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Am J Physiol Heart Circ Physiol 287: H1179-H1185, 2004. First published April 8, 2004; doi:10.1152/ajpheart.00725.2003
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Activation of IP prostanoid receptors prevents cardiomyocyte hypertrophy via cAMP-dependent signaling

Rebecca H. Ritchie,1,2 A. C. Rosenkranz,1 L. P. Huynh,1 T. Stephenson,2 D. M. Kaye,2 and G. J. Dusting1

1Howard Florey Institute and 2Baker Heart Research Institute, Melbourne, Victoria 8008, Australia

Submitted 5 August 2003 ; accepted in final form 7 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The antihypertrophic action of angiotensin-converting enzyme inhibitors in the heart results partly from local potentiation of bradykinin. We have demonstrated that the antihypertrophic action of bradykinin is mediated by the release of nitric oxide from endothelium and elevation of cardiomyocyte cGMP. Whether other paracrine factors derived from the coronary endothelium, such as prostacyclin (PGI2), may act to prevent hypertrophy has not been explored. In the vasculature, activation by PGI2 of IP and EP1 prostanoid receptors elicits vasodilatation (via cAMP-dependent signaling) and vasoconstriction, respectively. The present objective was to determine whether IP prostanoid receptor activation has antihypertrophic actions in adult rat cardiomyocytes (ARCM). The selective IP agonist cicaprost (1 µM) virtually abolished the increase in [3H]phenylalanine incorporation (a marker of hypertrophy) induced either by endothelin-1 (ET-1; 60 nM, n = 10, P < 0.005) or by angiotensin II (1 µM, n = 6, P < 0.005). Cicaprost also inhibited ET-1 induction of c-fos mRNA expression, an additional marker of hypertrophy in ARCM (n = 5, P < 0.005). In the absence of hypertrophic stimuli, cicaprost alone did not significantly influence either marker. The antihypertrophic actions of cicaprost were mimicked by the dual IP/EP1 agonist iloprost (1 µM) in the presence of the EP1 antagonist AH-6809 (3 µM). Furthermore, cicaprost modestly but significantly increased cardiomyocyte cAMP content by 13 ± 6% (P < 0.05, n = 4), and the antihypertrophic effect of cicaprost was lost in the presence of the cAMP-dependent protein kinase inhibitor H-89 (1 µM, n = 5, P < 0.05). However, ET-1 also induced increases in the activity of the intracellular growth signals ERK1 (by 3-fold) and ERK2 (by 5-fold) in ARCM, and these were not inhibited by cicaprost (P < 0.01, n = 5). Activation of IP receptors thus represents a novel approach to prevention of hypertrophy, and this effect is linked to cAMP-dependent signaling.

prostacyclin; endothelin-1; protein synthesis; cardiac hypertrophy

LEFT VENTRICULAR HYPERTROPHY is a complication that accompanies many cardiovascular diseases, including hypertension and heart failure, and is an independent risk factor for cardiac morbidity and mortality (17). In this context, therapeutic approaches targeting the renin-angiotensin system, angiotensin-converting enzyme (ACE) inhibitors, and angiotensin type-1 receptor antagonists are of considerable benefit (23). Local potentiation of bradykinin is thought to be involved in the antihypertrophic effect of these inhibitors and antagonists, both in vivo and in vitro (16, 26). We (24) have previously demonstrated that bradykinin blocks cardiomyocyte hypertrophy in vitro, but only in the presence of endothelial cells. Furthermore, the antihypertrophic action is exerted, at least in part, by stimulating the release of nitric oxide (NO) from adjacent endothelial cells (25), and elevation of the second messenger cGMP in cardiomyocytes is a key mediator of this antihypertrophic action (26). However, it is not known whether other paracrine factors derived from the cardiac endothelium have a role in the prevention of left ventricular hypertrophy, particularly prostacyclin (PGI2), which is also released by bradykinin (5).

PGI2 has been shown to play an important role in vascular protection, through actions such as vasodilatation (5, 18) and antiplatelet actions (5, 14, 18, 31). It is one of the most potent inhibitors of platelet activation identified (33). In vascular and human airway smooth muscle cells, PGI2 also exerts antiproliferative actions (2, 5). It is not known whether PGI2 has growth-inhibiting actions in cardiac muscle. PGI2 acts at both IP and EP1 prostanoid receptors, and mRNA for both receptors is detected in cardiac preparations (20). IP prostanoid receptor activation by PGI2 is coupled to adenylate cyclase and thus leads to vasodilatation via stimulation of cAMP-dependent protein kinase. In contrast, EP1 prostanoid receptor activation leads to signaling via inositol (1,4,5)-trisphosphate and diacylglycerol, thereby inducing vasoconstriction (3, 21). In addition to the endogenous ligand, IP and EP1 prostanoid receptors can be activated by a number of stable PGI2 mimetics, including iloprost (28) and cicaprost (31). Iloprost, like PGI2, is a nonselective receptor agonist with affinity at both IP and EP1 receptors. In contrast, cicaprost is more selective for and more potent at IP receptors than iloprost (12). However, selective IP receptor antagonists, or subtypes of IP receptors, have not been identified (12).

The objective of the present study was to search for antihypertrophic actions of IP prostanoid receptor agonists in adult rat cardiomyocytes, using both cicaprost and iloprost, and elucidate the intracellular signaling responsible. Adult (mature) cells are less likely to dedifferentiate in culture and do not exhibit the marked upregulation of atrial natriuretic peptide (ANP) or contractile proteins or other changes seen in the neonatal phenotype (10), and hypertrophic signaling in these cells is more likely to reflect the in vivo situation than neonatal cardiomyocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Angiotensin II (ANG II), human/porcine endothelin-1 (ET-1), and IBMX were purchased from Sigma (St. Louis, MO). Cicaprost and iloprost were gifts from Schering (Berlin, Germany). All reagents for cell culture were of tissue culture grade. Reagents for Western blot analysis and for real-time PCR analysis were of molecular biology grade. Forward and reverse primers, fluorogenic probes, TaqMan Universal PCR master mix, and TaqMan RT reagents were purchased from Applied Biosystems (Scoresby, Australia). All other materials were purchased from Sigma except where indicated and were of analytic grade or higher.

Isolation of adult rat cardiomyocytes. Male Sprague-Dawley rats (200–280 g) were anesthetized intraperitoneally with 100 mg/kg ketamine hydrochloride and 12 mg/kg xylazine before removal of the heart. Cardiomyocytes were then isolated as previously described (24, 25). Freshly dissociated cardiomyocytes were resuspended in serum-free bicarbonate-buffered medium 199 (M199; with Earle's salts and 25 mM HEPES, JRH Biosciences; Lenexa, KS) supplemented with 0.2% albumin (bovine fraction V), 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 25 µg/ml gentamicin (Life Technologies; New York, NY), 100 U/ml penicillin, and 100 µg/ml streptomycin (CSL Biosciences; Parkville, Australia). Cardiomyocytes were plated onto laminin (10 µg/ml, Collaborative Biomedical Products; Bedford, MA)-coated six-well plates (Falcon/Becton Dickinson; Lincoln Park, NJ) at a density of ≥8 x 104 cells per 35-mm well and incubated at 37°C (5% CO2 in air) overnight before study. Serum-free medium was replaced with fresh medium on the day of study. This technique yields <7% nonmyocyte contamination, as previously described (24, 25). The protocol was approved by the Animal Experimentation Ethics Committee of the Howard Florey Institute.

Cellular markers of myocyte hypertrophy. Conventional hypertrophic markers in isolated cardiomyocytes include de novo protein synthesis, increased protein content, and expression of immediate-early and fetal genes. In the present study, cardiomyocyte hypertrophy induced by ANG II was measured as increased protein synthesis (using [3H]phenylalanine incorporation) at 2 h. To demonstrate that this is an appropriate time point to determine hypertrophic responses, we first conducted a pilot study comparing basal and ANG II-stimulated levels of three hypertrophic markers, specifically protein synthesis and expression of the fetal gene ANP, in addition to protein synthesis at 2 and 24 h in adult rat cardiomyocytes, as described below.

[3H]phenylalanine incorporation. Cardiomyocytes were incubated for 2 h at 37°C in serum-free M199 containing 2 µCi/ml of L-[2,3,4,5-3H]phenylalanine (NEN Lifesciences; Boston, MA) with or without ET-1 (60 nM) or ANG II (1 µM). Pilot studies demonstrated that these were the lowest concentrations of ET-1 or ANG II that reproducibly increased [3H]phenylalanine incorporation. For the determination of antihypertrophic actions of PGI2 mimetics, medium was also supplemented with cicaprost (1 µM) or iloprost (1 µM) with or without the cAMP-dependent protein kinase inhibitor N-{2-[(p-bromocinnamyl)amino]ethyl}-5-isoquinoline sulfonamide (H-89; 1 µM, Calbiochem) or the EP1 prostanoid receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH-6809; 3 µM, Calbiochem). All pharmacological tools were present for the full 2-h incubation. At the end of the incubation period, culture medium was removed, and cardiomyocytes were rinsed three times with ice-cold PBS before trichloroacetic acid precipitation of DNA and protein and resuspension in 0.3 M sodium hydroxide (24, 25). [3H]phenylalanine incorporation (a measure of de novo protein synthesis and hence an in vitro marker of hypertrophy) was determined by liquid scintillation counting. DNA content was determined fluorimetrically using PicoGreen reagent (Molecular Probes; Eugene, OR). Results for [3H]phenylalanine incorporation were normalized to nanograms of DNA per sample (to correct for number of cells per sample), as previously described (24, 25). Within each experiment, each treatment group was studied at least in triplicate and the average result was taken. Data were then normalized (%) to the measurement of the averaged paired control wells for each experiment. For the pilot study comparing 2 and 24 h of ANG II incubation, total protein was also determined in these samples via Bradford assay, with results also expressed relative to nanograms of DNA per sample.

Accumulation of cAMP. Cardiomyocytes were incubated for 30 min at 37°C in serum-free M199 containing IBMX (1 mM, to prevent degradation of cAMP) ± ET-1 (60 nM) ± cicaprost (1 µM) and were then washed with ice-cold PBS (pH 7.4) before precipitation in 1.0 ml ice-cold 100% methanol. All pharmacological tools were present for the full 30-min incubation. All myocytes, including control, were exposed to 0.4% dimethylsulfoxide (IBMX vehicle). Well contents were then scraped into glass vacutainer tubes (Becton Dickinson) and dried under a stream of air at room temperature. Pellets were frozen at –20°C until the time of acetylated-cAMP [125I] radioimmunoassay (kit from Perkin-Elmer Lifesciences; Boston, MA) following the manufacturer's instructions. Within each experiment, each treatment group was studied at least in triplicate, and the average result was taken. cAMP content was determined as femtomoles of cAMP per 100-µl sample. Data were then normalized (%) to the measurement of the averaged paired control wells for each experiment.

Expression of c-fos and ANP mRNA by quantitative real-time PCR. Real-time PCR was used to measure mRNA expression of the immediate-early gene c-fos, an additional marker of cardiomyocyte hypertrophy, using the {Delta}{Delta}Ct method (27) relative to the internal standard 18S rRNA. Cardiomyocytes were incubated for 15 min with serum-free M199 alone or ET-1 (60 nM). The IP prostanoid agonist cicaprost (1 µM) or iloprost (1 µM ± 3 µM AH-6809) was added 30 min before ET-1. Total RNA was then extracted from cardiomyocytes using RNAWiz (Ambion, TX) and reverse transcribed using TaqMan RT reagents (Applied Biosystems) (27). The final real-time PCR mix (total volume 25 µl) comprised 5 ng cDNA template, 1x TaqMan Universal PCR mastermix, primers, and fluorogenic probes, all designed from rat-specific sequences published on GenBank and used at previously determined optimal concentrations. Forward and reverse primers for c-fos were 5'-CAACGAGCCCTCCTCTGACT-3' and 5'-TGCCTTCTCTGACTGCTCACA-3', respectively (both 300 nM); corresponding sequences for 18S rRNA were 5'-CGGCTACCACATCCAAGG-3' and 5'-GCTGGAATTACCGCGGCT-3' (both 120 nM). Probes were 5'-labeled with the reporter dyes FAM (c-fos: FAM-CTGAGCTCGCCCACACTGCTAGCC-TAMRA, 100 nM) or VIC (18S rRNA: VIC-TGCTGGCACCAGACTTGCCCTC-TAMRA, 125 nM) and 3'-labeled with the quencher molecule TAMRA. Both c-fos and 18S rRNA were amplified in the same tube to determine relative increases in c-fos transcripts relative to 18S. Reactions were performed in the ABI Prism 7700 sequence detection system (Applied Biosystems). The average fold induction of c-fos expression in the paired control samples was thus set as 1.0 (27). For the pilot study comparing 2 and 24 h of ANG II incubation, ANP gene expression was also determined using real-time PCR using rat-specific primer and probe sequences as previously described (26).

Activation of ERK1/2. Activation of ERK1/2, an intracellular signal implicated in the induction of cardiomyocyte growth by many hypertrophic stimuli (6), was determined using Western blot analysis of ERK1/2 protein in cardiomyocytes, using an antibody selective for the phosphorylated (active) form of the enzyme. Cardiomyocytes were incubated for 5 min with serum-free M199 alone or with ET-1 (60 nM). The IP prostanoid receptor agonist cicaprost (1 µM) or iloprost (1 ± 3 µM AH-6809) was added 30 min before ET-1. Cardiomyocytes were then lysed in 300 µl cell lysis buffer comprising 10 mM Tris·HCl, 50 mM NaCl, 1% Triton X-100, 30 mM sodium pyrophosphate, 50 mM NaF, 5 µM ZnCl2, 0.1 mM sodium orthovanadate, 1 mM dithiothreitol, 5 nM okadaic acid, and Complete protease inhibitor cocktail (as per the manufacturer's instructions, Roche Diagnostics). After 20 min of centrifugation (13,000 rpm, 4°C), total protein was determined in cytosolic fractions using the Bradford assay. Samples were then diluted 5:1 in 6x sample loading buffer, comprising 35.6 mM SDS, 0.93 g dithiothreitol, 1.2 mg bromophenol blue, and 3 ml glycerol in 1 M Tris·HCl, and stored at –80°C until required. On the day of analysis, thawed samples were boiled for 5 min to denature protein and loaded onto a 10% SDS-polyacrylamide gel (20 µg protein/lane). After electrophoresis, proteins were transferred to a nitrocellulose membrane and incubated for 1 h in blocking buffer [Tris-buffered saline (TBS) containing 5% nonfat dried milk and 0.05% Tween 20] before overnight incubation at 4°C with rabbit polyclonal antibody to either phospho-p44/42 MAPK (also known as ERK1/2) or to total 44/42 MAPK (1:1,000, Cell Signaling Technology). The blot was washed and incubated for 2 h with the secondary antibody, horseradish peroxidase-labeled goat anti-rabbit immunoglobulin (1:1,000). Proteins were visualized by enhanced chemiluminescence (kit from Amersham; Buckinghamshire, UK), anticipating a double band of the predicted size for ERK1/2 protein of 44 and 42 kDa.

Statistical analysis. The n value for each set of results represents the number of myocyte preparations studied. Results are expressed as means ± SE. Statistical comparisons with control (where n ≥ 4) utilized the Mann-Whitney rank sum test (for comparison of two nonnormally distributed groups), one-way ANOVA with the Bonferroni correction for {alpha} (for comparison of more than two normally distributed groups), or Kruskal-Wallis one-way ANOVA on ranks with the Student-Newman-Keuls test for multiple comparisons (for comparison of more than two nonnormally distributed groups). Values of P < 0.05 were accepted as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cellular markers of myocyte hypertrophy. For the pilot study comparing time points of ANG II administration, cardiomyocytes stimulated with ANG II exhibited increased phenylalanine incorporation, to 128 ± 3% and 141 ± 7% of the paired control at 2 and 24 h, respectively (both n = 3). Cardiomyocyte total protein content was increased at both time points, to 120 ± 12% and 134 ± 4% of the paired control at 2 and 24 h, respectively. However, ANG II only induced gene expression of ANP at 2 h (by 1.8 ± 0.3-fold of the paired control, n = 3) but not at 24 h (expression of 0.8 ± 0.3-fold of the paired control, n = 3). The induction of multiple hypertrophic markers by ANG II at 2 h indicated that [3H]phenylalanine incorporation at this time point was an appropriate measure of cardiomyocyte hypertrophy for all further studies.

Effect of cicaprost on ET-1- and ANG II-induced hypertrophy. ET1 significantly stimulated [3H]phenylalanine incorporation by 25 ± 4% in adult rat cardiomyocytes (Fig. 1A; P < 0.005, n = 10); this was virtually abolished by the IP prostanoid receptor agonist cicaprost (1 µM, P < 0.05 vs. ET-1 alone). In a second series of experiments, ANG II significantly stimulated cardiomyocyte [3H]phenylalanine incorporation by 33 ± 6% (Fig. 1B; P < 0.005, n = 6); again, this was abolished by 1 µM cicaprost (P < 0.05 vs. ANG II alone). Although the concentration-response relationship between cicaprost and its antihypertrophic actions was not examined in the present study, the selective IP prostanoid agonist did show evidence of antihypertrophic action at lower concentrations: 0.1 µM cicaprost prevented the ANG II-induced increase in cardiomyocyte [3H]phenylalanine incorporation, reducing it to 87 ± 9% of that observed in paired control cardiomyocytes (P < 0.05 vs. ANG II alone, Kruskal-Wallis analysis, n = 6). However, all further studies utilized 1 µM cicaprost, which did not significantly influence [3H]phenylalanine incorporation in the absence of hypertrophic stimuli (104 ± 6% of the control, n = 13, results not shown). No differences in cardiomyocyte morphology were observed with the various treatments (visual analysis under low-power magnification, results not shown).



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  		Fig. 1. Cicaprost (1 µM) blocked increases in [3H]phenylalanine incorporation by adult rat cardiomyocytes stimulated by endothelin (ET)-1 (60 nM; A) or ANG II (1 µM; B). *P < 0.05 vs. control; #P < 0.05 vs. hypertrophic stimulus.

 
Effects of cicaprost on cAMP signaling. In the vasculature, the vasodilator actions of IP receptor activation by PGI2 are attributed predominantly to cAMP-dependent signaling (3, 33). Cicaprost (1 µM) modestly, but significantly, increased cAMP content in adult rat cardiomyocytes by 13 ± 6% (Fig. 2A; P < 0.05, n = 4). Moreover, the inhibitory effect of cicaprost on ET-1-stimulated [3H]phenylalanine incorporation was lost in the presence of the cAMP-dependent protein kinase inhibitor H-89 (1 µM): [3H]phenylalanine incorporation remained elevated at 35 ± 15% above control (Fig. 2B; P < 0.05, n = 5). H-89 alone did not significantly influence [3H]phenylalanine incorporation (results not shown).



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  		Fig. 2. Role of cAMP-dependent signaling. A: influence of cicaprost on cardiomyocyte accumulation of cAMP. B: cAMP-dependent protein kinase inhibitor H-89 (1 µM) restores the [3H]phenylalanine incorporation response of adult rat cardiomyocytes to ET-1. *P < 0.05 vs. control.

 
Effects of iloprost. PGI2 activates both IP and EP1 prostanoid receptors in many cell types (3). In the present study, the nonselective IP/EP1 receptor agonist iloprost (1 µM) partially attenuated ET-1-stimulated [3H]phenylalanine incorporation, from 33 ± 4% above control to 18 ± 5% above control, but the addition of the selective EP antagonist AH-6809 (3 µM) was required to completely abolish the ET-1 response (Fig. 3; P < 0.005, n = 7). AH-6809 alone did not significantly influence [3H]phenylalanine incorporation (results not shown). As an additional marker of hypertrophy, the exposure of cardiomyocytes to ET-1 (15 min) also increased c-fos mRNA expression by 55 ± 13% (P < 0.005, n = 5; Fig. 4) compared with control adult rat cardiomyocytes. Activation of IP prostanoid receptors, either with cicaprost or with the combination of iloprost + AH-6809, when added 30 min before ET-1, blocked this ET-1-stimulated increase in c-fos mRNA, to 10 ± 11% and 14 ± 9% above control, respectively.



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  		Fig. 3. Iloprost (1 µM) requires the addition of the EP1 antagonist AH-6809 (3 µM) to block increases in [3H]phenylalanine incorporation by adult rat cardiomyocytes stimulated by ET-1 (60 nM). *P < 0.05 vs. control.

 


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  		Fig. 4. IP receptor activation blocks increases in c-fos mRNA expression stimulated by ET-1 (60 nM) in adult rat cardiomyocytes. *P < 0.05 vs. control.

 
Effects on ERK1/2 signaling. To further elucidate the mechanism by which activation of the IP prostanoid receptor attenuates ET-1-mediated cardiomyocyte hypertrophy, we investigated the role of the ERK1/2 MAPK pathway. ET-1 increased the activation of the intracellular growth signals ERK1 and ERK2 after 5-min incubation with adult rat cardiomyocytes. With the use of Western blot analysis for the phosphorylated forms of the enzyme, ET-1 increased the activation of ERK1 and ERK2 by 3.3 ± 0.6-fold and 5.3 ± 0.9-fold of paired control myocytes, respectively (P < 0.01, n = 5; Fig. 5A). However, the activation of IP prostanoid receptors with cicaprost failed to inhibit these ET-1 responses when added 30 min before ET-1. No changes in total ERK1 or ERK2 protein expression were observed with any of these treatments (Fig. 5B).



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  		Fig. 5. Influence of cicaprost (1 µM) on cardiomyocyte activation of ERK1/2 by ET-1 (60 nM). A: phosphorylated ERK1/2. B: total ERK1/2. Top: representative blots; bottom: pooled data. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding of the present study is that activation of IP prostanoid receptors exerts an antihypertrophic action in adult rat cardiomyocytes. Because cicaprost was equally effective in blocking ANG II- and ET-1-induced responses, its actions were likely directly antihypertrophic rather than affecting stability of the hypertrophic peptide stimuli. This is the first evidence that PGI2 or its mimetics possess growth-inhibiting actions in cardiac muscle. Because of its antiplatelet and vasodilator actions, PGI2 was initially evaluated for its therapeutic potential in thromboembolic disorders, cardiopulmonary bypass, acute myocardial infarction, unstable angina, peripheral vascular disease, congestive heart failure, and pulmonary hypertension (5, 22, 33). However, the lack of receptor selectivity and short half-life of this prostanoid prompted the development of stable PGI2 analogs such as cicaprost (22, 33). We now describe how the selective IP agonist cicaprost substantially inhibited ET-1-induced increases in two different markers of cardiomyocyte hypertrophy ([3H]phenylalanine incorporation and c-fos mRNA expression). These antihypertrophic actions were mimicked by the dual IP/EP1 agonist iloprost (in the presence of an EP1 antagonist). Although cicaprost increased cardiomyocyte cAMP content only modestly, inhibition of the cAMP-dependent protein kinase abolished the antihypertrophic effect of cicaprost. Activation of IP receptors by PGI2 mimetics may thus represent a novel approach to prevention of hypertrophy, via cAMP-dependent signaling.

Cyclooxygenase (COX) generates PGH2 from arachidonic acid, which is then converted to PGI2 via the enzyme PGI2 synthase (3, 5). Vascular endothelial cells are the most active producers of PGI2, but the underlying vascular smooth muscle cells, cardiomyocytes, and fibroblasts also produce PGI2 (5, 32, 33, 35). The growth-inhibiting action of PGI2 in vascular cells is well characterized (29), but there is little information regarding its actions on cardiac muscle growth. Both COX-1 and COX-2 isoforms contribute to PGI2 synthesis. Although the potential roles of COX in cardiac hypertrophy have not been fully elucidated, COX-2-deficient mice exhibit both reduced prostanoid synthesis and marked cardiac fibrosis (4). COX-derived PGI2 might thus serve as an endogenous defence against worsening cardiac enlargement and dysfunction. This is supported by the observation that nonselective COX inhibition abolishes the endothelium-dependent antihypertrophic actions of bradykinin (25). Disruption of PGI2 synthase expression results in elevated systolic blood pressure and vascular hypertrophy (34); the present study suggests that cardiac hypertrophy might also be predicted in settings of PGI2 deficiency, evidence supported by gene therapy with PGI2 synthase, in which right ventricular hypertrophy secondary to pulmonary hypertension is reduced (19). Deficiency of IP prostanoid receptors is also associated with augmented growth responses in the vasculature (8) as well as potentiated right ventricular hypertrophic responses to pulmonary hypertension (11). Systolic pressure overload in IP receptor-deficient animals might result in potentiation of the left ventricular hypertrophic response.

We have previously demonstrated that the dual IP/EP1 agonist iloprost does not block increases in cardiomyocyte protein synthesis induced by ANG II (25). Both IP and EP prostanoid receptors are detected in the heart (3, 21). In the vasculature, these receptors have opposing effects on smooth muscle contraction. Our results suggest that in cardiac muscle IP but not EP1 prostanoid receptor activation has an antihypertrophic effect and that in the case of the dual agonist iloprost, blocking its activity at EP1 receptors improves its protective action. Until recently, knowledge pertaining to the IP receptor included its relative abundance in various organs, amino acid sequence, and vascular and platelet actions (21). IP receptors are primarily thought to couple to Gs to activate adenylate cyclase and hence elevate cAMP (33). We have previously demonstrated the key role of cGMP as an antihypertrophic mediator, in both cardiac myocytes and whole hearts (26, 27). Whether a similar protective role for cAMP is evident in the heart has been difficult to elucidate. Our previous studies with the stable analog 8-bromo-cAMP have suggested cAMP might even promote hypertrophy in cardiomyocytes (25). In the present study, the antihypertrophic action of cicaprost was critically dependent on cAMP-dependent signaling, yet only modest increases in total cardiac myocyte cAMP content were observed. We speculate that instability of cAMP and/or its intracellular sublocalization might explain why a more marked effect on cAMP accumulation was not observed in the present study. Not all agonists that increase cAMP in the heart elicit antihypertrophic actions, for example, {beta}-adrenergic receptor agonists. Recently, it has been suggested that cAMP-dependent protein kinase is compartmentalized within the cell, such that high concentrations of cAMP and cAMP-dependent protein kinase are only required within specific intracellular locations, which contain discrete pools of cAMP-dependent protein kinase (13).

Our results indicate that IP receptor activation blocks cardiomyocyte growth, evidenced by induction of immediate-early gene expression and de novo protein synthesis, via cAMP-dependent signaling. Because ERK1/2, a member of the MAPK signaling family, is thought to transduce critical components of the cardiac growth response to the nucleus (6), we sought to determine whether cicaprost prevented its activation. Although we were able to demonstrate a marked activation of both ERK1 and ERK2 in response to ET-1, this was not prevented by cicaprost. This observation raises the possibility that IP prostanoid receptor activation specifically inhibits ERK-independent hypertrophic signaling, such as calcineurin/nuclear factors of activated T cells-activated responses (15). Alternatively, IP activation might target hypertrophic responses downstream of ERK1/2. Aside from IP and EP1 receptors, the only other receptors known to be activated by PGI2 mimetics are nuclear peroxisome proliferator-activated receptors (PPAR). Although activation of the insulin-sensitizing PPAR-{gamma} may elicit antihypertrophic responses (1), only iloprost and PGI2 (not cicaprost) are ligands at these receptors (7).

In conclusion, we have clearly demonstrated for the first time that IP prostanoid receptor activation inhibits cardiomyocyte hypertrophy in vitro, acting via cAMP-dependent signaling but without modulation of ERK1/2. At present, the major clinical use of PGI2 and its mimetics is in chronic treatment of severe pulmonary hypertension (30). Cicaprost, iloprost, and the parent compound PGI2 also elicit some cardioprotective actions in acute myocardial infarction (5, 14, 31). Furthermore, the antihypertrophic actions of ACE inhibitors in vivo are accompanied by threefold increases in left ventricular PGI2 concentration (9). Taken together with our in vitro observations, activation of cardiac IP receptors may represent a novel approach to prevention of hypertrophy, and we propose that in vivo studies specifically determining whether chronic activation of IP receptors prevents and/or regresses left ventricular hypertrophy in experimental models are now warranted.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
This study was supported by the National Health and Medical Research Council of Australia and the High Blood Pressure Research Foundation of Australia. A. C. Rosenkranz was supported by an Australian Postgraduate Award.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. H. Ritchie, Baker Heart Research Institute, PO Box 6492, St. Kilda Rd. Central, Melbourne, Victoria 8008, Australia (E-mail: rebecca.ritchie{at}baker.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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