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(Hypertension. 1996;27:1090-1096.)
© 1996 American Heart Association, Inc.

Articles

Cytosolic Calcium Changes Induced by Angiotensin II in Neonatal Rat Atrial and Ventricular Cardiomyocytes Are Mediated via Angiotensin II Subtype 1 Receptors

Rhian M. Touyz; Pavol Sventek; Richard Larivière; Gaétan Thibault; Jeannette Fareh; Timothy Reudelhuber; Ernesto L. Schiffrin

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

	Abstract

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Abstract We determined the effects of angiotensin II (Ang II) on cytosolic free calcium concentrations ([Ca²⁺]_i) in the absence and presence of the selective angiotensin subtype 1 (AT₁) receptor antagonist losartan or the selective AT₂ antagonist PD 123319 in cultured neonatal rat atrial and ventricular cardiomyocytes. We also assessed Ang II receptor density, affinity, and mRNA expression. [Ca²⁺]_i was measured in single cells microphotometrically and by fluorescent digital imaging with fura 2 methodology. Receptor parameters were assessed by competitive binding studies with ¹²⁵I-[Sar¹,Ile⁸]Ang II in the presence of increasing concentrations of [Sar¹,Ile⁸]Ang II, losartan, and PD 123319. AT₁ receptor (types AT_1A and AT_1B) mRNA abundance was measured by reverse transcription–polymerase chain reaction. Ang II produced concentration-dependent increases in [Ca²⁺]_i. Basal [Ca²⁺]_i values in atrial and ventricular cells were similar but Ang II (10^-9 mol/L)–induced [Ca²⁺]_i changes were significantly greater in atrial compared with ventricular cells. Ang II responses were blocked by losartan (10^-7 mol/L) but not PD 123319 (10^-7 mol/L). Binding studies demonstrated a single class of high-affinity Ang II binding sites on cardiomyocyte membranes (K_d=0.71±0.11 µmol/L). ¹²⁵I-[Sar¹,Ile⁸]Ang II was displaced by losartan but not by PD 123319. AT₁ receptor mRNA was detected by reverse transcription–polymerase chain reaction in cells from atria and ventricles. In atrial cardiomyocytes, both AT_1A and AT_1B receptor genes were expressed, whereas in ventricular cardiomyocytes, only the AT_1A receptor gene was expressed. These data demonstrate that neonatal cardiomyocytes possess Ang II receptors of the AT₁ receptor subtype that are linked to [Ca²⁺]_i signaling pathways. The different Ang II–induced [Ca²⁺]_i responses between atrial and ventricular cells may be related to differences in the distribution of AT₁ receptor subtype subvariants.

Key Words: cardiomyocytes • calcium, intracellular • angiotensin II • receptor, angiotensin

	Introduction

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The potent vasoactive peptide Ang II exerts effects directly and indirectly on the cardiovascular system.¹ Ang II directly stimulates cardiomyocyte excitation-contraction coupling, inducing positive inotropic and chronotropic responses in cardiac muscle,^{2 3} and indirectly influences these parameters by facilitating adrenergic neurotransmission.⁴ In addition to these physiological actions, Ang II has been associated with pathological consequences such as myocardial ischemia, hypertrophy, and pressure overload.^{5 6} The multiple cardiac actions of Ang II are mediated by changes in [Ca²⁺]_i.¹ Ang II activates inwardly directed Ca²⁺ currents,⁷ giving rise to increased Ca²⁺-induced Ca²⁺ release from cardiac sarcoplasmic reticular stores.⁸ This peptide also stimulates phospholipase C, which results in activation of protein kinase C and mobilization of intracellular Ca²⁺.^{9 10} Increased [Ca²⁺]_i may contribute to the positive inotropic effect of Ang II¹ ; however, not all studies have documented positive effects of Ang II. In neonatal rat ventricular cardiomyocytes, Ang II induces negative inotropic and positive chronotropic effects.⁷ These responses have been attributed to a rapid increase in [Ca²⁺]_i followed by a reduction in amplitude of the [Ca²⁺]_i transients.⁸ Furthermore, protein kinase C activation results in a negative inotropic effect in some myocardial preparations, including rat ventricular cardiomyocytes.¹¹ Most studies have examined the effects of Ang II in ventricular cells. Whether [Ca²⁺]_i responses in atrial cells are similar to those in ventricular cells is unknown.

Ang II–mediated effects occur via specific membrane receptors. Two Ang II receptor subtypes (AT₁ and AT₂) have been identified by ligand binding studies with nonpeptide Ang II antagonists.¹² AT₁-R is blocked by DuP 753 (losartan), and AT₂-R is blocked by PD 123177 and CGP 42112A.¹² AT₁-R has been cloned, and its second messenger system includes phospholipase C activation, Ca²⁺ mobilization, and protein kinase C stimulation,^{12 13} whereas the functional role of AT₂-R remains obscure.¹² Two subvariants of the AT₁-R, AT_1A and AT_1B, have been identified in the rat through DNA sequencing.¹⁴ These subvariants exhibit small differences (5%) in amino acid sequence but otherwise give identical binding results with Ang II analogues, and both utilize the Ca²⁺ pathway as a cytosolic second messenger.¹⁵ In the rat heart, Ang II receptor expression is increased during the neonatal period and decreases with maturation.¹⁶ The proportions of AT₁-R and AT₂-R are similar in the rat myocardium and do not change through development.^{17 18} The heart contains two major cell types: cardiomyocytes and fibroblasts.¹⁹ Although high-affinity Ang II binding sites have been demonstrated in cardiac tissues,¹⁷ which cardiac cell type is responsible for the binding sites is unclear. Some studies have demonstrated that fibroblasts express AT₁-R rather than AT₂-R,²⁰ whereas others have reported that isolated adult and neonatal rat cardiomyocytes exclusively exhibit AT₁-R.^{8 21} It has also been suggested that the Ang II receptor is expressed primarily in fibroblasts and not in cardiomyocytes.²² Thus, the receptor distribution in isolated cardiac cells appears distinct from that in cardiac tissue.

We designed this study to evaluate the effects of Ang II on [Ca²⁺]_i transients in primary cultured neonatal atrial and ventricular cardiomyocytes and to determine which Ang II receptor subtype mediates these responses. We demonstrate for the first time that responses to Ang II differ in atrial and ventricular neonatal cardiomyocytes and that these differential responses may be due to the presence of AT_1A-R and AT_1B-R in atrial cells but only AT_1A-R in ventricular cells.

	Methods

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Atrial and Ventricular Cardiomyocyte Culture
Cardiomyocyte cultures were prepared from 4-day-old neonatal Sprague-Dawley rats (Charles River, St Constant, Quebec, Canada) according to previously described methods.^{23 24} The rats were first injected intraperitoneally with 50 µL (50 U) heparin sulfate (Hepalean, Organon Canada Ltd). After decapitation, they were dipped in 70% ethanol, the chest was opened, and the hearts (from 65 rats) were removed with surgical forceps. Atria and ventricles were dissected and prepared separately. They were washed in 10 mL Joklik medium supplemented with 250 U heparin sulfate to remove red blood cells and were digested at 37°C in 25 mL Joklik medium containing 0.1% collagenase (CLS 2, Worthington Biochemical Corp) and 0.01% DNAse for 20 minutes with agitation (120 cycles per minute).

After incubation, the cells were dispersed by pipetting, and the remaining tissue was digested for another 20 minutes in 15 mL collagenase-DNAse solution. The dispersed cells were suspended in fetal calf serum, filtered through a nylon mesh, centrifuged, and resuspended in complete serum-free medium (CSFM-1) plus 10% Nu-Serum IV (Collaborative Research Inc). They were counted in a hematocytometer, diluted, and seeded in plastic six-well multidishes that contained round glass coverslips (25 mm diameter, Fisher Scientific Inc) at a density of 1.25x10⁵ cells per centimeter squared. The plated cells were grown on the glass coverslips at 37°C in 5% CO₂/95% air. Unlike the method described by Shields and Glembotski²⁴ in which plated cells were maintained for the first 24 hours in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, we maintained cells in CSFM-1²⁵ with 10% Nu-Serum IV. The day after seeding, the medium was replaced with CSFM-2 supplemented with cytosine arabinofuronoside, which was removed from the medium after 3 days. All studies were performed 5 days after isolation when the cells were contracting spontaneously and synchronously.

Measurement of Cardiomyocyte [Ca²⁺]_i
The cells attached to the glass coverslips were washed three times with warmed (37°C) modified Hanks' buffered saline solution containing (mmol/L) NaCl 137, NaHCO₃ 4.2, Na₂HPO₄ 3, KCl 5.4, KH₂PO₄ 0.4, CaCl₂ 1.3, MgCl₂ 0.5, MgSO₄ 0.8, glucose 10, and HEPES 5 (pH 7.4). The washed cells were loaded with fura 2–AM (4 µmol/L) that was dissolved in dimethyl sulfoxide with 0.02% pluronic F-127 and incubated at 37°C for 30 minutes in a humidified incubator (5% CO₂/95% air). The loaded cells were then washed with the modified Hanks' buffer. Four glass rings (diameter, 4 to 5 mm) were placed on the coverslip containing cells, and a vacuum seal was formed between the ring and the coverslip with vacuum grease (Dow Corning Co). Each ring was filled with 50 µL modified Hanks' buffer. This method physically isolated four areas on the glass coverslip and allowed for four separate experiments per coverslip. Two methods were used for measurement of [Ca²⁺]_i: in isolated cells by microphotometry and in cell clusters by fluorescent digital imaging. The advantage of microphotometric measurements is that the frequency of data point collection is faster than that for digital imaging. The advantages of digital imaging are that multiple individual cells can be examined simultaneously and that the cells under investigation can be imaged throughout the experiment. Although the systems were calibrated by different methods, microphotometric [Ca²⁺]_i results obtained in basal and stimulated cells were comparable to those obtained from digital imaging analysis.

For microphotometric determinations, the coverslip containing cells and rings was placed in a Leiden chamber (Medical System Corp) on top of the stage of an inverted microscope equipped for epifluorescence with a x40 oil-immersion objective (Diaphot TMD, Nikon). Fluorescence measurements of single cells were made microphotometrically according to previously described methods.²⁶ [Ca²⁺]_i was calculated according to the formula of Grynkiewicz et al.²⁷ In addition, [Ca²⁺]_i was measured in multiple cells (8 to 16 cells per experiment) simultaneously by fluorescent digital imaging with the Axiovert 135 inverted microscope and Attofluor Digital Fluorescence System (Zeiss) using excitatory wavelengths of 343 and 380 nm. This system was calibrated by viewing fura 2 solutions containing zero and saturating calcium concentrations and including these data in the ratio calculations for construction of a standard curve relating calcium concentration to the 343/380 ratio. For [Ca²⁺]_i measurement by imaging microscopy, the chamber containing the glass coverslip with adherent cells was mounted on the stage of the Axiovert inverted microscope, which was equipped for fluorescence measurements. Video images of fluorescence at 520 nm emission were obtained by an intensified CCD camera system (Zeiss) with the output digitized to a resolution of 512x480 pixels. The Attofluor hardware and software are capable of digitizing and displaying the video at the RS170 video rate of 4 to 30 frames per second. Images of fluorescence ratios were then obtained by dividing, pixel by pixel, the 343-nm image after background subtraction by the 380-nm image after background subtraction. With prior in vitro calibration, the Attofluor system allowed for direct intracellular free calcium measurement, which was graphed and analyzed by the AttoGraph for Windows program (Zeiss).

[Ca²⁺]_i effects of various Ang II concentrations (10^-11 to 10^-5 mol/L) were determined. Cells were used for single experiments and exposed to one peptide concentration only. This was achieved by addition of 50 µL of the appropriate Ang II concentration to the 50 µL of buffer contained within the glass ring that isolated the cells under investigation. The maximum peak ratio recorded was considered as the maximal response of the agonist. Responses occurred within 15 seconds after the agonist had been introduced.

Cardiomyocyte Membrane Preparations
Methods for preparation of membranes of cultured cardiomyocytes were based on previously described techniques.²³ In brief, atria and ventricles were homogenized in 0.05 mol/L NaHCO₃, pH 7.4, and 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF) with a polytron (Brinkmann Instruments) (two times for 10 seconds) and centrifuged at 3000g for 10 minutes. Atrial and ventricular membranes were prepared separately. The pellet was rehomogenized, and both supernatants were pooled and centrifuged at 40 000g for 15 minutes. The pellet was washed in 0.05 mol/L Tris-HCl, pH 7.4, recentrifuged at the same speed, and resuspended in the same buffer.

Cultured cardiomyocytes in 10-cm plastic dishes were washed twice with 0.02 mol/L NaHCO₃, pH 7.4, and 0.1 mmol/L PMSF and scraped in the same buffer (1 mL/60 cm²). The cells were homogenized in a Potter-Elvehjem homogenizer at 750 rpm with 10 strokes and centrifuged at 40 000g for 15 minutes. The pellet was resuspended in 0.05 mol/L Tris-HCl, pH 7.4. All membrane preparations were kept at -40°C until used.

Ang II Binding Assay
Ang II receptor subtypes were assessed by radioligand binding studies. [Sar¹,Ile⁸]Ang II was radiolabeled by the lactoperoxidase method.²⁸ The specific activity of labeled [Sar¹,Ile⁸]Ang II (1500 Ci/mmol) was assessed by self-displacement.²⁹ For competition analysis,²³ membrane proteins (30 to 50 µg) were incubated with 50 000 cpm of ¹²⁵I-[Sar¹,Ile⁸]Ang II in the presence of increasing concentrations (10^-13 to 10^-6 mol/L) of peptide, losartan, or PD 123319. After 60 minutes of incubation at room temperature, the volume was aspirated and filtered on a No. 34 glass filter (Schleicher & Schuell) with a cell harvester (Brandel). The filters were presoaked and washed with 0.05 mol/L Tris, pH 7.2, and 0.15 mol/L NaCl. Radioactivity on the filters was counted in a gamma counter (LKB, Wallac). Data were analyzed by the EBDA and LIGAND software of McPherson³⁰ (Biosoft).

RT-PCR of AT_1A-R and AT_1B-R and Southern Blot Analysis
Total RNA was extracted from frozen cell pellets of atrial and ventricular cardiomyocytes and from fresh frozen aorta and mesenteric artery tissue from Sprague-Dawley rats by the guanidinium isothiocyanate/phenol/chloroform extraction method.³¹ The RNA samples were resuspended in diethylpyrocarbonate-treated H₂O, and the optical density was determined at 260 and 280 nm. First strand cDNA was synthesized with 5 µg total RNA in a 50-µL reaction mixture containing (mmol/L) Tris-HCl 50 (pH 8.3), KCl 50, MgCl₂ 10, dithiothreitol 10, dNTPs 1, and spermidine 0.5 as well as 1 µg oligo NOT 1-dT₁₈ primer (Pharmacia) and 10 U/µg RNA avian myeloblastosis virus reverse transcriptase (Promega) for 1 hour at 42°C. The tubes were boiled, and bovine pancreas ribonuclease (Pharmacia) was added to a final concentration of 1 µg/mL. Five microliters of first strand cDNA was used for PCR amplification in a 50-µL reaction mixture containing (mmol/L) Tris-HCl 10 (pH 8.3), MgCl₂ 1.5, and KCl 50 as well as 0.1% Triton X-100, 200 µmol/L dNTPs, 100 pmol of each primer, and 2 U Taq DNA polymerase (Promega). PCR amplification was performed in an Easy Cycler (Ericomp, Inc) programmed as follows: 5 minutes initial denaturation at 95°C, 30 cycles each including 1 minute denaturation at 95°C, 1 minute annealing at 55°C, and 1 minute elongation at 72°C followed by a final elongation for 10 minutes at 72°C. A 351-bp AT_1A-R–specific PCR product was obtained with the forward primer 5'-GCAGCCTCTGACTAAATG-3' and the reverse primer 5'-AGTTGAACAGAACAAGTG-3' located at nucleotides 1398 to 1416 and 1730 to 1748, respectively, of the 3' untranslated region.^{32 33} A 427-bp AT_1B-R–specific PCR product was obtained with the forward primer 5'-CCATTTGGGCTAAGCAGC-3' and the reverse primer 5'-CAATGGTGTCATAGTCAC-3', located at nucleotides 1716 to 1734 and 2124 to 2142, respectively, of the 3' untranslated region.^{14 34} As a control for the RT analysis of RNA samples and genomic DNA contamination, a ß-actin PCR amplification was performed simultaneously. A 234-bp PCR product was obtained with the forward primer 5'-AGACCTCTATGCCAACACACT-3' and the reverse primer 5'-ATGGAGGGGCGGACTCATC-3' located within exons D and E, respectively, of the ß-actin gene.³⁵ Contamination of total RNA extracts with genomic DNA would result in a ß-actin PCR product of 357 bp because of a 125-bp spacing intron. After PCR, a 15-µL sample of the amplified products was separated by electrophoresis on a 1% agarose gel. The DNA was denatured and transferred to a nylon membrane (Hybond-N, Amersham) by capillary action according to the manufacturer's instructions. The membrane was dried and the DNA fixed by UV irradiation with a UV Stratalinker (Stratagene). The membrane was prehybridized for 3 hours at room temperature in 50 mmol/L phosphate buffer (pH 7.0) containing 5x SSC (20x SSC: 3 mol/L NaCl, 0.3 mol/L sodium citrate), 1% sodium dodecyl sulfate, 100 µg/mL yeast tRNA, 5x Denhardt's solution (50x Denhardt's solution: 1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin), 50% formamide, and 100 µg/mL sonicated salmon sperm DNA. An antisense oligonucleotide common to both amplified AT₁-R fragments (5'-TGTTCCTTTTGATTTCCAC-3'; underlined base is an A in AT_1B cDNA) was used as a probe for verification of the identity of the AT_1A and AT_1B amplified products. The oligonucleotide was labeled with T4 polynucleotide kinase (Pharmacia) and [-³²P]ATP (3000 Ci/mmol, Amersham). The ³²P-labeled oligonucleotide was purified by chromatography with an NACS cartridge (Gibco-BRL). Hybridization was carried out with 5x10⁶ cpm (10 mL total) of the ³²P-labeled probe for 18 to 20 hours at room temperature. The membranes were washed three times for 20 minutes at room temperature in 6x SSC, dried with paper towels, wrapped in plastic wrap, and exposed to Reflection film (DuPont) with intensifying screens at -70°C for 6 hours.

Statistical Analysis
For each experiment, at least four measurements were made by microphotometry and at least six by fluorescent digital imaging. Data obtained from digital imaging studies in which multiple cells were examined in each experiment were calculated as mean [Ca²⁺]_i per experiment and then as the mean of multiple experiments. Since results in isolated single cells (measured microphotometrically) and in cell clusters (measured by digital imaging) were similar, data obtained by the two systems were pooled, and the mean [Ca²⁺]_i value was determined.

Data are presented as mean±SE. Comparison of mean values was performed by ANOVA or Student's t test as appropriate. 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)].

	Results

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Characterization of Cultured Neonatal Cardiomyocytes
Primary cultures of cardiomyocytes may be contaminated by other cell types, especially fibroblasts. The purity of our cardiomyocyte preparation was greater than 95%, judged on the basis of the following criteria: (1) morphological features, (2) ANF antiserum immunostaining, and (3) spontaneous [Ca²⁺]_i oscillations. Morphologically, neonatal cardiomyocytes in primary culture are flat cells of variable shape and size that contract spontaneously. Cardiomyocytes, but not noncardiomyocytes, secrete ANF and stain with ANF immunostaining.²³ We have previously demonstrated that in cell isolates prepared according to the method described in the present study, more than 95% of the cells stained positive with ANF antiserum, indicating that the majority of plated cells were cardiomyocytes.²³ Fluorescent digital imaging allows for direct visualization of cells under investigation and demonstrated that cells studied had typical morphological and functional features (spontaneous contractions and oscillatory [Ca²⁺]_i waves) of cardiomyocytes. For the above reasons, cells studied in the present investigations were cardiomyocytes and not contaminating cells.

Basal [Ca²⁺]_i in Neonatal Cardiomyocytes
[Ca²⁺]_i measurements are presented as diastolic, systolic, and mean values according to previously described definitions.⁸ Diastolic [Ca²⁺]_i was determined as the average of the lowest point of each tracing 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 1). The mean value was calculated as the midpoint between the systolic and diastolic values, and the [Ca²⁺]_i amplitude was taken as the difference between systolic and diastolic values (Fig 1). The frequency of cell beating corresponded to the frequency of [Ca²⁺]_i spikes and was determined over 1 minute. Diastolic [Ca²⁺]_i was 62±2.4 nmol/L in atrial cells and 60±2.1 nmol/L in ventricular cells. The corresponding systolic [Ca²⁺]_i values were 135±2.1 and 123±1.9 nmol/L in atrial and ventricular cells, respectively. The mean [Ca²⁺]_i values ([systolic+diastolic]/2) were 99±1.3 nmol/L (atrial cells) and 90±1.1 nmol (ventricular cells). The [Ca²⁺]_i transient amplitudes were 73±4 nmol/L (atrial cells) and 63±2 nmol/L (ventricular cells). The frequencies of [Ca²⁺]_i spikes were 26±0.5 beats per minute (atrial cells) and 20±2 beats per minute (ventricular cells).

View larger version (39K): [in this window] [in a new window]   Figure 1. Effects of Ang II (ANG, 1 nmol/L), PD 123319 (PD, 0.1 µmol/L), and losartan (Los, 0.1 µmol/L) on mean atrial (open bars) and ventricular (shaded bars) cell [Ca2+]i. *P<.01 vs ANG and PD+ANG groups; P<.01 vs atrial [Ca2+]i. Inset: Representative tracing demonstrating effects of Ang II on atrial cell [Ca2+]i. The arrow indicates time of Ang II (10-9 mol/L) addition. The y axis is the fluorescence ratio (343/380). A indicates systolic [Ca2+]i; B, diastolic [Ca2+]i; A-B, amplitude of [Ca2+]i transient; and (A+B)/2, mean [Ca2+]i.

Effects of Ang II on Ca²⁺ in Neonatal Cardiomyocytes
Ang II increased [Ca²⁺]_i responses in a dose-dependent manner (Fig 2). The pD₂ values were 8.2±0.22 (atrial cells) and 8.0±0.22 (ventricular cells). In subsequent experiments, Ang II was used at a fixed concentration of 10^-9 mol/L because this is a low pharmacological dose corresponding to an EC₃₀ that elicits significant [Ca²⁺]_i responses. Ang II (10^-9 mol/L) significantly increased [Ca²⁺]_i responses in cells derived from the atrium and ventricle. Mean [Ca²⁺]_i values were significantly increased (P<.01) to 249±5 nmol/L (atrial cells) and 229±6 nmol/L (ventricular cells) (Fig 1). Ang II significantly increased (P<.01) [Ca²⁺]_i amplitude (164±8 nmol/L, atrial cells; 102±4 nmol/L, ventricular cells) as well as the frequency of [Ca²⁺]_i spikes (38±1 beats per minute, atrial cells; 34±0.8 beats per minute, ventricular cells). Ang II–induced mean [Ca²⁺]_i and [Ca²⁺]_i amplitude were significantly greater (P<.05) in atrial compared with ventricular cells (Fig 1). Time-course analysis demonstrated that within 100 seconds after Ang II addition, [Ca²⁺]_i responses returned to baseline values (Fig 3).

View larger version (15K): [in this window] [in a new window]   Figure 2. Line graph shows dose-response curve for Ang II in atrial cardiomyocytes from neonatal rats. Cardiomyocyte [Ca2+]i is presented as the mean [Ca2+]i value, where mean [Ca2+]i=(systolic [Ca2+]i-diastolic [Ca2+]i)/2. Values are mean±SE; n=5 to 8 experiments per concentration, with each experimental field comprising 8 to 16 cells.

View larger version (14K): [in this window] [in a new window]   Figure 3. Line graph shows time course of [Ca2+]i recovery to baseline after Ang II (1 nmol/L) stimulation. Peak [Ca2+]i was taken at 0 seconds and recovery to basal [Ca2+]i was measured thereafter. Results are mean±SE; n=5 to 8 experiments, with each experimental field comprising 8 to 16 cells.

Effects of [Sar¹,Ile⁸]Ang II, Losartan, and PD 123319 on Ang II–Stimulated [Ca²⁺]_i Transients in Neonatal Cardiomyocytes
Exposure of cells to 10^-7 mol/L [Sar¹,Ile⁸]Ang II, a specific Ang II receptor blocker, abolished the Ang II–induced [Ca²⁺]_i effects. When cells were preexposed to 10^-7 mol/L losartan, a specific AT₁-R blocker, responses to 10^-9 mol/L Ang II were significantly blocked (Fig 1). Preincubation with PD 123319 (10^-5 to 10^-8 mol/L), an AT₂ antagonist, failed to block the Ang II–induced [Ca²⁺]_i effects.

Ang II Receptor Binding Studies
Competition binding curves indicated the presence of a single population of high-affinity binding sites for Ang II in ventricular and atrial cells. The apparent dissociation constant for atrial cells (K_d) was 0.71±0.11 µmol/L and B_max was 54±3.4 fmol/mg protein (n=3), which was similar to that for ventricular cells. Losartan caused complete displacement of ¹²⁵I-[Sar¹,Ile⁸]Ang II, whereas PD 123319 had no effect (Fig 4).

View larger version (18K): [in this window] [in a new window]   Figure 4. Competition binding curves of 125I-[Sar1,Ile8]Ang II with [Sar1,Ile8]Ang II, losartan, and PD 123319 in atrial membranes. These curves show complete displacement by [Sar1,Ile8]Ang II and losartan but no displacement by PD 123319. Kd=0.71±0.11 µmol/L; Bmax=54±3.4 fmol/mg protein.

RT-PCR Analysis of AT_1A-R and AT_1B-R Expression
We performed RT-PCR analysis to determine whether the AT_1A-R and AT_1B-R genes are expressed in rat neonatal cardiomyocytes. Fig 5 (top) shows a PCR amplification product of 351 bp specific for the AT_1A-R in both atrial (lanes 1 and 3) and ventricular (lane 5) rat neonatal cardiomyocyte preparations. The amplification product of 427 bp specific for the AT_1B-R subtype was only evident in atrial cells (lanes 2 and 4) and in repeated experiments (n=7 from different cell preparations) was absent from ventricular neonatal cardiomyocytes (lanes 6, 8, 9, and 10). Southern blot analysis of the amplified products with the use of specific ³²P-labeled oligonucleotide probes independent of the PCR primers (Fig 5, bottom) confirmed that the PCR bands were consistent with the AT_1A-R and AT_1B-R sequences. As positive controls, we also performed RT-PCR analysis in rat aortic and mesenteric vascular tissue. AT_1A-R and AT_1B-R mRNAs were expressed in both aorta and mesenteric vessels (data not shown). PCR amplification of ß-actin indicates the efficiency of the cDNA synthesis by RT of RNA samples obtained from the cardiomyocytes. Furthermore, the presence of a single 234-bp ß-actin PCR amplification product from these preparations and absence of a 357-bp ß-actin PCR amplification product that would have been present if genomic DNA had been amplified indicates that the AT₁-R PCR amplification products from atrial and ventricular cardiomyocytes were derived specifically from reverse transcribed mRNA transcripts and were not due to amplification of genomic DNA.

View larger version (77K): [in this window] [in a new window]   Figure 5. RT-PCR analysis of AT1A-R and AT1B-R mRNA expressions in rat neonatal atrial and ventricular cells. Top, Ethidium bromide–stained 1% agarose gel shows the specific PCR amplification products of AT1A-R (lanes 1 and 3, atrial cells; lane 5, ventricular cells) and AT1B-R (lanes 2 and 4, atrial cells). There was no amplification product of AT1B-R in ventricular cells (lanes 6, 8, 9, and 10). Lane 7 represents the amplification product of ß-actin from ventricular cells for control and size orientation. The relative sizes of the PCR amplification products were evaluated with DNA size markers (lane M; DRI gest III, Pharmacia). Positions of the bands of 2322 and 603 bp are marked. Positions of the AT1B-R product of 427 bp, of AT1A-R of 351 bp, and of ß-actin of 231 bp are indicated. Bottom, Southern blot analysis of PCR amplification products resolved in the above agarose gel and hybridized with a specific 32P-labeled oligonucleotide probe common for both AT1A-R and AT1B-R located between the primers used for PCR amplification. Bands for AT1A-R can be observed in lanes 1 and 3 for atrial cells and lane 5 for ventricular cells, and bands for AT1B-R in lanes 2 and 4 for atrial cells. There was no hybridization with the AT1B-R probe in ventricular cells (lanes 6, 8, 9, and 10).

	Discussion

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Depending on the animal species and Ang II concentration studied, Ang II induces both negative and positive inotropic effects on the myocardium.^{1 7 8 36} These effects appear to be mediated by changes in [Ca²⁺]_i.^{8 36} Results from the present study demonstrate that Ang II increases atrial and ventricular cardiomyocyte [Ca²⁺]_i and the amplitude and frequency of [Ca²⁺]_i transients. These stimulatory actions support the findings of increased chronotropic and inotropic effects reported for Ang II.^{1 2} Our results are in partial agreement with those of Kem et al,⁸ who demonstrated two distinct Ang II–induced effects in neonatal ventricular cells. The first was a significant rise in diastolic [Ca²⁺]_i and an increase in [Ca²⁺]_i transient frequency, similar to our findings, and the second Ang II–induced effect was a reduction of the amplitude of the [Ca²⁺]_i transients.⁸ Reduced [Ca²⁺]_i amplitude was associated with a decrease in cell shortening and decrease in cardiac contraction,³⁷ suggesting that Ang II has a negative inotropic effect on the heart.⁷ In our study, however, Ang II increased [Ca²⁺]_i amplitude and frequency in both atrial and ventricular cells, consistent with positive ionotropic and chronotropic effects of Ang II. Underlying mechanisms of these Ang II actions may be related to responsiveness of myofilaments to elevated [Ca²⁺]_i and also to increased myofilament sensitization to [Ca²⁺]_i.^{1 36}

Basal [Ca²⁺]_i was similar in cells derived from the atrium and ventricle, but [Ca²⁺]_i responsiveness to Ang II was significantly greater in atrial cells. To the best of our knowledge, no other studies have compared Ang II–induced [Ca²⁺]_i responses between atrial and ventricular cells. The implications of these differential effects may be related to the functional variability of the atrium and ventricle. Increased Ang II–evoked [Ca²⁺]_i transients in atria may contribute to the active atrial conducting system, whereas the lower Ang II–induced [Ca²⁺]_i responses in ventricles may be associated with cardiac contraction. These proposals await elucidation. A possible underlying mechanism for the different [Ca²⁺]_i responses between atrial and ventricular cells may be the differences in expression patterns of AT₁-R subvariants, specifically the presence of AT_1A-R and AT_1B-R in neonatal atrial cardiomyocytes and of AT_1A-R only in neonatal ventricular cardiomyocytes.

[Sar¹,Ile⁸]Ang II, a specific Ang II receptor antagonist, abolished the [Ca²⁺]_i effects of Ang II, demonstrating that the responses were receptor mediated. The selective AT₁-R antagonist losartan completely blocked the Ang II–mediated increase in [Ca²⁺]_i, whereas PD 123319, the AT₂-R antagonist, even at high concentrations, had no effect. Thus, Ang II–induced [Ca²⁺]_i responses in neonatal atrial and ventricular cardiomyocytes are mediated via the AT₁-R subtype. Binding studies confirmed these findings and demonstrated a single population of high-affinity binding sites for Ang II. These results are consistent with data from preparations in other species as well as from the rat.^{38 39} We have also demonstrated for the first time that the AT_1A-R and AT_1B-R genes are expressed in neonatal rat atrial cardiomyocytes, whereas only the AT_1A-R gene is expressed in ventricular cells. [Ca²⁺]_i responsiveness to Ang II was greater in atrial compared with ventricular cells, and this may be related to differential AT₁-R subtype distribution in atrial and ventricular cells. Other studies have also demonstrated both subtypes of the AT₁-R in cardiac tissue, but none have examined atrial and ventricular cardiomyocytes separately. Matsubara et al¹⁶ recently reported that in neonatal rat cardiomyocytes (combined atrial and ventricular cells), the AT_1B-R mRNA level was 1.5-fold higher than the AT_1A-R mRNA level, whereas Iwai et al¹⁵ showed that in adult rat ventricular cells, the proportion of AT_1A mRNA to AT_1B mRNA was 1 to 2. It may be possible that AT_1A-R and AT_1B-R expressions are developmentally regulated and that with cardiac maturation, cell and subtype distribution change.

In conclusion, this study demonstrates that Ang II increases the amplitude and frequency of [Ca²⁺]_i transients in neonatal rat cardiomyocytes via Ang II receptors of the AT₁-R subtype. [Ca²⁺]_i responsiveness to Ang II was greater in atrial compared with ventricular cells, and this could be related to the expression of AT_1A-R and AT_1B-R subvariants in neonatal atrial cardiomyocytes but only of the AT_1A subvariant in neonatal ventricular cardiomyocytes.

	Selected Abbreviations and Acronyms

 

ANF = atrial natriuretic factor Ang II = angiotensin II AT1, AT2 = angiotensin subtype 1, subtype 2 [Ca2+]i = cytosolic free calcium concentration PCR = polymerase chain reaction R = receptor RT = reverse transcription

	Acknowledgments

 
This study was supported by a group grant from the Medical Research Council of Canada (MRC) to the Multidisciplinary Research Group on Hypertension. R.M. Touyz and J. Fareh are recipients of fellowships from the MRC and from the Canadian Hypertension Society and the MRC, respectively. R. Larivière was a Scholar of the Fonds de la Recherche en Santé du Québec. The authors thank André Turgeon for technical help.

	Footnotes

 
Reprint requests to Ernesto L. Schiffrin, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec H2W 1R7, Canada.

Received June 16, 1995; first decision September 19, 1995; accepted January 10, 1996.

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