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(Circulation Research. 2003;93:874.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Apoptosis Signal-Regulating Kinase 1 Plays a Pivotal Role in Angiotensin II–Induced Cardiac Hypertrophy and Remodeling

Yasuhiro Izumiya, Shokei Kim, Yasukatsu Izumi, Kaoru Yoshida, Minoru Yoshiyama, Atsushi Matsuzawa, Hidenori Ichijo, Hiroshi Iwao

From the Department of Pharmacology (Y.I., S.K., Y.I., K.Y., H. Iwao), Osaka City University Graduate School of Medical Science, Osaka, Japan; Department of Internal Medicine and Cardiology (M.Y.), Osaka City University Graduate School of Medical Science, Osaka, Japan; Laboratory of Cell Signaling (A.M., H. Ichijo), Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.

Correspondence to Shokei Kim, Department of Pharmacology, Osaka City University Graduate School of Medical Science, 1-4-3 Asahimachi, Abeno, Osaka 545-8585, Japan. E-mail kims{at}med.osaka-cu.ac.jp

	Abstract

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Multiple lines of evidence establish that angiotensin II (Ang II) induces not only hypertension but also directly contributes to cardiac diseases. Apoptosis signal-regulating kinase 1 (ASK1), one of mitogen-activated protein kinase kinase kinases, plays a key role in stress-induced cellular responses. However, nothing is known about the role of ASK1 in cardiac hypertrophy and remodeling in vivo. In this study, by using mice deficient in ASK1 (ASK1^-/- mice), we investigated the role of ASK1 in cardiac hypertrophy and remodeling induced by Ang II. Left ventricular (LV) ASK1 was activated by Ang II infusion in wild-type mice, which was mediated by angiotensin II type 1 receptor and superoxide. Although Ang II-induced hypertensive effect was comparable to wild-type and ASK1^-/- mice, LV ASK1 activation by Ang II was not detectable in ASK1^-/- mice, and p38 and c-Jun N-terminal kinase (JNK) activation was lesser in ASK^-/- mice than in wild-type mice. Elevation of blood pressure by continuous Ang II infusion was comparable between ASK1^-/- and wild-type mice. However, Ang II–induced cardiac hypertrophy and remodeling, including cardiomyocyte hypertrophy, cardiac hypertrophy–related mRNA upregulation, cardiomyocyte apoptosis, interstitial fibrosis, coronary arterial remodeling, and collagen gene upregulation, was significantly attenuated in ASK1^-/- mice compared with wild-type mice. These results provided the first in vivo evidence that ASK1 is the critical signaling molecule for Ang II–induced cardiac hypertrophy and remodeling. Thus, ASK1 is proposed to be a potential therapeutic target for cardiac diseases.

Key Words: mitogen-activated protein kinase • mice • reactive oxygen species • signal transduction • gene expression

	Introduction

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Cardiac hypertrophy is an adaptive response of the heart to several forms of cardiac disease. Although sustained hypertrophy is an initial compensatory mechanism to preserve cardiac function, it is an independent major risk factor of cardiac morbidity and mortality.¹ Therefore, identifying the molecular mechanisms involved in cardiac hypertrophy and remodeling is an important challenge of cardiovascular biology and medicine.

Multiple lines of experimental and clinical evidence indicate that angiotensin II (Ang II) induces not only hypertension but also directly contributes to pathophysiological cardiac hypertrophy and remodeling and heart failure.^2–9 Therefore, the molecular mechanisms underlying Ang II–induced cardiac diseases are clinically of great interest. However, the detailed molecular mechanism of Ang II–induced cardiac diseases remains to be defined.^10–13 Among a number of signal transduction pathways, mitogen-activated protein kinase (MAPK) signaling cascades are proposed to play an important role in cardiac hypertrophy and remodeling.^10–13 In MAPK signaling cascades, MAPK kinase kinase (MAPKKK) phosphorylates and thereby activates MAPK kinase (MAPKK), and the activated form of MAPKK in turn phosphorylates and activates MAPK.^14,15 Thus, investigation of the role of MAPKKK in cardiac hypertrophy and remodeling is very important issue. However, scarce information is available on the role of MAPKKK in cardiac hypertrophic remodeling in vivo.^16–18 Apoptosis signal-regulating kinase 1 (ASK1) is a recently identified MAPKKK that is activated in response to various cytotoxic stresses and relays those signals to c-Jun N-terminal kinase (JNK) and p38 specifically.^19–22 However, nothing is known about the in vivo role of ASK1 in pathological cardiac hypertrophy and remodeling.

In the present study, we investigated the role of ASK1 in Ang II–induced cardiac hypertrophy, apoptosis, fibrosis, and coronary arterial remodeling in vivo by using mice deficient in ASK1 (ASK1^-/- mice). We obtained the first in vivo evidence that Ang II activates cardiac ASK1 via angiotensin II type 1 (AT₁) receptor in superoxide-mediated manner and this ASK1 activation plays a pivotal role in Ang II–induced cardiac hypertrophy and remodeling.

	Materials and Methods

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Animal Preparation
Generation of ASK1^-/- mice has been described previously.²³ They were backcrossed into the C57BL/6J background at least 10 generations to reduce genetic variation. Experiments were performed with age-matched (8 to 10 weeks) C57BL/6J mice (wild-type) and ASK1^-/- mice. Ang II (200 ng/kg per minute) dissolved in saline or saline alone (control) was continuously infused subcutaneously into mice via osmotic minipump (ALZA Co), and the experiments described were performed. All procedures were in accordance with institutional guidelines for animal research.

Effect of Continuous Ang II Infusion on Cardiac Superoxide Production and ASK1, MAPK Activation, and Cardiac Hypertrophy in Wild-Type Mice
The first experiments were performed to examine the effect of Ang II infusion on LV superoxide production and LV ASK1 and MAPK activation in wild-type mice. Mice were divided into 5 groups, including (1) saline-infused group (control group), (2) Ang II–infused and vehicle-treated (0.5% carboxymethylcellulose solution) group, (3) Ang II–infused and valsartan (AT₁ receptor blocker, 20 mg/kg per day)-treated group, (4) Ang II–infused and amlodipine (calcium channel blocker, 1 mg/kg per day)-treated group, (5) Ang II–infused and tempol (metal-independent superoxide dismutase mimetic, 1.5 mmol/kg per day)-treated group. Valsartan and amlodipine and vehicle were given to mice orally by gastric gavage once a day from 3 days before Ang II infusion. Tempol, dissolved in saline, was intraperitoneally injected once a day from 3 days before Ang II infusion. Thirty minutes after Ang II infusion, mice were anesthetized, and the heart was excised and separated into the upper and lower portions. The upper portion was immediately frozen in Tissue-Tek O.C.T. embedding medium (Sakura Finetek) and placed at -80°C until confocal micrographic analysis for detection of superoxide production in the heart. The lower portion was immediately frozen and stored until protein extraction for Western blot analysis or kinase assay.

The second experiments were performed to examine the effect of valsartan, amlodipine, or tempol on Ang II–induced LV hypertrophy in wild-type mice. Ang II or saline were continuously infused for 2 weeks. Valsartan (20 mg/kg per day), amlodipine (1 mg/kg per day), or vehicle was given to mice orally by gastric gavage once a day from 1 day before infusion to the end of infusion. Tempol (1 mmol/L), dissolved in drinking water, was given from 24 hours before the start to the end of infusion. Blood pressure (BP) of the conscious mice was measured with the tail-cuff method (BP98A; Softron Co) at 7 and 14 days after start of Ang II infusion. At the end of infusion, mice in each group were anesthetized, and the heart was excised and weighed. The heart was fixed in 4% paraformaldehyde overnight and embedded in paraffin. Five-µm-thick sections were cut and stained for measurement of cardiomyocyte cross-sectional area (CSA).

Comparison Between Wild-Type and ASK1^-/- Mice in Ang II–Induced Cardiac ASK1 and MAPK Activations and Cardiac Hypertrophy and Remodeling
The first experiments were performed to compare the time course of Ang II (200 ng/kg per minute)–induced LV ASK1 and MAPK activation between ASK1^-/- mice and wild-type mice. Ang II was infused into mice for 0, 15, 30, and 60 minutes, and 1, 3, 7, and 14 days. At specified times after Ang II infusion, mice were anesthetized, and the heart was rapidly excised. LV tissue was separated from the right ventricle (RV) and atria, and stored until protein extraction for Western blot analysis or kinase assay.

The second experiments were undertaken to examine Ang II–induced cardiac hypertrophy– and fibrosis-related gene expression in wild-type and ASK1^-/- mice. Three days after start of Ang II infusion, mice were anesthetized, and the heart was rapidly excised. LV tissue was stored until the extraction of total tissue RNA for Northern blot analysis.

The third experiments were performed to evaluate cardiac hypertrophy caused by continuous Ang II infusion in wild-type and ASK1^-/- mice. BP and HR were measured with the tail-cuff method before and 3, 7, and 14 days after start of Ang II infusion. At the end of Ang II infusion, the heart was excised and LV was separated from RV and atria and weighed.

The fourth experiments were performed to investigate Ang II–induced cardiac hypertrophy and fibrosis in wild-type and ASK1^-/- mice. At the end of Ang II infusion, mice were anesthetized, subjected to transthoracic echocardiographic studies, and then the heart was removed. The heart was fixed, embedded, and stained for measurement of CSA, fibrosis, and apoptosis.

Furthermore, we also infused a higher dose of Ang II infusion (1000 ng/kg per minute) in wild-type and ASK1^-/- mice, and compared between both strains, regarding blood pressure, cardiac hypertrophy, and coronary arterial remodeling.

Measurement of Arterial Blood Pressure
Mice were anesthetized with intraperitoneal injection of sodium pentobarbital (40 mg/kg), and PE10 tubing was inserted into the right carotid artery and the right jugular vein. Mean blood pressure (MBP) was continuously monitored by a pressure transducer (P231D; Nihon Kohden) and recorded on a polygraph (RM 6100; Nihon Kohden) during the following experiments. Mice were given a bolus injection of Ang II at doses of 0.1, 1, and 10 µg/kg via jugular vein, and arterial blood pressure response to Ang II was compared between wild-type and ASK^-/- mice.

Statistical Analysis
All data are presented as mean±SEM. Statistical significance was determined with one-way ANOVA, followed by Duncan multiple range comparison test using Super ANOVA (Abacus Concepts, Inc). Statistical analysis of time course of protein kinase activity (Figure 2) was performed by two-way ANOVA followed by the least-squares mean test. Differences were considered statistically significant at a value of P<0.05.

View larger version (55K): [in this window] [in a new window]   Figure 2. Time course of Ang II (200 ng/kg per minute)–induced LV ASK1 and MAPK activation in wild-type and ASK1-/- mice. A, LV ASK1 activation by Ang II infusion, and the top and bottom panels show representative Western blot analysis and the quantitative data, respectively. m indicates minutes; d, days; -/-, ASK1-/-. B, LV MAPK activation by Ang II infusion, and the top and bottom panels show representative Western blot analysis and the quantitative data, respectively. WT indicates wild-type. Other abbreviations are the same as A. In both A and B, each bar represents mean±SEM (n=4 to 5). Mean values in control wild-type mice are expressed as 1. P<0.05, *P<0.01 vs control wild-type mice; +P<0.05 vs control ASK1-/- mice.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

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Effect of Ang II on Cardiac Superoxide Production and ASK1, MAPK Activation, and Cardiac Hypertrophy in Wild-Type Mice
We examined the effect of valsartan, amlodipine, or tempol on Ang II–induced superoxide production, ASK1, and MAP kinase activation. As shown in Figure 1A, Ang II generates superoxide in mice heart in vivo. However, valsartan or tempol treatment, but not amlodipine, abolished Ang II–induced cardiac superoxide production. As shown in Figure 1B, Ang II–induced activation of LV ASK1, p38, and JNK was significantly inhibited by treatment with valsartan or tempol but not with amlodipine.

View larger version (53K): [in this window] [in a new window]   Figure 1. Production of superoxide (A), activation of ASK1 or MAPK (B), and cardiac hypertrophy (C) in wild-type mice heart by Ang II infusion. A, Representative confocal fluorescence photomicrographs of dihydroethidium-treated LV section from control group (Con) and Ang II–infused groups treated with vehicle (Veh), valsartan (Val), amlodipine (Aml), or tempol (Tem). Original magnification, x200 (bar=100 µm). Same experiments were repeated 5 times and similar results were obtained. B, Effect of valsartan, amlodipine, or tempol on LV ASK1, p38, JNK, and ERK activation at 30 minutes after Ang II infusion, and the top and bottom panels show representative Western blot analysis and the quantitative data, respectively. P-ASK indicates phospho-ASK; P-p38, phospho-p38; and P-ERK, phospho-ERK. Other abbreviations are the same as A. Each bar represents mean±SEM (n=9 to 10). Mean values in control group are represented as 1. *P<0.01 vs Con; #P<0.01 vs Veh. C, Effect of valsartan, amlodipine, or tempol on LV cardiac hypertrophy by 2 weeks of Ang II infusion. LV indicates left ventricle; BW, body weight. Other abbreviations are the same as A. Each bar represents mean±SEM (n=6 to 7). *P<0.01 vs Con.

Figure 1C shows the effect of valsartan, amlodipine, or tempol on Ang II–induced LV hypertrophy. Blood pressure of mice from control group, and Ang II–infused groups treated with vehicle, valsartan, amlodipine, and tempol was 100±1, 115±2, 101±2, 102±2, and 116±2 mm Hg, respectively, at 7 days and 100±1, 122±2, 106±1, 102±1, and 120±3 mm Hg, respectively, at 14 days. Thus, blood pressure elevation by Ang II was completely inhibited by treatment with valsartan and amlodipine, although not significantly affected by tempol. Ang II infusion for 14 days significantly increased LV weight/body weight (BW) and cardiomyocyte CSA compared with control (3.45±0.07 versus 3.05±0.02 mg/g BW, P<0.01; 300.8±19.3 versus 222.5±6.8 µm², P<0.01, respectively), and these effects of Ang II were inhibited by valsartan or tempol, although not by amlodipine. On the other hand, in mice not subjected to Ang II infusion, valsartan, amlodipine, or tempol did not alter blood pressure, LV weight/BW, and cardiomyocyte CSA.

Comparison Between Wild-Type and ASK1^-/- Mice in Basal Body Weight, Blood Pressure, Heart Rate, and Cardiac Functions
As shown in the Table, basal BW, BP, and HR were similar between wild-type and ASK1^-/- mice. Basal cardiac dimensions and functions, including LV diameters, wall thickness, contractile function, and E wave/A wave (E/A) were not different between wild-type and ASK1^-/- mice, as evaluated by echocardiography.

View this table: [in this window] [in a new window]   Table 1. Body Weight, Blood Pressure, Heart Rate, and Echocardiographic Measurements at 6 Weeks After Infusion in Wild-Type and ASK1-/- Mice

Time Course of LV ASK1 and MAPK Activities in Wild-Type and ASK1^-/- Mice During Ang II Infusion
As shown in Figure 2A, in wild-type mice, LV ASK1 was activated at both the acute and chronic phases after Ang II infusion, whereas ASK1 was not detected in ASK1^-/- mice. As shown in Figure 2B, LV p38 and JNK showed similar biphasic activation to ASK1 in Ang II–infused wild mice. On the other hand, in ASK1^-/- mice, LV p38 was not at all activated by Ang II infusion and LV JNK activation was significantly diminished. LV ERK was not apparently activated by Ang II infusion in either ASK^-/- mice or wild-type mice.

Pressor Response to Ang II in ASK1^-/- Mice
Figure 3A shows direct basal MBP and its acute response to intravenous bolus injection of Ang II in wild-type and ASK1^-/- mice. There was no significant difference in the basal MBP and the pressor response to Ang II between the two strains.

View larger version (17K): [in this window] [in a new window]   Figure 3. Blood pressure in wild-type and ASK1-/- mice. A, Comparison of dose-response curves of Ang II–induced acute increase in direct mean arterial pressure from wild-type and ASK1-/- mice. Ang II was bullously intravenous injected into the mice at the dose of 0.1, 1, and 10 µg/kg. B, Time course of blood pressure in wild-type and ASK1-/- mice subjected to saline (control) or Ang II infusion via osmotic minipump for 14 days. Values represent mean±SEM (n=6 to 8). *P<0.01 vs saline-infused (control) wild-type mice or ASK1-/- mice.

Figure 3B indicates BP in wild-type and ASK1^-/- mice subjected to continuous Ang II infusion for 14 days. The increase in blood pressure was comparable between the two strains throughout Ang II infusion.

Effects of Ang II on Cardiac Hypertrophy in ASK1^-/- Mice
Fourteen days after continuous Ang II infusion, cardiac hypertrophy in wild-type and ASK1^-/- mice was compared. As shown in Figure 4A, LV weight/BW was not significantly different between saline-infused wild-type and ASK1^-/- mice. However, LV weight/BW in Ang II–infused wild-type mice was significantly increased compared with saline-infused wild-type mice (3.29±0.04 versus 3.08±0.01 mg/g BW; P<0.01) and was higher than that in Ang II–infused ASK1^-/- mice (3.29±0.04 versus 3.13±0.03 mg/g BW; P<0.01). Figure 4B shows cardiomyocyte CSA in wild-type and ASK1^-/- mice subjected to Ang II infusion. No significant difference in cardiomyocyte CSA was seen between saline-infused wild-type and ASK1^-/- mice. However, cardiomyocyte CSA in wild-type mice was significantly increased by Ang II infusion (289.4±3.8 versus 218.2±4.8 µm²; P<0.01) and was larger than Ang II–infused ASK1^-/- mice (289.4±3.8 versus 250.8±3.0 µm²; P<0.01).

View larger version (46K): [in this window] [in a new window]   Figure 4. Cardiac hypertrophy by Ang II infusion in wild-type and ASK1-/- mice. A, Top panels show representative images of light micrographs of cross section of midportion of the heart. Original magnification, x2 (bar=1 mm). Bottom bar graph shows left ventricular weight corrected for body weight. B, Top panels show representative images of fluorescence micrographs of LV cardiomyocyte CSA on LV free-wall sections. Original magnification, x400 (bar=20 µm). Bottom bar graph shows quantitative analysis of LV cardiomyocyte CSA. Each bar represents mean±SEM (n=6 to 7). *P<0.01 vs saline-infused wild-type mice; #P<0.01 vs Ang II–infused wild-type mice; P<0.01 vs saline-infused ASK1-/- mice.

We also examined the effect of longer term (6 weeks) of Ang II infusion in wild-type and ASK1^-/- mice. Six weeks after Ang II infusion, LV weight/body weight (3.54±0.11 versus 3.28±0.15 mg/g BW; P<0.01) and cardiomyocyte CSA (316.1±4.2 versus 275.1±3.15 µm²; P<0.05) were significantly larger in wild-type mice than in ASK1^-/- mice. And as shown in the Table, intraventricular septum (IVS) and posterior wall (PW) thickness estimated by echocardiography were also significantly larger in wild-type mice than in ASK1^-/- mice. Moreover, E/A, assessed by Doppler echocardiography, was significantly increased by 6 weeks of Ang II infusion in wild-type mice compared with control (2.31±0.19 versus 1.73±0.11; P<0.05), whereas in ASK1^-/- mice, this parameter remained unchanged by Ang II (1.82±0.08 versus 1.62±0.09; NS).

Furthermore, we examined the effect of a higher dose of Ang II infusion (1000 ng/kg per minute) on cardiac hypertrophy in wild-type and ASK1^-/- mice. This high dose of Ang II infusion significantly increased BP from 102±1 to 137±2 mm Hg in wild-type mice and from 103±1 to 138±2 mm Hg in ASK1^-/- mice at 7 days, and from 100±1 to 139±2 mm Hg in wild-type mice and from 102±4 to 140±2 mm Hg in ASK1^-/- mice at 14 days. In wild-type mice, LV weight/BW and cardiomyocyte CSA was clearly increased by Ang II compared with control at 14 days after infusion (3.83±0.05 versus 3.01±0.05 mg/g BW, P<0.01; 386.4±27.8 versus 234.4±6.8 µm², P<0.01, respectively). However, LV weight/BW and cardiomyocyte CSA in Ang II–infused ASK1^-/- mice were significantly smaller than those in Ang II–infused wild mice (3.39±0.06 versus 3.83±0.05 mg/g BW; P<0.01, 318.9±11.9 versus 386.4±27.8 µm²; P<0.01, respectively).

Effects of Ang II on Cardiomyocyte Apoptosis and Myocardial Interstitial Fibrosis in ASK^-/- Mice
As shown in Figure 5A, TUNEL-positive nuclei/cardiomyocyte area was not significantly different between saline-infused wild-type and ASK1^-/- mice. In wild-type mice, TUNEL-positive nuclei/cardiomyocyte area was significantly increased by Ang II infusion by 2.7-fold (P<0.01) and 5.9-fold (P<0.01) at 2 and 6 weeks, respectively. These effects of Ang II were significantly attenuated in ASK1^-/- mice compared with wild-type mice.

View larger version (44K): [in this window] [in a new window]   Figure 5. Cardiomyocyte apoptosis and myocardial interstitial fibrosis by Ang II infusion in wild-type and ASK1-/- mice. A, Top panels show representative images of light micrographs of TUNEL-positive nuclei on LV free wall sections at 6 weeks after Ang II infusion. Arrows indicate TUNEL-positive nuclei. Original magnification, x400 (bar=25 µm). Bottom bar graph shows quantitative analysis of TUNEL-positive nuclei corrected for total LV cardiomyocyte area (mm2). B, Top panels show representative images of light micrographs of myocardial interstitial fibrosis on LV free-wall sections at 6 weeks after infusion. Original magnification, x200 (bar=100 µm). Bottom bar graph shows relative area of fibrosis. In both A and B, each bar represents mean±SEM (n=6 to 7). *P<0.01 vs saline-infused wild-type mice; #P<0.01 vs Ang II–infused wild-type mice; P<0.01 vs saline-infused ASK1-/- mice.

As shown in Figure 5B, myocardial interstitial fibrosis was not significantly different between saline-infused wild-type and ASK1^-/- mice. Ang II infusion for 2 and 6 weeks significantly increased myocardial interstitial fibrosis in wild-type mice by 1.8-fold (P<0.01) and 3.0-fold (P<0.01), respectively, whereas Ang II–induced fibrosis was significantly diminished in ASK1^-/- mice.

Effect of Ang II on Coronary Arterial Remodeling in ASK1^-/- Mice
As shown in Figure 6, coronary arterial thickening and perivascular fibrosis of large and small coronary arteries in LV were not significantly different between saline-infused wild-type and ASK1^-/- mice. However, all these parameters in Ang II–infused wild-type mice were larger than either saline-infused wild-type mice or Ang II–infused ASK1^-/- mice at 2 weeks after Ang II infusion. Furthermore, we also examined the effect of 6 weeks of Ang II infusion on coronary arterial thickening and perivascular fibrosis and found that coronary arterial thickening and perivascular fibrosis by Ang II were attenuated in ASK1^-/- mice compared with wild-type mice, as in the case of 2 weeks of Ang II infusion (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 6. Coronary arterial thickening and perivascular fibrosis in wild-type and ASK1-/- mice subjected to 2 weeks of Ang II infusion. Top panel shows representative images of light micrographs of cross section of coronary artery on LV free-wall sections. Original magnification, x400 (bar=50 µm). Bottom bar graph shows quantitative results of coronary arterial thickening (A) and perivascular fibrosis (B). Each bar represents mean±SEM (n=6 to 7). *P<0.01 vs saline-infused wild-type mice; #P<0.01 vs Ang II–infused wild-type mice; +P<0.05, P<0.01 vs saline-infused ASK1-/- mice.

Effect of Ang II on Cardiac Gene Expression in ASK^-/- Mice
Figure 7 shows Ang II–induced cardiac hypertrophy– and fibrosis-related gene expression in wild-type and ASK1^-/- mice. LV mRNA levels for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), closely related to cardiac hypertrophy, in wild-type mice were increased by 5.6- and 2.8-fold (P<0.01), respectively, by Ang II infusion. The upregulation of these mRNAs by Ang II was significantly smaller in ASK1^-/- mice than wild-type mice. mRNA levels for LV collagen I, collagen III, and plasminogen activator inhibitor-1 (PAI-1) closely related to cardiac fibrosis and remodeling, in wild-type mice were increased by 8.9-, 5.5-, and 3.5-fold (P<0.01), respectively, by Ang II infusion, and these increases in wild-type mice were greater than those in ASK1^-/- mice.

View larger version (36K): [in this window] [in a new window]   Figure 7. Cardiac hypertrophy– and fibrosis-related gene expression in wild-type and ASK1-/- mice. Right top panel shows representative autoradiograms of LV mRNAs for ANP, BNP, collagen I, collagen III, PAI-1, and GAPDH. Bar graph shows each mRNA value, corrected for GAPDH mRNA value. Mean values in saline-infused wild-type mice group is represented as 1. Each bar represents mean±SEM (n=7 to 8). *P<0.01 vs saline-infused wild-type mice; #P<0.01 vs Ang II–infused wild-type mice; +P<0.05, P<0.01 vs saline-infused ASK1-/- mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Among a number of signal transduction pathways, MAPK signaling cascades are proposed to play a pivotal role in Ang II–induced cardiac hypertrophy and remodeling.^2,10–13 In the MAPK signaling cascades, MAPKKK phosphorylates and activates MAPKK, and in turn MAPKK phosphorylates and activates MAPK.^14,15 Thus, investigation of the role of MAPKKK in cardiac hypertrophy and remodeling is very important issue. However, at present, there have been only few studies concerning in vivo role of MAPKKK in cardiac hypertrophy and remodeling.^16–18 Zhang et al¹⁶ have examined the role of TGF-ß-activated kinase (TAK1) in cardiac hypertrophy induced by pressure overload. TAK1 is one member of MAPKKK family and stimulates JNK and p38 pathways. They have found that TAK1 is activated in mouse myocardium after aortic constriction and is sufficient to trigger p38 activation, and that transgenic mice expressing excess amount of TAK1 display significant cardiac hypertrophy, interstitial fibrosis, severe cardiac dysfunction, fetal gene expression, apoptosis, and early lethality, thereby proposing the involvement of TAK1 in cardiac remodeling. MAPK/ERK kinase kinase-1 (MEKK1) is another member of MAPKKK, and preferentially activates JNK pathway and also influences ERK activation. By overexpressing the G protein Gq in MEKK1-deficient mice, Minamino et al¹⁷ have demonstrated that MEKK1 is implicated in Gq-mediated pathological cardiac hypertrophy and dysfunction. On the other hand, Sadoshima et al¹⁸ have also reported the role of MEKK1 in pressure overload-induced cardiac hypertrophy and apoptosis by using the same mice deficient in MEKK1 as Minamino et al.¹⁷ Interestingly, in contrast to the detrimental role of MEKK1 in pathological cardiac hypertrophy induced by Gq,¹⁷ they showed that cardiac MEKK1 plays a protective role in pressure overload–induced heart failure and sudden death.¹⁸ Therefore, the role of MAPKKK in cardiac hypertrophy and remodeling depends on the context of stress stimulant.

ASK1 was recently identified as a member of the MAPKKK family and is known to preferentially activate p38 and JNK. Although ASK1 was originally found to function as a proapoptotic signaling intermediate,^19,23 it has become apparent that ASK1 mediates signals leading to cell survival.²⁴ Thus, ASK1 has a broad range of biological function depending on cell type and stresses. However, there is no available report on the role of ASK1 in cardiac hypertrophy and remodeling in vivo. Thus, in our present study, by using ASK1^-/- mice, we examined the role of ASK1 in cardiac hypertrophy and remodeling in Ang II infused model. In this work, mice were infused the low dose of Ang II causing a small and gradual elevation of blood pressure and gradual development of cardiac hypertrophy, which is more physiological condition like human essential hypertension than acute pressure overload model with aortic constriction.

Previous reports have shown that ASK1^-/- mice were indistinguishable in appearance from wild-type mice, and no developmental abnormalities were observed in histological analysis.²³ In our present study, we found that basal cardiac dimensions and functions, as evaluated by echocardiography, were not significantly different between wild-type and ASK1^-/- mice. These results confirm that ASK1 does not play an essential role in heart development, in contrast to the involvement of TAK1 in cardiac development.¹⁶ In cardiovascular system, basal BP, HR, and pressor response to Ang II were not significantly different between wild-type and ASK1^-/- mice, indicating that the absence of ASK1 does not affect BP and HR regulation and Ang II–induced blood pressure elevation.

In this study, Ang II–induced LV ASK1, p38, JNK activation, and LV hypertrophy were significantly inhibited by AT₁ receptor blocker or superoxide dismutase mimetic but not by amlodipine, indicating that LV ASK1 is directly activated by Ang II via AT₁ receptor in superoxide-mediated manner. Hirotani et al²⁵ have recently reported that Ang II directly generates ROS in cultured neonatal cardiac myocytes and subsequently activates ASK1, leading to cardiomyocyte hypertrophy. Therefore, the superoxide production and subsequent activation of ASK1 in the whole heart, observed in our present work, seems to be at least in part due to cardiomyocytes. However, it cannot be excluded that cardiac fibroblasts were also partially involved in Ang II–induced superoxide production and activation of ASK1, because our present study is based on whole heart analysis. Further study is needed to elucidate this point.

The absence of LV ASK1 activation in ASK1^-/- mice by Ang II infusion was accompanied by much lesser activation of p38 and JNK than wild-type mice throughout the infusion, thereby showing that Ang II–induced LV ASK1 activation plays a pivotal role in LV p38 and JNK activation. On the other hand, ERK was not activated by low dose of Ang II in either wild or ASK1^-/- mice. This absence of ERK activation by Ang II (200 ng/kg per minute) infusion in our present study is in good agreement with our previous findings, showing that the activation of ERK has a higher threshold than JNK.²⁶

As estimated by LV weight/BW, cardiomyocyte CSA, echocardiographic measurements, and LV ANP and BNP mRNA levels, cardiac hypertrophy by Ang II (200 ng/kg/min) infusion for 2 weeks was significantly attenuated in ASK1^-/- mice than in wild-type mice, independently of blood pressure. Similar attenuation of cardiac hypertrophy in ASK1^-/- mice by Ang II (200 ng/kg per minute) was also observed in 6 weeks of infusion. Furthermore, in higher dose (1000 ng/kg per minute) of Ang II infusion causing the remarkable blood pressure elevation, ASK^-/- mice exhibited the significant attenuation of cardiac hypertrophy compared with wild-type mice. Taken together with recent in vitro data that Ang II–induced rat neonatal cardiomyocyte hypertrophy is attenuated by antioxidant or overexpression of a dominant-negative mutant of ASK1, our present in vivo observations demonstrates that ASK1 plays a critical role in Ang II–induced cardiac hypertrophy. However, it is not clear whether ASK1 is involved in cardiac hypertrophy caused by high blood pressure. Therefore, further studies are needed to elucidate this point.

Besides cardiac myocyte hypertrophy, we also examined the possible involvement of ASK1 in cardiac apoptosis, cardiac fibrosis, and coronary arterial remodeling. Of note, cardiomyocyte apoptosis in wild-type mice was increased by 2 and 6 weeks of Ang II infusion, and this increase was significantly lessened in ASK1^-/- mice. These results provided the evidence that cardiac ASK1 is involved in Ang II–induced cardiac apoptosis. Furthermore, Ang II–induced myocardial interstitial fibrosis, gene expression of collagen I, collagen III, and PAI-1, and coronary arterial thickening and perivascular fibrosis were significantly attenuated in ASK1^-/- mice than wild-type mice. These results provided the first in vivo evidence that ASK1 is involved in cardiac interstitial fibrosis and coronary arterial remodeling. The important role of ASK1 in vascular remodeling in vivo is also supported by our other report that ASK1 is directly involved in vascular smooth muscle cell proliferation in vitro and in vivo.²⁷

In the present study, either cardiac p38 or JNK was activated by Ang II, but p38 had higher and longer activation than JNK. The absence of LV ASK1 activation in ASK1^-/- mice by Ang II infusion was associated with the attenuation of p38 or JNK activation, thereby suggesting that these kinases play some role in Ang II–induced cardiac changes. However, our present study did not allow us to determine whether and how JNK and p38 contribute to hypertrophic response stimulated by Ang II. In the next step of work, the role of p38 and JNK in Ang II–induced cardiac changes needs to be determined to elucidate the molecular mechanism underlying ASK1-mediated cardiac hypertrophy and remodeling.

In conclusion, our present work provided the first in vivo evidence that cardiac ASK1 is activated by Ang II via AT₁ receptor, in superoxide-mediated manner. ASK1 is a critical signaling molecule responsible for Ang II–induced cardiac hypertrophy, apoptosis, fibrosis, and coronary arterial remodeling. We propose that ASK1 seems to be the promising therapeutic target for cardiac hypertrophy and remodeling.

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid for Scientific Research (14370036 and 14570083) from the Ministry of Education, Science, Sports and Culture and the Hoh-ansha Foundation.

	Footnotes

 
Original received June 2, 2003; revision received September 25, 2003; accepted October 1, 2003.

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