and R is the ratio of fluorescence obtained with excitation at 343 and 380 nm, with min and max subscripts denoting the ratios obtained under Ca²⁺-free and Ca²⁺-saturated conditions, respectively. Maximal and minimal fluorescence intensities were obtained for each experiment by exposure of cells to 10 µmol/L ionomycin and 3 mmol/L EGTA, respectively. R_min and R_max values for SHR and WKY cardiomyocytes and fibroblasts are presented in Table 1. Video images of fluorescence were obtained with an intensified charge-coupled device (CCD) camera system (Zeiss) with the output digitized to a resolution of 512x480 pixels. Images of fluorescence ratios were obtained by dividing, pixel by pixel, the 343-nm image after background subtraction by the 380-nm image after background subtraction. After a 10- to 15-minute equilibration period, cultured cells were exposed to a single concentration of Ang II or ET-1 (10^-12 to 10^-5 mol/L) at room temperature. For determination of the Ang II receptor subtype that mediates Ang II–induced [Ca²⁺]_i responses, effects of Ang II (1 nmol/L) were assessed in cells that had been preincubated for 10 minutes with 1 µmol/L [Sar¹,Ile⁸]-Ang II (a nonselective Ang II receptor blocker), DuP 753 (a selective AT₁-R blocker), or PD 123319 (a selective AT₂-R blocker). For determination of whether cardiomyocytes possess functional ET_B-R, [Ca²⁺]_i effects of sarafotoxin S6c (10^-8 to 10^-6 mol/L) were assessed. The maximal peak ratio recorded was considered to be the maximal response of the agonist.

View this table: [in this window] [in a new window]   Table 1. Maximal and Minimal Fluorescence Ratios in Cardiomyocytes and Fibroblasts From Wistar-Kyoto and Spontaneously Hypertensive Rats

Ang II Receptor Binding Assays
Ang II binding studies were performed in duplicate in serum-free culture medium at room temperature for 90 minutes. [Sar¹,Ile⁸]-Ang II was radiolabeled (¹²⁵I) by the lactoperoxidase method and purified by high-performance liquid chromatography.³⁹ Increasing concentrations (10^-12 to 10^-6 mol/L) of [Sar¹,Ile⁸]-Ang II, DuP 753, or PD 123319 and 100 to 120 pmol/L of ¹²⁵I–[Sar¹,Ile⁸]-Ang II (2200 Ci/mmol) were used for Ang II receptor characterization. Nonspecific binding was determined with unlabeled [Sar¹,Ile⁸]-Ang II at 10^-6 mol/L. For cardiomyocyte binding studies, the reaction was stopped with 3.5 mL of 50 mmol/L Tris-HCl (pH 7.2) and 0.15 mol/L NaCl. The preparation was rapidly filtered through glass filters (Schleicher & Schuell) with a cell harvester (Brandel). Filters were rinsed with the Tris-HCl solution. After the binding reaction, attached fibroblasts were washed twice with 0.5 mL of culture medium (DMEM) and digested by 0.5 mL of 1N NaOH. Radioactivity on the filters or on digested cells was counted in a gamma counter with 80% efficiency (LKB Wallac). Binding data were analyzed with EBDA and LIGAND software of McPherson (Biosoft).

Statistical Analysis
Data are reported as mean±SE. For [Ca²⁺]_i measurements, 4 to 10 experiments were performed for each peptide concentration, with each experimental field comprising many cells. Statistical significance was evaluated by unpaired Student's t test or ANOVA when appropriate followed by Tukey-Kramer's correction to compensate for multiple testing. Concentration-response curves were fitted by nonlinear regression, and the concentration giving 50% of the maximal response (EC₅₀) was determined and pD₂ calculated as -log[EC₅₀ (mol/L)]. Differences were considered significant at a value of P<.05.

	Results

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Blood Pressure, Body Weight, and Heart Weight
Systolic pressure was significantly higher (P<.01) in SHR than WKY (Table 2). SHR were significantly lighter (P<.01) than their normotensive counterparts (Table 2). Heart weight (P<.05) and the ratio of heart weight to body weight (P<.01) were significantly greater in SHR than WKY (Table 2).

View this table: [in this window] [in a new window]   Table 2. Systolic Pressure, Body Weight, and Heart Weight in Wistar-Kyoto and Spontaneously Hypertensive Rats

Characterization of Cardiac Cells
Primary cell cultures can be contaminated by other cell types. The purity of our cardiomyocyte and fibroblast preparations was greater than 95%, determined on the basis of morphological features and immunochemical staining of the cells. Morphologically adult cardiomyocytes are rod shaped and striated and contract spontaneously, whereas fibroblasts are noncontractile flat cells of variable shape and size. We performed immunochemical staining in our previous studies to estimate cell purity in cardiomyocyte and fibroblast preparations.¹⁶ Cardiac fibroblasts in the fibroblast preparation were identified by anti-vimentin and demonstrated greater than 90% positive staining. Immunostaining of cells in the cardiomyocyte preparation with anti-actin and anti-desmin demonstrated that more than 95% of the cells stained positive, indicating that our cardiomyocyte preparation was of a high purity.

[Ca²⁺]_i studies and binding assays were performed in primary cultured cardiomyocytes that had been in culture for 12 to 14 hours and in primary cultured unpassaged fibroblasts that had been in culture for 6 days. At these times, light microscopy revealed that both cell types were morphologically similar to freshly plated cells. Additionally, we have previously reported that [Ca²⁺]_i responsiveness in cultured cells is similar to that in freshly isolated cells³⁵ and that ET-1 saturation and competition binding analyses gave the same results in freshly isolated cells as in cells cultured for 20 hours (unpublished data, 1995). These data suggested that cardiomyocytes studied at 12 to 14 hours and fibroblasts studied 6 days after plating did not exhibit significant phenotypic and functional changes in culture.

Basal [Ca²⁺]_i in Cardiomyocytes and Fibroblasts
In the resting and stimulated state, cardiomyocytes have oscillatory [Ca²⁺]_i waves that are absent in fibroblasts (Fig 1). In the present study, cardiomyocyte [Ca²⁺]_i data are presented as the diastolic, systolic, and spike [Ca²⁺]_i values according to previously described definitions.³⁸ Diastolic [Ca²⁺]_i was determined as the average of the lowest point of [Ca²⁺]_i oscillations recorded over a 30-second interval (Fig 1). The systolic value was taken as the average of the maximal points corresponding to the diastolic [Ca²⁺]_i (Fig 2). The frequency of [Ca²⁺]_i spikes was determined over 60 seconds. Basal fibroblast and cardiomyocyte systolic [Ca²⁺]_i values were significantly higher (P<.05) in SHR than WKY (Fig 2). Basal [Ca²⁺]_i spike frequency did not differ in cardiomyocytes from SHR and WKY (Fig 2).

View larger version (21K): [in this window] [in a new window]   Figure 1. Representative tracings of [Ca2+]i measurements in WKY cardiomyocytes and fibroblasts loaded with fura 2-acetoxymethyl ester and stimulated with 10-8 mol/L ET-1. Top, Tracing from cardiomyocytes that demonstrates [Ca2+]i oscillations; inset, tracing of oscillations measured over 20 seconds. A indicates the lowest points of [Ca2+]i oscillations, corresponding to diastolic [Ca2+]i; B indicates the maximal points of [Ca2+]i oscillations, corresponding to systolic [Ca2+]i. Bottom, Tracing from fibroblasts demonstrating that the [Ca2+]i response is smooth and nonoscillatory. [Ca2+]i is expressed in nmol/L.

View larger version (29K): [in this window] [in a new window]   Figure 2. Basal [Ca2+]i in WKY and SHR cardiomyocytes and fibroblasts. Cardiomyocyte [Ca2+]i values are expressed as diastolic, systolic, and spike frequency. Diastolic [Ca2+]i corresponds to the average of the lowest point of [Ca2+]i oscillations over 30 seconds; systolic [Ca2+]i is the average of the maximal points corresponding to diastolic [Ca2+]i. [Ca2+]i spike frequency corresponds to the oscillatory spikes over 60 seconds. Numbers in parentheses indicate number of experiments, with each experimental field comprising 6 to 10 cells for cardiomyocytes and 10 to 20 cells for fibroblasts. *P<.05, **P<.01 vs WKY.

Ang II–Induced [Ca²⁺]_i Responses in Cardiomyocytes and Fibroblasts
Effects of Ang II on cardiomyocyte and fibroblast [Ca²⁺]_i are presented in Fig 3. Ang II at concentrations less than 10^-7 mol/L had no effect on [Ca²⁺]_i in WKY cardiomyocytes. At 10^-6 and 10^-7 mol/L, Ang II increased systolic [Ca²⁺]_i in WKY cardiomyocytes by 78±6 and 104±6 nmol/L, respectively (Fig 3). In SHR cardiomyocytes, Ang II increased diastolic, systolic, and [Ca²⁺]_i spike frequency in a concentration-dependent manner (Fig 3). Cardiomyocyte [Ca²⁺]_i responses and [Ca²⁺]_i sensitivity to Ang II were significantly greater (P<.01) in SHR than WKY (pD₂: 7.9±0.2 versus 6.4±0.2, SHR versus WKY). Ang II–stimulated [Ca²⁺]_i spike frequency did not differ between SHR and WKY.

View larger version (28K): [in this window] [in a new window]   Figure 3. Concentration-response curves for Ang II in WKY and SHR cardiomyocytes and cardiac fibroblasts. [Ca2+]i results are presented as maximal diastolic, systolic, and spike frequency [Ca2+]i responses elicited by each Ang II concentration. Definitions are as in Fig 2 legend. Values are mean±SE, with each data point being the mean of four to six experiments and each experimental field comprising 4 to 8 cells for cardiomyocytes and 10 to 18 cells for fibroblasts. *P<.05, **P<.01, +P<.001 vs WKY.

[Ca²⁺]_i responses to Ang II in WKY and SHR fibroblasts were dose dependent. In WKY fibroblasts, the pD₂ of the response curve to Ang II was 8.4±0.2, and in SHR fibroblasts, the pD₂ was 9.1±0.1. In SHR fibroblasts, absolute [Ca²⁺]_i responses and net [Ca²⁺]_i changes elicited by Ang II at concentrations greater than 10^-8 mol/L were significantly lower (P<.01) than in WKY fibroblasts (Fig 3).

Effects of DuP 753 and PD 123319 on Ang II–Induced Effects in Cardiomyocytes and Fibroblasts
To determine the Ang II receptor subtype through which Ang II mediates its [Ca²⁺]_i responses in cardiac cells, we assessed Ang II effects in the presence of [Sar¹,Ile⁸]-Ang II (an Ang II receptor blocker that does not differentiate receptor subtypes), DuP 753 (a selective AT₁-R blocker), and PD 123319 (a selective AT₂-R blocker) (Fig 4). We conducted these experiments using a fixed concentration of 1 nmol/L Ang II (corresponding approximately to EC₃₀). Since WKY cardiomyocytes failed to respond to 1 nmol/L Ang II, these cardiomyocytes were not used for examination of Ang II receptor subtypes. In SHR cardiomyocytes and SHR and WKY fibroblasts, [Sar¹,Ile⁸]-Ang II and DuP 753 completely blocked the Ang II–induced [Ca²⁺]_i response. PD 123319 had no significant effect on Ang II–elicited [Ca²⁺]_i responses (Fig 4).

View larger version (30K): [in this window] [in a new window]   Figure 4. Fibroblast and cardiomyocyte systolic [Ca2+]i effects of 1 nmol/L Ang II in the absence and presence of 1 µmol/L [Sar1,Ile8]-Ang II (Sar) (nonspecific Ang II receptor blocker), losartan (Los) (selective AT1-R blocker), and PD 123319 (PD) (selective AT2-R blocker). +P<.001 vs other groups. Numbers in parentheses indicate number of experiments, with each experimental field comprising 8 to 15 cells. WKY cardiomyocytes were not studied because 1 nmol/L Ang II did not induce a significant effect on [Ca2+]i.

Ang II Receptor Binding Studies
Cardiomyocytes from control rats exhibited very little specific binding, suggesting a low number of Ang II receptors. Total and specific Ang II binding were significantly greater in SHR cardiomyocytes, suggesting a greater density of Ang II receptors on cardiomyocytes from SHR than WKY (Fig 5). Because of the low receptor density, we could not perform Ang II competitive studies on cardiomyocytes. Fibroblast Ang II receptor subtypes were assessed with [Sar¹,Ile⁸]-Ang II, DuP 753, and PD 123319. [Sar¹,Ile⁸]-Ang II and DuP 753 completely displaced the tracer, whereas PD 123319 had no effect, indicating that Ang II receptors on ventricular fibroblasts were exclusively of the AT₁-R subtype. The maximal binding densities (B_max) and apparent dissociation constants (K_d) for WKY and SHR fibroblasts were not significantly different (Table 3).

View larger version (15K): [in this window] [in a new window]   Figure 5. Total, specific, and nonspecific Ang II binding on WKY and SHR cardiomyocytes. Data were calculated as cpm for 50 000 cells. Values are mean±SE; n=5 rats per strain. *P<.05 vs WKY.

View this table: [in this window] [in a new window]   Table 3. Angiotensin II Binding Parameters of Primary Cultured Ventricular Fibroblasts From Wistar-Kyoto and Spontaneously Hypertensive Rats

ET-1–Induced [Ca²⁺]_i Responses in Cardiomyocytes and Fibroblasts
ET-1 increased [Ca²⁺]_i in a dose-dependent manner in SHR and WKY cardiomyocytes and fibroblasts. The concentration-response curve of ET-1 was not significantly modified in either SHR cell type (Fig 6). [Ca²⁺]_i sensitivity to ET-1 was similar in SHR (pD₂=7.2±0.3) and WKY (pD₂=7.8±0.3) cardiomyocytes and in SHR (8.2±0.4) and WKY (7.2±0.6) fibroblasts.

View larger version (25K): [in this window] [in a new window]   Figure 6. Concentration-response curves for ET-1 in WKY and SHR cardiomyocytes and cardiac fibroblasts. Cardiomyocyte [Ca2+]i results are presented as maximal diastolic, systolic, and spike frequency [Ca2+]i responses elicited by each ET-1 concentration. Definitions are as in Fig 2 legend. Values are mean±SE, with each data point being the mean of four to six experiments and each experimental field comprising 5 to 10 cells for cardiomyocytes and 10 to 20 cells for fibroblasts.

In addition to assessing cardiomyocyte [Ca²⁺]_i effects of ET-1 (which stimulates both ET_A-R and ET_B-R), we determined the effects of sarafotoxin S6c, a highly selective ET_B-R agonist. Fig 7 demonstrates that sarafotoxin S6c increased systolic [Ca²⁺]_i in WKY and SHR cardiomyocytes in a dose-dependent fashion, indicating that cardiomyocytes contain ET_B-R. The absolute and net [Ca²⁺]_i change elicited by sarafotoxin S6c was not significantly different between WKY and SHR. We did not perform ET-1 receptor binding assays in the present study because we have recently reported that the ET-1 receptor status is similar in WKY and SHR hearts.²⁴

View larger version (20K): [in this window] [in a new window]   Figure 7. Effects of sarafotoxin S6c (S6c), the ETB-R agonist, on systolic [Ca2+]i in WKY and SHR cardiomyocytes. Values are mean±SE. Numbers in parentheses indicate number of experiments, with each experimental field comprising 5 to 10 cells. *P<.05, **P<.01 vs basal.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates that cultured cardiomyocytes and fibroblasts from hypertrophied hearts of SHR exhibit differential responses to Ang II and ET-1. In SHR cardiomyocytes, Ang II–induced [Ca²⁺]_i responses, [Ca²⁺]_i sensitivity to Ang II, and Ang II binding are significantly increased, whereas ET-1–elicited responses, which are mediated by ET_A-R and ET_B-R subtypes, are unaltered. In SHR fibroblasts, [Ca²⁺]_i responsiveness to Ang II is reduced and responses to ET-1 are normal. The accuracy of measuring [Ca²⁺]_i depends on accurate calibration of the hydrolyzed fura 2. In the present investigation, we used an in situ calibration technique to obtain [Ca²⁺]_i values and to demonstrate that hydrolysis of fura 2 in the two rat strains was similar. Thus, the interstrain differences reported here are true [Ca²⁺]_i differences and are not due to differential fura 2 loading. Since the purpose of our study was to determine underlying cellular events in the pathophysiology of cardiac hypertrophy, we examined isolated cultured cells rather than intact heart. Primary cultured cardiomyocytes and fibroblasts represent excellent models for the study of cellular and biochemical events in cardiac cells because several features of in vivo behavior are retained in culture.⁴⁰ We investigated cardiomyocytes that had been in culture for 12 hours and fibroblasts that had been in culture for 6 days. At the time of experimentation, these cells exhibited morphological and biochemical characteristics similar to those of freshly plated cells,³⁵ suggesting that they have not undergone significant phenotypic or functional change in culture.

Established hypertension in adult SHR is associated with vascular structural alterations as well as cardiac hypertrophy.⁴¹ In our study, heart weight and the ratio of heart weight to body weight were greater in SHR than in WKY, indicating significant cardiac hypertrophy in SHR. Cardiac mass increases early in SHR, and already at 3 weeks of age, there is hypertrophy and hyperplasia, as evidenced by increased DNA and protein content.⁴¹ Factors underlying the genesis of cardiac hypertrophy in genetically hypertensive rats are multiple and include genetic, hemodynamic, and local modulation systems, such as the cardiac renin-angiotensin system. Ang II, the major mediator of the renin-angiotensin system, acts directly on cardiac tissue and may be involved in the development of cardiac hypertrophy. In vivo studies have demonstrated that Ang II has a hypertrophic action on heart tissue separate from indirect effects mediated through increases in blood pressure.¹ Angiotensin-converting enzyme inhibitors prevent or regress established left ventricular hypertrophy in SHR, whereas chronic infusion of Ang II into rats increases left ventricular mass even when subpressor doses of Ang II are used or when the pressor activity of Ang II is blocked.¹⁸ In vitro studies have also implicated Ang II in the cardiac hypertrophy associated with hypertension. In cultured cardiomyocytes, Ang II induces cellular hypertrophy, increases protein synthesis, and increases proto-oncogene mRNA, whereas Ang II receptor blockade blocks these effects.^{42 43} The biochemical processes by which Ang II stimulates cardiac hypertrophy in SHR are unclear, but [Ca²⁺]_i, a major intracellular signaling messenger, may play a role. In experimentally induced cardiac hypertrophy in rats, cardiac Ca²⁺ handling is altered. In the present study, we demonstrate, as we did previously,^{16 35} that cardiomyocytes from normal adult rats have low Ang II receptor binding and that [Ca²⁺]_i responses are significantly elevated only at high Ang II concentrations, suggesting that functional Ang II receptors are present but in very low numbers. In cardiomyocytes from hypertrophied hearts of genetically hypertensive SHR, basal [Ca²⁺]_i is elevated, Ang II–induced [Ca²⁺]_i responses are exaggerated, [Ca²⁺]_i sensitivity to Ang II is enhanced, and Ang II binding is increased. This augmentation of Ang II–related effects may be attributed to upregulation of Ang II receptors or to more efficient coupling of the peptide to its receptor. Our data in this pressure-overload model are in agreement with our previous study performed in volume-overload hypertrophy¹⁶ as well as other studies that demonstrated more pronounced sensitivity to Ang II and enhanced Ang II–stimulated phospholipid signaling in hypertrophied hearts.^{17 44}

In addition to studying cultured cardiomyocytes, we also examined cardiac fibroblasts, which constitute the major noncardiomyocyte cell type of the heart. Ang II increased [Ca²⁺]_i in a dose-dependent manner in WKY and SHR fibroblasts. Although receptor status in SHR was not significantly different from that of WKY and [Ca²⁺]_i sensitivity to Ang II was unchanged, [Ca²⁺]_i responses elicited by high concentrations of Ang II were significantly blunted in SHR. Underlying mechanisms for this attenuation are unclear, but it may be possible that in the presence of high Ang II concentrations, receptors are desensitized, ligand-receptor coupling is altered, or receptor distribution is changed. We have previously reported reduced Ang II–stimulated responsiveness and decreased receptor density in fibroblasts in volume-overload cardiac hypertrophy.¹⁶ The difference between the previous study¹⁶ and the present one, in which Ang II receptor density on cardiac fibroblasts was not significantly altered, could be because downregulation of Ang II receptors in fibroblasts may occur only in severe forms of cardiac hypertrophy. In the less severe forms such as in SHR, in which Ang II receptor status is unchanged, altered ligand-receptor coupling could explain attenuated [Ca²⁺]_i responses to Ang II. With progression of cardiac hypertrophy, as in our previous work in volume-overload hypertrophy,¹⁶ fibroblast Ang II receptor downregulation may underlie further attenuation of fibroblast calcium responses to Ang II.

We assessed the receptor subtype through which Ang II mediates its intracellular events both pharmacologically and by binding studies. We demonstrate here that rat cardiac Ang II receptors are exclusively of the AT₁-R subtype and that these receptors are linked to the Ca²⁺ signaling pathway, in agreement with other reports.⁴⁵ AT₁-R mediate cell proliferation and myocardial fibrosis and may play a pathophysiological role in the development of cardiac hypertrophy in SHR because this effect in response to Ang II is inhibited both in vivo and in vitro by the selective AT₁-R blocker DuP 753.^{1 46}

ET-1 has also been implicated in cardiac hypertrophy, either as an endogenously generated intermediate of the Ang II–induced hypertrophic response or as an independent, directly acting agent. ET-1, which binds to ET_A-R and ET_B-R, and sarafotoxin S6c, which selectively binds to ET_B-R, elicited large dose-dependent [Ca²⁺]_i increases in cardiomyocytes and fibroblasts from both rat strains. These results indicate the presence of functional endothelin receptors of both subtypes on rat cardiac cells and confirm our earlier data derived from binding studies.²⁴ In contrast to enhanced Ang II–stimulated effects in SHR cardiac cells, ET-1–induced effects were not significantly different in either cardiomyocytes or fibroblasts of WKY and SHR. These findings parallel our previous, less in-depth study in which we demonstrated that cardiac ET-1 content and endothelin receptor density and affinity were similar in whole hearts of WKY and SHR.²⁴ Delbridge et al⁴⁷ also failed to demonstrate differences in contractile responses evoked by ET-1 in WKY and SHR ventricular cardiomyocytes. These results suggest that ET-1 may not play a crucial role in established cardiac hypertrophy in SHR. This agrees with our previous reports showing that chronic treatment of SHR with endothelin antagonists during the developmental phase⁴⁸ or in the phase of established hypertension⁴⁹ did not affect cardiac hypertrophy. It should also be noted that in a model of endothelin-dependent hypertension, the deoxycorticosterone acetate–salt hypertensive rat, even when blood pressure rose significantly less in rats treated chronically with endothelin receptor antagonists, cardiac hypertrophy was unaffected,⁵⁰ suggesting a small role for endothelins in hypertrophy of the heart in hypertensive rats.

In conclusion, the present study demonstrates that in cardiac hypertrophy associated with spontaneous hypertension, Ang II and ET-1 elicit distinct and differential responses in a cell-specific manner. Whereas Ang II–mediated effects are enhanced in cardiomyocytes, responses are blunted in fibroblasts, and ET-1–related actions are not modified. Stimulation of cardiomyocytes by Ang II in hypertrophied hearts associated with pressure overload in genetic hypertension suggests that Ang II modulates the function of cardiomyocytes of SHR but not those of WKY, whereas cardiac actions of ET-1 do not change with the development of hypertension.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1-R, AT2-R = angiotensin type 1, type 2 receptor(s) [Ca2+]i = intracellular free calcium concentration ET-1 = endothelin-1 SHR = spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This study was supported by a group grant to the Multidisciplinary Research Group on Hypertension from the Medical Research Council (MRC) of Canada. Drs Touyz and Fareh are recipients of fellowships from the MRC and Canadian Hypertension Society/MRC, respectively. The authors thank Carole Tremblay for secretarial assistance.

	Footnotes

 
Reprint requests to Rhian M. Touyz, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, Quebec, Canada H2W 1R7.

Received May 3, 1996; first decision May 15, 1996; accepted June 7, 1996.

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