©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Endothelin-1 Is Involved in Mechanical Stress-induced Cardiomyocyte Hypertrophy (*)

(Received for publication, August 24, 1995; and in revised form, December 1, 1995)

Tsutomu Yamazaki (1) (2) Issei Komuro (1)(§) Sumiyo Kudoh (1) Yunzeng Zou (1) Ichiro Shiojima (1) Yukio Hiroi (1) Takehiko Mizuno (1) Koji Maemura (1) Hiroki Kurihara (1) Ryuichi Aikawa (1) Hiroyuki Takano (1) Yoshio Yazaki (1)

From the  (1)Third Department of Internal Medicine, University of Tokyo School of Medicine and the (2)Health Service Center, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have recently shown that mechanical stress induces cardiomyocyte hypertrophy partly through the enhanced secretion of angiotensin II (ATII). Endothelin-1 (ET-1) has been reported to be a potent growth factor for a variety of cells, including cardiomyocytes. In this study, we examined the role of ET-1 in mechanical stress-induced cardiac hypertrophy by using cultured cardiomyocytes of neonatal rats. ET-1 (1010M) maximally induced the activation of both Raf-1 kinase and mitogen-activated protein (MAP) kinases at 4 and 8 min, respectively, followed by an increase in protein synthesis at 24 h. All of these hypertrophic responses were completely blocked by pretreatment with BQ123, an antagonist selective for the ET-1 type A receptor subtype, but not by BQ788, an ET-1 type B receptor-specific antagonist. BQ123 also suppressed stretch-induced activation of MAP kinases and an increase in phenylalanine uptake by approximately 60 and 50%, respectively, but BQ788 did not. ET-1 was constitutively secreted from cultured cardiomyocytes, and a significant increase in ET-1 concentration was observed in the culture medium of cardiomyocytes after stretching for 10 min. After 24 h, an 3-fold increase in ET-1 concentration was observed in the conditioned medium of stretched cardiomyocytes compared with that of unstretched cardiomyocytes. ET-1 mRNA levels were also increased at 30 min after stretching. Moreover, ET-1 and ATII synergistically activated Raf-1 kinase and MAP kinases in cultured cardiomyocytes. In conclusion, mechanical stretching stimulates secretion and production of ET-1 in cultured cardiomyocytes, and vasoconstrictive peptides such as ATII and ET-1 may play an important role in mechanical stress-induced cardiac hypertrophy.


INTRODUCTION

Cardiac hypertrophy, a major underlying cause of heart diseases such as myocardial infarction and cardiac arrhythmias(1) , is formed when increased external stimuli such as hemodynamic overload and neurohumoral factors are continuously imposed on cardiac myocytes(2, 3) . These external stimuli are generally transduced into the nucleus through protein kinase cascades of phosphorylation(4) , and Raf-1 kinase (Raf-1) (^1)(5) and mitogen-activated protein (MAP) kinases (6, 7) are important components in these cascades. We have recently reported that stretching of cardiomyocytes sequentially activates Raf-1 and MAP kinases, followed by an increase in protein synthesis(8) . Interestingly, all of these events were partially suppressed by a specific antagonist of the angiotensin II (ATII) type 1 receptor, CV11974(9) . These results suggest that mechanical stress exemplified by stretching might stimulate the secretion of ATII from cardiomyocytes and that ATII participates in the activation of the protein kinase cascades and the production of cardiomyocyte hypertrophy through the ATII type 1 receptor. However, because the inhibition of these hypertrophic events by CV11974 is incomplete, factors other than ATII should also be involved in cardiomyocyte hypertrophy induced by mechanical stress.

Endothelin-1 (ET-1) is a vasoactive peptide that contains 21 amino acids with two intramolecular disulfide bond and was initially identified and purified from porcine aortic endothelial cell cultures (10) . This peptide is produced by endothelial and epithelial cells, macrophages, fibroblasts, and many other types of cells, including cardiac myocytes (for a review, see (11) ), and is not only a potent vasoconstrictor, but also a potent growth factor for a variety of cells, including cardiac myocytes(12, 13, 14, 15) . It has been reported that ET-1 increases the protein synthesis and the surface area of cardiomyocytes without cell proliferation. Like many other vasoactive peptides, ET-1 increases phosphoinositide turnover and elevates diacylglycerol levels in rat cardiomyocytes(15) . Two distinct ET-1 receptor subtypes (ET(A) and ET(B)), which have seven transmembrane-spanning regions and belong to the superfamily of G protein-coupled receptors, have been cloned from cDNA libraries of various cell types(16, 17, 18, 19, 20, 21) , and both receptor subtypes have been shown to be expressed in the heart(16, 17, 18, 21) .

In this study, we first elucidated that ET-1 induces the activation of hypertrophic signals such as Raf-1 and MAP kinases through the ET(A) receptor, followed by an increase in protein synthesis in neonatal rat cardiomyocytes. These responses were quite similar to those observed when mechanical stress was imposed on cardiac myocytes (8) . Then, we examined the involvement of ET-1 in mechanical stretch-induced hypertrophic responses by using ET-1 receptor-specific antagonists. The ET(A) receptor-specific antagonist, BQ123, significantly inhibited stretch-induced activation of Raf-1 and MAP kinases and uptake of phenylalanine into cells. We have further demonstrated that ET-1 is constitutively secreted from the cultured cardiomyocytes of neonatal rats and that mechanical stretching enhances the ET-1 release from the cells. Moreover, mRNA levels of ET-1 were also increased by stretching of cardiomyocytes. These results suggest that not only ATII, but also ET-1 plays an important role in mechanical stress-induced cardiac hypertrophy.


EXPERIMENTAL PROCEDURES

Materials

[-P]ATP, [alpha-P]UTP, and [^3H]phenylalanine were purchased from DuPont NEN. Dulbecco's modified Eagle's medium was from Life Technologies, Inc. Syntide-2, a peptide substrate for Raf-1, was from Santa Cruz Biotechnology, Inc. RNazol was from Biotecx Laboratories (Houston, TX). Other reagents were purchased from Sigma.

Cell Culture of Cardiomyocytes

Primary cultures of cardiomyocytes were prepared from ventricles of 1-day-old Wistar rats as described previously (22) according to the method of Simpson and Savion (23) with minor modifications. Stretching of cardiomyocytes was conducted as described previously(22, 24) . In brief, cardiomyocytes were plated at a field density of 1 times 10^5 cells/cm^2 on 35-mm culture dishes or silicone rubber culture dishes (20 times 40 mm) with 2 ml of culture medium. The culture medium was changed 24 h after seeding to a solution consisting of Dulbecco's modified Eagle's medium. Cardiomyocytes were stretched by 20% and lysed on ice with buffer A (25 mM Tris-HCl, 25 mM NaCl, 1 mM sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 10 nM okadaic acid, 0.5 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride).

MAP Kinase Assay in Myelin Basic Protein (MBP)-containing Gels

MAP kinase activities were measured using MBP-containing gels as described previously(25) . In brief, MAP kinases were immunoprecipitated with polyclonal antibodies against MAP kinases, alphaY91(26) , in the presence of 0.15% SDS, and the immunoprecipitates were electrophoresed on an SDS-polyacrylamide gel containing 0.5 mg/ml MBP. MAP kinases in the gel were denatured in 6 M guanidine HCl and renatured in 50 mM Tris-HCl (pH 8.0) containing 0.04% Tween 40 and 5 mM 2-mercaptoethanol. Phosphorylation of MBP was assayed by incubating the gel with [-P]ATP. After incubation, the gel was washed extensively, dried, and then subjected to autoradiography.

Western Blot Analysis of MAP Kinases

The immunoprecipitates of cardiomyocyte lysates with alphaY91 were separated by SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were electrotransferred onto Immobilon-P membrane and incubated with alphaY91. After washing, the membrane was incubated for 1 h with alkaline phosphatase-coupled second antibody and developed using the ProtoBlot immunoblotting system according to the manufacturer's instructions (Sigma).

Assay of MAP Kinase Kinase Kinase Activity of Raf-1

The activities of Raf-1 were assayed by measuring the phosphorylation of syntide-2, a peptide substrate for Raf-1. Total cell lysates or immunoprecipitates with Raf-1-specific antibody (27) were incubated with the substrate (10 µg of syntide-2) and 2 µCi of [-P]ATP in buffer B (25 mM Tris-HCl (pH 7.4), 10 mM MgCl(2), 1 mM dithiothreitol, 40 µM ATP, 2 µM protein kinase inhibitor peptide, and 0.5 mM EGTA) for 30 min at 25 °C. After incubation, syntide-2 was collected using P-81 paper, and the paper was washed five times for 10 min each in 0.5% phosphoric acid, washed in acetone, dried, and measured by Cerenkov counting.

Amino Acid Uptake into Cardiomyocytes

After culture in serum-free Dulbecco's modified Eagle's medium for 48 h, cardiomyocytes were stretched for 24 h. One µCi/ml [^3H]phenylalanine was added to the culture medium 2 h before harvest. The cells were rapidly rinsed four times with ice-cold phosphate-buffered saline (10 mM sodium phosphate and 0.85% NaCl (pH 7.4)) and incubated for >20 min on ice with 1 ml of 20% trichloroacetic acid. The total radioactivity in each dish was determined by liquid scintillation counting.

Measurement of ET-1 Concentration in Media

Preparation of a neutralizing monoclonal antibody against ET-1 (HPE37B11) (IgG(1) subtype) was carried out as described previously (28) . The levels of ET-1 concentration in media were determined by specific enzyme immunoassay for ET-1 as previously reported(29) .

Northern Blot Analysis

The cDNA for rat ET-1 was cloned by polymerase chain reaction using oligonucleotide primers derived from the rat ET-1 cDNA sequence(30) . The antisense RNA probe for ET-1 was prepared from the linearized plasmid containing the cloned 1.0-kilobase fragment of ET-1 cDNA by transcribing with RNA polymerase in the presence of [alpha-P]UTP. Total cellular RNA was extracted from cardiomyocytes using RNazol. RNA samples were electrophoresed through 1.2% agarose-formaldehyde gels and transferred to Hybond-N nylon membrane. The membranes were hybridized for 24 h at 65 °C in 6 times SSC, 50% formamide, 0.5% SDS, 2.5 times Denhardt's solution, 0.25 mg/ml salmon sperm DNA, and P-labeled probes. Membranes were washed twice in 2 times SSC and 0.1% SDS at 65 °C for 15 min and twice in 0.1 times SSC and 0.1% SDS at 65 °C for 15 min and exposed to Kodak X-Omat AR film at -80 °C for 24 h.


RESULTS

ET-1 Activates MAP Kinases

Many lines of evidence have suggested that MAP kinases are key molecules in intracellular signal transduction pathways and play essential roles in cellular proliferation and differentiation(31, 32) . As described in previous reports, MAP kinases are activated by mechanical stress in cardiac myocytes(8, 9, 25) , and MAP kinase activity is a sensitive and quantitative marker for hypertrophic responses to external stimuli of cardiac myocytes(8, 9) . To elucidate the role of ET-1 in mechanical stress-induced hypertrophy, we first examined whether ET-1 evokes the activation of MAP kinases in cardiac myocytes. Cardiac myocytes were incubated with various concentrations (10 to 10M) of ET-1 for 8 min. No significant increase in MAP kinase activities was observed at 10M ET-1. A slight increase in MAP kinase activities was observed at 10M, and the maximal activation of MAP kinases was obtained at 1010M ET-1 (Fig. 1, A and B). Western blot analysis showed that almost the same amount of MAP kinase protein was present in each sample (Fig. 1A, upper panel), which suggests that the increase in phosphorylation activities is due to the activation of MAP kinases. Fig. 2(A-C) shows the time course of ET-1-induced MAP kinase activation. The increase in MAP kinase activities induced by 10M ET-1 was detectable from as early as 2 min, and the activities reached a peak at 8 min. The activities decreased sharply and returned to control levels at 30 min after stimulation with ET-1.


Figure 1: Dose dependence of ET-1-induced MAP kinase activation. A, neonatal rat cardiomyocytes were stimulated with various doses of ET-1 for 8 min. Cardiomyocyte lysates were immunoprecipitated with an antibody against MAP kinases, alphaY91, and the immunoprecipitates were subjected to Western blot analysis (upper panel) or were electrophoresed on an SDS-polyacrylamide gel containing MBP. After denaturation and renaturation of MAP kinases, phosphorylation of MBP was assayed by incubating the gel with [-P]ATP. After incubation, the gel was dried and subjected to autoradiography (lower panel). B, the data presented are an average from two independent experiments. The activity of 44-kDa MAP kinase in control lysates is designated as 1.0.




Figure 2: Time course of ET-1-induced MAP kinase activation. A and B, cardiomyocytes were exposed to 10M ET-1 for the indicated periods of time. MAP kinase activities were measured as described in the Fig. 1legend. C, the data are presented as -fold increases as compared with the activity of 44-kDa MAP kinase without stimulation (1.0, 0 min) from four independent experiments (mean ± S.E.). Statistical analysis was performed using the paired sample t test with p values corrected by the Bonferroni method (see (46) ). *, p < 0.05 versus control.



ET-1 Induces Raf-1 Activation

Raf-1 activates MAP kinases through phosphorylating MAP kinase kinase ((5) ; for a review, see (33) ). Many laboratories have reported that ET-1 activates protein kinase C (for a review, see (11) ), and protein kinase C has been shown to activate Raf-1 in fibroblasts(34) . Therefore, we analyzed the Raf-1 activities in cardiomyocytes after the addition of ET-1. From as early as 1 min, ET-1 (10M) dramatically increased the phosphorylating activity of syntide-2, which is a good substrate for Raf-1. Maximal activation was observed at 4 min, and the activities decreased thereafter and returned to control levels 30 min after stimulation (Fig. 3). To verify whether the phosphorylation of this peptide was done by Raf-1, immunoprecipitates with Raf-1-specific antibody (27) were subjected to the syntide-2 phosphorylation assay after 1-30 min of stimulation with 10M ET-1. The time course and -fold activation of immunoprecipitates were almost the same as those of lysates without immunoprecipitation. Maximal phosphorylation (2.6-fold) was also observed at 4 min after stimulation, and the activities returned to basal levels at 30 min after stimulation. These results suggest that the peptide is mainly phosphorylated by Raf-1 and that ET-1 increases the Raf-1 activity in cardiac myocytes.


Figure 3: Activation of Raf-1 by ET-1 stimulation. Cardiac myocytes were stimulated with 10M ET-1 for the indicated periods of time. Aliquots of the cardiomyocyte lysates were incubated with substrate (syntide-2) and [-P]ATP. After terminating the reaction, the reaction mixture was spotted on P-81 paper. The incorporation of P into syntide-2 was assessed by Cerenkov counting. The data are presented as means ± S.E. from four independent experiments compared with controls (100%). *, p < 0.05 versus control.



Both Neutralizing Antibody against ET-1 and Specific Antagonist of ET(A) Receptor Block ET-1-induced MAP Kinase Activation

To examine whether the MAP kinase activation by ET-1 is specific, cardiac myocytes were stimulated with ET-1 after pretreatment with various concentrations of a neutralizing antibody against ET-1 (HPE37B11)(28) . One-hundred mg/ml HPE37B11 almost completely blocked 10M ET-1-induced MAP kinase activation, as shown in Fig. 4. Excess ET-1 (10M)-induced MAP kinase activation was not completely blocked by preincubation of ET-1 with 100 mg/ml HPE37B11, and 10M ET-1-induced MAP kinase activation was not blocked by control mouse IgG, suggesting that ET-1 specifically activates MAP kinases in cardiac myocytes. The two main subtypes of ET-1 receptors (ET(A) and ET(B)) have been identified pharmacologically and have recently been molecularly cloned(16, 17, 18, 19, 20, 21) . To examine which receptor subtype mediates MAP kinase activation by ET-1 in cardiomyocytes, we preincubated cardiac myocytes with BQ123 (an antagonist of the ET(A) receptor) or BQ788 (an antagonist of the ET(B) receptor) and exposed cardiomyocytes to 10M ET-1 for 8 min. It has been reported that 10M BQ123 specifically blocks the ET(A) receptor, but not the ET(B) receptor, and that 10M BQ788 works as a specific inhibitor of the ET(B) receptor(35, 36) . BQ123 (10M) almost completely suppressed the activation of MAP kinases induced by ET-1 (Fig. 5, A and B, BQ123+ET-1), whereas BQ788 (10M) had no inhibitory effects on ET-1-induced MAP kinase activation (Fig. 5, A and B, BQ788+ET-1). These results suggest that the induction of MAP kinase activation by ET-1 is mediated through the ET(A) receptor.


Figure 4: Effect of ET-1-neutralizing antibody on ET-1-induced MAP kinase activation. ET-1 was added to cardiac myocytes after pretreatment with a neutralizing antibody against ET-1 (HPE37B11) at the indicated concentrations or with control mouse IgG (100 µg/ml) for 30 min. MAP kinase assays were performed as described in the Fig. 1legend. A representative autoradiogram from three independent experiments is shown.




Figure 5: Effect of ET(A) or ET(B) receptor antagonist on ET-1- or stretch-induced MAP kinase activation. A, after pretreatment with BQ123 (10M) or BQ788 (10M) for 30 min, cardiomyocytes were stimulated with 10M ET-1 for 8 min. MAP kinase assays were performed as described in the Fig. 1legend. B, the intensities of the 42- and 44-kDa bands were measured by densitometric scanning of the autoradiogram. Values represent the average from two independent experiments. The intensity of 44-kDa MAP kinase in unstimulated myocytes is designated as 1.0. C, after pretreatment with BQ123 (10M) and CV11974 (10M) for 30 min, cardiomyocytes were stretched by 20% for 8 min, and MAP kinase activities were measured as described in the Fig. 1legend. A representative autoradiogram from three independent experiments is shown. D, the intensities of the 42- and 44-kDa bands in the autoradiogram (C) were measured by densitometric scanning. The data represent the average percentage of the controls (100%) from three independent experiments (mean ± S.E.). *, p < 0.05 versus control.



ET(A) Receptor Antagonist Suppresses Stretch-induced MAP Kinase Activation

Our (9, 38) and other (37) laboratories have recently reported that mechanical stretching induces the secretion of ATII from cardiac myocytes and that the secreted ATII induces c-fos gene expression and MAP kinase activation. We have examined whether ET-1, as well as ATII, is involved in stretch-induced activation of MAP kinases. After pretreatment with BQ123 (10M) for 30 min, cardiomyocytes were stretched by 20% for 8 min. Stretching of cardiomyocytes increased the activities of both 42- and 44-kDa MAP kinases by approximately 6- and 5-fold, respectively, and the BQ123 pretreatment reduced stretch-induced MAP kinase activation by 60% (Fig. 5. C and D, BQ123+Stretch). Consistent with the previous report(9) , an ATII type 1 receptor-specific antagonist, CV11974, showed similar inhibitory effects on stretch-induced MAP kinase activation (Fig. 5. C and D, CV11974+Stretch). In addition, pretreatment with both BQ123 and CV11974 at the same time more potently but incompletely blocked the stretch-induced MAP kinase activation (Fig. 5. C and D, BQ123+CV11974+Stretch). These results suggest that MAP kinase activation induced by mechanical stress is partially dependent on ET-1 through the ET(A) receptor as well as on ATII through the ATII type 1 receptor.

ET(A) Receptor Antagonist Blocks Stretch-induced Increase in Phenylalanine Uptake into Cardiac Myocytes

We have previously shown that stretching of cardiac myocytes increases amino acid incorporation into proteins, suggesting that mechanical stress directly induces cardiomyocyte hypertrophy(22, 24) . We examined whether ET-1 induces cardiac hypertrophy by measuring the relative amount of protein synthesis with the use of [^3H]phenylalanine. Stimulation of cultured cardiac myocytes with ET-1 (10M) for 24 h caused a significant increase (204% of unstimulated control) in phenylalanine incorporation, and this increase was completely abolished when cardiomyocytes were pretreated with 10M BQ123 (105% of control). In contrast, BQ788 had no inhibitory effects on the increase in amino acid incorporation by ET-1 (10M), suggesting that ET-1 induces cardiomyocyte hypertrophy through the ET(A) receptor. Stretching by 20% stimulated an increase in amino acid incorporation into proteins by 1.51-fold, as reported previously(22, 24) . This increase was partially suppressed by pretreatment with 10M BQ123 (1.24-fold of control), whereas pretreatment with 10M BQ788 showed no effects on the stretch-induced increase in phenylalanine uptake. These results suggest that part of stretch-induced cardiomyocyte hypertrophy is also dependent on ET-1 through the ET(A) receptor.

Stretching of Cardiomyocytes Directly Stimulates Secretion of ET-1

The above results suggest that stretching stimulates the secretion of ET-1 from cardiac myocytes. To prove this hypothesis, the culture media of cardiomyocytes were collected from the dishes after stretching by 20% for 1 min to 24 h, and the levels of ET-1 concentration were determined by enzyme immunoassay using a specific antibody against ET-1(29) . The levels of ET-1 in the culture media of unstretched cardiomyocytes were under detectable levels (i.e. <1.0 pM) from 1 to 120 min, but after 24 h, they reached 1.48 ± 0.21 pM, suggesting that cultured cardiac myocytes are constitutively secreting a small amount of ET-1. On the other hand, stretching of cardiomyocytes for 10 min significantly increased the ET-1 levels (3.21 ± 0.31 pM). The concentration levels were gradually elevated, and the maximal level was obtained when cardiomyocytes were stretched for 24 h (4.83 ± 0.25 pM) (Fig. 6A).


Figure 6: Release of ET-1 in culture medium and ET-1 gene expression in stretched myocytes. A, after changing the medium, cardiomyocytes were stimulated with stretching (20%) for the indicated periods of time. The amount of ET-1 was determined by enzyme immunoassay as previously reported(29) . The results are indicated as means ± S.E. for six independent experiments. Asterisks indicate undetectable levels (<1 pM). B, cardiomyocytes were stretched by 20% for the indicated periods of time. Northern blot hybridization was performed using P-labeled ET-1 riboprobe. Similar results were obtained from three independent experiments, and a representative autoradiogram is shown.



Mechanical Stretching Increases ET-1 Gene Expression

To ascertain whether enhanced ET-1 release by stretching cardiomyocytes is accompanied by changes in gene expression, we examined ET-1 mRNA levels after stretching. Northern blot analysis revealed slight basal expression of the ET-1 gene in unstretched cardiomyocytes. The expression of the ET-1 gene was rapidly and transiently increased following mechanical stretching by 20%. The maximal increase in ET-1 mRNA levels was observed at 30 min after stretching, and the levels decreased thereafter (Fig. 6B). These results suggest that not only the secretion, but also the production of ET-1 is stimulated by mechanical stress in cardiac myocytes.

ET-1 and ATII Synergistically Activate MAP Kinases and Raf-1 in Cardiac Myocytes

It has been reported that the secretion of ATII is also induced by mechanical stress(9, 37) . Another report has shown that ATII induces the release of ET-1 from cultured cardiomyocytes of neonatal rats and that the secreted ET-1 evokes cardiomyocyte hypertrophy(39) . Therefore, we investigated whether ATII activates MAP kinases via ET-1 secretion. After pretreatment with 10M BQ123 for 30 min, cardiomyocytes were stimulated with 10M ATII for 8 min, and MAP kinase activities were analyzed. As shown in Fig. 7A, ATII-induced MAP kinase activation was not inhibited by the ET(A) receptor antagonist, suggesting that ATII activates MAP kinases independently of the secreted ET-1. Moreover, the ATII receptor antagonist, CV11974, did not suppress ET-1-induced MAP kinase activation (Fig. 7B). This result suggests that the activation of MAP kinases by ET-1 is also independent of ATII.


Figure 7: ATII and ET-1 independently induce MAP kinase activation, and both agents synergistically activate MAP kinases and Raf-1. A and B, after pretreatment with 10M BQ123 (A) or 10M CV11974 (B) for 30 min, cardiac myocytes were stimulated with 10M ATII (AngII; A) or 10M ET-1 (B) for 8 min. Analysis of MAP kinase activities was performed as described in the Fig. 1legend. A representative autoradiogram from two independent experiments is shown. C, cardiomyocytes were stimulated with ET-1 (10M) and/or with ATII (10M) for 8 min, and MAP kinase activities were determined. A representative autoradiogram from two independent experiments is shown in the upper panel, and the densitometric scanning data of the 42- and 44-kDa bands are shown in the lower panel. Values represent the means from two independent experiments. The intensity of 44-kDa MAP kinase in unstimulated cardiomyocytes is designated as 1.0. D, after stimulation with ET-1 (10M) and/or with ATII (10M) for 4 min, Raf-1 activities were determined in cardiomyocytes. The data represent the average percentage of the controls (100%) from four independent experiments (mean ± S.E.) *, p < 0.05 versus control.



We next investigated the mutual effects of ET-1 and ATII. Although 10M ET-1 did not significantly activate MAP kinases by itself, the presence of 10M ET-1 dramatically augmented ATII (10M)-induced MAP kinase activation (Fig. 7C). With regard to Raf-1, similar synergistic effects were observed. The addition of either 10M ET-1 or 10M ATII slightly activated Raf-1; however, the simultaneous addition of ET-1 and ATII markedly activated Raf-1 (Fig. 7D).


DISCUSSION

We (9, 38) and others (37) have reported that endogenous ATII plays an important role in stretch-induced cardiomyocyte hypertrophy. However, incomplete inhibitions of stretch-induced hypertrophic responses by ATII receptor antagonists suggest the involvement of other factors in producing hypertrophy(9, 38) . ET-1 has been reported to be not only a vasoconstrictor, but also a potent hypertrophy-promoting factor. ET-1 produces cardiomyocyte hypertrophy in vitro as well as in vivo(13, 14, 15, 40, 41) . Therefore, to elucidate the involvement of ET-1 in stretch-induced cardiac hypertrophy, we first investigated whether ET-1 activates the protein kinase cascade of phosphorylation and induces cardiac hypertrophy. ET-1 activated MAP kinases and Raf-1 in a dose-dependent manner through the ET(A) receptor, followed by an increase in protein synthesis. We next examined the involvement of ET-1 in stretch-induced cardiac hypertrophy by using ET-1 receptor antagonists. The ET(A) receptor-specific antagonist, BQ123, significantly inhibited stretch-induced Raf-1 and MAP kinase activation, suggesting that ET-1 mediates stretch-induced cardiomyocyte hypertrophy through the ET(A) receptor. ET-1 was constitutively secreted from cardiomyocytes, and the secretion was enhanced by mechanical stress. In addition, Northern blot analysis revealed that mRNA levels of ET-1 were also increased by stretching. Finally, we have demonstrated that ET-1 and ATII synergistically activate Raf-1 and MAP kinases.

Recently, Ito et al.(41) have reported that continuous administration of BQ123 in rats with aortic banding blocks both cardiac hypertrophy and the increase in skeletal alpha-actin and atrial natriuretic peptide gene expression. These results suggest that ET-1 may play an important role in producing cardiac hypertrophy during pressure overload in vivo. It remains uncertain, however, how ET-1 is involved in mechanical stress-induced cardiac hypertrophy. We have demonstrated for the first time by using deformable silicone dishes that ET-1 is constitutively released from cardiomyocytes and that the release is enhanced by mechanical stress. Recently, Ito et al.(39) have shown that ATII not only up-regulates prepro-ET-1 mRNA levels at 30 min, but also stimulates ET-1 release from neonatal rat cardiomyocytes at 60 min. They hypothesized that ATII activates the transcription of the ET-1 gene, resulting in the increased secretion of the ET-1 protein. In the present study, we have shown that ET-1 is constitutively secreted from cardiomyocytes and that mechanical stretching enhances the secretion from as early as 10 min after stretching (Fig. 6A). We have also observed the increase in ET-1 mRNA levels at 30 min after stretching (Fig. 6B). Collectively, these results suggest that ET-1 may be stored in cardiomyocytes like ATII (37) and that mechanical stretching directly induces the secretion of ET-1 as well as increases the production of ET-1. It has been thought that ET-1 is synthesized and released from the cell surface by exocytosis without prior concentration and storage in secretory granules(42) . Namely, ET-1 production is thought to be regulated at the level of transcription rather than at the level of protein secretion. To elucidate the intracellular localization of ET-1 in cultured cardiomyocytes, we attempted an immunohistochemical analysis using ET-1-specific antibodies, but failed to detect ET-1 in secretory granules (data not shown). Some recent reports, however, have shown that ET-1 is stored in secretory granules after segregation from the Golgi cisterns of bone cells(43) , endothelial cells of umbilical veins(44) , and aortic endothelial cells(45) . Further studies are necessary to elucidate the existence of ET-1 in secretory granules of cardiac myocytes and the mechanism of the increase in ET-1 secretion during stretch-induced cardiomyocyte hypertrophy.

In this study, we have shown that mechanical stretching increases the secretion of ET-1 from cardiomyocytes and that stretch-induced activation of Raf-1 and MAP kinases is in part dependent on ET-1 through the ET(A) receptor. It has been reported that unlike ATII, the secretion of ET-1 is not enhanced by stretching(37) . Although we do not know the reason for the discrepancy, our very sensitive assay for ET-1, as well as the experiment using the specific antagonist, strongly suggests that ET-1 is increased in the medium after stretching. We have not determined exactly the local concentrations of ET-1 around the cultured cardiac myocytes; however, the levels of ET-1 after stretching were not as high as ATII levels reported previously(37) . As shown in Fig. 7(C and D), however, even a low level of ET-1 was able to induce hypertrophic responses in the presence of ATII. We have shown that ET-1 activates Raf-1 and MAP kinases in a synergistic manner with ATII in cultured cardiac myocytes. Interaction between ET-1 and several growth factors has been shown to have synergistic stimulatory effects on proliferation of vascular smooth muscle cells or fibroblasts in culture (11) . Although it has been well established that both ET-1 and ATII activate protein kinase C in cardiac myocytes, other signal transduction pathways such as Ca-calmodulin and tyrosine kinases have been demonstrated in other cell types. The mechanisms underlying the synergistic action of ET-1 with different growth factors have yet to be established; however, ET-1 may induce the hypertrophic responses through different pathways compared with ATII.

Although the precise mechanisms by which the secretion of ET-1 and ATII is enhanced in stretched cardiomyocytes and by which ET-1 and ATII synergistically activate hypertrophic responses remain unknown, this study shows that not only ATII, but also ET-1 is involved in stretch-induced cardiac hypertrophy. In conclusion, these results suggest that vasoactive peptides play an important role in mechanical stress-induced cardiac hypertrophy, and this new finding may pave the way to develop new therapeutic strategies for cardiac hypertrophy.


FOOTNOTES

*
This work was supported by a grant-in-aid for scientific research and developmental scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, a grant from the Japan Cardiovascular Foundation and the Sankyo Life Science and Study Group of Molecular Cardiology, Japan (to I. K.), and Japan Heart Foundation Research Grant for 1994 (to T. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 3127); Fax: 81-3-3815-2087; :komuro-tky{at}umin.u-tokyo.ac.jp.

(^1)
The abbreviations used are: Raf-1, Raf-1 kinase; MAP, mitogen-activated protein; ATII, angiotensin II; ET-1, endothelin-1; ET(A), endothelin-1 type A; ET(B), endothelin-1 type B; MBP, myelin basic protein.


ACKNOWLEDGEMENTS

We thank Toru Suzuki for critical reading of the manuscript. We acknowledge Banyu Pharmaceutical Co., Ltd. (Tokyo, Japan) for providing BQ123 and BQ788, Takeda Chemical Industries, Ltd. (Osaka, Japan) for providing CV11974, and Yamasa Corp. (Tokyo, Japan) for providing HPE37B11.


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