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Am J Physiol Heart Circ Physiol 293: H1662-H1672, 2007. First published June 8, 2007; doi:10.1152/ajpheart.00159.2007
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Developmental changes in gene expression of Epac and its upregulation in myocardial hypertrophy

Coskun Ulucan,1,2 Xu Wang,2 Erdene Baljinnyam,2 Yunzhe Bai,1 Satoshi Okumura,1 Motohiko Sato,1 Susumu Minamisawa,1 Shinichi Hirotani,2 and Yoshihiro Ishikawa1,2,3

1Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama, Japan; and 2Cardiovascular Research Institute, Departments of Cell Biology and Molecular Medicine, and 3Department of Medicine (Cardiology), New Jersey Medical School, Newark, New Jersey

Submitted 7 February 2007 ; accepted in final form 4 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although it has been shown that Epac1 mRNA is expressed ubiquitously and Epac2 mRNA predominantly in the brain and endocrine tissues, developmental and pathophysiological changes of these molecules have not been characterized. Developmental changes were analyzed in murine heart, brain, kidneys, and lungs by RT-PCR analysis, which revealed more drastic developmental changes of Epac2 mRNA than Epac1. Only the Epac2 mRNA in kidney showed a transient expression pattern with dramatic decline into adulthood. In addition to developmental changes, we found that Epac gene expression was upregulated in myocardial hypertrophy induced by chronic isoproterenol infusion or pressure overload by transverse aortic banding. Both Epac1 and Epac2 mRNA were upregulated in isoproterenol-induced left ventricular hypertrophy, whereas only Epac1 was increased in pressure overload-induced hypertrophy. Stimulation of H9c2, cardiac myoblast cells, with fetal calf serum, which can induce myocyte hypertrophy, upregulated Epac1 protein expression. We also demonstrated that Epac was the limiting moiety, relative to Rap, in the Epac-Rap signaling pathway in terms of stoichiometry and that Epac stimulation led to the activation of ERK1/2. Our data suggest the functional involvement of Epac in organogenesis and also in physiological as well as pathophysiological processes, such as cardiac hypertrophy. Furthermore, our results suggest the importance of the stoichiometry of Epac over that of Rap in cellular biological effects.

ontogeny; rap; adenosine 3',5'-cyclic monophosphate, pressure overload; isoproterenol

VARIOUS EFFECTS OF THE SECOND MESSENGER cAMP on multiple crucial functional processes in the cell, such as metabolism, muscle contraction, neuronal plasticity, and gene transcription, have been known to mainly depend on the activation of a serine/threonine-specific protein kinase A (PKA) (2). Besides direct modulation of certain ion channels by cAMP, however, a new PKA-independent signaling pathway has been established with the discovery of "Epacs," guanine nucleotide exchange factors that are directly activated by cAMP (7, 17). Two isoforms, Epac1 and Epac2, which are coded by different genes, mediate the cAMP signal transduction to activate the small-molecular-weight G protein Rap to regulate various cellular functions (11). Although Epac1 and Epac2 have a distinct structure in the number of cyclic nucleotide binding (CNB) domain (7, 17) and its affinity to cAMP (6), both proteins possess the COOH-terminal catalytic region consisting of a Ras-exchanger-motif (REM) domain, a Ras-association (RA) domain, and a CDC25 homology, as the catalytic domain, and activate Rap. Epac has been shown to be involved in cellular processes such as secretion (23), cell adhesion (14), cell-cell junction formation (32), Ca2+ signaling (36), apoptosis (20), hypertrophy (9, 28), proliferation (9), differentiation (4), and gene expression (21), although the role of Epac2 is less well known relative to that of Epac1 and has been studied in insulin secretion or exocytosis (16). BLAST analysis of the amino acid sequence of Epac2 revealed relatively high homology to Epac1 with 49% identity and 66% positives. Even the domains are highly similar, and the effects of both isoforms might be different due to altered protein interactions and subcellular localization.

Important components in regulating cAMP signaling are the upstream G protein-coupled receptor (GPCR), Gs protein, and adenylyl cyclase (AC) (25). The interaction of these components determines the efficacy of the stimulus, whereas the interaction is dependent on the available number of each of the components. Gs is excessively more highly expressed than GPCR and AC, whereas the amount of AC expression is slightly higher than that of GPCR (1, 26). This composition implicates that AC and GPCR are the determinants for the generation of cAMP in the GPCR-Gs-AC signaling pathway (22). However, there are no stoichiometric data available about the downstream effectors of cAMP such as PKA, Epac, and Rap. Northern blot analysis of the gene expression pattern of Epac revealed that Epac1 mRNA was highly expressed in a variety of tissues such as the heart, kidneys, ovaries, thyroid glands, and the corpus callosum of the brain, whereas Epac2 mRNA was detectable most notably in the brain, pituitary, and adrenal gland (17), although their developmental or pathophysiological changes have been poorly understood.

The aim of this study was to analyze in detail the developmental mRNA expression pattern of both Epac1 and Epac2 in different organs and to examine pathophysiological changes in Epac in various cardiac hypertrophic models. We have also addressed the importance of the stoichiometry of this molecule compared with its downstream target, Rap1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and reagents. COS-7, H9c2, and HepG cells (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and a 1% solution of penicillin-streptomycin at 37°C in 5% CO2. At least 24 h before experimental processing, cells were subcultured into 60-mm dishes. The Epac-specific cAMP analog 8-para-methoxyphenylthio-2'-O-methyl-cAMP (8-pMe-OPT) was purchased from Axxora Life Science (San Diego, CA), and insulin was obtained from Sigma-Aldrich (St. Louis, MO).

Culture of neonatal mice ventricular myocytes. Spontaneously beating neonatal mice cardiomyocytes were isolated and purified as described previously by our group (13). Briefly, hearts were harvested from 1- to 2-day-old C57BL/6 mice. Ventricles were chopped to small pieces and incubated three times with 0.1% collagenase II (Worthington, Lakewood, NJ) for 15 min, where the supernatant was removed at each interval. To remove fibroblasts, cells were preplated onto culture dishes (Falcon 3803; BD Biosciences, San Jose, CA) for 45 min, and the medium with nonattached cells was replated on culture dishes. Cardiomyocytes were cultured for 24 h in DMEM with the above-mentioned supplements plus 0.1 mM bromodeoxyuridine.

Adenovirus infection and plasmid transfection. Recombinant adenovirus containing human Epac1, Epac2, and Rap1 was constructed according to the manufacturer's protocol (Adeno-X Expression System 1; BD Biosciences Clontech, Mountain View, CA). Human Epac1 cDNA was kindly provided by Dr. J. L. Bos (University Medical Center Utrecht, Utrecht, The Netherlands) and Epac2 by Dr. A. M. Graybiel (Massachusetts Institute of Technology, Cambridge, MA), and human Rap1 plasmid was purchased from Invitrogen. Briefly, the corresponding encoding sequence was cloned into pShuttle2 (Clontech) to obtain a mammalian expression cassette, which was then excised and ligated to BD Adeno-X viral DNA. The recombinant vector was introduced into human embryonic kidney (HEK)-293 cells to recover infectious adenovirus, which were propagated in HEK-293 cells and purified using the BD Adeno-X virus purification kit (BD Biosciences Clontech). The viral titer was determined using the Adeno-X rapid titer kit (BD Biosciences Clontech). Cells were infected with adenoviruses at appropriate multiplicities of infection as indicated. Rap1 GTPase-activating protein 1 (RapGAP) sequence expressed in pCMV-SPORT6 was purchased from Invitrogen and cloned into pShuttle2 for transfection assays.

Western blotting. Cells were lysed in lysis buffer containing 25 mM Tris·HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 1 mM DTT, 5% glycerol, protease inhibitor cocktail (Sigma-Aldrich), 1 mM PMSF, 1 mM NaF, and 1 mM Na3VO4. Protein concentration was measured by the Bradford method using bovine serum albumin as standard. Proteins were separated on SDS-polyacrylamide gel, and the gel was blotted onto polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA). Detection of proteins was made using antibodies anti-Rap1 (Pierce, Rockford, IL), anti-Epac1, anti-PKA C{alpha}, anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Akt, anti-p44/42 MAPK (ERK1/2), and the phospho-specific antibodies against Akt and ERK1/2 (Cell Signaling Technology, Danvers, MA).

Rap1 activation assay. Rap1 activity was measured using the EZ-Detect RAP1 activation kit (Pierce, Rockford, IL) according to manufacturer's protocol. Briefly, cell lysates were incubated with Rap-binding domain RalGDS-RBD fused to a glutathione S-transferase disk. After repeated washing steps, bound GTP-Rap1 was removed from the disk by boiling in a SDS sample buffer and analyzed by Western blotting using Rap1 antibody.

Animals. Hearts, brains, kidneys, and lungs were dissected from C57BL/6 mice at the ages of embryonic day 19 (fetus), around the day of birth (neonates), 3 wk after birth, and 12 wk after birth. To protect RNA from degradation, freshly dissected specimens were immediately submerged in RNA stabilization reagent (RNAlater; Ambion, Austin, TX) and stored at 4°C until further processing as described in Real-time quantitative RT-PCR. All animal cares and study protocols were approved correspondingly by the Animal Care and Use Committee at New Jersey Medical School and the Animal Ethics Committee of the Yokohama City University School of Medicine.

Adenylyl cyclase type 5 knockout mice. The generation of the adenylyl cyclase type 5 (AC5) knockout mice with 129/SvJ-C57BL/6 genetic background was described in detail previously (29). All mice used in this study were backcrossed to C57BL/6.

Transgenic mice. AC5 transgenic mice were generated (FVB/NJ background) using cDNA of canine AC5 (15) driven by the {alpha}-myosin heavy chain promoter to achieve cardiac-specific expression (12).

Transverse aortic constriction. Transverse aortic constriction (TAC) was performed as described previously in our laboratory (29, 31). Briefly, 15-wk-old C57BL/6 male mice were anesthetized by intraperitoneal application of Avertin (tribormoethanol; Sigma- Aldrich) at 250 mg/kg body wt. Open-chest surgery was performed under air ventilation with a tidal volume of 0.2 ml and a respiratory rate of 100 breaths/min. Constriction of 0.4-mm in diameter was obtained by ligation of the transverse aorta against a 27-gauge needle. Sham-operated mice underwent a similar surgical procedure without constriction of the aorta. Two weeks after TAC, animals were killed and hearts were dissected. These hearts showed a mild degree of cardiac hypertrophy without major deterioration in cardiac function as shown in multiple studies from our laboratory (29, 31). The left ventricle (LV) was separated, fresh frozen in liquid nitrogen, and stored at –80°C for RNA isolation.

Chronic infusion of isoproterenol. Chronic infusion of isoproterenol (Calbiochem, San Diego, CA) was performed as previously reported by our group (19). Mini-osmotic pumps (model 2001; Alzet, Cupertino, CA) were implanted in anesthetized 13-wk-old C57BL/6 male mice. Pumps were filled with either isoproterenol or vehicle (0.5 mM ascorbic acid in PBS) to deliver isoproterenol at a dose of 60 µg·g–1·day–1 for 7 days. The heart was dissected from mice, and the LV was separated from the heart. Tissues were quickly frozen in liquid nitrogen and stored at –80°C.

Real-time quantitative RT-PCR. RNA was extracted using Tri reagent (Sigma-Aldrich) according to the manufacturer's protocol. The RNA concentrations and quality were determined by spectrophotometry. To remove contaminating DNA, samples were treated with recombinant DNase I (DNA-free; Ambion). RNA was reverse transcribed with TaqMan RT reagents (Applied Biosystems, Foster City, CA) according to the manufacturer's instruction.

Epac1, Epac2, Gs{alpha}, PKA {alpha}-catalytic subunit (PKA C{alpha}), PKA beta-catalytic subunit (PKA Cbeta), atrial natriuretic factor (ANF), Rap1A, and Rap2A mRNA expressions were quantified by real-time PCR (qPCR) using the ABI PRISM 7700 Sequence Detector (Perkin-Elmer/Applied Biosystems). Real-Time PCR was performed using PCR Master Mix (ABI) including SYBR green in a 30-µl volume. 18S ribosomal RNA (18S rRNA control kit; Eurogentec, San Diego, CA) was used as internal standard in each tissue and developmental age. In addition to Epac1 and Epac2 primers, which were designed with Primer Sequencer (Applied Biosystems), primers were designed using Primer3 Input software (30) (Table 1). The cycling condition was as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles consisting of 15 s of denaturation at 95°C and 1 min of combined annealing/extension at 60°C. To identify specific PCR products generated in the presence of SYBR green, a melting curve analysis was performed following amplification. The relative amounts of genes of interest were generated using the standard curve method and normalized to 18S rRNA. Mean mRNA expressions were expressed as percentages of the mean value of fetus or control mice.


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  		Table 1. Primers for real-time quantitative RT-PCR

 
Statistical analysis. The data were analyzed for significant group differences by using one-way ANOVA followed by Tukey's post test. Two-group comparisons were performed with two-tailed Student's t-test or Mann-Whitney test, accordingly. Statistical significance was assumed at P < 0.05. Analysis for the presence or absence of correlation between the test parameters was performed, and a Pearson correlation coefficient of r ≥ 0.7 was taken to indicate the presence of strong correlation (GraphPad Prism; GraphPad Software).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Developmental changes of Epac1 and Epac2 mRNA expression in the heart, brain, kidneys, and lungs. We quantified the gene expression of Epac1 (Fig. 1) and Epac2 (Fig. 2) mRNA using qPCR in the heart, brain, kidneys, and lungs of mice at the ages of embryonic day 19 (fetus), the day of birth (neonate), 3 wk after birth, and 12 wk after birth.


Figure 1
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  		Fig. 1. Developmental changes in Epac1 mRNA expression in the heart (A; n = 4), brain (B; n = 4–5), kidneys (C, n = 4–5), and lungs (D; n = 4–5) of mice at the ages of embryonic day 19 (fetus), around the day of birth (neonate), and 3 and 12 wk postnatal were measured by quantitative real-time PCR (qPCR). Values are means ± SE of mRNA levels, expressed as percentages of normalized expression levels of fetus. vs. fetus and neonate. +P < 0.05; #P < 0.01 vs. fetus. {ddagger}P < 0.001 vs. neonate. *P < 0.05; **P < 0.01; ***P < 0.001 vs. fetus and neonate.

 

Figure 2
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  		Fig. 2. Developmental changes in Epac2 mRNA expression in the heart (A; n = 4), brain (B; n = 4–5), kidneys (C; n = 4–5), and lungs (D; n = 4–5) of mice at ages indicated in Fig. 1 were analyzed by qPCR. Epac2 expression levels changed in a greater scale than Epac1 expression during development. Contrary to other tissues and Epac1, kidneys showed a dramatically decrease in Epac2 mRNA expression after the young adult stage. Values are means ± SE of mRNA levels, expressed as percentages of normalized expression levels of fetus. #P < 0.01 vs. fetus. *P < 0.05; {ddagger}P < 0.001 vs. neonate. **P < 0.01; ***P < 0.001 vs. fetus and neonate. &P < 0.001 vs. 3 wk.

 
Comparing the early developmental stages of fetus and neonates with the adult stages of 3- and 12-wk-old animals, we found modest, but statistically significant, increases of Epac1 mRNA expression in the heart, brain, and kidneys. Epac1 mRNA expression in the heart of 12-wk-old animals increased by 42% (P < 0.01) (Fig. 1A) compared with that in the fetus. It also increased by 71% (P < 0.05) in the brain and 55% (P < 0.001) in kidneys (Fig. 1, B and C). Lungs showed a significant increase in 3-wk-old animals by 59% (P < 0.05), which declined thereafter to insignificant values (Fig. 1D).

Developmental changes in Epac2 mRNA expression in all four tissues were on a greater scale than those seen in Epac1. Cardiac Epac2 gene expression showed steep increases at each stage, with an increase by 176% (P < 0.01) in 3-wk-old animals and by 366% (P < 0.001) in 12-wk-old animals compared with that in fetus (Fig. 2A). The mRNA expression in the brain was increased in both 3- and 12-wk-old animals, by 310% (P < 0.001) and 348% (P < 0.001), respectively (Fig. 2B), compared with that in fetus. In lungs, developmental changes were also similar to those of Epac1, which showed an increase in 3-wk-old animals by 99% (P < 0.01) and then a decrease in 12-wk-old animals to the value in fetus (Fig. 2D). In kidneys, however, the Epac2 gene expression decreased considerably by 94 and 96% in 3- and 12-wk-old (P < 0.001 vs. fetus and neonate, each), respectively, indicating that Epac2 expression almost disappeared in adult kidneys (Fig. 2C).

Interestingly, when we calculated the ratio of Epac1 to Epac2 mRNA expression (Epac1/Epac2), it decreased in the heart of 3- and 12-wk-old mice to 0.50 and 0.31 of the fetal values (P < 0.001 vs. fetus and neonate, each), respectively (Fig. 3A). A similar decrease of Epac1/Epac2 was calculated for the brain of neonate, 3-wk-old, and 12-wk-old mice to 0.61, 0.44, and 0.40 of the fetal values (P < 0.01, fetus vs. neonate; P < 0.001, fetus vs. 3- and 12-wk-old mice), respectively, whereas in the lungs, Epac1/Epac2 was increased 2-and 1.9-fold in 12-wk-old mice compared with neonate and 3-wk-old mice, respectively (Fig. 3, B and C). These results suggest that Epac2, relative to Epac1, became dominant in the adult brain and heart, but not in lungs, compared with fetal organs.


Figure 3
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  		Fig. 3. Ratios of Epac1 to Epac2 mRNA during development in the heart (A), brain (B), and lungs (C) are shown as relative changes from the value for fetus. Values are means ± SE. *P < 0.01; #P < 0.001 vs. fetus. **P < 0.01 vs. neonate and 3 wk. ***P < 0.001 vs. fetus and neonate.

 
Developmental changes in Gs{alpha}, PKA C{alpha}, and PKA Cbeta mRNA expression in the heart. To compare the above developmental changes in Epac1 and Epac2 expression with those of other molecules involved in cAMP signal, we examined the mRNA expression of Gs{alpha}, PKA C{alpha}, and PKA Cbeta in the heart by using qPCR (Fig. 4). In contrast to Epac expression, the mRNA expression levels of Gs{alpha} slightly decreased during development by 23 and 18% in 3- and 12-wk-old mice (P < 0.001 vs. fetus, each), respectively (Fig. 4A). PKA C{alpha} remained unchanged during development, whereas PKA Cbeta mRNA showed a transient decrease at 3 wk by 30% (P < 0.05 vs. neonate) (Fig. 4, B and C).


Figure 4
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  		Fig. 4. Gene expression levels of Gs{alpha}, PKA {alpha}-catalytic subunit (C{alpha}), and PKA beta-catalytic subunit (Cbeta) during development in the heart. Gs{alpha} (A), PKA C{alpha} (B), and PKA Cbeta (C) mRNA expression were measured by qPCR in the heart (n = 4 each) of mice during development at indicated ages. Only Gs{alpha} expression was slightly decreased from the age of 3 wk, whereas PKA C{alpha} remained unchanged through development and PKA Cbeta decreased transiently at 3 wk compared with the neonatal stage. Values are means ± SE of mRNA levels, expressed as percentages of normalized expression levels of fetus. {ddagger}P < 0.001 vs. fetus. *P < 0.05; #P < 0.01 vs. neonate.

 
Influence of AC5 expression on developmental Epac1 and Epac2 mRNA. Because AC plays a pivotal role in cAMP signaling, we were interested in investigating the effects of genetic ablation of type 5 AC (AC5), a major cardiac AC isoform, on cardiac Epac gene expression. We analyzed Epac1 and Epac2 mRNA expression in the heart of AC5 knockout (AC5KO) mice at the ages of embryonic day 19 (fetus), around the day of birth (neonate), 3 wk after birth, and 12 wk after birth.

Epac1 expression appeared to decrease, but nonsignificantly, in neonates compared with that in fetus, followed by an increase in 3 wk by 116% (P < 0.001 vs. neonate), and remained increased compared with that in neonates (P < 0.01 vs. neonate) (Fig. 5A). The above changes were essentially similar to those in wild type (WT) (Fig. 1A). Epac2 developmental changes were also similar to those in WT (Fig. 2A). From the neonatal stage to 3 wk, Epac2 mRNA expression increased by 121% (P < 0.05). A further large increase occurred in 12-wk-old mice by 249% compared with 3-wk-old mice (P < 0.001) (Fig. 5B). The ratio of cardiac Epac1 to Epac2 mRNA expression in AC5KO also decreased in adults, but not until the age of 12 wk (to 0.38 of the fetal value, P < 0.01) (Fig. 5C) compared with WT (Fig. 3A).


Figure 5
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  		Fig. 5. Epac1 and Epac2 mRNA expression in adenylyl cyclase type 5 (AC5) knockout and overexpressing (AC5OE) mice. Epac1 (A; n = 3–4) and Epac2 (B; n = 4) mRNA expression were analyzed by qPCR in the heart of AC5 knockout mice (AC5KO) at indicated ages (A and B), as well as changes in the ratio between Epac1 and Epac2 (C) and in AC5-overexpressing mice (AC5OE) in adults (n = 8) (D). Values are means ± SE, expressed as percentages of expression levels in fetus for AC5KO mice (A and B), as relative difference from value in fetus for Epac1/Epac2 mRNA ratio (C), and as percentages of expression in nontransgenic mice for AC5 transgenic mice (D). &P < 0.01 vs. fetus. *P < 0.05; #P < 0.01; **P < 0.001 vs. neonate. {ddagger}P < 0.01; ***P < 0.001 vs. fetus, neonate, and 3 wk.

 
Thus genetic disruption of AC5 did not induce major changes in Epac expression in the heart. Epac expression was not changed in the heart of AC5-overexpressing mice, as well (Fig. 5D), suggesting that the amount of AC5 expression did not play a major role in regulating the expression of Epac in the heart.

Effect of pressure overload on cardiac Epac gene expression. To further understand the role of Epac in intact animals, we analyzed Epac1 and Epac2 gene expression in LV tissues of mice that underwent transverse aortic banding (TAC) to induce LV hypertrophy by pressure overload. Two weeks after TAC, the ratio of LV weight to tibial length rose significantly by 46.2% (P < 0.001 vs. sham), and the mRNA level of the fetal gene ANF increased as expected (P < 0.01 vs. sham) (Fig. 6A). We found that Epac1 mRNA expression was increased mildly, but significantly, by 26% (P < 0.05 vs. sham), whereas Epac2 mRNA expression was unchanged (Fig. 6B). Calculation of Epac1/Epac2 mRNA in each animal showed an increase to 1.4 times the sham value (P < 0.01) (Fig. 6C). In addition to Epac1, Rap1A and Rap2A mRNA, effectors of Epac, also increased by 38% (P < 0.05 vs. sham) and 42% (P < 0.01 vs. sham), respectively. Correlation analysis of all animals demonstrated that the upregulation of both ANF and Epac1 mRNA was associated significantly with LV weight (Fig. 6, E and F).


Figure 6
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  		Fig. 6. Pressure overload induced by transverse aortic constriction (TAC) increased left ventricular (LV) Epac1 and Rap1/2 gene expression. LV gene expression of atrial natriuretic factor (ANF; A), Epac1 and Epac2 (B), and Rap1 and Rap2 (D) were analyzed by qPCR in sham and TAC mice. A: TAC increased expression of the fetal gene ANF (sham, n = 6; TAC, n = 5). B: Epac1 mRNA was increased compared with sham animals, whereas Epac2 expression level was unchanged. C: the ratio of Epac1 to Epac2 mRNA in each analyzed tissue was also increased. D: both Rap1 and Rap2 gene expression were increased in a manner similar to Epac1 gene expression. Values are means ± SE, expressed as either percentages or relative expression levels of sham mice. *P < 0.05; **P < 0.01 vs. sham. Correlation calculations of gene expression for LV weight in sham and TAC mice (n = 11) showed a positive correlation with LV hypertrophy for both ANF (E) and Epac1 (F). Normalized gene expression values are shown on the y-axis, whereas LV weight is shown in the x-axis. The Pearson correlation coefficient (r) and the corresponding P value are indicated.

 
Effect of isoproterenol infusion on cardiac Epac gene expression. To examine changes in Epac1 mRNA expression in another model of LV hypertrophy, we used mice with chronic isoproterenol infusion using osmotic minipumps, which developed cardiac hypertrophy as shown previously by our group (19). Chronic isoproterenol induced a significant increase in the ratio of LV weight to tibial length by 40.7% (P < 0.001 vs. control) and increased both Epac1 and Epac2 gene expression by 59% (P < 0.05 vs. control) and 36% (P < 0.05 vs. control), respectively (Fig. 7A). The association between LV weight and Epac2 mRNA expression was positively and significantly related (P < 0.05) (Fig. 7B).


Figure 7
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  		Fig. 7. LV Epac1 and Epac2 mRNA expression were increased during isoproterenol (ISO)-induced hypertrophy. Mice were treated with saline (control, CTRL) or ISO (60 µg·g–1·day–1) for 7 days (CTRL, n = 4; ISO, n = 5). A: LV Epac gene expression was analyzed by qPCR and normalized to 18S rRNA. B: correlation between Epac2 and LV weight of these mice was demonstrated to be significant by Pearson correlation coefficient (r) and the corresponding P value. Values are means ± SE, expressed as percentages of expression levels in control mice. *P < 0.05 vs. CTRL.

 
Serum stimulation induced upregulation of Epac1 protein expression. Although we tried to demonstrate increased Epac protein expression in the above hypertrophied hearts, Epac was not detectable in cardiac tissues by immunoblotting. We found, however, that Epac protein was readily detectable in a cardiac myogenic cell line, H9c2 cells. Although incubation of myoblasts in medium containing a low concentration of serum induces myotube formation, H9c2 cells remained undifferentiated in our experiments due to a relatively short exposure time to a low concentration of serum. Fetal calf serum (FCS) is known to contain a variety of growth hormones and has a hypertrophic effect on cardiomyocytes (8). FCS stimulation for 24 h upregulated the Epac1 protein expression to 1.6 times that in unstimulated cardiac myoblasts (P < 0.01) (Fig. 8, A and B), whereas that of Rap1 and PKA C{alpha} remained unchanged (Fig. 8C).


Figure 8
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  		Fig. 8. Fetal calf serum (FCS) increased Epac1 protein expression in H9c2 cells. A: cells preconditioned with 5% FCS were subjected to either serum starvation or incubation with 20% FCS for 24 h (n = 4 each). B: densitometric analysis, presented as relative changes in serum-starved cells (means ± SE) revealed a 1.6-fold increase of Epac1 protein expression in FCS-treated cells. C: cells were stimulated with increasing concentrations of FCS as indicated for 30 h (n = 2 each). Equal amounts of cell lysates were analyzed by immunoblotting with Epac1, Rap1, PKA C{alpha}, and GAPDH antibodies. **P < 0.01 vs. 0% FCS.

 
Stoichiometric significance of Epac1 in Rap1 activation. Because the above findings demonstrated relatively large changes in developmental and spatiotemporal changes in Epac expression, we hypothesized that the amount of Epac might play a more important role than Rap1 in the Epac-Rap pathway. Thus we investigated the stochiometrical significance of Epac1/Rap1 with adenovirus-mediated overexpression of these proteins in cultured cells. To stimulate Epac selectively, we used an Epac-specific cAMP analog, 8-pMe-OPT (5). The effectiveness of Epac1 overexpression was confirmed in Rap activation assays in cardiomyocytes and COS-7 cells in an Epac dose- and a postinfection time-dependent manner (Fig. 9, A–C) as well as in a 8-pMe-OPT dose-dependent manner (Fig. 9D).


Figure 9
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  		Fig. 9. Epac1 adenovirus infection increased Rap1 activity in a dose- and time-dependent manner. A: COS-7 cells were infected with adenovirus harboring Epac1 at different multiplicities of infection (MOI) for 24 h and stimulated with 50 µM 8-pME-OPT-2'-O-Me-cAMP (8-pMe-OPT-2) for 15 min, followed by Rap1 activation assays. Band C: at indicated times after Epac1 adenovirus infection, COS-7 cells (B) and cardiomyocytes (C) were stimulated with 100 µM 8-pMe-OPT, followed by Rap1 activation assays. D: COS-7 cells were infected with Epac1 adenovirus at an MOI of 10 for 24 h and treated with 8-pMe-OPT at indicated concentrations for 15 min, followed by Rap1 activation assays. Equal amounts of cell lysates were analyzed for activation of Rap1 by using GTP-bound Rap1 (Rap1-GTP) assay as described in METHODS.

 
Overexpression of Rap1 or Epac1 in cardiomyocytes induced different effects on Rap1 activity (Fig. 10A). Overexpression of either Epac1 or Rap1 proteins in cardiomyocytes increased the amount of activated Rap1 to a similar extent at base, i.e., in the absence of 8-pMe-OPT stimulation (Fig. 10A). However, after stimulation with 8-pMe-OPT, Rap1 activity was increased in cells overexpressing Epac1 to a much greater degree than in Rap1 overexpressing cells (Fig. 10A), suggesting that the amount of activated Epac1 played an important role in Rap activation. Figure 10B shows that the amount of Rap1 in cells, which were infected with Epac1 adenovirus, compared with that in noninfected cells was similar. Likewise, the adenovirus-mediated overexpression of Rap1 did not affect endogenous Epac1 protein expressions.


Figure 10
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  		Fig. 10. Stoichiometric importance of Epac1 vs. Rap1. A: cardiomyocytes were infected for 24 h with adenovirus expressing either Rap1 at an MOI of 50 or Epac1 at an MOI of 2 as indicated. Indicated cells were stimulated with 100 µM 8-pMe-OPT-cAMP for 15 min. B: overexpression of Epac1 or Rap1 did not change the endogenous expression of Epac1 or Rap1. C: fibroblasts were infected for 48 h with adenovirus harboring either Rap1 at an MOI of 50 or Epac1 at an MOI of 2. Cells were transfected with pShuttle plasmid containing RapGAP for 24 h. Specified cells were stimulated with 50 µM 8-pMe-OPT-cAMP for 15 min. D: H9c2 cells were infected for 48 h with Epac1 or Rap1 adenovirus at an MOI of 2 or an MOI of 50, respectively, and stimulated with 50 µM 8-pMe-OPT for 15 min. Cell lysates were assayed for GTP-bound Rap1 by pull-down assays. E: densitometric analysis of data in D, shown as relative changes (means ± SE) of unstimulated Epac1-overexpressing H9c2 cells. Under basal conditions, the activation of Rap1 was greater with Rap1 overexpression than Epac1 overexpression. However, under 8-pMe-OPT stimulation, the Rap1 activity in Epac1 adenovirus-infected cells exceeded that in Rap1-overexpressing cells. *P < 0.01 vs. unstimulated Epac1. **P < 0.001 vs. unstimulated Epac1 and unstimulated and stimulated Rap1. #P < 0.001 vs. unstimulated Epac1 and unstimulated Rap1.

 
To confirm that Epac1 had a greater stoichiometric significance in Rap activation than Rap itself, we coinfected cells with Rap1GAP, which inactivates Rap1 but not Epac1, by catalyzing the hydrolysis of Rap1-GTP to GDP. When Rap1 was overexpressed and stimulated with 8-pMe-OPT, Rap activity was increased significantly. This increase was readily attenuated by co-overexpressing Rap1GAP, indicating that Rap1GAP inactivated Rap1 as expected. When Epac1 was overexpressed and stimulated with 8-pMe-OPT, Rap1 activity was similarly increased. However, co-overexpression of Rap1GAP did not decrease Rap1 activity to a significant degree (Fig. 10C), suggesting that activated Epac1 dominated the regulation of Rap1 activation over Rap1GAP in the stimulated cAMP-Epac-Rap1 signaling pathway and that activated Epac1 level plays a more important role than the Rap1 availability for the cellular Rap1 activation. To further address the hypothesis that during stimulation of the Epac-Rap signaling pathway, the amount of available Epac is of greater importance than that of Rap1, statistical analysis was performed on Rap1-GTP protein quantity in H9c2 cells (Fig. 10, D and E). Without cAMP stimulation, the Rap1 activity in Rap1-overexpressing cells was 4.8-fold higher than that in Epac1-overexpressing cells (P < 0.01). However, after stimulation with 8-pMe-OPT, the Rap1 activity in cells overexpressing Epac1 increased 16.9-fold (P < 0.001) and was 1.7-fold greater than that in Rap1-overexpressing cells (P < 0.001), which showed only a 2-fold increase compared with basal conditions (Fig. 10E). Together, these results suggest the superior role of Epac over Rap1 in the stimulated cAMP-Epac-Rap1 signaling pathway. This is reminiscent of the relationship between beta-adrenergic receptors and G protein. Because G protein exists to a stoichiometric excess to the receptor, the amount of beta-adrenergic receptors plays a more important role in the stimulation of beta-adrenergic receptor-G protein-cAMP signaling pathway. Accordingly, it is tentative to speculate that Rap1 may exist to a stoichiometric excess to Epac1 in cells.

Epac1 activates p44/42 MAPK (ERK1/2) and Akt. Because H9c2 cells expressed a relatively high level of endogenous Epac1, we stimulated H9c2 cells with 8-pMe-OPT, which activated both Akt and ERK in a time-dependent manner (Fig. 11A). Similarly, Epac1 overexpression in HepG cells showed an increase in phosphorylated Akt as well as in phosphorylated ERK1/2 (Fig. 11B), which was further increased in the presence of 8-pMe-OPT (Fig. 11C). 8-pMe-OPT by itself did not significantly increase ERK1/2 phosphorylation, presumably because of a relatively low level of endogenous Epac expression in HepG cells (data not shown). Insulin induced ERK1/2 activation, as previously shown (35), and was further enhanced by Epac1 at different time points of insulin stimulation (Fig. 11, C and D). These results demonstrate that Epac activation leads to activation of MAPK/ERK, one of the downstream molecules in insulin signal, suggesting the presence of cross talk between cAMP and insulin signal.


Figure 11
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  		Fig. 11. Epac1 activated Akt and p44/42 MAPK (ERK1/2) and enhanced insulin-induced ERK1/2 activation in intact cells. A: after 6 h of serum depletion, H9c2 cells were stimulated with 50 µM 8-pMe-OPT for the indicated times, followed by immunoblotting. B: HepG cells were infected with adenovirus harboring Epac1 at an MOI of 1 for 72 h and serum starved for 6 h before harvesting, followed by immunoblotting. C: HepG cells were infected with Epac1 adenovirus at an MOI of 2 for 2 days as indicated. The cells were then serum deprived for 16 h, followed by insulin stimulation at 10 µM with or without preincubation with 8-pME-OPT at 10 µM for 15 min, or cells were left untreated. D: HepG cells were infected with Epac1 adenovirus at an MOI of 1 for 3 days and stimulated after 12 h of serum starvation with insulin at 10 µM for the indicated times. Equal protein amounts of cell lysates were used for Western blotting with antibodies against Akt, phosphorylated Akt, ERK1/2, phosphorylated ERK1/2, and Epac1.

 

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Our data have suggested the role of Epac in the development of organs, such as the heart, brain, kidneys, lungs, and adrenal glands, among others, and its involvement in pathophysiological changes as shown in various cardiac hypertrophic models. Furthermore, activation of Epac may be the limiting factor in the stimulated cAMP-Epac-Rap1 signaling pathway, which also leads to the activation of ERK1/2, a common downstream effecter shared with insulin signal.

Although Epac mRNA expression was shown in a variety of fetal and adult tissues, little is known about the ontogeny of the Epac gene expression in such tissues. Our results show that expression of both Epac1 and Epac2 is under developmental regulation. The Epac mRNA analysis in the heart, brain, kidneys, and lungs by RT-PCR revealed that the developmental gene expression of Epac2 changed more dramatically than that of Epac1. Epac1 mRNA reached its maximum level of expression in mice at the age of 3 wk in all analyzed tissues without further significant developmental changes. By contrast, Epac2 gene expression increased further into adulthood in the heart and decreased to fetal levels in lungs. In kidneys, Epac2 mRNA almost disappeared after the young adult stage, suggesting that Epac2 plays an important role in fetal, but not adult, renal function. In addition, we have demonstrated the change in the ratio of the two isoforms, i.e., decrease in the Epac1/Epac2 ratio, with cardiac development. This was interesting, because similar developmental changes in the two major AC isoforms AC5 and AC6, i.e., the AC6/AC5 ratio, have been shown in the heart (33), although the presence or absence of AC5 does not seem to change the expression of Epac (Fig. 5).

We have demonstrated that the cAMP-Epac-Rap1 pathway was responsive in the adaptation to pressure overload as well as catecholamine-induced cardiac hypertrophy. Interestingly, in the pressure overload model, the gene expression of only the Epac1 isoform was increased, whereas isoproterenol treatment induced an increase of both Epac1 and Epac2. Increased expression of Epac1 at the level of protein was shown in H9c2 cells when cells were stimulated with FCS. Morel et al. (28) demonstrated that Epac adenovirus infection of cardiac myocytes induced hypertrophy and the expression of hypertrophy gene markers. On the other hand, the stimulation of rat neonatal ventriculocytes with Epac-selective analog blocked the hypertrophic effect of leukemia-inhibiting factor (9). We do not know whether Epac is required for the development of cardiac hypertrophy or whether its expression is increased as a result of hypertrophy at this time. The development of transgenic models overexpressing Epac or mice with disrupted Epac expression is necessary to address such issues in the future.

Although the involvement of Epac in activation of the mitogen-activated protein kinase ERK1/2 was demonstrated in a variety of cell lines (18, 21), other reports showed Epac-independent regulation of ERK (10, 34). We have demonstrated that Epac activated ERK as well as Akt and enhanced the insulin-induced ERK phosphorylation. Because there is cross talk between the phosphatidylinositol 3 (PI3) kinase/Akt and Ras/ERK pathway via PKC (24), and Epac may be involved in PI3 kinase/Akt activation (3, 27), it is possible that the phosphorylation of ERK1/2 was mediated through PI3 kinase activation by PKC. Wang et al. (34) demonstrated in PC12 cells that Epac-dependent ERK activation was determined by the plasma membrane localization of Epac. Thus the role of Epac in ERK1/2 activation might depend on subcellular localization as well as cell-specific abundance of participant molecules, including PKA, Rap, and Ras. In particular, the role of Epac1/Epac2 stoichiometry in the cross talk of cAMP signaling pathways needs to be further evaluated.

An important aspect in a signaling pathway is to determine the stoichiometry among the participating molecules. The limiting component in the interaction of Epac with Rap was examined by overexpressing both proteins and was found to be Epac under conditions in which the cAMP-Epac-Rap1 signaling pathway is stimulated. This indicates that the Epac-Rap pathway is more effectively modified by increasing or decreasing the amount of Epac rather than that of the Rap protein itself. Epac may thus serve as a potential target of pharmacotherapy for various diseases, including cardiac hypertrophy.


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This work was in part supported by National Institutes of Health Grants R01 GM067773 and HL059139, the Ministry of Education, Science, Sports and Culture of Japan, and the Kitsuen Research Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Ishikawa, Cardiovascular Research Institute, Depts. of Cell Biology and Molecular Medicine and Dept. of Medicine (Cardiology), New Jersey Medical School, UMDNJ, Newark, NJ 07103 (e-mail: ishikayo{at}umdnj.edu)

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.


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