Differential Activation of Cardiac c-Jun Amino-Terminal Kinase and Extracellular Signal-Regulated Kinase in Angiotensin II–Mediated Hypertension

From the Department of Pharmacology, Osaka City University Medical School, (M.Y., S.K., Y.I., S.Y., H.I.), and Environmental Biological Life Science Research Center (BILIS), Inc (M.Y.), Shiga, Japan.

Correspondence to Shokei Kim, MD, Department of Pharmacology, Osaka City University Medical School, 1-4-54 Asahimachi, Abeno, Osaka 545-8585. Japan. E-mail kims{at}msic.med.osaka-cu.ac.jp

	Abstract

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Abstract—Two subgroups of mitogen-activated protein kinases, c-jun NH₂-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), are thought to be involved in cultured cardiac myocyte hypertrophy and gene expression. To examine the in vivo activation of these kinases, we measured cardiac JNK and ERK activities in conscious rats subjected to acute or chronic angiotensin II (Ang II) infusion, by using in-gel kinase methods. About 50 mm Hg rise in blood pressure by Ang II (1000 ng · kg^-1 · min^-1) infusion caused larger activation of left ventricular JNK than ERK, via the AT₁ receptor. In spite of short duration (about 30 minutes) of maximal blood pressure elevation by Ang II, JNK sustained the peak value (more than 5-fold increase) from 15 minutes up to at least 3 hours. Similar activation of JNK was seen in the right ventricle. Thus, cardiac JNK activation by Ang II seems to be in part mediated by its direct action via the AT₁ receptor. The dose-response relationships for Ang II–induced rises in blood pressure and cardiac JNK and ERK activation indicated that cardiac JNK or ERK was not activated by a mild increase in blood pressure and that cardiac JNK was activated by Ang II–mediated hypertension in a more sensitive manner than ERK. Cardiac hypertrophy, induced by chronic Ang II infusion, was preceded by JNK activation without ERK activation. Furthermore, gel mobility shift analysis showed that cardiac JNK activation was followed by increased activator protein-1 DNA binding activity due to c-Fos and c-Jun. These results provided the first evidence for the preferential activation of cardiac JNK in Ang II–induced hypertension and suggested that JNK might play some role in Ang II–induced cardiac hypertrophic response in vivo. However, further study is needed to elucidate the role of JNK in cardiac hypertrophy in vivo.

Key Words: c-jun NH₂-terminal kinase • extracellular signal-regulated kinase • activator protein-1 • angiotensin II • cardiac hypertrophy

	Introduction

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Extracellular signal-regulated kinases (ERKs),^{1 2 3} composed of p42ERK and p44ERK, are one main subgroup of mitogen-activated protein (MAP) kinases and are important mediators of the signal-transduction pathway responsible for cell differentiation and growth, by activating activator protein-1 (AP-1) or regulating numerous gene expressions. Accumulating in vitro evidence shows that ERKs in cultured neonatal rat cardiac myocytes are rapidly and transiently activated by mechanical stretch^{4 5} or various hypertrophic factors,^{6 7 8} such as angiotensin II (Ang II), endothelin-1, or phenylephrine. The activation of ERK has been reported to be involved in cardiac myocyte hypertrophy⁹ and the gene expression of atrial natriuretic factor (ANF), the fetal gene reexpressed during cardiac hypertrophy,^{9 10} while a minor role of ERK activation in cardiac hypertrophic response has been also reported,¹¹ indicating that the role of ERK in cardiac hypertrophy is controversial. However, most of the information on ERK has come from in vitro studies, and the activating mechanism and role of ERK in vivo are poorly understood.

c-Jun NH₂-terminal kinases (JNKs)^{12 13 14 15} (consisting of p46JNK and p55JNK), also called stress-activated protein kinases, are another major subgroup of MAP kinases identified several years ago and are known to phosphorylate c-Jun and ATF-2 and play a key role in cell growth or apoptosis.^{16 17} In vitro studies show that in contrast to the preferential activation of ERK by growth factors and vasoactive peptides, JNKs are generally activated mainly by inflammatory cytokines and cellular stresses, such as heat shock, osmotic shock, or ultraviolet irradiation.^{12 13 14 15 16} However, very recently it has been demonstrated that JNKs in cultured neonatal rat cardiac myocytes are significantly activated by mechanical stretch¹⁸ or Ang II.¹⁹ Furthermore, JNK has been reported to be responsible for the hypertrophic response of cultured cardiac myocytes.^{20 21} Thus, JNK, as well as ERK, may contribute to the development of cardiac hypertrophy or cardiac gene expression. However, as in the case of ERK, the in vivo regulation and significance of cardiac JNK remain unclear.

To examine the significance of MAP kinases in hypertensive cardiac hypertrophy in vivo, we have recently determined cardiac ERK and JNK activities in stroke-prone spontaneously hypertensive rats (SHRSP) and obtained the evidence that both cardiac ERK and JNK activities are significantly increased in SHRSP compared with control normotensive Wistar-Kyoto rats.²² Furthermore, the prevention of cardiac hypertrophy in SHRSP by an angiotensin-converting enzyme inhibitor was associated with the decrease in cardiac JNK activity but not with ERK, suggesting that the increased cardiac JNK activity in SHRSP may be implicated in cardiac hypertrophy or gene expression.²² Thus, detailed investigation of the in vivo model seems essential to elucidate the role of MAP kinases in hypertensive pathological cardiac hypertrophy.

A growing body of in vitro and in vivo evidence supports the hypothesis that Ang II plays a key role in cardiac hypertrophy and gene expression induced by pressure overload such as in hypertension.^{6 23 24 25 26 27} However, it is unknown whether cardiac MAP kinases are indeed activated by Ang II in vivo and whether these kinases participate in Ang II–induced cardiac hypertrophy and gene expression in vivo. Therefore, in the present study, we investigated the acute and chronic effects of Ang II infusion on cardiac MAP kinases in conscious rats. We obtained the first in vivo evidence that Ang II–mediated hypertension activates cardiac JNK in a more sensitive manner than ERK, suggesting that JNK may play some role in Ang II–induced cardiac hypertrophic response in vivo.

	Materials and Methods

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Materials
Synthetic Ang II was purchased from Peptide Institute Inc. RNH-6270, which is a potent and specific AT₁ receptor antagonist, and CS-866, which is a prodrug type of AT₁ receptor antagonist and is deesterified to RNH-6270 after oral administration, were gifted from Sankyo Co, Ltd (Tokyo, Japan).²⁸ All antibodies used were purchased from Santa Cruz Biotechnology, Inc and were as follows: polyclonal rabbit anti-p44ERK (ERK-1) IgG (c-16); polyclonal rabbit anti-p42ERK (ERK-2) IgG (c-14); polyclonal rabbit anti-p46JNK (JNK-1) IgG (c-17), which recognizes not only p46JNK but also p55JNK; and polyclonal rabbit anti-p55JNK (JNK-2) IgG (FL). Recombinant protein A–agarose (50%, vol/vol) was purchased from Upstate Biotechnology.

Animals and Experimental Design
All procedures were in accordance with institutional guidelines for the care and use of laboratory animals. Nine-week-old male Sprague-Dawley rats (Clea Japan) were used in this study. They were fed a standard laboratory chow (CE-2, Clea Japan) and given tap water ad libitum.

The first experiments were performed to examine the time course of cardiac and aortic JNK and ERK activities in rats subjected to continuous infusion of a pressor dose of Ang II (100 or 1000 ng · kg^-1 · min^-1). Rats were anesthetized with sodium pentobarbital (50 mg/kg IP), and polyethylene catheters were inserted into the left femoral vein and artery for continuous infusion of Ang II and for direct measurement of arterial pressure, respectively. Both catheters were tunneled through the subcutaneous tissue to exteriorize in the dorsal midcervical region of the neck. The rats were then allowed to recover from anesthesia and surgical procedure for about 20 hours until start of the experiments. All experiments were performed on conscious rats. Ang II dissolved in saline or saline alone (control) was continuously infused to rats via the femoral vein catheter by syringe infusion pump (Harvard Apparatus, Ltd) at a flow rate of 2 mL/h for specified times. To infuse rats with Ang II for 24 hours, an Alzet osmotic minipump (Alza Corp) containing Ang II dissolved in saline or saline alone (control) was connected to the catheter inserted into the femoral vein and implanted subcutaneously in the rat. Blood pressure was monitored by a pressure transducer (P231D, Nihon Kohden) and recorded on a polygraph (RM 6100, Nihon Kohden). At specified times after start of Ang II infusion, rats were decapitated and the heart and thoracic aorta were rapidly excised and immersed in precooled phosphate-buffered saline (pH 7.4) containing 2.5 mmol/L EDTA, 2 mmol/L ß-glycerophosphate, 10 mmol/L NaF, 1 mmol/L Na₃VO₄, and 1 mmol/L PMSF, as described.²² Then the left and right ventricles were rapidly separated from the atria in the above buffer on ice, immediately frozen in liquid nitrogen, and stored at -80°C until use. The thoracic aorta was also rapidly dissected from adherent fat and connective tissues in the above buffer on ice, frozen in liquid nitrogen, and stored at -80°C until use. Extreme care was taken to be certain that the aorta was not stretched on dissection.

The second experiments were undertaken to examine the dose-response relations for an Ang II–induced rise in blood pressure and cardiac and aortic MAP kinase activation. All experiments were performed on conscious rats subjected to the insertion of a catheter into the femoral artery and vein, as described in the first experiments. Ang II dissolved in saline, at a dose of 1, 10, 100, or 1000 ng · kg^-1 · min^-1, or saline alone (control) was continuously infused to rats for 15 minutes via the femoral vein catheter by syringe infusion pump at a flow rate of 2 mL/h. Direct blood pressure was continuously monitored throughout the infusion. After 15 minutes of infusion, rats were decapitated and the left ventricle and thoracic aorta were rapidly taken, trimmed, and frozen in liquid nitrogen in the same manner as in the first experiments. To examine the contribution of the AT₁ receptor to MAP kinase activation in vivo, 0.1 mg/kg RNH-6270 dissolved in saline was injected via the femoral vein catheter 30 minutes before start of 1000 ng · kg^-1 · min^-1 Ang II infusion.

In the third experiments, chronic Ang II infusion, causing cardiac hypertrophy, was carried out to examine the role of MAP kinases and AP-1 in cardiac hypertrophy. An Alzet osmotic minipump containing saline-dissolved Ang II or saline alone (control) was implanted subcutaneously in the rat under ether anesthesia, and Ang II (400 ng · kg^-1 · min^-1, SC) was infused into rats for 15 and 30 minutes; 1, 3, 6, and 24 hours; and 3 and 7 days. Systolic blood pressure of conscious rats was measured by the tail-cuff method. After the infusion, rats were decapitated and the heart was immediately excised. The left ventricle was separated from atria and right ventricle, as described above, weighed, and separated into 2 portions. Half of the left ventricle was frozen and stored at -80°C until measurement of MAP kinase activities. The other half was rapidly homogenized to prepare nuclear protein extracts for gel mobility shift assay, as described below. To examine the role of the AT₁ receptor in cardiac MAP kinase activity and AP-1 DNA binding activity, CS-866 (10 mg · kg^-1 · d^-1), a long-acting specific AT₁ receptor antagonist that is a prodrug of RNH-6270, suspended with 0.5% carboxymethylcellulose, was orally given to rats once a day from 1 day before start of Ang II infusion to the end of the infusion.

Measurement of Cardiac and Aortic MAP Kinase Activities
Cardiac and aortic MAP kinase activities were measured by using in-gel kinase method, as previously described in detail.^{22 29} In our previous study,²² we demonstrated that cardiac ERK and JNK can be successfully quantified by direct in-gel kinase assay. Glutathione S-transferase (GST)–c-Jun(1–79) and myelin basic protein were used as the substrate for JNK and ERK assays, respectively. In brief, frozen left or right ventricle or thoracic aorta from each rat was homogenized on ice with a polytron homogenizer (PCU-11, Kinematica AG) in 20 mmol/L HEPES (pH 7.2), containing 25 mmol/L NaCl, 2 mmol/L EGTA, 50 mmol/L NaF, 1 mmol/L Na₃VO₄, 25 mmol/L ß-glycerophosphate, 0.2 mmol/L DTT, 1 mmol/L PMSF, 60 µg/mL aprotinin, 2 µg/mL leupeptin, and 0.1% Triton X-100, and incubated on ice for 30 minutes. After the homogenates were sonicated on ice for 15 seconds (SONIFIER 250, Branson Ultrasonics Co), protein extracts were obtained by centrifugation for 30 minutes at 15 000 rpm at 4°C. Cardiac or aortic protein extracts (40 µg and 10 µg, for JNK and ERK assay, respectively), denatured in Laemmli sample buffer, were electrophoresed on SDS-polyacrylamide (12%) gel containing 0.1 mg/mL glutathione S-transferase–c-Jun(1–79) for JNK assay or 0.5 mg/mL myelin basic protein for ERK assay. After electrophoresis, protein kinases in the gels were denatured by guanidine-HCl and renatured in Tris-HCl (pH 8.0), and the gels were incubated with [-³²P]ATP, washed extensively, dried, and subjected to autoradiography. The densities of autoradiograms were measured by using a bioimaging analyzer (BAS-2000, Fuji Photo Film Co).

Immune Complex In-Gel Kinase Assay
We also performed in-gel kinase assays after immunoprecipitation of cardiac ERK and JNK with their specific antibodies. Immunoprecipitation of ERK and JNK was performed as previously described.²² In brief, the samples of cardiac protein extracts (100 µg of protein) were preabsorbed with 10 µL of recombinant protein A–agarose (50%, vol/vol) at 4°C for 2 hours and then centrifuged at 10 000g at 4°C for 15 minutes. The resulting supernatants were incubated with combined anti-p42ERK IgG (6 µg) and anti-p44ERK IgG (0.3 µg) for ERK assays, or combined anti-p46JNK IgG (1 µg) and anti-p55JNK IgG (0.5 µg) for JNK assays at 4°C for 2 hours, and were then added to 40 µL of recombinant protein A–agarose, followed by incubation at 4°C for 12 hours. After centrifugation at 800g for 10 minutes, the pellets were washed 4 times with lysis buffer containing 0.5 mol/L NaCl. Finally, the pellets were suspended with 25 µL of lysis buffer. The immunoprecipitates were boiled for 5 minutes in Laemmli sample buffer containing 1 mmol/L Na₃VO₄, centrifuged, and the resulting supernatants subjected to in-gel kinase assay of ERKs or JNKs, as described above.

Gel Mobility Shift Assay
For gel mobility shift assay, left ventricular nuclear protein extracts were rapidly prepared from chronically Ang II–infused rats, according to the method of Schreiber et al,³⁰ with minor modification. Left ventricular tissue was rapidly homogenized with a Dounce homogenizer in 10 vol of 10 mmol/L HEPES (pH 7.9), containing 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1.5 mmol/L MgCl₂, 10 mmol/L NaF, 1 mmol/L Na₃VO₄, 1 mmol/L DTT, 20 mmol/L ß-glycerophosphate, 0.5 mmol/L PMSF, 60 µg/mL aprotinin, and 2 µg/mL leupeptin, and was incubated on ice for 15 minutes. After the addition of Nonidet P-40 to 0.6%, the homogenate was vigorously vortex-mixed for 10 seconds. The nuclear fraction was precipitated by centrifugation at 5000 rpm for 10 minutes at 4°C and then resuspended in 20 mmol/L HEPES (pH 7.9), 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1.5 mmol/L MgCl₂, 20% glycerol, 10 mmol/L NaF, 1 mmol/L Na₃VO₄, 0.2 mmol/L DTT, 20 mmol/L ß-glycerophosphate, 0.5 mmol/L PMSF, 60 µg/mL aprotinin, and 2 µg/mL leupeptin. The mixture was incubated on ice for 15 minutes, centrifuged at 15 000 rpm at 4°C for 10 minutes, and the resulting supernatant was stored at -80°C until use.

The detailed procedure of gel mobility shift assay has been previously described.³¹ In brief, the samples of ventricular nuclear extracts (10 µg protein) were incubated with 10 fmol of a ³²P-labeled oligonucleotide probe containing the consensus AP-1 binding sequence (5'-CGCTTGATGACTCAGCCGGAA-3') at room temperature for 20 minutes, in 20 µL of binding buffer, consisting of (mmol/L) HEPES (pH 7.9) 20, EDTA 0.2, EGTA 0.2, NaCl 80, MgCl₂ 0.3, DTT 1, PMSF 0.2, 6% glycerol, and 2 µg of poly[dI-dC] (Pharmacia) as a nonspecific competitor. For competition experiments, mutant AP-1 oligonucleotide competitor (5'-CGCTTGATGACTTGGCCGGAA-3') was also used. The DNA-protein complexes were electrophoresed on 4% nondenaturing polyacrylamide gels, and the gels were then dried, subjected to autoradiography, and analyzed with a bioimaging analyzer (BAS-2000). Supershift assays were performed with rabbit polyclonal anti–c-Fos IgG or anti–c-Jun IgG (Santa Cruz Biotechnology, Inc). Each antibody (1 µg) was added to samples after the initial binding reaction between ventricular nuclear extracts and ³²P-labeled consensus AP-1 oligonucleotide, and the reaction was allowed to proceed at room temperature for 1 hour and subjected to electrophoresis as described above.

Statistical Analysis
Data are expressed as mean±SEM. Statistical significance between more than 2 groups was tested using 1-way ANOVA followed by the Student-Newman-Keuls test. If the data exhibited significant heterogeneity of variance, statistical analysis was performed on log-transformed data. Statistical analysis of blood pressure (Figure 1A) was performed by 2-way ANOVA followed by the least-squares means test (SuperANOVA, Abacus Concepts). Statistical significance between 2 groups was tested using the unpaired Student's t test. Differences were considered statistically significant at a value of P<0.05.

View larger version (30K): [in this window] [in a new window]   Figure 1. Time course of blood pressure and left ventricular JNK and ERK activities in Ang II (1000 ng · kg-1 · min-1)–infused rats. A, Line graph showing time course of mean blood pressure in Ang II– or saline-infused rats. The start of Ang II infusion is indicated by the arrow. B, The upper panel shows representative autoradiograms of p46JNK and p55JNK activities at each time point and the bar graph shows left ventricular p46JNK and p55JNK activities. The mean value of each JNK isoform at 0 minutes is represented as 1. C, The upper panel shows representative autoradiograms of p42ERK and p44ERK activities at each time point and the bar graph shows left ventricular p42ERK and p44ERK activities. The mean value of each ERK isoform at 0 minutes is represented as 1. Each value represents mean±SEM (n=6 to 8). *P<0.01 and P<0.05 compared with 0 minutes.

	Results

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Time Course of Blood Pressure and Cardiac JNK and ERK Activities in Ang II (1000 ng · kg^-1 · min^-1)–Infused Rats
As shown in Figure 1A, blood pressure of conscious rats was rapidly increased after start of Ang II infusion (1000 ng · kg^-1 · min^-1), peaked at 5 to 15 minutes (about 50 mm Hg increase), thereafter gradually decreased, and returned to almost control level by 24 hours. As shown in Figure 1B, left ventricular p46JNK and p55JNK activities rapidly increased, reached the peak (6.7- and 5.7-fold increase, respectively) at 15 minutes, sustained the peak value up to at least 3 hours, and returned to control value by 24 hours. Thus, left ventricular JNK retained the peak value for a much longer period than did blood pressure. As shown in Figure 1C, left ventricular p42ERK had very high basal activities in vivo compared with p44ERK, although it is unknown whether this high basal activity of p42ERK was due to its high phosphorylation rate or the increased protein expression compared with p44ERK. The increase in left ventricular ERK activity by Ang II infusion was smaller than that in JNK. p44ERK activity reached the peak (about 3.1-fold increase) at 15 minutes and retained the peak value until 3 hours, while p42ERK activity transiently increased by 2.2-fold only at 15 minutes.

Figure 2 shows right ventricular JNK and ERK activities in Ang II (1000 ng · kg^-1 · min^-1)–infused rats. Both p46JNK and p55JNK activities in the right ventricle significantly increased at either 15 minutes or 3 hours and returned to control value at 24 hours, similar to left ventricular JNK activity. On the other hand, right ventricular p44ERK or p42ERK activity was not significantly changed by Ang II infusion at any time point examined.

View larger version (27K): [in this window] [in a new window]   Figure 2. Time course of right ventricular JNK and ERK activities in Ang II (1000 ng · kg-1 · min-1)–infused rats. A, The upper panel shows representative autoradiograms of JNK activities at each time point and the bar graph shows right ventricular p46JNK and p55JNK activities. The mean value of each JNK isoform at 0 minutes is represented as 1. B, The upper panel shows representative autoradiograms of ERK activities at each time point and the bar graph shows right ventricular p42ERK and p44ERK activities. The mean value of each ERK isoform at 0 minutes is represented as 1. Each value represents mean±SEM (n=6 to 8). *P<0.01 and P<0.05 compared with 0 minutes.

Time Course of Aortic JNK and ERK Activities in Ang II (1000 ng · kg^-1 · min^-1)–Infused Rats
As shown in Figure 3, in the aorta, JNK and ERK activities were significantly increased by Ang II infusion (1000 ng · kg^-1 · min^-1), with the peak at 5 to 15 minutes (more than several-fold increase), but thereafter rapidly decreased and returned to control levels by 3 hours. Thus, the duration of aortic JNK activation by Ang II infusion in vivo was shorter than that of cardiac JNK.

View larger version (33K): [in this window] [in a new window]   Figure 3. Time course of aortic JNK and ERK activities in Ang II (1000 ng · kg-1 · min-1)–infused rats. A, The upper panel shows representative autoradiograms of JNK activities at each time point and the bar graph shows aortic p46JNK and p55JNK activities. The mean value of each JNK isoform at 0 minutes is represented as 1. B, The upper panel shows representative autoradiograms of ERK activities at each time point and the bar graph shows aortic p42ERK and p44ERK activities. The mean value of each ERK isoform at 0 minutes is represented as 1. Each value represents mean±SEM (n=6 to 8). *P<0.01 and P<0.05 compared with 0 minutes.

Dose-Response Relationships for Ang II–Induced Increase in Blood Pressure and Activation of Left Ventricular and Aortic JNK and ERK
Figure 4 shows the dose-response relations of Ang II and blood pressure. Continuous Ang II infusion at the dose of 1 ng · kg^-1 · min^-1 did not change blood pressure throughout 15 minutes of the infusion. Ang II infusion at the dose of 10, 100, and 1000 ng · kg^-1 · min^-1 significantly increased blood pressure in a dose-dependent manner, by 23, 41, and 47 mm Hg, respectively, at 15 minutes. Pretreatment with RNH-6270, a selective AT₁ receptor antagonist, almost completely blocked Ang II (1000 ng · kg^-1 · min^-1)–induced increase in blood pressure.

View larger version (19K): [in this window] [in a new window]   Figure 4. Dose dependency of Ang II–induced rise in blood pressure. Ang II (1), Ang II (10), Ang II (100), and Ang II (1000) indicate continuous Ang II infusion at doses of 1, 10, 100, and 1000 ng · kg-1 · min-1, respectively. Ang II (1000)+RNH indicates 1000 ng · kg-1 · min-1 Ang II with pretreatment of bolus intravenous injection of RNH-6270 (0.1 mg/kg). Values represent mean±SEM (n=5 to 10). *P<0.01 and P<0.05 compared with 0 minutes.

Figure 5 illustrates the dose dependency of activation of left ventricular JNK and ERK. Fifteen minutes of Ang II infusion at 1 ng · kg^-1 · min^-1 (a subpressor dose, as shown in Figure 4) or 10 ng · kg^-1 · min^-1 (a mild pressor dose, as shown in Figure 4) did not significantly increase left ventricular JNK or ERK activities. Ang II infusion at 100 ng · kg^-1 · min^-1 (causing about 40 mm Hg rise in blood pressure, as shown in Figure 4) increased left ventricular p46JNK and p55JNK activities by 5.2- and 4.5-fold, respectively. On the other hand, unlike JNK, left ventricular p42ERK or p44ERK activity was not increased by this dose of Ang II infusion. Both JNK and ERK were significantly activated by 1000 ng · kg^-1 · min^-1 Ang II, which was completely blocked by pretreatment with RNH-6270. Thus, the in vivo activation of left ventricular JNK by Ang II infusion occurred in a more sensitive manner than that of ERK.

View larger version (39K): [in this window] [in a new window]   Figure 5. Relationship between the dose of Ang II infusion and activation of left ventricular JNK and ERK. Ang II, at a dose of 1, 10, 100, or 1000 ng · kg-1 · min-1, was continuously infused to conscious rats for 15 minutes, and left ventricular tissue was taken for measurement of ERK and JNK activities. The upper panel shows representative autoradiograms of JNK and ERK activities from control rats (saline infused) and various doses of Ang II–infused rats. The bar graph shows left ventricular p46JNK, p55JNK, p42ERK, and p44ERK activity. The mean value of each JNK and ERK isoform from control rats is represented as 1. Each bar represents mean±SEM (n=6 to 12). *P<0.01 compared with control group.

Furthermore, to confirm that Ang II (100 ng · kg^-1 · min^-1) infusion does not significantly increase cardiac ERK activities, we measured cardiac ERK activities from rats subjected to intravenous infusion of Ang II (100 ng · kg^-1 · min^-1) for 5, 15, and 30 minutes, using the immune complex in-gel kinase assay. Figure 6A and 6B showed that the binding activity of anti-p44ERK IgG (C-16) was greater than that of anti-p42ERK IgG (C-14). Based on these results, cardiac protein extracts (100 µg) were immunoprecipitated with 0.3 µg of anti-p44ERK IgG and 6 µg of anti-p42ERK IgG, for in-gel kinase assay. As shown by the immune complex in-gel kinase assay in Figure 6C, left ventricular p42ERK or p44ERK activity was not increased by 5, 15, or 30 minutes of Ang II (100 ng · kg^-1 · min^-1) infusion, confirming the observations obtained by direct in-gel kinase assay (Figure 5). We also measured left ventricular JNK activity in Ang II (100 ng · kg^-1 · min^-1)–infused rats, using the immune complex in-gel kinase assay, and found that left ventricular p46JNK and p55JNK activities increased by 3.4- and 2.1-fold (n=3; P<0.01), respectively, after 15 minutes of Ang II infusion and by 4.9- and 3.1-fold (n=3; P<0.01), respectively, after 30 minutes, consistent with the findings obtained by direct in-gel kinase assay (Figure 5).

View larger version (20K): [in this window] [in a new window]   Figure 6. Cardiac ERK activities from control and Ang II (100 ng · kg-1 · min-1)–infused rats, measured by the immune complex in-gel kinase assay. A, Control rat cardiac extracts (200 µg protein) were immunoprecipitated with 10 µg of normal rabbit IgG (1) and anti-p44ERK IgG (C-16), 0.3 µg (2), 1 µg (3), 3 µg (4), and 10 µg (5) and were subjected to in-gel kinase assay, as described in Materials and Methods. p44ERK in 200 µg cardiac protein extracts could be sufficiently immunoprecipitated even with 0.3 µg of anti-p44ERK IgG (C-16). The same experiments were also performed on Ang II–infused rat cardiac extracts, and similar results were obtained. B, Control rat cardiac extracts (200 µg protein) were immunoprecipitated with 10 µg of normal rabbit IgG (1) and anti-p42ERK IgG (C-14), 0.3 µg (2), 1 µg (3), 3 µg (4), and 10 µg (5) and were subjected to in-gel kinase assay. In contrast to anti-p44ERK IgG, anti-p42ERK IgG dose dependently immunoprecipitated p42ERK in 200 µg cardiac protein extracts. When 200 µg cardiac protein extracts were used, the maximum immunoprecipitation of p42ERK was attained by 3 or 10 µg anti-p42ERK IgG. The same experiments were also performed on Ang II–infused rat cardiac extracts, and similar results were obtained. C, Cardiac protein extracts (100 µg) from control rats and rats infused with 5, 15, or 30 minutes of Ang II (100 ng · kg-1 · min-1) were immunoprecipitated with anti-p44ERK IgG (C-16), 0.3 µg, and anti-p42ERK IgG (C-14), 6 µg, followed by in-gel kinase assay, as described in Materials and Methods. The upper panel shows representative autoradiograms of ERK activities from control and Ang II–infused rats. The mean value of each ERK isoform in the control group (0 minutes) is represented as 1. Each bar represents mean±SEM (n=4 to 5).

Figure 7 depicts the dose dependency of activation of aortic JNK and ERK. As in the left ventricle, Ang II infusion at 1 or 10 ng · kg^-1 · min^-1 did not activate aortic JNK or ERK. In contrast to the case of left ventricular ERK, 100 ng · kg^-1 · min^-1 Ang II significantly activated not only aortic JNK but also aortic ERK. Aortic JNK and ERK activation by Ang II (1000 ng · kg^-1 · min^-1) was prevented by RNH-6270.

View larger version (39K): [in this window] [in a new window]   Figure 7. Relationship between the dose of Ang II infusion and activation of aortic JNK and ERK. Ang II, at a dose of 1, 10, 100, or 1000 ng · kg-1 · min-1, was continuously infused to conscious rats for 15 minutes, and the aorta was taken for measurement of ERK and JNK activities. The upper panel shows representative autoradiograms of JNK and ERK activities from control rats (saline infused) and rats infused with various doses of Ang II. The bar graph shows aortic p46JNK, p55JNK, p42ERK, and p44ERK activity. The mean value of each JNK and ERK isoform from control rats is represented as 1. Each bar represents mean±SEM (n=6 to 12). *P<0.01 and P<0.05 compared with control group.

Effect of Chronic Ang II Infusion on Blood Pressure, Left Ventricular Weight, and MAP Kinases
To examine the possible involvement of MAP kinases in Ang II–induced cardiac hypertrophy and remodeling in vivo, Ang II (400 ng · kg^-1 · min^-1, SC) was chronically infused into rats by osmotic minipump and the effects on left ventricular JNK and ERK activities were examined. As shown in Figure 8A, blood pressure of Ang II–infused rats was significantly increased compared with saline-infused rats (control) at 3 days (143±3 versus 119±4 mm Hg; P<0.01) and 6 days (155±9 versus 120±4 mm Hg; P<0.01). Treatment with CS-866 completely blocked Ang II–induced elevation of blood pressure. As shown in Figure 8B, left ventricular weight of Ang II–infused rats was significantly increased compared with control at 3 days (2.33±0.04 versus 2.00±0.02 mg/g body weight; P<0.01) and 7 days (2.60±0.06 versus 2.07±0.04 mg/g body weight; P<0.01), which was completely prevented by CS-866 treatment.

View larger version (25K): [in this window] [in a new window]   Figure 8. Time course of blood pressure (A) and left ventricular weight corrected for body weight (B) of chronically Ang II–infused rats. Control indicates saline-infused group; Ang II, Ang II–infused group; and Ang II+CS-866, Ang II–infused and CS-866–treated group. Each bar represents mean±SEM (n=4 to 6). *P<0.01 compared with Ang II.

As shown in Figure 9A, left ventricular p46JNK and p55JNK activities were gradually increased after start of continuous Ang II infusion (400 ng · kg^-1 · min^-1, SC) by osmotic minipump, reached the peak (2.8- and 2.7-fold increase, respectively; P<0.05) after 3 hours, thereafter gradually returned to control levels, and were inversely decreased to 49% and 54%, respectively, of control levels at 7 days. On the other hand, as shown in Figure 9B, left ventricular p42ERK or p44ERK activities were not changed throughout 7 days of Ang II infusion.

View larger version (50K): [in this window] [in a new window]   Figure 9. Effect of chronic Ang II infusion on left ventricular JNK (A) and ERK (B) activities. C indicates saline-infused group (control); A, Ang II–infused group. Ang II (400 ng · kg-1 · min-1) or saline was subcutaneously infused to rats by osmotic minipump for 15 or 30 minutes; 1, 3, 6, or 24 hours; or 3 or 7 days. Each bar represents mean±SEM (n=4 to 6). The mean value of each JNK or ERK isoform in the control group at each time point is represented as 1. *P<0.01 and P<0.05 versus control (saline-infused group).

Effect of Chronic Ang II Infusion on Left Ventricular AP-1 DNA Binding Activity
We examined the effect of chronic Ang II infusion (400 ng · kg^-1 · min^-1, SC) on left ventricular AP-1 DNA binding activity. As shown in Figure 10A, gel mobility shift assay of left ventricular nuclear extracts, using labeled AP-1 consensus oligonucleotide probe, produced 2 major bands. The upper band, designated with a half bracket, was efficiently competed for increasing concentrations of a cold AP-1 oligonucleotide, but not by a mutant AP-1 probe, indicating that this band represented a specific AP-1 DNA binding. Furthermore, the addition of anti–c-Fos or anti–c-Jun antibody to the binding reaction resulted in the decrease in this band, accompanied by the appearance of supershifted complexes, indicating that specific AP-1 DNA binding complex contained c-Fos and c-Jun proteins. As shown in Figure 10B, left ventricular AP-1 DNA binding activity was increased by 1.7-fold at 6 hours after start of Ang II infusion (400 ng · kg^-1 · min^-1, SC), reached the peak value (3.2-fold) at 24 hours, and thereafter decreased. Treatment with CS-866 completely inhibited Ang II–induced increase in AP-1 binding activity.

View larger version (46K): [in this window] [in a new window]   Figure 10. Effect of chronic Ang II infusion on left ventricular AP-1 DNA binding activity. A, Left ventricular nuclear extracts from rats infused with Ang II for 24 hours by osmotic minipump were incubated with a 32P-labeled AP-1 consensus oligonucleotide probe in the absence of unlabeled AP-1 oligonucleotide probe (–), and in the presence of 10-, 30-, 100-, and 200-fold molar excess of unlabeled AP-1 probe (x10, x30, x100, and x200, respectively) and 200-fold molar excess of unlabeled mutant AP-1 probe (x200 mutant AP-1). Furthermore, supershift assays were performed with anti–c-Fos IgG (Anti-c-Fos) and anti–c-Jun IgG (Anti-c-Jun). The upper band designated with a half bracket indicates specific AP-1 DNA binding complexes. AP-1 oligo indicates unlabeled AP-1 oligonucleotide competitor; NS, nonspecific binding; and F, free probe. The arrows indicate supershifted complexes. B, The upper panel shows a representative autoradiogram of gel mobility shift assay of left ventricular specific AP-1 DNA binding activity from 3 groups of rats at 24 hours. The lower panel shows left ventricular AP-1 DNA binding activity from Ang II–infused rats for 6 and 24 hours and 3 and 7 days. Control indicates saline-infused group; Ang II, Ang II–infused group; Ang II+CS-866, Ang II–infused and CS-866-treated group. Each bar represents mean±SEM (n=4 to 6). The mean value of AP-1 binding activity in the control group at each time point is expressed as 1. *P<0.01 and P<0.05 versus control (saline-infused group).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although the mechanism of activation of MAP kinases has been extensively studied in cultured cells, the regulation and function of MAP kinases in vivo are poorly understood. Furthermore, previous information on cardiac hypertrophy and MAP kinases has been almost limited to the data obtained from neonatal rat cardiac myocytes, and it is an open question whether the mechanism of hypertrophy in neonatal rat heart can apply to adult heart.³² Recently, Xu et al,³³ who have examined the in vivo effects of hypertension on ERK2 (p42ERK) and JNK1 (p46JNK) activities in various tissues of rats by immunocomplex kinase assay, have found that either restraint-induced persistent hypertension or acute hypertension induced by a bolus injection of hypertensive agents (eg, Ang II) significantly activates vascular p42ERK and p46JNK to a similar extent. In contrast to the significant activation of p42ERK and p46JNK in vascular tissue, these investigators have found no activation of cardiac p42ERK or p46JNK by restraint, which causes the continuous elevation of blood pressure by about 40 mm Hg.³³ However, in their work, cardiac MAP kinase activities were examined only in restraint-induced hypertensive rats, but not in hypertensive agent–induced hypertensive rats, which encouraged us to examine the effects of Ang II–elicited hypertension on cardiac MAP kinases in detail. Furthermore, in our present study, by successfully using in-gel kinase assay instead of immunocomplex kinase assay, we could measure not only p42ERK and p46JNK but also p44ERK and p55JNK.

Multiple lines of evidence indicate that Ang II play a key role in the development of pathological cardiac hypertrophy.^{23 24 25 26} Continuous infusion of Ang II to rats causes cardiac hypertrophy and fibrosis.^{34 35 36} Furthermore, we have previously examined the effects of continuous Ang II infusion on cardiac gene expressions of rats and demonstrated that Ang II infusion, via the AT₁ receptor, increases cardiac expression of fetal phenotypes of genes such as ANF, skeletal -actin, and ß-myosin heavy chain and cardiac fibrosis–related genes such as transforming growth factor-ß1 and collagen type I or type III.³⁴ However, the signal-transduction pathway underlying cardiac hypertrophy and gene expressions by Ang II infusion in vivo remains to be determined. In our present work, to elucidate the possible activation of cardiac MAP kinases by Ang II, we examined the time course of cardiac MAP kinase activities in Ang II–infused rats at the dose of 1000 ng · kg^-1 · min^-1, which is known to cause about a 3-fold increase in plasma Ang II levels (within pathophysiological levels).³⁷ We obtained the first evidence that left ventricular p46JNK, p55JNK, p42ERK, and p44ERK are significantly activated by Ang II infusion. The inhibition of cardiac JNK and ERK activations by RNH-6270 showed AT₁ receptor-mediated activation of these kinases. Interestingly, in the right ventricle not affected by hypertension-induced hemodynamic stress, JNK was significantly activated by Ang II infusion, whereas there was no activation of ERK. These observations suggest that cardiac JNK activation by Ang II infusion may be in part due to its direct action via the AT₁ receptor, whereas cardiac ERK activation may be exclusively mediated by the elevation of blood pressure.

In cultured neonatal rat cardiac myocytes, mechanical stretch and Ang II significantly activates both JNK^{18 19} and ERK.^{4 5 6} However, at present, it is unclear which kinase is more readily activated by these hypertrophic stimuli, JNK or ERK. To determine how cardiac JNK and ERK are sensitive to Ang II in vivo, we examined the dose-response relationships for Ang II–induced rise in blood pressure and cardiac ERK and JNK activations. We found no activation of cardiac JNK and ERK by a subpressor dose (1 ng · kg^-1 · min^-1) of Ang II infusion or a mild pressors dose (10 ng · kg^-1 · min^-1) of Ang II infusion, with about 20 mm Hg rise in blood pressure, indicating that mild changes in blood pressure, which healthy humans often encounter, cannot activate cardiac JNK or ERK. Of note are the observations that Ang II infusion at 100 ng · kg^-1 · min^-1, causing moderate rise in blood pressure (about 40 mm Hg increase), significantly activated cardiac p46JNK and p55JNK but failed to activate cardiac p42ERK or p44ERK (Figures 5 and 6), demonstrating that Ang II–induced cardiac activation of JNK in vivo occurs in a more sensitive manner than that of ERK. Furthermore, left ventricular hypertrophy, induced by chronic Ang II infusion, was preceded by the significant activation of JNK without ERK activation. These observations, taken together with our recent findings that the prevention of cardiac hypertrophy in SHRSP by an angiotensin-converting enzyme inhibitor is accompanied by a decrease in cardiac p46JNK and p55JNK activities but not by a decrease in p42ERK or p44ERK activity,²² support the notion that JNK may play some role in Ang II–induced cardiac hypertrophic response in vivo. However, the involvement of p42ERK cannot necessarily be excluded, because cardiac p42ERK had very high basal activity in vivo, as shown by in-gel kinase assay.

In the present study, we found that left ventricular AP-1 DNA binding activity was significantly increased by Ang II infusion via the AT₁ receptor. Supershift analysis showed that c-Fos and c-Jun contributed to the increased AP-1 binding activity. Interestingly, this AP-1 activation in Ang II–infused rats was preceded by JNK activation. JNK is well known to increase c-Jun transactivational activity by phosphorylating c-Jun on 2 critical N-terminal serines^{12 15} and to induce c-fos gene expression.³⁸ Therefore, it has been well established that JNK is involved in the activation of transcription factor, AP-1.³⁹ These findings suggest that AP-1 activation in Ang II–induced cardiac hypertrophy may be in part mediated by JNK activation, although our present study did not provide direct evidence for it.

AP-1 importantly regulates the expression of various genes by binding AP-1 consensus sequences present in their promoter region.^{16 39} Interestingly, fetal phenotypes of cardiac genes such as skeletal -actin⁴⁰ and ANF³⁹ and cardiac fibrosis–associated genes such as transforming growth factor-ß1⁴¹ and collagen type I⁴² have an AP-1–responsive sequence in their promoter region. Indeed, AP-1 activation has been demonstrated to lead to the increased promoter activity of skeletal -actin⁴⁰ and transforming growth factor-ß1.⁴¹ Furthermore, we have previously reported that the expression of the above-mentioned cardiac genes is significantly enhanced in Ang II–infused rats, via the AT₁ receptor.³⁴ However, further detailed work must be awaited to demonstrate the possibility that the activation of AP-1 by Ang II infusion may be responsible for the above-mentioned cardiac hypertrophy-associated gene expressions.

Of note, in spite of the rapid decline of the peak value of blood pressure by Ang II infusion (1000 ng · kg^-1 · min^-1), the maximal activation of cardiac p46JNK, p55JNK, and p44ERK lasted for much longer. On the other hand, the increase in aortic JNK and ERK activity rapidly returned to control levels as rapidly as blood pressure. Very recently, we have examined the effect of balloon injury on rat arterial JNK and ERK activities and found that arterial JNK and ERK activities are increased by balloon injury but rapidly returned to control levels,³¹ similar to our present results on activation of aortic JNK and ERK by Ang II infusion. These findings suggest that there may be differential activation kinetics of JNK and p44ERK between cardiac and vascular tissues and that long duration of JNK and p44ERK activation by Ang II infusion may be the unique event in the heart. Furthermore, although 100 ng · kg^-1 · min^-1 Ang II infusion activated JNK but not ERK in the heart, this dose of Ang II activated ERK as much as JNK in the aorta, thereby showing that the preferential activation of JNK by Ang II in vivo is specific for the heart. Thus, the regulatory mechanism and role of ERK and JNK in vivo seem to differ between cardiac and vascular tissues.

p38,^{2 3 16} also known as protein kinase HOG1, is the third member of the MAP kinase family and is shown to be activated by cell stresses such as endotoxins, osmotic shock, or metabolic inhibitors, although it is not commonly activated by mitogens. Very recently, Zechner et al⁴³ have shown that the activation of p38 in neonatal rat cardiac myocytes, by transfection with MKK6, augments cell size, enhances ANF and skeletal -actin promoter activities, and elicits sarcomeric organization, supporting the important role of p38 in myocardial cell hypertrophy. However, there is no report on the effect of Ang II on cardiac p38 activity in vitro or in vivo. Therefore, further investigation on p38, as well as ERK and JNK, is important to elucidate the molecular mechanism of cardiac hypertrophy in vivo.

In conclusion, our present study provided the first evidence that both cardiac JNK and ERK are activated by Ang II in vivo but that JNK activation occurs in a more sensitive manner than ERK activation. Furthermore, AP-1 activation in Ang II–induced cardiac hypertrophy may be in part due to JNK activation. Our in vivo observations, taken together with the previous in vitro evidence for the important contribution of JNK in the hypertrophic response of cultured cardiac myocytes,^{20 21} suggest that JNK may play some role in the Ang II–induced cardiac hypertrophic response. However, further in vivo study is needed to demonstrate our proposal.

	Acknowledgments

 
This work was supported in part by Grants-in-Aid for Scientific Research 09670101 and 09470527 from the Ministry of Education, Science, Sports and Culture. RNH-6270 and CS-866 were gifted from Sankyo Co, Ltd, Tokyo, Japan. The authors are grateful to Eriko Gomi for technical assistance and Kazuko Tsukahara for in-gel kinase assays.

Received January 7, 1998; accepted July 9, 1998.

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