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(Circulation Research. 1996;78:525-535.)
© 1996 American Heart Association, Inc.

Articles

Extracellular ATP Inhibits Adrenergic Agonist–Induced Hypertrophy of Neonatal Cardiac Myocytes

Jing-Sheng Zheng, Marvin O. Boluyt, Xilin Long, Lydia O'Neill, Edward G. Lakatta, Michael T. Crow

From the Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Md.

Correspondence to Michael T. Crow, PhD, Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Gerontology Research Center, 4940 Eastern Ave, Baltimore, MD 21224.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract We have previously shown that extracellular ATP, like norepinephrine (NE) and many other hypertrophyinducing agents, increases expression of the immediate-early genes c-fos and junB in cultured neonatal cardiac myocytes but that the intracellular signaling pathways activated by ATP and responsible for these changes differ from those stimulated by NE. Furthermore, whereas NE increases incorporation of [¹⁴C]phenylalanine (¹⁴C-Phe) and cell size in neonatal cardiomyocytes, ATP does not. Since ATP is coreleased with NE from sympathetic nerve endings in the heart, we investigated whether ATP could modulate cardiac hypertrophy induced by adrenergic agonists, such as NE. We report in the present study that extracellular ATP inhibited the increase in incorporation of ¹⁴C-Phe into cellular protein and the increase in cell size in neonatal rat cardiac myocytes that was induced by NE, phenylephrine (PE), basic fibroblast growth factor, or endothelin-1. This inhibition was dose dependent, occurred predominantly through P₂ purinergic receptors, and was observed even when cells were treated with ATP for as little as 1 hour before the addition of the hypertrophy-inducing agent. ATP also selectively affected changes in gene expression associated with hypertrophy. It prevented PE-stimulated increases in atrial natriuretic factor and myosin light chain-2 mRNA levels, while appearing to augment basal and PE-stimulated skeletal -actin mRNA levels. ATP alone increased sarcoplasmic reticulum Ca²⁺-ATPase mRNA levels but had no effect when added with PE. ATP did not significantly affect the level of the constitutively expressed mRNA for GAPDH. Neither the PE-stimulated increase in immediate-early gene expression nor the initial induction of mitogen-activated protein kinase activity by PE was inhibited by ATP. These results demonstrate that extracellular ATP can inhibit hypertrophic growth of neonatal cardiac myocytes and differentially alter the changes in gene expression that accompany hypertrophy.

Key Words: ATP • purinergic receptors • cardiac hypertrophy • norepinephrine • cardiac myocytes

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cultured neonatal cardiomyocytes are a useful experimental model system for elucidating the cellular and molecular mechanisms regulating cardiac cell growth and hypertrophy.^{1 2} In response to stretch or hypertrophic agonists such as NE, ET, or bFGF, neonatal cardiac myocytes undergo substantial increases in ¹⁴C-Phe incorporation and cell size without a concomitant increase in cell number. These same reagents also cause increases in the expression of immediate-early genes (eg, c-fos and junB), upregulation of the expression of various contractile protein genes, such as MLC-2, selective upregulation of fetal/neonatal isoforms of the contractile proteins, such as skeletal -actin and bMHC, and upregulation of noncontractile protein genes, such as ANF. Many of these same changes in gene expression are also characteristic of in vivo models of cardiac hypertrophy, such as that resulting from aortic coarctation.¹

It has been known for many years that ATP is stored and released with NE from adrenal chromaffin cells and sympathetic nerve endings.^{3 4} In the mammalian vas deferens, ATP and NE act as synergistic neurotransmitters via postjunctional modulation, whereas ATP and adenosine inhibit NE release from sympathetic nerves supplying vas deferens via activation of P₁ purinergic receptors.⁵ In adult cardiac myocytes, NE acts synergistically to potentiate the ATP-induced increase in intracellular calcium.⁶

We have previously shown that both NE and ATP increase the expression of the immediate-early genes c-fos and junB in neonatal cardiac myocytes and that in the case of c-fos expression, this increase occurs, at least in part, through activation of different intracellular signaling pathways. Pretreatment with an intracellular Ca²⁺ chelator significantly inhibits the ATP-induced expression of c-fos mRNA but not the NE-induced expression of c-fos. Furthermore, we showed that whereas NE increases incorporation of ¹⁴C-Phe and cell size in cardiac myocytes, ATP does not, indicating that the activation of c-fos and junB alone is not sufficient to stimulate hypertrophy in these cells.⁷

Since ATP is released with NE from sympathetic nerve terminals and since the intracellular signaling pathways activated by ATP are different from those activated by NE, it was of interest to determine what effect ATP might have on the hypertrophic response of neonatal cardiac myocytes. Accordingly, the purpose of the present study was to investigate whether ATP altered the increase in ¹⁴C-Phe incorporation and changes in gene expression brought about by hypertrophyinducing agents. We report in the present study that extracellular ATP inhibited the increase in ¹⁴C-Phe incorporation and expression of various contractile and noncontractile genes normally observed in response to a variety of hypertrophy-inducing agents. On the other hand, expression of the skeletal -actin gene, which is normally increased after the administration of hypertrophy-inducing agents, such as PE, was unchanged by ATP. ATP increased the level of SERCA mRNA but had no significant effect when added in conjunction with PE. Surprisingly, the continued presence of ATP was not required for the observed inhibitory effects on hypertrophy, since a 1-hour pretreatment with ATP before the addition of PE was sufficient to inhibit changes in ¹⁴C-Phe incorporation for at least 3 days.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
ATP disodium salt, adenosine (free base), ATP--S, NE (arterenol), PE, PIA, and 8-PT were purchased from Sigma Chemical Co. ATP, adenosine, and ATP--S were dissolved in water, aliquoted, and then stored at -70°C until use. Each aliquot was used only once. NE and PE were dissolved in ascorbic acid (final concentration, 100 µmol/L), aliquoted, and used only once. 8-PT was dissolved in dimethyl sulfoxide and stored at -20°C. Recombinant human bFGF was purchased from Collaborative Biomedical Products and dissolved in DMEM and 0.1% BSA (type V, Sigma). It was aliquoted and stored at -70°C, and each aliquot was used only once. ET-1 was purchased from Peninsula Laboratories and used immediately after resuspending in water. PicoGreen reagent was obtained from Molecular Probes, Inc. Radiolabeled compounds were obtained from New England Nuclear and Amersham.

Cell Culture
Neonatal ventricular myocytes were cultured as previously described⁸ with some modifications. Hearts from 1- to 3-day-old Wistar rats were removed, and the ventricles were trisected and then digested with collagenase type II (0.5 mg/mL, Worthington Biochemical) and pancreatin (0.6 mg/mL, Sigma) for 20 minutes at 37°C. Cells were collected by centrifugation and resuspended in horse serum. The above steps were repeated 7 to 10 times until the hearts were completely digested. The cells from all digestions were combined, washed, and then subjected to centrifugation through a discontinuous Percoll gradient of 1.050, 1.062, and 1.082 g/mL, respectively. The band at the 1.062/1.082 interface was collected and used as the source of purified myocytes. Myocytes were resuspended in culture media consisting of a 4:1 (vol/vol) mixture of DMEM containing 4.5 g/L glucose and medium 199 (Earle's salts, GIBCO Laboratories) supplemented with 10% preselected horse serum, 5% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL, GIBCO Laboratories). Myocytes were then plated on gelatin-coated 100-mm plastic tissue culture plates at a density of 1.1x10⁵ cells per square centimeter or six-well culture plates at a density of 2.5x10⁴ cells per square centimeter. Plating efficiency was such that cell-cell contact was minimal at the time the cells were harvested for analyses. Myocyte cultures were maintained in serum-containing media at 37°C for 24 hours in humidified air with 5% CO₂. The medium was then changed to serum-free medium (DMEM/medium 199 containing antibiotics and supplemented with 1 µmol/L insulin, 5 µmol/L transferrin, and 10 nmol/L selenium). All experiments were initiated 24 hours after the change to serum-free conditions (48 hours after plating). To inhibit fibroblast proliferation, 10^-4 mol/L bromodeoxyuridine (Boehringer Mannheim) was added to all cultures at the time of plating and was maintained in the culture media throughout the experiment. The purity of the cultures was determined 48 hours after plating by immunocytochemistry with antibodies to sarcomeric myosin (McAb F59)⁹ or sarcomeric actin (Sigma). For each experiment, one plate was stained for either sarcomeric actin or MHC, this plate having been treated in a manner identical to the others in the experiment. On average, myocyte preparations were 92.2% myocytes 48 hours after plating. This average included a few cultures that were between 60% and 90% myocytes. These batches of cells were not used for any of the experiments described in the present study. Because of the background incorporation of ¹⁴C-Phe in fibroblasts that is stimulated by the agents used in the present study, we were only able to obtain reliable data that represent true hypertrophy (ie, ¹⁴C-Phe incorporation in the absence of [³H]thymidine incorporation) in cultures in which myocyte content was >90% purity.

The number of cells in the cultures that contracted at a rate >10 bpm was 4.0±1.1% for control (untreated) myocytes and was not different among the various experimental groups 72 hours after treatment. To determine whether ATP affected the attachment of cells, cell number was routinely monitored 72 hours after incubation with the appropriate agents. Four low-power fields (x125) were randomly selected per dish, and the number of cells was counted in each field.

¹⁴C-Phe Incorporation and Measurements of Cell Size
The incorporation of ¹⁴C-Phe was assessed as described by the method of Simpson¹⁰ with the following modifications. After cell isolation, the myocytes were cultured in serum-containing medium and plated in six-well tissue culture plates at a density of 2.5x10⁴ cells per square centimeter. After 1 day in serum-containing medium, the cells were switched to serum-free medium (see above). After 1 additional day in serum-free medium, ¹⁴C-Phe (0.1 µC/mL) with or without ATP or various hypertrophy-inducing agents was added to the cultures. Cells were then analyzed for changes in cell size and ¹⁴C-Phe incorporation into cellular protein 3 days later. For cell size measurements, two or three fields were randomly chosen and photographed at high power (x400), and 150 individual cell areas were measured by planimetry. For ¹⁴C-Phe incorporation into cellular protein, the amount of radiolabel incorporated into TCA-insoluble material was determined by first rinsing the cultures with Dulbecco's PBS three times and then incubating the cultures in ice-cold 10% TCA for at least 1 hour at 4°C. The plates were then rinsed three times with 10% TCA, and the TCA-insoluble material left on the plate was dissolved in 1 mL of 1% SDS. The radiolabel in this solution was then quantified by liquid scintillation counting. In some experiments, an aliquot of the SDS solution obtained from each well was used for measurement of DNA concentration using the PicoGreen reagent as described by the manufacturer (Molecular Probes, Inc).

[¹⁴C]Uridine Incorporation Into Total RNA
As an indicator for changes in RNA synthesis, [¹⁴C]uridine incorporation was measured exactly as described above for ¹⁴C-Phe incorporation, except that cells were harvested 48 hours after the addition of 0.2 µCi/mL [¹⁴C]uridine (NEC 598; specific activity, 521 mCi/mmol; New England Nuclear) and various treatments. Results were then normalized to total DNA content as described above.

RNA Blotting
Total RNA was isolated from cultured ventricular myocytes using the guanidinium isothiocyanate method.¹¹ Briefly, cells were rinsed in Dulbecco's PBS and then scraped directly into 8 mL of 4 mol/L guanidinium isothiocyanate, 0.5% sodium lauryl sarcosine, 25 mmol/L sodium citrate (pH 7), and 100 mmol/L 2-mercaptoethanol. After homogenization with a Polytron, the extract was clarified by centrifugation at 8000g for 20 minutes. The clarified homogenate was then layered onto a 2-mL cushion of 5.7 mol/L cesium chloride, 25 mmol/L sodium acetate (pH 5.4), and 2 mmol/L EDTA and subjected to centrifugation at 30 000 rpm in a Beckman SW41 rotor for 18 hours. The pelleted RNA was dissolved in water, extracted first with phenol/chloroform/isoamyl alcohol (25:24:1) and then with chloroform/isoamyl alcohol alone, and precipitated after adding sodium acetate (pH 5.2) to a final concentration of 0.3 mol/L. After denaturation in formamide and formaldehyde, equal amounts of total RNA (10 µg per lane) were size-fractionated by electrophoresis through 1% agarose gels containing 3% formaldehyde. The fractionated RNA was electrophoretically transferred to nylon membranes (Duralon, Stratagene Cloning Systems) at 5 V/cm, cross-linked by ultraviolet irradiation (120 mJ), and then hybridized at 63.5°C as described by Church and Gilbert¹² with ³²P-radiolabeled oligonucleotide and cDNA probes. Probes for the translated region of rat ANF and the 3'-untranslated region of rat skeletal -actin were prepared as described previously.¹³ The rat c-fos probe was a cDNA probe generated by polymerase chain reaction using published nucleotide sequences of the rat c-fos cDNA¹⁴ and cDNA derived from the RNA of serum-stimulated rat aortic vascular smooth muscle cells. The probe encompasses the nucleotide sequence from amino acid 125 to amino acid 293. The MLC-2 probe was a cDNA probe encompassing the entire translated region of rat cardiac myosin light chain-2 (pMLC-D2) and was generously provided by Dr K.R. Chien (University of California at San Diego).¹⁵ The GAPDH probe encodes 400 bp of the conserved coding region of the rat GAPDH cDNA and was generated by polymerase chain reaction as described previously.¹⁶ The probe for cardiac SERCA mRNA was a synthetic end-labeled oligonucleotide complementary to the terminal 33 bases of the translated region of the rat cDNA¹⁷ with the following sequence: 5' CTC CAG TAT TGC AGG CTC CAG GTA GTT TCG GGC 3'. The probe for 18S rRNA was a synthetic oligonucleotide previously described.¹⁸ cDNA probes were radiolabeled using the random priming method^{19 20} ; oligonucleotides were radiolabeled by tailing with [-³²P]dATP.²¹

Measurement of MAPK Activity
Neonatal cardiac myocytes were cultured in six-well plates at a density of 1.2x10⁵ cells per square centimeter for assays of MAPK, which was measured after a modification of the procedure of Bogoyevitch et al.²² Cell extracts from a single well of a six-well plate were used for each time point. Myocytes were rinsed with ice-cold Dulbecco's PBS and then scraped into 100 mL lysis buffer (20 mmol/L Tris [pH 8.0], 137 mmol/L NaCl, 1 mmol/L Na₃PO₄, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL aprotinin). The extract was then clarified by centrifugation, and the supernatant was diluted 1:1 with 2x SDS-PAGE sample buffer (125 mmol/L Tris [pH 6.8], 4% SDS, 20% glycerol, and 10% ß-mercaptoethanol) and separated on a 10% SDS-polyacrylamide gel containing 0.5 mg/mL myelin basic protein. After electrophoresis, the gels were washed three times for 20 minutes each in 20% isopropanol and 50 mmol/L Tris (pH 8.0), followed by three additional washes (20 minutes each) in 50 mmol/L Tris (pH 8.0) and 5 mmol/L ß-mercaptoethanol. The gels were washed two times (30 minutes each) in 6 mol/L guanidine HCl, 50 mmol/L Tris (pH 8.0), and 5 mmol/L ß-mercaptoethanol. Proteins were renatured by washing the gels overnight at 4°C in at least five changes of 50 mmol/L Tris (pH 8.0), 5 mmol/L ß-mercaptoethanol, and 0.04% Tween 40. The gels were then washed two times (30 minutes each) in 40 mmol/L HEPES (pH 8.0), 10 mmol/L MgCl₂, and 2 mmol/L dithiothreitol. In situ (ie, in gel) phosphorylation of myelin basic protein was performed by layering 10 mL of gel buffer (40 mmol/L HEPES [pH 8.0], 0.5 mmol/L EGTA, and 10 mmol/L MgCl₂) containing 0.05 mmol/L ATP and 25 µCi [-³²P]ATP on top of each gel at room temperature. The reaction was stopped by washing the gels in 5% TCA and 1% sodium pyrophosphate with numerous changes. Gels were then dried, and signals were quantified on a Betascope Phosphorimager (Betagen).

HPLC Monitoring of ATP Hydrolysis and Degradation in Culture Media
The nucleotides in culture media were separated on a Waters HPLC model 441 system with 5-mm reverse-phase C18 columns using a method previously described.²³ The mobile phase was 50 mmol/L ammonium phosphate (pH 6.5). The retention times and amplitudes of 5 nmol ATP, ADP, or AMP alone and in combination were determined and used to identify ATP and its hydrolysis products present in 50 µL of conditioned medium.

Statistical Evaluations and Comparisons
Each preparation of myocytes was derived from 150 to 200 neonatal hearts, and numerous separate analytical determinations were made from each preparation. The number of different preparations from which the data are derived is indicated in the legend of each table or figure. For statistical analyses, however, each dish or well was considered an independent observation and is represented as an n value. Although a substantial portion of the variance of each measurement comes from factors other than those associated with possible differences among a given preparation, this choice of the n value must be acknowledged as a limitation of the study, since individual dishes from a single preparation are not completely independent.

Differences in plating efficiency were accounted for either by normalizing results to an internal standard such as DNA, RNA, or protein or by arbitrarily assigning the control dish a value of 1 and expressing other values relative to control. Data were expressed as the mean±SEM. For mRNA and some ¹⁴C-Phe incorporation experiments, comparisons between a treatment group and the control group were made with one-sample t tests, whereas comparisons among treatment groups were made with two-sample t tests.²⁴ P values were corrected by the Bonferroni method. For experiments in which ¹⁴C-Phe incorporation measurements were normalized to DNA concentration and for cell size measurements, the mean values of the control and experimental groups were compared by ANOVA where indicated. Post hoc comparisons between groups were made with the Tukey procedure. Tests of linear trends across ordered groups were made according to Altman.²⁵ For all these tests, a value of P<.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Extracellular ATP Inhibits Adrenergic Agonist–Stimulated Increases in ¹⁴C-Phe Incorporation
To determine whether extracellular ATP affects the adrenergic agonist–stimulated hypertrophy of neonatal cardiac myocytes, the incorporation of ¹⁴C-Phe into cellular protein was measured in the presence and absence of the ₁-adrenergic receptor agonist PE. After 3 days of incubation in 20 µmol/L PE, ¹⁴C-Phe incorporation normalized to total DNA content increased 1.8-fold over control levels (Fig 1). Extracellular ATP (100 µmol/L) added at the time of PE stimulation inhibited the increase in PE-stimulated ¹⁴C-Phe incorporation. ATP inhibited both NE- and PE-stimulated increases in ¹⁴C-Phe incorporation in a dose-dependent manner, with an IC₅₀ of 8 µmol/L and a maximal effective dose of 100 µmol/L (Fig 2). As reported by others,²⁶ PE also increased the incorporation of [¹⁴C]uridine into cellular RNA after 48 hours of treatment by 1.7-fold. Extracellular ATP blocked this increase in [¹⁴C]uridine incorporation (Fig 3).

View larger version (21K): [in this window] [in a new window]   Figure 1. Extracellular ATP inhibits increased 14C-Phe incorporation into total cellular protein in cultured neonatal cardiac myocytes by 1-adrenergic agonists. Serum-free cultures were incubated with PE (20 µmol/L) alone or in combination with ATP (100 µmol/L), ADP (100 µmol/L), AMP (100 µmol/L), or PIA (100 µmol/L). The incorporation of 14C-Phe into cellular protein over the next 3 days was measured and normalized to total DNA content for each dish. Cells were washed with Dulbecco's PBS, incubated with ice-cold 10% TCA for 1 hour, and then solubilized with 1% SDS. 14C-Phe incorporation was determined by liquid scintillation counting. Data are expressed as the mean±SEM (n=6 dishes per bar, two dishes each from three different culture preparations). *P<.05 vs untreated control (CON); #P<.05 vs PE-treated cells (one-way ANOVA, Tukey procedure). Among the groups treated with PE plus ATP, ADP, AMP, or PIA (the four rightmost bars), there was a statistically significant trend consistent with the pharmacological definition of P2 purinergic receptor responses (P<.05).

View larger version (13K): [in this window] [in a new window]   Figure 2. Dose-response plot of the effect of ATP on the NE (2 µmol/L)–stimulated incorporation of 14C-Phe into TCA-precipitable protein. Serum-free cell cultures were treated with NE and different concentrations of ATP for 3 days. 14C-Phe incorporation into total protein was measured as described in the legend to Fig 1 and in "Materials and Methods." A single experiment with PE as the agonist and varying concentrations of ATP yielded similar results. Data are expressed relative to untreated control (arbitrarily assigned a value of 1) and are graphed as the mean±SEM (n=8 dishes per point, four preparations per point). Control value was 8649±2669 cpm per dish.

View larger version (16K): [in this window] [in a new window]   Figure 3. The effect of ATP (100 µmol/L) on incorporation of [14C]uridine into cellular RNA. Serum-free cultures were treated with various agents and incubated for 48 hours in the presence of [14C]uridine. Cells were harvested as indicated in the legend to Fig 1, and [14C]uridine incorporation was determined by liquid scintillation counting. Values are mean±SEM for 12 dishes per bar. Data are derived from two separate preparations of myocytes. *P<.05 vs control myocytes; #P<.05 vs PE-treated myocytes (one-way ANOVA, Tukey procedure).

The effect of ATP on PE-induced hypertrophy in the neonatal cardiomyocytes was primarily mediated by P₂ purinergic receptors. The data in Fig 1 for the effects of ATP, ADP, AMP, and PIA, a nonhydrolyzable analogue of adenosine, on the inhibition of PE-stimulated increases in ¹⁴C-Phe incorporation show a significant trend across ordered groups consistent with the pharmacological definition of purinergic P₂ receptors (ATP>ADP>AMP>adenosine) (P<.05).²⁷ In addition, the ATP-mediated inhibition of the PE-induced increase in incorporation of ¹⁴C-Phe was not affected by coincubation with 8-PT, a specific P₁ purinergic receptor antagonist (Table 1).²⁸ Although not as effective as ATP, 100 µmol/L adenosine also inhibited PE-induced ¹⁴C-Phe incorporation. In this case, 8-PT blocked the moderate effect of adenosine (Table 1). Together, these results suggest that stimulation of P₂ purinergic receptors by ATP is sufficient to account for the inhibition of the PE-induced increase in ¹⁴C-Phe incorporation. Adenosine may also affect ¹⁴C-Phe incorporation, although the magnitude of the effect is much smaller than that of ATP and, in contrast to ATP, appears to be mediated by P₁ purinergic receptors. Since only a few purinergic receptors have been cloned, it is also possible that a novel receptor subtype with pharmacological properties different from the "classic" subtypes may, in fact, be responsible for the effects we have observed.²⁹

View this table: [in this window] [in a new window]   Table 1. Effect of Purinergic Nucleotides on PE-Stimulated 14C-Phe Incorporation Into Neonatal Cardiomyocytes

To determine the generality of these observations with respect to other hypertrophy-inducing agents, we examined the effect of ATP on ¹⁴C-Phe incorporation during incubation of the myocytes with 2 µmol/L NE, 100 nmol/L ET-1, and 10 ng/mL bFGF. NE, ET-1, and bFGF each caused a significant increase in ¹⁴C-Phe incorporation into nondividing myocytes after 3 days of incubation, and these increases were blocked by coincubation with 100 µmol/L ATP (Table 2).

View this table: [in this window] [in a new window]   Table 2. Effect of Purinergic Nucleotides on Adrenergic Receptor Agonist– and Growth Factor–Stimulated 14C-Phe Incorporation Into Neonatal Rat Cardiomyocyte Protein

Normalizing ¹⁴C-Phe incorporation to total DNA content obscured the fact that incubation of the cultures with ATP alone decreased basal ¹⁴C-Phe incorporation values. This is shown in the "ATP" data of Table 2, which contains ¹⁴C-Phe incorporation data expressed per dish rather than normalized to total DNA content. This decrease is likely due to the strong trend for ATP to reduce the number of cells in unstimulated cultures (76% of control, Fig 4), although this trend did not reach statistical significance. Normalizing to total DNA content would automatically compensate for any differences in cell number between the treatment groups. The loss in cell number may also explain the differences observed in total RNA yield from the control and ATP-treated groups. The yield of RNA after 24 hours of treatment was as follows (µg per 100-mm dish): control, 17.8±2.9 (mean±SEM); ATP, 9.2±1.9; PE, 14.2±2.4; and PE/ATP, 14.0±1.7 (with the effect of ATP alone being significantly different [P<.05] from control values). In three experiments in which cultures were harvested after 48 hours of treatment, total RNA yield exhibited a similar tendency to decrease when treated with ATP alone, whereas cultures treated with PE exhibited a 1.4-fold increase in RNA yield, and those treated with both PE and ATP exhibited a total RNA yield similar to control values.

View larger version (27K): [in this window] [in a new window]   Figure 4. Effect of ATP (100 µmol/L) and PE (20 µmol/L) on total cell number. Cell number was estimated 72 hours after incubation with the appropriate agents by averaging the cell counts from four randomly selected low-power fields (x125). Values are mean±SEM for 12 dishes per bar and are derived from four different preparations of myocytes. VEH indicates vehicle. There were no significant differences among the groups, although there was a trend for ATP to reduce the number of myocytes attached to the dish (ANOVA).

To determine whether ATP could inhibit cellular hypertrophy that was already in progress, ATP (100 µmol/L) was added to cultures at various times after PE addition, and the effect on ¹⁴C-Phe incorporation was measured (Fig 5). Bar C shows the PE-stimulated increase in ¹⁴C-Phe incorporation over 3 days of 2.3 times the control value (bar A). Bars D, E, and F show the effect of adding ATP for 1, 2, or 3 days (at the time of PE addition), respectively. Analysis of the results shown in bars C, D, E, and F revealed a significant negative trend across ordered groups (P<.05). These results show that ATP was effective in inhibiting the increase in ¹⁴C-Phe incorporation already initiated by PE (bars D and E) and that the extent of this inhibition was greater either the longer ATP was present or the earlier it was added.

View larger version (31K): [in this window] [in a new window]   Figure 5. Effects of ATP added at different times on the PE-stimulated incorporation of 14C-Phe into TCA-precipitable protein. Cultured neonatal cardiac myocytes were untreated (bar A, control) or treated with 100 µmol/L ATP (bar B) or 20 µmol/L PE (bar C) for 3 days. Bars D through F illustrate the effects of adding ATP after stimulation of protein synthesis with PE at the times indicated. Bar G illustrates the effect on PE-stimulated 14C-Phe incorporation of preincubation of the cultures for 1 hour with ATP followed by washout of the ATP before adding PE. All cultures were harvested after 3 days of 14C-Phe incorporation. Data are expressed relative to control (arbitrarily assigned a value of 1) as mean±SEM (n=7 to 10 dishes per bar). Data are derived from three to six different preparations of myocytes. Control value was 2038±408 cpm per dish. One-hour pretreatment with ATP (bar G) was significantly different from treatment with PE alone (bar C, two-sample t test, P<.05). Among bars C, D, E, and F, a significant negative trend across ordered groups was observed (P<.05).

Measurement by HPLC of the ATP present in culture media indicated that ATP levels were not stable throughout the entire incubation period (Fig 6). In some experiments, therefore, ATP levels were periodically replenished, and in other experiments, the effects of the nonhydrolyzable analogue, ATP--S, on ¹⁴C-Phe incorporation were tested (Table 3). The effects observed under these various conditions were identical to those seen with a single application of ATP.

View larger version (9K): [in this window] [in a new window]   Figure 6. HPLC analysis of adenine nucleotides present in 50 µL conditioned medium of neonatal cardiomyocytes at 1 (B), 4 (C), and 8 (D) hours after addition of 100 µmol/L ATP. The different peaks were identified by comparison with the elution of purified ATP, ADP, and AMP shown in panel A. The data shown are representative of four measurements from four different myocyte preparations.

View this table: [in this window] [in a new window]   Table 3. Effect of Supplemented Purinergic Nucleotides on PE-Stimulated 14C-Phe Incorporation Into Neonatal Cardiomyocytes

The above experiments suggested that the continued presence of ATP may not be required to achieve a maximum inhibitory effect on ¹⁴C-Phe incorporation. To directly test this hypothesis, cultures were first pretreated with ATP for 1 hour, and then the medium containing ATP was removed and replaced with fresh serum-free medium containing PE. ¹⁴C-Phe incorporation was then measured over the next 3 days. Under these conditions, the extent of ATP inhibition was similar to that when ATP was present throughout the 3-day period of PE treatment (Fig 5, bar G). Dishes treated for 1 hour with ATP alone did not differ from the control condition in their incorporation of ¹⁴C-Phe over the next 72 hours (Table 3). When data from the 1-hour ATP pretreatment experiment were normalized to total DNA content, the incorporation values were as follows (cpm/µg DNA): untreated control, 1841±178 (mean±SEM for six dishes each; three preparations of myocytes); PE, 3349±278; and ATP (1 hour pretreatment), 2017±123. The values after 1-hour pretreatment with ATP were not significantly different from the control values but were significantly lower than values for PE-treated dishes (P<.05, ANOVA, Tukey procedure).

ATP Inhibits the Increase in Cell Size After Adrenergic Stimulation
ATP also inhibited the increase in cell size observed after adrenergic stimulation. Serum-starved neonatal cardiomyocytes were incubated with or without NE (2 µmol/L), PE (20 µmol/L), and ATP (100 µmol/L) for 3 days. Cell area was measured by planimetry at high power (x400). Both NE and PE alone significantly increased cell size by 65% and 51%, respectively (P<.001), whereas ATP alone had no statistically significant effect on cell size (Table 4). When added with either NE or PE, however, ATP completely inhibited the agonist-stimulated increases in cell size.

View this table: [in this window] [in a new window]   Table 4. Effects of ATP and PE on Individual Cardiomyocyte Size

ATP Selectively Inhibits Hypertrophy-Associated Changes in Gene Expression
The mRNA levels for ANF, which normally decreases in the ventricle after birth, and MLC-2 have both been shown to increase in adult and neonatal ventricular myocytes during hypertrophy induced by NE or PE.³⁰ Both ANF and MLC-2 mRNA levels were also increased in our neonatal myocyte cultures after 24 to 48 hours of PE stimulation (Figs 7, 8A, and 8B). ATP added at the time of PE stimulation completely inhibited the increases in ANF and MLC-2 gene expression.

View larger version (51K): [in this window] [in a new window]   Figure 7. Effect of ATP on basal and PE-stimulated changes in the expression of ANF, MLC-2, skeletal -actin (-SK), and SERCA2 mRNA levels. Representative Northern blot of the effects of ATP (100 µmol/L) on the PE (20 µmol/L)–stimulated increase in ANF, MLC-2, -SK, SERCA2, and GAPDH mRNA. A single blot was stripped and reprobed sequentially for each mRNA and subsequently for the 18S rRNA. Cultures were treated as described in Fig 1, except that cells were plated in 100-mm dishes, and total RNA was harvested after 48 hours of treatment.

View larger version (19K): [in this window] [in a new window]   Figure 8. Quantitative analysis of the effect of ATP on hypertrophy-induced changes in cardiac mRNA levels. A, ANF; B, MLC-2; C, skeletal -actin (a-SK); D, SERCA; and E, GAPDH. Cell cultures were treated as described in "Materials and Methods" and harvested after 24 to 48 hours of treatment. Radioactive signals were quantified using a Betascope Phosphorimager (model 603, Betagen) and are expressed as the ratio of specific mRNA to 18S rRNA to compensate for possible variations in RNA loading. In most cases, each blot was hybridized with each probe as shown in Fig 7. In a few cases, an individual lane or several lanes for a given probe were uninterpretable because of artifacts, in which case the data were not used. Data are expressed relative to control (CON, arbitrarily assigned a value of 1) as the mean±SEM. Each bar represents the mean of four to eight observations (one observation is one sample of total RNA derived from two to five dishes; each observation is from an independent preparation of myocytes). *P<.05 vs CON (one-sample t test).

In contrast, ATP had no effect on the increase in skeletal -actin mRNA levels that occurred in response to PE. Figs 7 and 8C show that 24 hours of incubation with PE caused an 3.5-fold increase in skeletal -actin mRNA levels. Coincubation with ATP had no effect on the PE-induced changes in skeletal -actin mRNA accumulation, although there was a tendency for ATP alone to increase expression of skeletal -actin and, when coadministered, to augment the PE-stimulated increase in skeletal -actin expression. Another change in gene expression associated with myocyte hypertrophy in vivo and the growth factor stimulation of cultured cardiomyocytes is the downregulation of the cardiac-specific SERCA gene.^{31 32 33 34 35} In response to PE stimulation for 24 to 48 hours, the level of SERCA mRNA in our cultures also tended to decrease slightly, although this decrease was not significant (Figs 7 and 8D). Coincubation with ATP and PE had no effect on the levels of SERCA mRNA, but ATP alone significantly increased expression of SERCA mRNA.

To examine the effects of ATP on the expression of a constitutively expressed "housekeeping" gene, we studied the effects of ATP, both alone and in combination with PE, on the mRNA level of GAPDH (Figs 7 and 8E). There was no significant effect of any of the treatments on the level of GAPDH mRNA.

ATP Does Not Inhibit NE- or PE-Induced Expression of the c-fos Gene or Increased Activity of MAPK
We have previously shown that both ATP and NE increase expression of the immediate-early gene, c-fos.⁷ To determine whether the inhibitory effects of ATP were mediated by antagonizing NE-induced c-fos gene expression, Northern blot analyses were performed. Both ATP and NE increased c-fos mRNA to a similar level, as did the combination of ATP and NE (Fig 9).

View larger version (12K): [in this window] [in a new window]   Figure 9. Effects of ATP on NE-stimulated c-fos mRNA expression. Cultures were treated with ATP (100 µmol/L) and/or NE (2 µmol/L), and RNA was harvested 30 minutes after treatment. Blots were probed for c-fos mRNA, stripped, and then reprobed for 18S rRNA. The ratio of c-fos/18S rRNA is presented, and data are expressed relative to control (CON, arbitrarily assigned a value of 1) as mean±SEM (n=3 to 10 observations). Each observation consisted of total RNA harvested from two to five dishes per treatment; each observation is derived from a separate preparation of myocytes. Compared with CON, c-fos expression was significantly elevated after each of the three treatments (P<.05, t test, Bonferroni correction).

Since it has also been recently shown that a number of hypertrophic agents, which initially use dissimilar intracellular signaling pathways, converge at the activation of the MAPKs p44^ERK1 and p42^ERK2,²² we examined whether ATP affected MAPK activity stimulated by PE. MAPK activity, as measured by phosphorylation of myelin basic protein incorporated into the polyacrylamide gel matrix, was elevated at 10 and 30 minutes after treatment with either PE or ATP. There was no significant difference from PE-stimulated activity when ATP was coadministered (Fig 10). Therefore, ATP and PE either alone or in combination caused similar increases in MAPK activity in cultured neonatal myocytes at these time points. These data indicate that the mechanism of action of ATP in suppressing adrenergic-induced hypertrophy does not involve modifying the increase in MAPK activity that is seen early after PE stimulation. In addition, because ATP alone does not stimulate hypertrophy of neonatal cardiomyocytes,⁷ it may be inferred that increased MAPK activity at 10 to 30 minutes may be required but is apparently not sufficient to induce hypertrophy.

View larger version (36K): [in this window] [in a new window]   Figure 10. Effects of ATP on PE-stimulated MAPK activity. Cultured neonatal cardiac myocytes (83% myocytes at the time of study) were treated for the indicated times with ATP (100 µmol/L), PE (20 µmol/L), or a combination of ATP/PE and compared with untreated control cultures. Extracts were run on 10% polyacrylamide gels containing myelin basic protein and assayed for MAPK activity as described in "Materials and Methods." Values are mean±SEM for three or four dishes per bar (four different preparations of myocytes). *P<.05 compared with control (one-sample t test, Bonferroni correction).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A number of studies have shown that NE and PE stimulate cellular hypertrophy in neonatal cardiac myocytes and that for NE, which is an agonist at both - and ß-adrenergic receptors, this effect is primarily mediated by ₁-adrenergic receptor stimulation. Activation of ₁-adrenergic receptors induces expression of immediate-early genes, upregulates early developmental isogenes (eg, cardiac bMHC and skeletal -actin), increases the synthesis and release of ANF from ventricular myocytes, and increases overall ¹⁴C-Phe incorporation and cell size.^{10 30} We have previously shown that extracellular ATP, which is coreleased with NE from sympathetic nerve endings, increases immediate-early gene expression but does not increase ¹⁴C-Phe incorporation or induce cellular hypertrophy. Our data also indicate that ATP acts via an intracellular signaling pathway that is distinct from that stimulated by NE.⁷

In the present study, we have demonstrated that extracellular ATP inhibits the increases in ¹⁴C-Phe incorporation and cellular size in neonatal cardiac myocytes that are observed after the administration of a variety of hypertrophy-inducing agents (Fig 1 and Table 2). These were the adrenergic agonists NE and PE and the growth factors ET-1 and bFGF. This inhibition by ATP was dose dependent, exhibiting an IC₅₀ of 8 µmol/L (Fig 2). The complete inhibition of ¹⁴C-Phe incorporation by ATP, the failure of the nonhydrolyzable P₁ purinergic receptor agonist PIA to inhibit hypertrophy, and the failure of the P₁ purinergic antagonist 8-PT to block this effect indicate that the inhibition of ¹⁴C-Phe incorporation by ATP is mediated by P₂ purinergic receptors. Although stimulation of these receptors by ATP was sufficient to account for the changes in ¹⁴C-Phe incorporation that we observed, stimulation of P₁ purinergic receptors by adenosine also led to a significant inhibition of ¹⁴C-Phe incorporation, although the effect was not as great as that with ATP. ATP was also capable of inhibiting elevated ¹⁴C-Phe incorporation that was already in progress. Surprisingly, pretreatment of myocyte cultures with ATP for as little as 1 hour before the addition of PE was sufficient to inhibit subsequent PE-induced changes in ¹⁴C-Phe incorporation for up to 3 days (Fig 5, Table 3), demonstrating that the continued presence of ATP or of its degradation products was not required for its maximum inhibitory effect.

Others have shown that hypertrophy-inducing agents stimulate RNA synthesis and lead to an increase in total RNA levels.²⁶ We have also noted a trend for total RNA to increase after the addition of PE and have measured changes in [¹⁴C]uridine incorporation as an indicator of changes in total RNA synthesis, since this method is more sensitive for detecting changes in RNA synthesis. These results (Fig 3) show that PE significantly increases [¹⁴C]uridine incorporation and that coadministration of ATP blocks this increase.

When ¹⁴C-Phe incorporation was expressed per dish (Table 2) rather than normalized to total DNA content, it appeared that ATP affected basal ¹⁴C-Phe incorporation, suggesting that the effects of ATP may be due to a generalized inhibition of protein synthesis rather than selectively inhibiting the increases due to hypertrophic agonist stimulation. The fact, however, that there is no significant effect of ATP on basal levels of ¹⁴C-Phe incorporation when incorporation is normalized to total DNA content makes this unlikely and instead suggests that ATP may affect cell attachment or viability. In fact, we have noted a strong trend toward cell loss (76% of control, Fig 4) and a significant reduction in total isolatable RNA from myocyte cultures treated with ATP alone. Although this effect suggests that ATP and/or its hydrolysis products may have a toxic effect during long-term (3-day) incubation, the following observations indicate that this effect is unrelated to the ability of ATP to modulate hypertrophy. First, preincubation of myocytes with ATP for 1 hour followed by its washout does not inhibit basal ¹⁴C-Phe incorporation but is as effective as a 3-day treatment in inhibiting PE-stimulated ¹⁴C-Phe incorporation (Fig 5). Second, when hypertrophy is assessed independently of cell number, as was the case when changes in individual cell size were examined, there was no effect of ATP on control myocytes (Table 4). Finally, for reasons that are unclear, there is no cell loss and no loss in isolatable total RNA in cultures in which ATP and the hypertrophy-inducing agents are incubated together (see text and Fig 4).

ATP also inhibited the expression of several hypertrophy-associated genes whose mRNA levels are increased by adrenergic agonists. These include ANF and MLC-2 (Figs 7, 8A, and 8B) but not skeletal -actin (Figs 7 and 8C). The levels of all three mRNAs were increased by adrenergic agonists, but only the increases in ANF and MLC-2 mRNA were inhibited by ATP. There was a trend for ATP to increase skeletal -actin mRNA levels, although this did not reach statistical significance. We also examined whether ATP could alter mRNA levels of the cardiac-specific SERCA (SERCA2). Incubation with ATP for 24 hours led to a significant increase in SERCA2 mRNA levels (Figs 7 and 8D); PE alone had no significant effect on SERCA gene expression but did negate the stimulatory effect of ATP. There is ample evidence in the literature showing that SERCA2 gene expression is downregulated in vivo during cardiac hypertrophy induced by aortic constriction³⁴ and in some, but not all, models of heart failure.^{13 35} In addition, Bassani et al³⁶ have shown that contractile arrest following chronic verapamil treatment leads to upregulation of SERCA gene expression, whereas Schneider and Parker³⁷ have shown that bFGF causes a reduction in SERCA2 gene expression. ATP also did not affect the expression of the constitutively expressed housekeeping gene, GAPDH. These data suggest that the effects of ATP may be selective for a subset of cardiac-specific genes and that it probably does not affect the level of mRNAs for various housekeeping genes. To the best of our knowledge, this is the first report of a physiologically relevant molecule that can antagonize hypertrophy and have differential effects on gene expression.

At present, the mechanism(s) by which ATP exerts its inhibitory effect on the hypertrophy process is unknown. Upon binding to P₂ purinergic receptors on cardiac membranes, extracellular ATP has been shown to exert a variety of biochemical and physiological effects, including activation of nonselective cation channels and L-type Ca²⁺ channels, increased intracellular Ca²⁺, increased phosphatidylinositide turnover, and decreased cAMP production.^{38 39 40 41 42 43} The fact that ATP inhibits the hypertrophy induced by stimulation of different receptor types (eg, G protein–linked serpentine receptors and tyrosine kinase receptors), which at least initially stimulate different intracellular signaling pathways, argues that its effects must be on distal steps in these signal transduction pathways. Likewise, the fact that PE-induced changes in skeletal -actin mRNA levels were unaltered or possibly augmented by ATP argues that binding and at least some of the subsequent intracellular signaling activity initiated by adrenergic receptor stimulation are unaffected by ATP. Since ATP activates c-fos expression in our cultures via a Ca²⁺-dependent signaling pathway, whereas NE does so by a Ca²⁺-independent mechanism,⁷ it is possible that ATP exerts its inhibitory effects on hypertrophy via a Ca²⁺-dependent signaling pathway. This seems unlikely, however, since it has been reported that adrenergic stimulation of ANF production in neonatal cardiomyocytes requires Ca²⁺ influx and the activation of Ca²⁺-regulated kinases.⁴⁴ Another potential mechanism excluded by the present data is the possibility that ATP inhibits hypertrophy by inhibiting the early increase in MAPK activity that we observed for PE and that has been observed for other hypertrophy-inducing agents.²² Although ATP itself stimulated MAPK activity for at least 30 minutes, it is possible that ATP could inhibit hypertrophy by inhibiting PE-stimulated MAPK activity at later times (eg, 24 to 72 hours after PE).

The data in the present study indicate that the effects of ATP are evident rapidly after ATP addition and are sustained for a number of days after ATP removal. Pretreatment with ATP for as little as 1 hour is as effective as 3 days of continuous exposure in inhibiting the NE- and PE-stimulated increases in radiolabeled phenylalanine incorporation (Fig 5, Table 3). Although the rapid activation of a persistent inhibitory effect suggests that ATP may alter the pattern of immediate-early gene expression required for hypertrophy, the increased level of c-fos mRNA and activation of MAPK were both unaffected by ATP. Nevertheless, the Fos and Jun families consist of many different members whose homodimerization and heterodimerization products can lead to functionally different DNA binding complexes.^{45 46 47} Furthermore, although c-fos expression following its activation is quite short-lived, persistent activator protein-1 activation is generally required for the downstream effectors of immediate-early gene induction, such as cell proliferation by growth factors.⁴⁸ It is possible that ATP disrupts long-lived activator protein-1 complexes that are potentially necessary for hypertrophy. Since the 1-hour pretreatment experiment suggests that the mechanism for inhibiting hypertrophy can be initiated before the addition of the hypertrophic agent, an analysis of gene expression specifically induced by ATP in neonatal cardiomyocytes may provide clues into the mechanism of the ATP-mediated suppression of hypertrophy.

Extracellular ATP as a modulator of the hypertrophic process may be important in at least two physiological circumstances in which ATP might be expected to accumulate in the extracellular space surrounding cardiomyocytes. During sympathetic discharge, it has been demonstrated that ATP is coreleased with NE,⁴ and during ischemic injury to the heart, ATP may be released from injured myocytes as well as from degranulating platelets⁴⁹ that accumulate during reperfusion of the tissue.

	Selected Abbreviations and Acronyms

 

14C-Phe = [14C]phenylalanine 8-PT = 8-phenyltheophylline ANF = atrial natriuretic factor bFGF = basic fibroblast growth factor bMHC = ß-myosin heavy chain ET = endothelin HPLC = high-performance liquid chromatography MAPK = mitogen-activated protein kinase MHC = myosin heavy chain MLC-2 = myosin light chain-2 NE = norepinephrine PE = phenylephrine hydrochloride PIA = phenylisopropyl adenosine SERCA = sarcoplasmic reticulum Ca2+-ATPase TCA = trichloroacetic acid

	Acknowledgments

 
The authors gratefully acknowledge Chunlin Yang for his help in HPLC determinations and Kenneth R. Chien (University of California at San Diego) for the MLC-2 cDNA probe.

Received January 11, 1995; accepted December 29, 1995.

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