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Circulation Research. 1995;77:258-265

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(Circulation Research. 1995;77:258-265.)
© 1995 American Heart Association, Inc.

Articles

Angiotensin II Partly Mediates Mechanical Stress–Induced Cardiac Hypertrophy

Tsutomu Yamazaki, Issei Komuro, Sumiyo Kudoh, Yunzeng Zou, Ichiro Shiojima, Takehiko Mizuno, Hiroyuki Takano, Yukio Hiroi, Kohjiro Ueki, Kazuyuki Tobe, Takashi Kadowaki, Ryozo Nagai, Yoshio Yazaki

From the Third Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo, Japan.

Correspondence to Issei Komuro, The Third Department of Medicine, University of Tokyo School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail komuro-tky@umin.u-tokyo.ac.jp.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract We have previously shown that mechanical stress induces activation of protein kinases and increases in specific gene expression and protein synthesis in cardiac myocytes, all of which are similar to those evoked by humoral factors such as growth factors and hormones. Many lines of evidence have suggested that angiotensin II (Ang II) plays a vital role in cardiac hypertrophy, and it has been reported that secretion of Ang II from cultured cardiac myocytes was induced by mechanical stretch. To examine the role of Ang II in mechanical stress–induced cardiac hypertrophy, we stretched neonatal rat cardiac myocytes in the absence or presence of the Ang II receptor antagonists saralasin (an antagonist of both type 1 and type 2 receptors), CV-11974 (a type 1 receptor–specific antagonist), and PD123319 (a type 2 receptor–specific antagonist). Stretching cardiac myocytes by 20% using deformable silicone dishes rapidly increased the activities of mitogen-activated protein (MAP) kinase kinase activators and MAP kinases. Both saralasin and CV-11974 partially inhibited the stretch-induced increases in the activities of both kinases, whereas PD123319 showed no inhibitory effects. Stretching cardiac myocytes increased amino acid incorporation, which was also inhibited by approximately 70% with the pretreatment by saralasin or CV-11974. When the culture medium conditioned by stretching cardiocytes was transferred to nonstretched cardiac myocytes, the increase in MAP kinase activity was observed, and this increase was completely suppressed by saralasin or CV-11974. These results suggest that Ang II plays an important role in mechanical stress–induced cardiac hypertrophy and that there are also other (possibly nonsecretory) factors to induce hypertrophic responses.


Key Words: CV-11974 • signal transduction • MAP kinase • saralasin


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Elevated blood pressure causes sustained increase in hemodynamic overload, which ultimately leads to development of cardiac hypertrophy.1 Cardiac hypertrophy is not only a fundamental process of adaptation to an increased workload but also an important cause of increased morbidity and mortality.2 The hypertrophied heart exhibits impaired contraction and relaxation and reduced coronary reserve after transient ischemia, both of which lead to higher mortality in heart failure and ischemic heart disease.2 Many lines of evidence have suggested that mechanical stress plays a pivotal role in developing cardiac hypertrophy during hemodynamic overload without any involvements of humoral and/or neural factors.3 Thus, elucidation of the mechanism(s) of mechanical stress–induced cardiac hypertrophy is of utmost importance.

We have previously reported on the intracellular signaling pathway(s) of stretch-induced cardiac myocyte hypertrophy using an in vitro system of stretching deformable silicone dishes, in which we imposed mechanical stress on cultured cells under no influence of humoral factors.4 5 We have shown that mechanical loading on cultured myocytes activates protein kinase phosphorylation cascades (eg, PKC,5 MAP kinase, and S6 kinase6 ) and induces the expression of specific genes and increases in protein synthesis.4 5 These signaling pathways and events are highly reminiscent of those observed when humoral factors such as hormones and growth factors are added to cardiac myocytes.7 8

A growing body of data suggests that the local renin-angiotensin system plays an important role in the development of cardiac hypertrophy.9 All components of the renin-angiotensin system (eg, renin, angiotensinogen, and ACE) have been identified in the heart at both the mRNA and protein levels.10 Ang II has been reported to increase hydrolysis of phosphoinositides and to activate MAP kinases followed by gene expression and enhanced protein synthesis in cultured cardiac myocytes11 12 (Y. Kato, T. Yamazaki, and I. Komuro, unpublished observation). Many recent reports have also shown that the cardiac renin-angiotensin system is activated in experimental left ventricular hypertrophy induced by hemodynamic overload. Increases in angiotensinogen and ACE mRNAs have been reported in the hypertrophied left ventricle of rats.13 Subpressor doses of ACE inhibitors can cause regression of cardiac hypertrophy with no change in systemic systolic blood pressure.14 Moreover, an increase in left ventricular mass produced by abdominal aortic constriction was completely prevented by an ACE inhibitor without any change in afterload and plasma renin activity.15 These results suggest that the local renin-angiotensin system may play a critical role in cardiac hypertrophy induced by pressure overload and that Ang II may act to promote the growth of cardiac myocytes by autocrine or paracrine mechanisms.3

It was recently reported that mechanical stretch causes direct secretion of Ang II from the cytoplasmic granules of cultured cardiac myocytes and that stretch-induced hypertrophic responses are completely dependent on the secreted Ang II.16 Our preliminary studies, however, showed that a specific antagonist of the type 1 Ang II receptor CV-11974 only partially inhibited the activation of MAP kinases and c-fos gene expression and attenuated the stretch-induced increase in phenylalanine incorporation into cells.17 To elucidate this discrepancy, we extensively examined the relation between mechanical stress and Ang II using three Ang II receptor antagonists: saralasin (antagonist of type 1 and 2 Ang II receptors), CV-11974 (type 1 receptor–specific antagonist), and PD123319 (type 2 receptor–specific antagonist). In the present study, we show that mechanical stretch induces activation of MAP kinase kinase activators and MAP kinases and an increase in phenylalanine incorporation, all of which are significantly but only partially suppressed by the pretreatment with saralasin and CV-11974 but not PD123319. We also demonstrate that the medium conditioned by stretching of cultured cardiomyocytes induces activation of MAP kinases, which is completely suppressed by saralasin or CV-11974. These results suggest that Ang II partly mediates mechanical stress–induced hypertrophic responses and that other (possibly nonsecretory) factors may also be involved in these events.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
[3H]phenylalanine and [{gamma}-32P]ATP were purchased from Du Pont-New England Nuclear Co and Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) from Gibco BRL Co. Other reagents were purchased from Sigma Co. CV-11974 was a gift from Takeda Chemical Industries, Ltd. PD123319 was a gift from Parke-Davis Co.

Cell Culture and Stretching of Cardiac Myocytes
Primary cultures of cardiac myocytes were prepared from ventricles of 1-day-old Wistar rats as described previously4 according to the method of Simpson and Savion,18 and stretching of myocytes was conducted as described previously.4 5 In brief, cells were plated at a field density of 1x103 cells/mm2 on the silicone rubber culture dishes. At 24 hours after seeding, the culture medium was changed to a solution consisting of DMEM containing 0.1% FBS. Since, like c-fos gene expression,4 more definite hypertrophic responses were observed by 20% stretch than by 10% stretch (data not shown), uniaxial strain was applied by stretching the silicone dish by 20%. Stretch and control experiments were carried out simultaneously with the same pool of cells in each experiment to match for temperature, CO2 content, and pH of the medium for the stretched and control cells.

MAP Kinase Assays
MAP kinase activity was measured using phosphocellulose paper as described previously.6 After stimulation, cultured cardiac myocytes were lysed on ice with 0.2 mL of buffer A containing 25 mmol/L Tris-HCl, 25 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 10 nmol/L okadaic acid, 0.5 mmol/L ethylene glycol-bis (ß-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), and 1 mmol/L phenyl-methyl sulfonyl fluoride. Aliquots of the myocyte extracts were incubated with 2 µCi [{gamma}-32P]ATP and substrate (MBP) for 10 minutes at 25°C in 40 µL of kinase buffer (25 mmol/L Tris-HCl, pH 7.4, 10 mmol/L MgCl2, 1 mmol/L dithiothreitol [DTT], 40 µmol/L ATP, 2 µmol/L protein kinase inhibitor peptide, 0.5 mmol/L EGTA). The reaction was terminated by adding 10 µL of stopping solution containing 0.6% HCl, 1 mmol/L ATP, and 1% bovine serum albumin. Aliquots of the reaction mixture (15 µL) were spotted on 1.5x1.5-cm squares of P81 paper (Whatman). The paper was washed five times for at least 10 minutes each in 0.5% phosphoric acid and then dried. The incorporation of 32P into MBP was determined by Cerenkov counting, and the relative activity was presented compared with that of nontreated cells.

Kinase Assays in MBP-Containing Gels After SDS-PAGE
Kinase assays in MBP-containing gels were performed as described previously.6 Cardiac myocytes were lysed with buffer A as described for MAP kinase assays, and aliquots of the extracts were electrophoresed on an SDS-polyacrylamide gel containing 0.5 mg/mL MBP. SDS was removed from the gel by washing with two changes of 100 mL each of 20% 2-propanol in 50 mmol/L Tris-HCl (pH 8.0) for 1 hour and then 250 mL of 50 mmol/L Tris-HCl (pH 8.0) containing 5 mmol/L 2-mercaptoethanol for 1 hour at room temperature. The enzyme was denatured by treating the gel first with two changes of 100 mL of 6 mol/L guanidine/HCl at room temperature for 1 hour and then renatured with five changes of 250 mL each of 50 mmol/L Tris-HCl (pH 8.0) containing 0.04% Tween 40 and 5 mmol/L 2-mercaptoethanol at 4°C for 3 hours. After renaturation, the gel was immersed in 5 mL of 40 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 8.0) containing 2 mmol/L DTT and 10 mmol/L MgCl2 for 1 hour at 25°C. Phosphorylation of MBP was carried out by incubating the gel with 25 µCi [{gamma}-32P]ATP at 25°C for 1 hour in 5 mL of 40 mmol/L HEPES (pH 8.0), 0.5 mmol/L EGTA, 10 mmol/L MgCl2, 2 µmol/L protein kinase inhibitor, and 40 µmol/L ATP. After incubation, the gel was washed with a 7% acetic acid solution until the radioactivity of the washing solution became negligible. The washed gel was dried and then subjected to autoradiography.

Assay of MAP Kinase Kinase Activator Activity
The activity of MAP kinase kinase activators was assayed by measuring the phosphorylation of MAP kinase kinase using recombinant MAP kinase kinase fused to glutathione S transferase (rMAPKK).19 Cell lysates were incubated with 2 µCi [{gamma}-32P]ATP and substrate (100 µg rMAPKK) in buffer B containing 25 mmol/L Tris-HCl (pH 7.4), 10 mmol/L MgCl2, 1 mmol/L DTT, 40 µmol/L ATP, 2 µmol/L protein kinase inhibitor peptide, and 0.5 mmol/L EGTA for 30 minutes at 25°C. After incubation, rMAPKK was collected using glutathione beads and was electrophoresed on a 7% polyacrylamide gel. The gel was dried and subjected to autoradiography.

Amino Acid Incorporation Into Proteins
After 2 days in the serum-free medium, cardiac myocytes were stretched for 24 hours in the absence or presence of saralasin (10-6 mol/L) or CV-11974 (10-6 mol/L). The relative amount of protein synthesis was determined by assessing the incorporation of the radioactivity into a trichloroacetic acid (TCA)-insoluble fraction. One microcurie per milliliter of [3H]phenylalanine was added to the culture medium 2 hours before harvesting. The cells were rapidly rinsed four times with ice-cold phosphate-buffered saline (10 mmol/L sodium phosphate and 0.85% NaCl, pH 7.4) and incubated for 20 minutes on ice with 1 mL of 20% TCA. The total TCA-insoluble radioactivity in each dish was determined by liquid scintillation counting. We repeated the experiment five times in triplicate.

Statistics
Statistical comparison of the control group with treated groups was carried out using the paired-sample t test with P values corrected by the Bonferroni method. The accepted level of significance was P<.05.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Ang II Activates MAP Kinase
A number of reports have suggested that MAP kinase is a key molecule in intracellular signal transduction and plays an essential role in cellular proliferation and differentiation.20 21 As described in the previous study, MAP kinase is activated by mechanical stress in cardiac myocytes,6 and MAP kinase activity is a sensitive and quantitative marker for hypertrophic responses of cardiac myocytes. To clarify the relation between Ang II and mechanical stress, we first examined whether Ang II evokes activation of MAP kinases. We performed an assay of MBP phosphorylation using phosphocellulose paper, which is an established and highly sensitive method for quantifying MAP kinase activity.6 22 23 Cardiac myocytes were incubated with various concentrations (10-10 to 10-5 mol/L) of Ang II for 8 minutes. No significant increase in MAP kinase activity was observed at 10-10 mol/L Ang II as compared with the vehicle (control). We observed a slight increase in MAP kinase activity at 10-9 mol/L, and the maximal activation of MAP kinases was obtained at 10-7~10-6 mol/L of Ang II. The activity of MAP kinases was increased by 60% (10-8 mol/L) to 150% (10-6 mol/L) as compared with the vehicle (Fig 1Down). Although the reason is not clear at this moment, 10-5 mol/L Ang II showed less activation of MAP kinases than 10-6 mol/L Ang II. This high concentration of Ang II may cause injury to cardiac myocytes. Fig 2Down shows the time course of Ang II–induced MAP kinase activation. The increase in MAP kinase activity induced by 10-6 mol/L Ang II was detectable as early as 1 minute, and the activity reached a peak at 8 minutes. The activity decreased gradually and returned to the control level at 30 minutes after the addition of Ang II.



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  		Figure 1. Plot shows dose dependence of Ang II–induced MAP kinase activation. Neonatal rat cardiac myocytes were stimulated with various doses of Ang II for 8 minutes. Aliquots of the cardiomyocyte lysates were incubated with substrate (25 µg MBP) and [{gamma}-32P]ATP in kinase buffer A. After the reaction was terminated, the reaction mixture was spotted on P81 paper (Whatman), and after washing, the incorporation of 32P into MBP was assessed by Cerenkov counting. Data are presented as mean±SEM from six independent experiments compared with controls (=100%, vehicle). Statistical analysis was performed using the paired-sample t test with P values corrected by the Bonferroni method (*P<.05 and {dagger}P<.01 vs control).



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  		Figure 2. Plot shows time course of Ang II–induced MAP kinase activation. Cardiac myocytes were exposed to 10-6 mol/L Ang II for various periods of time. MAP kinase activity was measured as described in Fig 1Up. Data represent the average percentage of control (=100%, 0 minutes) from seven independent experiments (mean±SEM) (*P<.05 and {dagger}P<.01 vs control).

Ang II AT1 Receptor Antagonist Blocks Ang II–Induced MAP Kinase Activation
The two main Ang II receptor subtypes, AT1 and AT2 receptors, have been identified pharmacologically,24 and the cDNAs encoding both receptors recently have been isolated.25 26 27 28 To examine which receptor subtype mediates MAP kinase activation by Ang II in cardiac myocytes, we preincubated cardiac myocytes with saralasin (an antagonist of both AT1 and AT2 receptors), CV-11974 (an antagonist of AT1 receptor), and PD123319 (an antagonist of AT2 receptor) and exposed cardiac myocytes to 10-7 mol/L Ang II for 8 minutes (Fig 3Down). Both saralasin and CV-11974 blocked Ang II–induced MAP kinase activation in a dose-dependent manner. Both 10-6 mol/L saralasin and 10-6 mol/L CV-11974 completely suppressed maximal activation of MAP kinase induced at 10-7 mol/L Ang II. On the other hand, 10-6 mol/L PD123319 had no inhibitory effects on Ang II–induced MAP kinase activation. These results suggest that the induction of MAP kinase activation by Ang II is mediated by the AT1 receptor.



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  		Figure 3. Bar graph shows effect of Ang II receptor antagonists on Ang II–evoked MAP kinase activation. Cardiac myocytes were pretreated with saralasin, CV-11974, or PD123319 at the indicated concentration for 30 minutes and stimulated by 10-7 mol/L Ang II for 8 minutes. MAP kinase assays using P81 paper were performed as described in Fig 1Up. Data represent the average percentage of controls (=100%, vehicle) from four independent experiments (mean±SEM) (*P<.05 and {dagger}P<.01 vs control).

Ang II AT1 Receptor Antagonist Partially Blocks Stretch-Induced MAP Kinase Activation
We have previously demonstrated that stretching of cardiac myocytes increases MAP kinase activity.6 The time course of MAP kinase activation by mechanical stress mimics that by Ang II (Fig 2Up; Reference 66 ). We then investigated the involvement of Ang II in stretch-induced activation of MAP kinases. After pretreatment with saralasin or CV-11974, cardiac myocytes were stretched by 20% for 8 minutes (Fig 4Down). Stretch by 20% remarkably increased the activity of both 42-kDa and 44-kDa MAP kinases by 80%. Both Ang II receptor antagonists significantly reduced the activity of MAP kinases in a dose-dependent manner. Inhibitory effects, however, were not complete, and approximately 30% of the increased activity remained even after pretreatment with 10-6 mol/L saralasin or CV-11974. We then examined the time course of stretch-induced activation of MAP kinases in cardiac myocytes with or without CV-11974 pretreatment. Stretch-induced activation of MAP kinase was detectable at 2 minutes, peaked at 8 minutes, and returned to the control level at 30 minutes after stretch (Fig 5ADown). CV-11974 (10-6 mol/L) not only suppressed the maximum activation of MAP kinase induced by stretch at 8 minutes but also attenuated the activation from 1 minute to 20 minutes. The suppression, however, was incomplete throughout the time course (Fig 5ADown). In contrast, pretreatment with PD123319 did not inhibit the stretch-induced MAP kinase activation at any time (Fig 5BDown). These results suggest that MAP kinase activation induced by mechanical stress is partially dependent on Ang II through the AT1 receptor. To confirm this hypothesis, we measured MAP kinase activity using a more accurate method by performing kinase assays in MBP-containing gels after SDS-PAGE followed by denaturation and renaturation. As shown in Fig 6Down, only 42-kDa MAP kinase was slightly activated in nonstretched myocytes (lane a), and stretching myocytes remarkably activated both 42-kDa and 44-kDa-MAP kinases (lane b). This activation was also partially suppressed by pretreatment with 10-6 mol/L CV-11974 (lane c), which completely suppressed the maximum activation of MAP kinase induced by 10-7 mol/L Ang II (Fig 3Up). From these results, it is strongly suggested that there are alternative pathways that activate MAP kinases during mechanical stretch other than Ang II.



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  		Figure 4. Bar graph shows effect of Ang II receptor antagonists on stretch-evoked MAP kinase activation. After treatment with saralasin or CV-11974 at the indicated concentration for 30 minutes, cardiac myocytes were stretched by 20% for 8 minutes, and MAP kinase activity was measured using P81 paper. Data represent the average percentage of control (=100%, nonstretch) from five independent experiments (mean±SEM) (*P<.05 and {dagger}P<.01 vs control).



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  		Figure 5. Plots show time course of stretch-induced MAP kinase activation. After pretreatment with 10-6 mol/L CV-11974 (A) or 10-6 mol/L PD123319 (B) for 30 minutes, cardiac myocytes were stretched by 20% for 8 minutes, and MAP kinase activity was measured using P81 paper. Open and closed circles indicate activity of stretched myocytes without and with pretreatment, respectively. Data represent the average percentage of controls (=100%, 0 minutes) from four (A) or two (B) independent experiments (mean±SEM).



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  		Figure 6. MAP kinase assays using MBP-containing gels. Cardiac myocytes were stretched for 8 minutes with or without pretreatment by 10-6 mol/L CV-11974 (lanes b and c). The cells were lysed in situ, and the aliquots of the lysates were electrophoresed on SDS-polyacrylamide gels containing MBP. SDS was removed from the gel, and after denaturation with 6 mol/L guanidine/HCl and renaturation in a buffer containing 0.04% Tween 40, the gel was incubated with [{gamma}-32P]ATP. After washing, the gel was dried and subjected to autoradiography. Control samples were taken from the nonstretched cells (lane a). Data were taken from a representative experiment performed in triplicate.

Ang II AT1 Receptor Antagonist Partially Blocks Stretch-Induced Activation of MAP Kinase Kinase Activators
To examine whether the partial inhibition of stretch-activated signals by AT1 receptor antagonists is specific to MAP kinases or applicable to other kinases, we analyzed the MAP kinase kinase activators after stretching myocytes. Recently, MAP kinase kinase, the direct upstream enzyme of MAP kinases, has been isolated and shown to specifically phosphorylate the regulatory tyrosine and threonine residues of MAP kinases, causing their full activation. The MAP kinase kinase has been shown to be phosphorylated and activated by MAP kinase kinase activators (Raf-1 kinase and MEK kinase). The activity of MAP kinase kinase activators was measured by examining the phosphorylation of rMAPKK. After incubating rMAPKK with lysates of myocytes and [{gamma}-32P]ATP, purified rMAPKK was electrophoresed on an SDS-polyacrylamide gel, and phosphorylation of rMAPKK was analyzed by autoradiography. Although the control nonstretched cells did not show any activity of MAP kinase kinase activators (Fig 7aDown), stretching myocytes for 2 minutes dramatically induced activation of MAP kinase kinase activators (Fig 7bDown). Pretreatment of CV-11974 suppressed stretch-induced activation of MAP kinase kinase activators by approximately 50% (Fig 7cDown). These results suggest that the partial dependence of stretch-activated hypertrophic responses on Ang II is not specific to MAP kinases but may be a general phenomenon.



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  		Figure 7. Activation of MAP kinase kinase activators by stretching cardiac myocytes. Cardiac myocytes were pretreated with 10-6 mol/L CV-11974 (lane c) and stretched by 20% for 2 minutes (lanes b and c). The lysates were incubated with rMAPKK and [{gamma}-32P]ATP. rMAPKK was collected using glutathione beads and electrophoresed on a 7% polyacrylamide gel. After washing, the gel was dried and subjected to autoradiography. Lane a shows no activity of MAP kinase kinase activators in nonstretched cells. Similar results were obtained from five separate experiments.

Ang II Receptor Antagonists Incompletely Block Stretch-Induced Increase in Phenylalanine Incorporation Into Myocytes
We have previously shown that stretching myocytes increases phenylalanine incorporation into cells, suggesting that mechanical stress directly induces cardiac cellular hypertrophy.4 5 To examine whether Ang II receptor antagonists attenuate stretch-induced cardiac hypertrophy, we measured the relative amount of protein synthesis using [3H]phenylalanine. As shown in Fig 8Down, Ang II (10-6 mol/L) stimulated increases in amino acid incorporation by approximately 1.7-fold, which was completely suppressed by pretreatment with either saralasin (10-6 mol/L) or CV-11974 (10-6 mol/L). Stretch for 24 hours also caused a 42% increase in phenylalanine incorporation, as described previously.4 However, this increase was reduced by only 70% when the cells were pretreated with either saralasin or CV-11974. The increase in amino acid incorporation by Ang II or stretch was not suppressed by the pretreatment with PD123319 (data not shown). These data also suggest that a part of the stretch-induced cellular hypertrophy is dependent on Ang II through the AT1 receptor.



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  		Figure 8. Bar graph shows effect of Ang II receptor antagonists on Ang II–stimulated and stretch-evoked phenylalanine incorporation. After pretreatment with saralasin (10-6 mol/L) or CV-11974 (10-6 mol/L), cardiac myocytes were stimulated by Ang II or by stretching for 24 hours, and [3H]phenylalanine (1 µCi/mL) was added 2 hours before harvesting. The total radioactivity of incorporated [3H]phenylalanine was determined by liquid scintillation counting. Data represent the average percentage of control (=100%, no stimulus) from five independent experiments (mean±SEM) (*P<.05 and {dagger}P<.01 vs control).

Stretch-Conditioned Media Induce MAP Kinase Activation
Recently, Ang II has been reported to be secreted from stretched cardiac myocytes.16 Since there may be two pathways, Ang II–dependent and Ang II–independent pathways in mechanical stress–induced hypertrophic responses, we examined whether only Ang II was released into the culture medium after stretch using Ang II receptor antagonists. The culture media were transferred to cardiac myocytes cultured on regular culture dishes from the dish of cardiac myocytes that were stretched for 8 minutes. The addition of media conditioned by stretch significantly increased the activity of MAP kinases in recipient myocytes. The pretreatment of the recipient cells with saralasin (10-6 mol/L) or CV-11974 (10-6 mol/L) almost completely suppressed MAP kinase activation induced by the addition of conditioned media (Fig 9Down). These findings suggest that mechanical stress stimulates the secretion of Ang II from the cultured cardiocytes and that Ang II is the major molecule that evokes hypertrophic responses in cardiac myocytes as a secretory molecule.



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  		Figure 9. Bar graph shows activation of MAP kinases by stretch-conditioned media. The culture media of the cells that were not stretched (Non-Stretch) or stretched by 20% for 8 minutes (Stretch) were transferred to quiescent cardiac myocytes cultured in plastic culture dishes. Some of the cells were pretreated by saralasin (10-6 mol/L) (Stretch+Saralasin) or CV-11974 (10-6 mol/L) (Stretch+CV-11974) and then incubated with the stretch-conditioned media. MAP kinase activity was measured using P81 paper. Data represent the average percentage of controls (=100%, non–stretch-conditioned media) from six independent experiments (mean±SEM) (*P<.05 vs control).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we demonstrated the signaling pathways by which mechanical stress induces cardiac cellular hypertrophy. Stretching myocytes sequentially activated MAP kinase kinase activators and MAP kinases followed by an increase in protein synthesis. All of these events were significantly suppressed by specific antagonists of the Ang II receptor CV-11974 and saralasin but not by PD123319. Furthermore, we showed that stretch-conditioned media activate MAP kinases and that this activation is completely blocked by the same Ang II receptor antagonists. These results suggest that mechanical stress exemplified by stretch might stimulate the secretion of Ang II from cardiac myocytes and that Ang II evokes protein kinase activation and produces cardiac hypertrophy through the AT1 receptor. However, because neither saralasin nor CV-11974 can inhibit these hypertrophic events completely, factors other than Ang II might be involved in cardiac myocyte hypertrophy induced by mechanical stress.

Accumulating data have suggested that MAP kinases are activated by many growth factors and cytokines and play key roles in cell proliferation and differentiation.29 Both MAP kinase antisense RNA and interfering mutants of MAP kinase inhibited growth factor–stimulated gene expression and cell growth.20 In addition, Cowley et al21 showed by using interfering and constitutively activated mutants of MAP kinase kinase that the MAP kinase pathway mediates both the differentiation of PC12 pheochromocytoma cells and the transformation and mitogenic response of NIH 3T3 cells. We have previously shown that mechanical stress induces activation of PKC, MAP kinases, and S6 kinase in cultured cardiac myocytes followed by increases in specific gene expression and an amino acid incorporation into cells.5 6 It is known that both Raf-1 kinase30 31 32 and MEK kinase33 can phosphorylate and activate MAP kinase kinase. In other words, Raf-1 kinase and MEK kinase converge at MAP kinase kinase in the protein kinase network, mediating the activation of MAP kinases by external stimuli. We have demonstrated for the first time in the present study that stretching myocytes increases the activity of MAP kinase kinase activators. We have also observed that stretch stimulus activates MAP kinase kinase in cultured cardiac myocytes (unpublished data), suggesting that mechanical stress may evoke the sequential activation of the protein kinase cascade of phosphorylation and then induce cardiac hypertrophy. Cardiac myocytes of 1-day-old neonatal rats were used in the present study. Lazou et al34 showed that the activation of MAP kinases and MAP kinase kinases was provoked by potential hypertrophic agonists such as high coronary perfusion pressure, norepinephrine, and isoproterenol in perfused adult rat hearts. It remains to be determined whether stretch-induced hypertrophic responses observed in neonatal rat cardiocytes can be applied to adult heart cells.

Lack of a correlation between an elevated arterial pressure and an increased myocardial mass has been demonstrated in hypertensive cardiac hypertrophy of animals and humans.17 35 36 This dissociation suggests that humoral and/or neural factors as well as hemodynamic overload are responsible for the development and regression of cardiac hypertrophy. Many animal and human studies have shown that the local renin-angiotensin system plays an important role in the pathogenesis of left ventricular hypertrophy.9 10 Increased myocardial angiotensinogen mRNA levels and ACE activity are found in the models of hypertensive cardiac hypertrophy of rats.15 ACE inhibitors produce significant regression of left ventricular hypertrophy in human and spontaneously hypertensive rats.15 37 We have recently reported that the treatment of spontaneously hypertensive rats with an Ang II AT1 receptor antagonist, CV-11974, not only reduces the thickness of the left ventricular wall but also decreases both the relative amount of V3 myosin heavy chain and interstitial fibrosis.17 The regression of hypertrophy by ACE inhibitors and the Ang II receptor antagonist might be not only due to the decreased blood pressure but also due to inhibition of the tissue renin-angiotensin system, because vasodilators like hydralazine do not produce regression of hypertrophy despite adequate control of blood pressure and because ACE inhibitors and the Ang II receptor antagonist prevent and cause regression of cardiac hypertrophy with subpressor doses.14 17 These results suggest that the local renin-angiotensin system may play a critical role in cardiac hypertrophy induced by mechanical stress. In the present study, CV-11974 (an AT1 receptor–specific antagonist) and saralasin (an antagonist of both AT1 and AT2 receptors) significantly suppressed activation of MAP kinases and MAP kinase kinase activators and an increase in protein synthesis stimulated by stretch. On the other hand, PD123319 (AT2 receptor–specific antagonist) did not show any inhibitory effects on these events. These results suggest that mechanical stress–induced hypertrophy is at least in part dependent on Ang II through the AT1 receptor.

CV-11974 and saralasin only partially suppressed stretch-induced activation of MAP kinases (Figs 4Up, 5AUp, and 6Up) and MAP kinase kinase activators (Fig 7Up) as well as an increase in amino acid incorporation (Fig 8Up). Mechanical stress increased MAP kinase activity by 80% in cardiac myocytes (Fig 4Up), and the magnitude of an increase in MAP kinase activity induced by stretch was comparable to that induced by 10-8 mol/L Ang II (Fig 1Up). Less than 10-7 mol/L saralasin and 10-7 mol/L CV-11974 was enough to completely suppress MAP kinase activation induced by 10-8 mol/L Ang II (data not shown). Activation of MAP kinases induced by mechanical stress, however, was only partially suppressed with 10-6 mol/L antagonists, suggesting that factors other than Ang II are involved in this activation. This result is different from the previous report,16 in which increases in c-fos gene expression and phenylalanine incorporation were completely suppressed by the Ang II receptor antagonist saralasin and the AT1-specific antagonist losartan. In our study, 10-6 mol/L losartan (a gift from Merck Co) also showed partial suppression of these hypertrophic events (data not shown). Although we do not know the exact reason for this discrepancy, MAP kinase activity might be a more quantitative marker for hypertrophic responses than c-fos gene expression. Partial suppression of hypertrophic responses by Ang II receptor antagonists suggests two possibilities. One is that humoral factors other than Ang II are also secreted and evoke similar responses. Another possibility is that mechanical stress itself induces hypertrophic responses. The activation of MAP kinases by the addition of stretch-conditioned medium was almost completely blocked by pretreatment with saralasin or CV-11974, suggesting that Ang II may be the only secreted factor that activates hypertrophic events in cardiac myocytes. We therefore hypothesize that Ang II may play a critical role in MAP kinase activation as an "exogenous" factor and that there may be "endogenous" factor(s) in cardiac myocytes during mechanical stress. In other words, mechanical stress itself may evoke hypertrophic responses, and the secreted Ang II may induce similar signals and amplify the responses.

Judging from the degree of the activation of MAP kinases by the conditioned media, there might be {approx}5x10-10 mol/L Ang II in the culture media after stretch (Figs 1Up and 9Up). We measured Ang II concentration in the media after stretch by radioimmunoassay, but we could not get a consistent increase in Ang II (data not shown). Although we do not know at present whether this is due to a technical reason or other reasons, our observation that the activation of MAP kinases by the stretch-conditioned media was completely suppressed by Ang II receptor antagonists (Fig 9Up) suggests Ang II secretion after stretch. Recently, it was reported that Ang II is secreted from cardiac myocytes by stretching of cultured cardiac myocytes using a similar system.16 The mechanism by which Ang II is released into the culture medium is then open to question. Since Ang II was reported to exist in a small granular compartment,16 Ang II should be secreted from cardiac myocytes by regulated mechanisms. A number of reports have suggested that an increase in intracellular calcium or PKC activity plays an important role in regulated secretion. It has been reported that stretching myocytes induces entering of extracellular Ca2+ into the cells, which then induces Ca2+ release from Ca2+ stores in the myocytes.38 Although the stretch-induced activation of MAP kinases is partially dependent on transsarcolemmal influx of Ca2+,6 the chelation of extracellular Ca2+ with ethylene diamine tetraacetic acid basically had no effects on stretch-induced c-fos gene expression.39 40 On the contrary, c-fos gene expression induced by mechanical stress was completely blocked by PKC inhibitors and by the downregulation of PKC.5 These results suggest that PKC may be directly activated by mechanical stress, which may subsequently induce Ang II secretion. PKC is also known to activate Raf-1 kinase and MAP kinases.41 42 Taken together, mechanical stress may directly activate the protein kinase cascade of phosphorylation including PKC and MAP kinases, and the activated PKC may induce the secretion of Ang II. The secreted Ang II may amplify the signals directly evoked by mechanical stress. Further studies are necessary to understand the mechanisms by which cardiac myocytes receive mechanical stress and secrete Ang II.


*    Selected Abbreviations and Acronyms
 
ACE = angiotensin-converting enzyme
Ang II = angiotensin II
MAP = mitogen-activated protein
MBP = myelin basic protein
MEK = MAP kinase/extracellular signal-regulated kinase kinase
PAGE = polyacrylamide gel electrophoresis
PKC = protein kinase C
rMAPKK = recombinant MAP kinase kinase
SDS = sodium dodecyl sulfate


*    Acknowledgments
 
This study was supported by a grant-in-aid for scientific research and developmental scientific research from the Ministry of Education, Science, and Culture; a grant from Japan Heart Foundation Research for 1994 (T.Y.); and a grant from the Japan Cardiovascular Foundation, the Sankyo Life Science and the Study Group of Molecular Cardiology Japan (I.K.). We appreciate Toru Suzuki for critical reading and Fumiko Harima for excellent technical assistance.

Received December 8, 1994; accepted April 14, 1995.


*    References
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