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Research Institute (T.T., T.S., K.M., I.K., S.S., K.K.) and Department of Medicine (T.H., F.Y., Y.K.), National Cardiovascular Center, Suita, Osaka 565-8565, Japan; and Second Department of Internal Medicine (M.K.), Kagawa University Faculty of Medicine, Kagawa 761-0793, Japan
Address all correspondence and requests for reprints to: Takeshi Horio, M.D., Division of Hypertension and Nephrology, Department of Medicine, National Cardiovascular Center, 5-7-1, Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: thorio{at}ri.ncvc.go.jp.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelin-1 (ET-1) is a 21-amino-acid peptide secreted from many types of cells including cardiac nonmyocytes (8, 9). It is well established that ET-1 provokes cardiac hypertrophy both in vivo and in vitro (9, 10). In addition, it has been shown that endogenous ET-1 secreted from cardiac nonmyocytes regulates cardiac myocyte hypertrophy in a paracrine manner (8, 9). However, it is unclear whether CNP affects the stimulatory action of ET-1 on myocyte hypertrophy. On the other hand, previous studies suggested that some types of vasoactive agents inhibit CNP signaling in HEK293 cells and NIH3T3 cells (11, 12). However, little is known about the inhibitory effect of hypertrophic agents including ET-1 on CNP signaling in cardiac myocytes.
Therefore, we conducted the present study to investigate the suppressive effect of CNP on ET-1-induced cardiac myocyte hypertrophy and the converse inhibition by ET-1 of the effect of CNP on cardiac myocytes, through the interaction between CNP and ET-1 signaling pathways.
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Materials and Methods |
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Cell culture
Primary cultures of rat cardiac myocytes were prepared as previously described (13). Briefly, apical halves of cardiac ventricles from 1- to 2-d-old Wistar rats were separated, minced, and dispersed with 0.1% collagenase type II. To segregate myocytes from nonmyocytes, a discontinuous gradient of Percoll was prepared. After centrifugation, the upper layer consisted of a mixed population of nonmyocyte cell types, and the lower layer consisted almost exclusively of cardiac myocytes. After the myocytes were incubated twice on uncoated 10-cm culture dishes for 30 min to remove any remaining nonmyocytes, the nonattached viable cells were plated on gelatin-coated culture plates or culture dishes and then cultured in DMEM supplemented with 10% FCS. After 36 h of incubation in DMEM with 10% FCS, cardiac myocytes were serum starved for 24 h before the experiments. This purification procedure has well been established (8), and in fact, >95% of the cells obtained by this method were cardiomyocytes.
For preparation of nonmyocyte-conditioned medium, cardiac nonmyocytes were cultured on 10-cm dishes. After incubation in DMEM with FCS, the medium was changed to fresh serum-free DMEM and cells were incubated for 24 h. After this conditioning period, cells were incubated with or without CNP, ANP, or Br-cGMP for 24 h. The medium was then collected as nonmyocyte-conditioned medium.
Northern blot analysis
Total RNA was extracted from cultured cells with TRIzol LS reagents as previously described (5). Poly(A)+ RNA was prepared for the detection of GC-A and GC-B genes using Oligotex-dT30 Super (Takara Biomedicals, Shiga, Japan). Total or poly(A)+ RNA (15 µg/lane) was electrophoresed on a 1% agarose gel and then transferred to a nylon membrane. Hybridization and washing of the membrane were carried out with cDNA probes for rat ANP, BNP, 
-skeletal muscle isoform of actin (SK-actin), sarcoplasmic reticulum Ca2+-ATPase (SERCA2), GC-A, GC-B, and glyceraldehyde-3-phosphate dehydrogenase genes, according to methods previously reported (5, 7).
Measurement of cellular cGMP
After preincubation for selected periods, myocytes grown in 24-well plates were treated for 15 min with various agents in the presence of 5 x 10–4 mol/liter 3-isobutyl-1-methylxanthine. The intracellular cGMP levels were determined by a RIA performed with a cGMP assay kit (Yamasa Shoyu, Chiba, Japan), as previously described (5).
Protein synthesis
The protein synthesis in cultured cardiac myocytes was evaluated based on the incorporation of [3H]leucine into cells as reported previously (5). After the preconditioning period, various agents and 0.5 µCi of [3H]leucine were added. After the cells were incubated for 24 h, the radioactivity of aliquots of the trichloroacetic acid-insoluble material was determined with a liquid scintillation counter.
Measurement of immunoreactive ANP and ET-1
The culture medium was acidified with acetic acid, boiled for 5 min to inactivate intrinsic proteases, and lyophilized. The RIA for rat ANP was performed as described previously (5). The RIA for rat ET-1 was performed with the ET-1,2-[125I] high sensitivity RIA system (Amersham Bioscience, Tokyo, Japan).
EMSA
Nuclear proteins were extracted with NE-PER nuclear and cytoplasmic extraction reagents, and EMSA was performed with a LightShift chemiluminescent EMSA kit (Pierce, Rockford, IL). For EMSA, the binding reactions were performed for 20 min in 10 mmol/liter Tris-HCl (pH 7.5), 50 mmol/liter KCl, 5 mmol/liter MgCl2, 1 mmol/liter dithiothreitol, 50 ng/µl poly (dI-dC)(dI-dC), 0.05% Nonidet P40, 2.5% glycerol, biotin 3'-end-labeled double-stranded oligonucleotide, and 2 µg (GATA) or 10 µg (MEF-2) of nuclear protein extract. Samples were electrophoresed on a native 5% polyacrylamide gel and then transferred to a nylon membrane. The biotin end-labeled DNA was detected by chemiluminescence. The nucleotide sequence of the sense strand of the double-stranded oligonucleotides was as follows: for GATA, 5'-CAC TTG ATA ACA GAA AGT GAT AAC TCT-3'; for MEF-2, 5'-TGG GCT ATT TTT AGG GGT TGA CTG-3'. Supershift experiments were performed by incubating the nuclear proteins with 2 µg of GATA-4 or -6 or MEF-2 antibody.
Measurement of intracellular Ca2+ concentration ([Ca2+]i)
Changes in [Ca2+]i were measured using a fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) system as described previously (14). After preincubation for selected periods, myocytes grown in gelatin-coated 96-well black-wall microplates were loaded with Fluo-3 fluorescent calcium indicator dye as follows. The culture medium was removed by aspiration and replaced with 0.1 ml of dye-loading buffer containing 20 mmol/liter HEPES, 2.5 mmol/liter probenecid, and 4 µmol/liter Fluo-3. After incubation for 1 h in a CO2 incubator, the cells were washed four times with Hanks’ balanced salt solution (HBSS) containing 20 mmol/liter HEPES and 2.5 mmol/liter probenecid. After a final wash, the solution was aspirated to a residual volume of 100 µl ET-1, CNP, and/or Br-cGMP and diluted with HBSS containing 20 mmol/liter HEPES and 2.5 mmol/liter probenecid. The fluorometric imaging plate reader transfers 100 µl from the reagent microplate to the cells in 10 sec and makes fluorescence readings for up to 60 sec. The instrument’s software normalizes the fluorescent reading to give equivalent initial readings at time zero.
Ca2+/calmodulin-dependent kinase II (CaMKII) activity
The activities of CaMKII were assayed with a SignaTECT calcium/calmodulin-dependent protein kinase assay system (Promega, Madison, WI) according to the manufacturer’s instructions.
Western immunoblot analysis
After stimulation for selected periods, cells were rapidly rinsed with ice-cold PBS and harvested in a sample buffer (62.5 mmol/liter Tris-HCl, 2% sodium dodecyl sulfate, 10% glycerol, 0.01% bromophenol blue, and protease inhibitor cocktail, pH 6.8). Cell lysates were electrophoresed through a reducing SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk and incubated with an antibody for phosphospecific ERK42/44 or ERK42/44. The expression levels of the protein and phosphoprotein were detected with a Phototope-HRP Western blot detection system (Cell Signaling Technology).
Statistical analysis
All values are shown as the mean ± SD. Statistical significance between the two groups was determined using unpaired t test. For multiple comparisons, data were subjected to one-way ANOVA followed by Fisher’s multiple comparison test. P values of less than 0.05 were considered statistically significant.
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Results |
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). The expression level of GC-B was more intense than that of GC-A (Fig. 1B).
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). ANP also increased the cGMP level concentration dependently, but the effect was weaker than that of CNP.
Effect of CNP on protein synthesis and ANP secretion in cardiac myocytes
CNP dose-dependently (10–8–10–7 mol/liter) decreased the incorporation of [3H]leucine into cardiac myocytes under nonstimulated conditions, but ANP did not (Fig. 2A). Br-cGMP mimicked the effect of CNP of reducing the incorporation of [3H]leucine (Fig. 2B). Under ET-1-stimulated (10–10–10–7 mol/liter ET-1) conditions, CNP (10–7 mol/liter) also attenuated the protein synthesis, but the inhibitory effect decreased as the concentration of ET-1 increased (Fig. 2C). Therefore, the inhibition by CNP was most effective in the absence or at a low concentration (10–10 mol/liter) of ET-1 and was less effective at high ET-1 concentrations (10–8–10–7 mol/liter) (Fig. 2D). Br-cGMP (10–3 mol/liter) also attenuated ET-1-stimulated protein synthesis, but the inhibitory effect was not influenced by the doses of ET-1 (Fig. 2, C and D). CNP (10–7 mol/liter) inhibited the ET-1-stimulated secretion of ANP, but its inhibitory effect was limited at high concentrations of ET-1 (Fig. 2, E and F).
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SK-actin, and SERCA2. ET-1 dose-dependently increased ANP, BNP, and SK-actin mRNA expressions in cardiac myocytes (Fig. 3A). CNP (10–7 mol/liter) inhibited the expression of these genes, but its effects were somewhat attenuated in the presence of high concentrations of ET-1. The SERCA2 gene expression, which is known to decrease in response to hypertrophic stimulation in cardiac myocytes (15), was decreased by ET-1 in a dose-dependent manner. Although CNP up-regulated the SERCA2 gene expression, the stimulatory effect was suppressed at high concentrations of ET-1.
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, ET-1 significantly increased the activity of GATA with a maximal increase at 30 min. Competition of the DNA binding with unlabeled oligonucleotide indicated the specificity of the GATA binding complexes. A supershift analysis showed that the majority of the GATA binding protein in cardiac myocytes was GATA-4. CNP strongly suppressed the basal activity and partially suppressed the ET-1-stimulated activity of GATA-4 (Fig. 3C). ET-1 also increased the activity of MEF-2 with a maximal increase at 15 min (Fig. 3D). Competition with cold oligonucleotide and supershift analysis indicated the specificity of the MEF-2 binding complexes. CNP strongly suppressed the basal MEF-2 activity and partially suppressed the ET-1-stimulated activity (Fig. 3E). Br-cGMP mimicked these inhibitory effects of CNP on ET-1-induced GATA-4 and MEF-2 activation (Fig. 3, C and E).
Effect of CNP on [Ca2+]i transients and CaMKII activity in cardiac myocytes
Previous studies have demonstrated that an increase in [Ca2+]i transients and the activation of CaMKII play a critical role in ET-1-induced cardiac hypertrophy (17, 18). Consistent with these reports, the intracellular Ca2+ chelator BAPTA, calmodulin inhibitor W7, CaMKII inhibitor KN93, and L-type Ca2+ channel antagonists nifedipine and diltiazem inhibited ET-1-induced incorporation of [3H]leucine (Table 1). CNP significantly suppressed the L-type Ca2+ channel agonist BAY K 8644-induced [3H]leucine incorporation, and Br-cGMP reproduced this suppressive effect, suggesting that CNP inhibits myocyte growth triggered by Ca2+ entry via L-type Ca2+ channels through a cGMP-dependent mechanism (Table 2). Pretreatment with CNP or Br-cGMP significantly diminished the amplitude of the [Ca2+]i transient induced by ET-1 (Fig. 4A). ET-1 also activated CaMKII maximally 1 min after exposure (Fig. 4B). CNP strongly suppressed the basal CaMKII activity and partially suppressed the activity stimulated by 10–7 mol/liter ET-1 (Fig. 4C). Br-cGMP mimicked the inhibition by CNP of ET-1-induced CaMKII activation.
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, ET-1 phosphorylated ERK maximally within 5 min of exposure. The inhibition by CNP of ERK phosphorylation was most effective in the absence of ET-1, and was less effective at a high concentration (10–7 mol/liter) of ET-1 (Fig. 4E). This inhibitory effect of CNP on the ET-1-induced phosphorylation of ERK was also mimicked by Br-cGMP.
Effect of ET-1 on CNP-induced cGMP production in cardiac myocytes
To investigate the mechanism by which the antihypertrophic effect of CNP was diminished in the presence of high doses of ET-1, we examined the effect of ET-1 on the CNP-induced elevation of cGMP in cultured cardiac myocytes. Pretreatment with ET-1 (10–7 mol/liter) significantly reduced the CNP-induced cGMP elevation (Fig. 5A), and the inhibitory effect was concentration dependent (Fig. 5B). The involvement of protein kinase C (PKC) activation and increase in Ca2+ influx, which are two major downstream consequences of ET-1 receptor activation, in the ET-1-mediated inhibition of cGMP production was examined. Pretreatment with a direct PKC activator, PMA, or the Ca2+ ionophore ionomycin significantly decreased CNP-induced cGMP elevation, and the effects were dose dependent (Fig. 5, C and D). The inhibitory effect of PMA was stronger than that of ionomycin. Inhibition of PKC by GFX or removal of free Ca2+ by BAPTA restored the PMA- or ionomycin-suppressed cGMP elevation. BQ123, an ET-A receptor antagonist, almost completely blocked the ET-1-suppressed cGMP elevation (Fig. 5E). Inhibition of PKC with H7 or GFX significantly restored the ET-1-suppressed cGMP elevation. Inhibition of the increase in free Ca2+ by BAPTA and inhibition of CaMKII by KN93 also restored the ET-1-suppressed cGMP elevation, although these effects were partial. Treatment with GFX plus BAPTA completely restored the ET-1-suppresed cGMP elevation.
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), and this inhibition was mimicked by Br-cGMP (Fig. 6B). Nonmyocyte-conditioned medium markedly increased the incorporation of [3H]leucine into cardiac myocytes, and this increase was significantly blocked by BQ123 but not by BQ788, an ET-B receptor antagonist (Fig. 6C). This hypertrophic response was also attenuated by the pretreatment of nonmyocytes with CNP, ANP, or Br-cGMP (Fig. 6D). However, the inhibition of nonmyocyte-conditioned medium-stimulated protein synthesis by incubation of myocytes with CNP was less than that by pretreatment of nonmyocytes with CNP.
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Discussion |
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GATA-4 and MEF-2, which are prominent Ca2+-sensitive pathways, are involved in reactivation of the fetal gene program in response to hypertrophic stimulation (16). In the present study, CNP inhibited ET-1-induced GATA-4 and MEF-2 DNA binding activities, and the inhibitory effects of CNP were mimicked by Br-cGMP. Therefore, the inhibitory effect of CNP on the ET-1-induced increase in GATA-4 and MEF-2 DNA binding activities might be cGMP dependent. The ERK cascade has been shown to not only be involved in the cardiac hypertrophic response (24) but also play an essential role in ET-1-induced cardiac myocyte hypertrophy (19). A recent study also found that ERK activation is coupled with GATA-4 activation (25) and that CaMK stimulates MEF-2 (26). Furthermore, the present study demonstrated that CNP inhibited ET-1-induced ERK phosphorylation. These findings suggest that the inhibitory effect of CNP on ET-1-induced GATA-4 activation may be mediated at least partially via the inhibition of ERK phosphorylation. However, another report questioned the interaction between ERK and GATA-4 (27). Additional study is needed on the direct or indirect inhibitory effect of CNP on GATA-4 and MEF-2 activation.
In the present study, CNP strongly inhibited low-dose ET-1-induced hypertrophic responses, but the effect was weakened at high concentrations of ET-1, whereas the Br-cGMP-mediated antihypertrophic effect was not influenced by the doses of ET-1. Because ET-1 dose-dependently suppressed CNP-induced cGMP production, it is suggested that high doses of ET-1 overcame the antihypertrophic effect of CNP via inhibition of cGMP production-dependent action. The inhibition by ET-1 of CNP-induced cGMP production was mimicked by PMA and ionomycin. Moreover, this effect was blocked by pretreatment with ET-A receptor antagonist, PKC inhibitors, and CaMKII inhibitor. These results suggest that ET-1 inhibits CNP-induced cGMP production via ET-A receptor-mediated PKC activation, Ca2+ influx, and CaMKII activation. Although the precise mechanism of the inhibitory effect of ET-1 on CNP-induced cGMP production is unclear, one possibility is desensitization of the GC-B receptor by these signaling pathways. A previous report demonstrated that some types of growth factors desensitize the GC-B receptor via dephosphorylation of GC-B in other cell types (11).
It has been shown that cardiac nonmyocytes regulate myocyte hypertrophy at least partially via ET-1 secretion and that the interaction between myocytes and nonmyocytes plays an important role in the process of cardiac myocyte hypertrophy (8). We previously reported that CNP inhibits thrombin- and angiotensin II-stimulated ET-1 release from vascular endothelial cells (28). Therefore, we hypothesized that CNP inhibits cardiac myocyte hypertrophy via inhibition of ET-1 secretion from cardiac nonmyocytes. In the present study, CNP inhibited ET-1 secretion from cardiac nonmyocytes, and Br-cGMP mimicked this effect. Nonmyocyte-conditioned medium clearly stimulated the protein synthesis in cardiac myocytes, and an ET-A receptor antagonist partially but significantly inhibited this effect. In addition, pretreatment of nonmyocytes with CNP significantly inhibited the nonmyocyte-conditioned medium-induced protein synthesis in myocytes. These results suggest that CNP inhibits cardiac myocyte hypertrophy not only directly but indirectly via inhibition of ET-1 secretion from cardiac nonmyocytes.
It is known that the GC-A receptor interacts with both ANP and BNP, whereas the GC-B receptor is selectively stimulated by CNP (4). In the present study, the expression of GC-B receptor mRNA was significantly more intense than that of GC-A in neonatal cardiac myocytes. The dominance of CNP in cGMP accumulation and antihypertrophic effect compared with ANP in the present study might reflect these findings. The expression of GC-B and its potential in CNP-induced cGMP production in cardiac myocytes has been shown (3, 21, 29, 30). However, Lin et al. (3) demonstrated that the GC-A receptor was predominantly expressed in adult rat ventricular myocytes using an RT-PCR technique. Although the exact reason for these discrepant findings is unclear, other than the methods used to detect the gene expression of GC-A and GC-B, differences in characteristics between neonatal cells and adult cells and other experimental conditions might be involved. In addition, previous studies and our present study used normal animals (i.e. intact hearts), but in the hypertrophied left ventricle, a marked increase in the GC-B receptor mRNA expression has been reported (31). Additional investigation is necessary to clarify the pathophysiological role of the GC-B receptor in response to CNP stimulation, particularly during the process of hypertrophy and in hypertrophied hearts in vivo.
Concerning CNP-mediated antihypertrophic and antiproliferative effects, an important issue exists to be interpreted. That is, it is disputable whether CNP specifically inhibits ET-1-dependent hypertrophic and proliferative actions or whether it inhibits all growth-promoting effects regardless of the hormone that initiates the response. Previous studies revealed that CNP also inhibited angiotensin II-, oxidized low-density lipoprotein-, fetal bovine serum-, and platelet-derived growth factor-mediated cell growth (21, 32, 33, 34). In addition, CNP inhibited various activities under basal (nonstimulated) conditions, as shown in the present study and our recent study (7). These accumulative findings suggest that CNP may be a general antagonist of cell-growth responses.
The interactions between ET-1 and CNP-GC-B receptor pathways are summarized in Fig. 7. In summary, CNP directly inhibited the ET-1-induced hypertrophic response of cardiac myocytes via a cGMP-dependent mechanism and also inhibited the paracrine hypertrophic effect of cardiac nonmyocytes via the inhibition of ET-1 secretion from nonmyocytes. ET-1 attenuated the direct antihypertrophic effect of CNP via inhibition of cGMP production.
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Footnotes |
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Abbreviations: ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; Br-cGMP, 8-bromo-cGMP; CaMKII, Ca2+/calmodulin-dependent kinase II; CNP, C-type natriuretic peptide; ET-1, endothelin-1; FCS, fetal calf serum; GC, guanylyl cyclase; GFX, GF 109203 X; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SERCA2, sarcoplasmic reticulum Ca2+-ATPase; SK-actin, -skeletal muscle isoform of actin.
Received September 19, 2003.
Accepted for publication January 14, 2004.
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References |
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. Endocrinology 139:4576–45801-adrenergic stimulation on L-type Ca2+ current in rat ventricular myocytes. J Mol Cell Cardiol 30:1955–1965[CrossRef][Medline]
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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) and ANP (
) on the production of cellular cGMP in cardiac myocytes. Values are the mean ± SD of four measurements. *, P < 0.05 vs. control; 
, P < 0.05 vs. the same dose of ANP.
) on protein synthesis under ET-1-stimulated conditions. The mean radioactivity in the control wells was 2597 ± 54 cpm/well. *, P < 0.05 vs. control; 
