Cardiac-Specific Activation of Angiotensin II Type 1 Receptor–Associated Protein Completely Suppresses Cardiac Hypertrophy in Chronic Angiotensin II–Infused Mice
We cloned a novel molecule interacting with angiotensin II type 1 receptor, which we named ATRAP (for angiotensin II type 1 receptor–associated protein). Previous in vitro studies showed that ATRAP significantly promotes constitutive internalization of the angiotensin II type 1 receptor and further attenuates angiotensin II–mediated hypertrophic responses in cardiomyocytes. The present study was designed to investigate the putative functional role of ATRAP in cardiac hypertrophy by angiotensin II infusion in vivo. We first examined the effect of angiotensin II infusion on endogenous ATRAP expression in the heart of C57BL/6J wild-type mice. The angiotensin II treatment promoted cardiac hypertrophy, concomitant with a significant decrease in cardiac ATRAP expression, but without significant change in cardiac angiotensin II type 1 receptor expression. We hypothesized that a downregulation of the cardiac ATRAP to angiotensin II type 1 receptor ratio is involved in the pathogenesis of cardiac hypertrophy. To examine this hypothesis, we next generated transgenic mice expressing ATRAP specifically in cardiomyocytes under control of the α-myosin heavy chain promoter. In cardiac-specific ATRAP transgenic mice, the development of cardiac hypertrophy, activation of p38 mitogen-activated protein kinase, and expression of hypertrophy-related genes in the context of angiotensin II treatment were completely suppressed, in spite of there being no significant difference in blood pressure on radiotelemetry between the transgenic mice and littermate control mice. These results demonstrate that cardiomyocyte-specific overexpression of ATRAP in vivo abolishes the cardiac hypertrophy provoked by chronic angiotensin II infusion, thereby suggesting ATRAP to be a novel therapeutic target in cardiac hypertrophy.
Evidence suggests that the activation of angiotensin II (Ang II) type 1 receptor (AT1R) through the tissue renin-angiotensin system may play an important role in the development of cardiac hypertrophy. The carboxyl-terminal portion of AT1R is involved in the control of AT1R internalization independent of G protein coupling, and it plays an important role in linking receptor-mediated signal transduction to the specific biological response to Ang II.1,2
We previously cloned a novel AT1R-associated protein (ATRAP) that specifically interacts with the carboxyl-terminal domain of AT1R.3–6 We showed that ATRAP is broadly expressed in many tissues, as is AT1R, and suppresses Ang II–mediated pathological responses in cardiomyocytes and vascular smooth muscle cells by promoting the constitutive internalization of AT1R.7–9 However, the function of ATRAP in cardiac hypertrophy in vivo still remains to be demonstrated. Thus, the present study was carried out to investigate whether there is a role for ATRAP in the cardiac hypertrophy induced by chronic Ang II treatment in vivo. We first examined an effect of chronic Ang II infusion on endogenous cardiac expression of ATRAP in C57BL/6J wild-type (Wt) mice. Next, we examined whether cardiac ATRAP attenuates the pathological hypertrophic response provoked by chronic Ang II infusion using transgenic (Tg) mice with cardiomyocyte-specific overexpression of ATRAP.
Materials and Methods
This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. All of the animal studies were reviewed and approved by the animal studies committee of Yokohama City University.
Animals and Treatment
Male Wt mice were purchased from Charles River Laboratories. Tg mice expressing the ATRAP specifically in cardiomyocytes were generated on a C57BL/6J background using standard techniques. Littermates genotyped as Wt were used as the littermate control (LC) mice in this study. Mice aged 8 to 12 weeks were used in the present study.
Ang II (200 ng/kg per minute) or vehicle was continuously infused into mice subcutaneously via an osmotic minipump (model 1003D, 2001, 2001D, 2002; ALZET) for 0, 15, 30, and 60 minutes and 3 and 14 days. Olmesartan (RNH6270) was provided by Sankyo Pharmaceuticals. It was dissolved in drinking water for oral administration and given to Wt mice for 2 weeks. The olmesartan dosage (10 mg/kg per day) was determined from previous reports.10
Blood Pressure Measurements by Tail-Cuff Method and Echocardiography
Systolic blood pressure (BP; SBP) and heart rate were measured indirectly by the tail-cuff method (BP-monitor MK-2000; Muromachi Kikai Co), as described.7,11 Under anesthesia with an intraperitoneal injection of Avertin, transthoracic echocardiography was performed with an echo cardiographic system equipped with a 12.0-MHz phase-array transducer (Aplio SSA-700A; Toshiba), as described previously.12,13 Left ventricular (LV) diameter, wall thickness, and the ejection fraction were measured using M-mode tracings and averaged for 3 cycles.
BP Measurements by Radiotelemetry
Direct BP measurement was performed by a radiotelemetric method in which a BP transducer (PA-C10, Data Sciences International) was inserted into the left carotid artery. Ten days after transplantation, each mouse was housed individually in a standard cage on a receiver under a 12-hour light-dark cycle. Direct BP was recorded every minute by radiotelemetry, as described previously.14
Western Blot Analysis of ATRAP and AT1R
The characterization and specificity of the antimouse ATRAP antibody was described previously.7–9 The anti-AT1R antibody (sc-1173, lot E2508) was purchased from Santa Cruz Biotechnology, Inc.7 To examine the specificity of the antibody, an AT1R-selective blocking peptide (sc-1173p) was used. Western blot showed a single protein band of ≈42 kDa, which was abolished by an AT1R-selective blocking peptide (Figure S1, available in the online Data Supplement at http://hyper.ahajournals.org). Western blot analysis was performed as described previously.7,8 Briefly, tissue extracts were used for electrophoresis, and membranes (Millipore) were incubated with an anti-ATRAP antibody or an anti-AT1R antibody and subjected to enhanced chemiluminescence (Amersham Biosciences). The images were analyzed quantitatively using FUJI LAS3000 Image Analyzer (FUJI Film) for determination of the ATRAP and AT1R protein levels. To measure the cardiac expression ratio of ATRAP/AT1R, each ATRAP protein level was divided by the corresponding AT1R protein level obtained by reprobing and, thus, derived from the same extract.
After 2 weeks of vehicle or Ang II infusion, both the LC and Tg mice hearts were cleared by perfusion with PBS at 70 mm Hg through the coronary arteries and then fixed by perfusion with 4% paraformaldehyde. Tissue sections were stained with hematoxylin/eosin and immunohistochemical antibody (antidystrophin monoclonal antibody, Novocastra) for cell size measurement, because this antibody binds to myocardial cellular membranes. Cross-sectional area of cardiomyocytes in the LV free wall was measured digitally using Image-Pro Plus software, as described previously.15
Real-Time Quantitative RT-PCR Analysis
Total RNA was extracted from the LV with ISOGEN (Nippon Gene), and cDNA was synthesized using the SuperScript III First-Strand System (Invitrogen). Real-time quantitative RT-PCR was performed by incubating the reverse transcription product with TaqMan PCR Master Mix and a designed TaqMan probe (Applied Biosystems).11 RNA quantity was expressed relative to the 18S rRNA control.
Determination of Mitogen-Activated Protein Kinase Activity
Western blot analysis was performed for phosphorylated p38, extracellular signal-regulated protein kinase 1/2 (ERK), and c-Jun N-terminal kinase (JNK) using antiphospho-p38 antibody (V1211, Promega), antiphospho-ERK antibody (4370, Cell Signaling Technology), and antiphospho-JNK antibody (4668, Cell Signaling Technology), which recognize only activated p38, ERK1/2, and JNK, respectively, as described previously.8 To detect total p38, ERK, and JNK, the anti-p38 mitogen-activated protein kinase (MAPK) antibody (sc-728, Santa Cruz Biotechnology), anti-ERK antibody (4695, Cell Signaling Technology), and anti-JNK antibody (sc-571, Santa Cruz Biotechnology), were used.
For the statistical analysis of differences among groups, unpaired Student t test or ANOVA followed by Scheffe F test was used. All of the quantitative data are expressed as mean±SE. Values of P<0.05 were considered statistically significant.
Effects of Ang II Infusion on Cardiac Hypertrophy in Wt Mice
In the first experiment, age-matched Wt mice were divided into 3 groups: (1) a vehicle-infused group; (2) an Ang II (200 ng/kg per minute)–infused group without ARB treatment; and (3) an Ang II (200 ng/kg per minute)–infused group with ARB treatment. Ang II infusion significantly increased diastolic intraventricular septum and diastolic LV posterior wall thickness, as estimated by echocardiography and heart weight (HW)/body weight (BW) ratio, and these hypertrophic responses to Ang II treatment were completely prevented by angiotensin receptor blocker (ARB) treatment (Table 1). Ang II infusion also increased cardiac hypertrophy-related gene expression in Wt mice (Figure 1A). LV mRNA levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide were increased 2.2- and 2.6-fold by Ang II infusion, respectively, and the mRNA upregulation of these peptides was abolished by ARB treatment.
Effects of Ang II Infusion on Cardiac ATRAP and AT1R Expression in Wt Mice
We also examined the effects of Ang II infusion on endogenous ATRAP and AT1R protein expression in the hearts of Wt mice. With respect to the regulation of cardiac AT1R expression by Ang II infusion, previous studies reported increased, decreased, or unaltered cardiac AT1R levels after Ang II infusion, probably because of differences in the dose and duration of the Ang II infusion.16–18 In the present study, treatment with Ang II did not affect cardiac AT1R protein or mRNA levels (Figure 1B and 1C and data not shown). On the other hand, Ang II infusion significantly decreased the cardiac ATRAP protein level, thereby resulting in downregulation of the cardiac ATRAP/AT1R ratio (Figure 1B and 1C). However, ARB treatment by olmesartan (10 mg/kg per day) recovered the cardiac expression ratio of ATRAP to AT1R so as to be comparable with the vehicle-infused group.
Effects of Ang II Infusion on Cardiac Hypertrophy in Cardiac-Specific ATRAP Tg Mice
Because chronic Ang II treatment significantly decreased the endogenous ATRAP expression in the heart concomitant with the development of cardiac hypertrophy in Wt mice, we hypothesized that an increase in cardiac ATRAP expression might suppress it in vivo. Thus, to validate the antihypertrophic properties of ATRAP in vivo, we generated Tg mice with cardiac-specific overexpression of ATRAP by the use of mouse ATRAP cDNA linked to the α-major histocompatibility complex promoter.19 Quantitative analysis of ATRAP expression at the protein level revealed the highest and a moderate expression level of ATRAP in lines 52 and 46 (Tg52 and Tg46), respectively, among 10 obtained lines of Tg mice, and these 2 lines of Tg mice are characterized in Figure S2.
Age-matched LC and 2 independent lines of Tg mice (Tg46 and Tg52) were divided into 6 groups: (1) vehicle-infused LC mice; (2) Ang II (200 ng/kg per minute)–infused LC mice; (3) vehicle-infused Tg46 mice; (4) Ang II (200 ng/kg per minute)–infused Tg46 mice; (5) vehicle-infused Tg52 mice; and (6) Ang II (200 ng/kg per minute)–infused Tg52 mice. Although Ang II infusion significantly increased the diastolic intraventricular septum and diastolic LV posterior wall thickness, as estimated by echocardiography and HW/BW ratio in LC mice, these cardiac hypertrophic responses to Ang II infusion were completely suppressed in both Tg52 mice and Tg46 mice (Table 2). Thus, Tg52 mice were further characterized in comparison with LC mice.
The results of SBP measurement by the tail-cuff method did not result in significant Ang II–mediated BP responses in the LC or Tg mice (Table S1). Thus, to examine diurnal BP profiles and strictly compare the effects of Ang II infusion on BP, direct BP measurement by radiotelemetric devices was performed in LC and Tg mice. In LC mice, Ang II infusion for 2 weeks tended to increase SBP in the light period (118.2±4.0 versus 132.7±3.7 mm Hg; P=0.075) and significantly increased SBP in the dark period (129.3±2.7 versus 144.7±4.1 mm Hg; P<0.05; Figure 2A). Similarly, in Tg52 mice, Ang II infusion significantly increased SBP in both the light (115.2±2.8 versus 130.5±5.0 mm Hg; P<0.05) and dark (128.7±2.0 versus 143.3±4.6 mm Hg; P<0.05) periods. Although radiotelemetric SBP of the vehicle-infused Tg52 mice was significantly lower than that of the vehicle-infused LC mice at 12:00 am (107.2±2.7 versus 116.2±4.5 mm Hg; P<0.05) and 3:00 pm (112.0±2.0 versus 123.7±3.5 mm Hg; P<0.05; Figure 2B), SBP of the Ang II–infused Tg52 mice was comparable to that of the Ang II–infused LC mice throughout the light-dark cycle (Figure 2C). Regarding other parameters obtained by radiotelemetry, the mean BP and heart rate of Tg52 mice were comparable to those of LC mice with or without Ang II infusion (Figure S3).
These results of direct BP measurement by the radiotelemetric method confirmed no significant BP difference between the LC and Tg52 mice after Ang II infusion. With respect to histological analysis, Ang II infusion significantly increased the cross-sectional area of LC mice (251.5±6.7 versus 302.0±10.4 μm2; P<0.01) but not Tg52 mice (267.7±11.2 versus 277.3±11.5 μm2; Figure 3A). There were no significant increases in interstitial fibrosis in either the LC mice or Tg52 mice on Masson staining at this stage (data not shown). These results indicate that the cardiac hypertrophy effects induced by Ang II infusion were completely suppressed in Tg52 mice with cardiac-specific overexpression of ATRAP.
Effects of Ang II Infusion on Hypertrophy-Related Gene Expression in Cardiac-Specific ATRAP Tg Mice
We examined whether cardiac-specific overexpression of ATRAP attenuates the cardiac hypertrophy-related gene expression induced by chronic Ang II infusion. Although Ang II infusion in LC mice increased the LV mRNA expression levels of atrial natriuretic peptide and brain natriuretic peptide by 1.7- and 2.1-fold induction, the upregulation of these mRNAs in response to Ang II infusion was completely suppressed in Tg52 mice (Figure 3B). With respect to the inhibitory effect of Ang II treatment on the cardiac ATRAP/AT1R ratio, although Ang II infusion significantly decreased the cardiac ATRAP/AT1R ratio through suppression of cardiac ATRAP expression in LC mice (Figure 3C), Ang II treatment did not affect the ratio at all in Tg52 mice (Figure 3D).
Effects of Ang II Infusion on MAPK Activation in Cardiac-Specific ATRAP Tg Mice
As the downstream effector of the AT1R signaling pathway, MAPK plays an important role in the development of cardiac hypertrophy.20–22 Thus, ultimately we examined the time course of LV MAPK activation in LC and Tg52 mice during Ang II treatment. LV p38 was significantly activated after 30 minutes and 14 days of Ang II infusion in LC mice (Figure 4A). However, LV p38 was not at all activated in Tg52 mice during Ang II treatment. On the other hand, LV JNK and ERK were not significantly activated by Ang II infusion in either LC or Tg52 mice (Figure 4B and 4C).
This is the first report to our knowledge of a novel inhibitory function of cardiac ATRAP on cardiac hypertrophy in vivo. Activation of AT1R signaling through the tissue renin-angiotensin system provokes sequential activation of signaling pathways, which leads to cardiac hypertrophy,23–26 and chronic elevation of circulating Ang II causes sustained hypertension and associated cardiac hypertrophy.27 The carboxyl-terminal portion of AT1R is important for receptor internalization and activation of downstream signaling pathways.28,29 ATRAP was cloned as a specific interacting molecule with the carboxyl-terminal domain of the AT1 receptor by a yeast 2 hybrid screening system.3 The results of in vitro studies showed that ATRAP suppresses Ang II–induced hypertrophic and proliferative responses by promoting a constitutive internalization of AT1R and decreasing the p38 activity and transforming growth factor β production in cardiomyocytes and vascular smooth muscle cells, respectively, thereby suggesting that ATRAP is an endogenous inhibitor of AT1R signaling.8,9
In the present study, we first showed that chronic infusion of Ang II significantly decreased the cardiac expression ratio of ATRAP/AT1R with the development of cardiac hypertrophy, for which the effect was completely inhibited by an AT1R antagonist. We observed recently that treatment with an AT1R antagonist recovered a constitutive decrease in the ratio of cardiac expression of ATRAP/AT1R in spontaneously hypertensive rats, which was accompanied by a decrease in cardiac p38 activity and a suppression of cardiac hypertrophy.30 Previous studies have shown that increases in cardiac p38 activity through the activation of AT1R signaling are profoundly involved in cardiac hypertrophy and the damage incurred in genetic and experimental hypertension models, including spontaneously hypertensive rats and Ang II infusion.31,32
Because we had hypothesized that cardiac-specific upregulation of the ATRAP/AT1R ratio suppresses the cardiac hypertrophy induced by Ang II infusion, we produced the cardiomyocyte-specific ATRAP Tg mice, with a constitutively high expression of cardiac ATRAP and a resultant upregulation of the cardiac ATRAP/AT1R ratio, to examine this hypothesis. We obtained in vivo evidence that ATRAP attenuates the cardiac hypertrophy by chronic Ang II infusion concomitant with a suppression of cardiac p38 activation. Because the physiological parameters, such as BW, heart rate, radiotelemetric BP, cardiac functions, and HW/BW ratio, are comparable at baseline in LC and Tg mice, the effect of cardiac-specific overexpression of ATRAP is likely to be exerted only in the context of a hypertensive challenge.
The present study shows the enhancement of cardiomyocyte-specific ATRAP expression in vivo protects against the hypertrophic responses in mice induced by an Ang II–induced BP increase. Nevertheless, a limitation of the present study is that the results do not allow us to dissociate the direct effect of ATRAP enhancement on cardiac hypertrophy from the influence of pressure overload. In fact, several previous studies have indicated that cardiac hypertrophy can occur even in the absence of cardiac AT1R,33,34 that high doses of Ang II–released specifically in the heart do not result in cardiac hypertrophy,35 and that if the kidneys do not express AT1R, infusion of a very high dose of Ang II does not result in cardiac hypertrophy.36 Therefore, the role of ATRAP in the regulation of cardiac hypertrophy in vivo needs to be further investigated in other models of hypertension and/or pressure or volume overload.
Because the results obtained in this study are essentially derived from Tg mice, it is important to exercise caution in interpreting the finding to be relevant to the pathophysiology of human cardiac hypertrophy. Nevertheless, the findings of the present study provide important information for the further investigation of the in vivo functional roles of ATRAP in the pathogenesis of cardiac hypertrophy and suggest the potential benefit of an ATRAP activation strategy. Additional studies to elucidate the molecular mechanisms of the antihypertrophic properties of cardiac ATRAP may enable a clinical application of ATRAP in the near future, such as the use of activating ligands for more efficient inhibition of AT1R signaling in combination with inhibitors of the renin-angiotensin system.
Sources of Funding
This study was supported by grants from the Japan Society for the Promotion of Science; Yokohama Foundation for Advancement of Medical Science; Takeda Science Foundation; Salt Science Research Foundation (No. 0911); Kidney Foundation, Japan (JKFB09-25); Mitsubishi Pharma Research Foundation; and the Strategic Research Project of Yokohama City University. S.M. was supported by the High-Tech Research Center Project for Private Universities with a matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology.
- Received November 4, 2009.
- Revision received November 23, 2009.
- Accepted February 11, 2010.
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