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1Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215; 2Department of Developmental Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, South Carolina 29208; and 3Division of Molecular Cardiovascular Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229
Submitted 17 April 2003 ; accepted in final form 14 July 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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, PKC-
, and p70S6 kinase. These data demonstrate that ventricular myocyte-specific expression of AT2 receptors promotes the development of dilated cardiomyopathy and heart failure in vivo.
myosin light-chain promoter; cardiac disease; myocyte
However, the AT2 receptor is expressed in adult hearts under certain pathological conditions. Recent reports suggest that in experimental myocardial infarction in rodents, the AT2 receptor is acutely upregulated and AT2 receptor deficiency exacerbates early mortality (2). In humans with chronic coronary disease in the absence of severe heart failure, the AT2 receptor does not appear to be upregulated (21). In contrast, in experimental hypertrophy (17) and chronic severe heart failure in humans (4, 11, 30, 33), most studies report an increase in AT2 relative to AT1 receptors in the ventricle. In vitro, upregulation of the AT2 receptor is associated with complex effects on growth as well as promotion of apoptosis (9, 34). Whereas transient AT2 upregulation after myocardial infarction may be adaptive, the effects of chronic upregulation of AT2 on ventricular performance and morphology are not yet understood.
To test the hypothesis that chronic AT2 expression adversely affects chronic cardiac remodeling and function in vivo, we generated lines of transgenic (TG) mice with differing levels of AT2 receptor expression. AT2 receptors are not constantly upregulated in the atria of patients with cardiac disease (21, 27). In addition, expression of AT2 receptors driven by the -myosin heavy-chain promoter in both ventricles and atria in TG mice is associated with unexpected chronotropic effects (19) that may indirectly modify cardiac workload. These considerations prompted us to develop the reagents necessary to restrict TG expression to the ventricular compartment only. To study the effects of ventricular myocyte-specific expression in the absence of confounding effects in other cardiac chambers or cell compartments, AT2 expression was driven by the myosin light-chain (MLC-2v) promoter. The aim of this study was to determine whether ventricular myocyte-specific overexpression of AT2 receptors could induce heart failure.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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8.0-kb DNA fragment that consisted of the MLC-2v promoter and AT2 cDNA (Fig. 1A). The DNA fragment was purified and used to generate FVB/n TG mice. The transgene copy number was assessed by Southern blotting. Briefly, tail DNA was digested with HpaI and EcoRI. The resultant DNA fragments were separated on agarose gel, transferred to the membrane, and detected with a transgene-specific cDNA probe. The intensity of the hybridization signal corresponds to the copy number of the transgene. Subsequent studies were performed using 18-wk-old mice. All animal studies were in compliance with the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council, Washington, DC: National Academy Press, 1996).
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Quantitative RT-PCR. RNA was prepared from TG and nontransgenic (NTG) mice (n = 15–20/group) using TRI reagent (Sigma). RNA was treated with DNase I (GIBCO-BRL), and 1 µg was used for reverse transcription with random primers. Real-time PCR was carried out in a 7700 Sequence Detector (ABI Prism) using AT2-specific primers (sense, 5-ACATTACCAGCAGCCGTCCT-3; antisense, 5-TGAGCAATTAAAGGCGGACTC-3; Taqman probe, 5-TTGATAATCTCAACGCAACTGGCACCAA-3). Conditions were as follows: 35 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 30 s. Data were analyzed using Sequence Detector 1.7 software. RT-PCR was also carried out to determine expression of the AT2 transgene and AT1 receptor in TG and NTG mice using 18S as an internal control. PCR was carried out using AT2-specific primers (sense, 5-CCTGGCAAGCATCTTATGTAGTT-3; antisense, 5-TTAAGACACAAAGGTGTCCATTTC-3; fragment size, 623 bp) or AT1-specific primers (sense, 5-GCATCATCTTTGTGGTGGGAA-3; antisense, 5-GAAGAAAAGCACAATCGCCAT-3). We used 18S primers (Ambion) to amplify 18S as an internal control. Conditions were as follows: 1 cycle of 94°C for 4 min then 28 cycles of 94°C for 1 min, 55°C for 30 s, and 72°C for 30 s. At 28 cycles, band intensity increased exponentially (data not shown); thus the relative band intensity reflects the relative amount of substrate cDNA/mRNA.
Characterization of AT2 receptors. AT2 receptor protein was measured via Western blotting using anti-FLAG antibody (Chemicon) as well as anti-AT2 antibody (a gift from Dr. Robert M. Carey; n = 8–10/group). A radioligand binding assay was performed with crude membranes isolated from left ventricles (n = 25–30/group) as described by Akishita et al. (3). Membrane fractions (50 µg of protein) were incubated at 21°C for 2 h in 200 µl of 25 mmol/l Tris·HCl (pH 7.4) that contained 0.25% BSA and 0.2 nmol/l 125I-labeled [Sar1,Ile8]ANG II (PerkinElmer) in the absence (for the total count) or presence of 200 µmol/l PD-123319 (Sigma). Bound and free ligands were separated with GF/C membrane filters (Millipore). AT2 receptor binding was determined by subtracting the count of samples incubated with PD-123319, which is a specific AT2 antagonist, from the total count. The net radioactivity count was converted to molar values by use of specific activity of the ligand.
Western blot analyses of signaling molecules. Hearts from 18-day embryos and 3-day-, 2-wk-, and 18-wk-old mice were removed, and LV tissues were immediately frozen in liquid nitrogen. Because of the small size of the hearts and the paucity of LV tissue at the early time points, protein samples from LV tissues from 18-day embryos and 3-day- and 2-wk-old mice of NTG or TG mice with a high level of AT2 expression (
) were pooled for analysis (n = 3 or 4/group). Densitometry levels of signaling proteins were normalized to densitometry levels of GAPDH, which was used as an internal control. LV protein (50 µg) was loaded on SDS-PAGE gels, transferred to nitrocellulose membrane, and probed with phospho-PKC-/II (Cell Signaling), PKC-, PKC-
(BD Transduction Laboratories), phospho-ERK, ERK, phospho-p70S6 kinase (p70S6K), p70S6K (Cell Signaling), and GAPDH (CHEMICON) antibodies.
In vivo contractile function. Two-dimensional guided Mmode echocardiography was performed (8) with mice under conscious sedation (chloral hydrate, 200 mg/kg ip). At least 5 sequential beats were analyzed (n = 6–9/group). In vivo LV hemodynamics were assessed (8) by direct LV catheterization (n = 6–12/group).
Microscopic analyses. Myocyte area and length were measured in isolated myocytes as previously described (Ref. 13; n = 18–47 myocytes/animal from 5 animals/group). Confocal microscopic examination of phalloidin, cytochrome c, and terminal deoxynucleotidyl transferase end-labeling (TUNEL) staining was performed as previously described (Ref. 8; n = 5/group). For immunoelectron microscopy, hearts were prepared for electron microscopy via standard techniques (18). Thin sections were labeled with a rabbit polyclonal IgG antibody to cytochrome c (sc-7159, Santa Cruz) and tagged with 10 nM goat anti-rabbit colloidal gold. For quantitation of cytochrome c, point counting of 10 photographs of different sections of the ventricle was performed (n = 5 or 6 animals/group and 11–14 sections/animal).
Statistical analysis. Values are expressed as means ± SE. Comparisons among the groups were analyzed using ANOVA and Tukey's post hoc test. Statistical significance was accepted at the level of P < 0.05.
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RESULTS |
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To determine the effects of differing levels of ventricular-specific AT2 overexpression, we compared an line (copy number 18) and a line with a lower level of AT2 expression (copy number 9, 
). Quantitative real-time RT-PCR showed that AT2 mRNA levels in NTG ventricles were extremely low and could only be detected after 30 cycles of PCR. The mRNA levels of AT2 receptor in the TG line were 4.04-fold higher than in the TG line. There were no significant differences in LV AT1 receptor mRNA expression between , , and NTG animals (1.50 ± 0.18, 1.60 ± 0.07 vs. 1.61 ± 0.06 densitometric units; P = not significant).
Consistent with the quantitative real-time RT-PCR observation, LV AT2 protein was undetectable in NTG mice using anti-AT2 antibody. The relative level of AT2 protein in the line was 4.18- and 4.27-fold of that in the line using anti-FLAG antibody (Fig. 2A) and anti-AT2 antibody (data not shown), respectively. The size of AT2 protein in TG mice was 80 kDa, which is consistent with prior observations (25). In addition, the AT2 receptor binding activity was also examined in LV membrane fractions using the method of Akishita et al. (3). AT2-specific binding was extremely low in NTG mice (Fig. 2B), which corroborates the RT-PCR data and the immunoblotting of AT2 protein. AT2-specific binding was detected in the TG mice, and AT2-specific binding in mice was 1.25-fold of mice (Fig. 2B).
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In vivo LV function. In vivo LV function was assessed by hemodynamic measurements of 18-wk-old NTG and TG mice from the and lines. The time point of 18 wk was selected because initial pilot studies showed the development of clinical signs of overt heart failure (tachypnea and edema) in TG mice. There were no significant differences among the NTG mice from each line, and these data were pooled for analysis. Shown in Table 1, LV weight and LV wt/body wt were increased in TG compared with NTG mice, whereas these parameters were not increased in mice. In TG compared with NTG mice, LV systolic pressure and peak +dP/dt were decreased, and LV end-diastolic pressure was increased; LV developed pressure per gram of LV mass, which is an index of LV pressure development per force-developing unit, was depressed (434 ± 34 vs. 787 ± 34 mmHg/g; P < 0.01). In TG mice, the values of LV systolic and diastolic function were intermediate between the values of TG and NTG mice (Fig. 3). The left atria were dilated and left atrium wt/body wt was significantly increased in TG vs. NTG mice. Because AT2 transgene expression is ventricle restricted, the increase in left atrial mass is likely secondary to the elevation of diastolic filling pressures in both the left ventricle and the left atrium. This was not observed in TG mice, which had better cardiac function and lower LV end-diastolic pressure measurements.
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Echocardiography corroborated the presence of LV systolic dysfunction in both TG lines with more severe LV dysfunction in the TG mice (Fig. 4). As shown in Table 2, midwall fractional shortening, which is an echocardiographic index of LV systolic function, was depressed in TG mice (22 ± 1 vs. 26 ± 1%; P < 0.05) and severely depressed in TG mice (13 ± 2 vs. 26 ± 1%; P < 0.001) compared with NTG mice. Enlargement of LV chamber dimensions, LV wall thinning, and depression of the index of relative wall thickness were present in TG compared with NTG mice, which is indicative of dilated cardiomyopathy. The TG mice were similar to NTG mice with respect to LV dimensions and wall thickness.
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Microscopy observations. Analysis of myocyte area and length in isolated myocytes showed that both area and length were increased in myocytes from TG mice whereas myocyte size was not increased in myocytes from TG compared with NTG mice (see Table 1). Confocal microscopy showed that LV interstitial spaces from TG mice were enlarged (Fig. 5A, middle) with hypertrophied myocytes that frequently had abnormal intercalated discs evident by confocal microscopy (Fig. 5A, bottom and insets). These findings are similar to those previously reported in mice with severe pressure-overload cardiac hypertrophy (8). Scanning electron microscopy of the hearts from the TG animals also showed enlarged interstitial spaces with increased amounts of fibrillar collagen, which is characteristic of hypertrophied hearts (Fig. 5B, top and middle). Similar but not as extensive characteristics of hypertrophy were found in the TG mice. Initial studies on the occurrence of apoptosis detected by TUNEL staining and confocal microscopy indicated a low prevalence (0.1%) of apoptosis in the LV myocytes in the free wall of TG animals, but no evidence of apoptosis was observed in the TG or NTG mice (n = 5/group; data not shown).
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Consistent with the TUNEL staining, localization of cytochrome c using confocal microscopy suggested that there was a higher concentration of cytochrome c in the cytoplasm of TG animals compared with TG or NTG mice (Fig. 6). Transmission electron microscopy (TEM) showed an increased distribution of cytochrome c in the cytoplasm of TG myocytes by immunogold labeling (n = 5 or 6 animals/group and 11–14 sections/animal; Fig. 5B, bottom). Analysis (
2) of the localization of cytochrome c-positive points in the cytoplasm compared with those localized in the mitochrondria showed that a significantly higher number of positive points were localized in the cytoplasm of TG myocytes compared with TG or NTG mice (P < 0.05; data not shown). In addition, the TEM micrographs showed disarray of the Z-bands, intercalated discs, and contractile fibers; this is similar to the findings illustrated in Fig. 5A.
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Phosphorylation of cardiac signaling proteins. To assess potential signaling mechanisms in AT2 TG mice, we compared the levels and phosphorylation states of several proteins that are implicated in pathological hypertrophy. In 18-wk-old TG mice compared with NTG mice (Fig. 7), LV levels of phospho-PKC-/II, which are implicated in the activation of mitogen-activated protein kinase pathway and heart failure (24), were increased (229 ± 20 vs. 100 ± 8%; P < 0.01). Total PKC- levels were also increased (158 ± 9 vs. 100 ± 10%; P < 0.01). No change in the level of total PKC- was found (105 ± 3 vs. 100 ± 10%; P = not significant). In addition, phospho-ERK1/2 levels were increased in TG mice (129 ± 11 vs. 100 ± 4%; P < 0.05). Phosphorylated levels of its potential down-stream target p70S6K (Thr421/Ser424) were markedly elevated (151 ± 16 vs. 100 ± 7%; P < 0.01), and phosphorylated levels of p85S6K (Thr389), which is an isoform of p70S6K, were increased (175 ± 14 vs. 100 ± 6%; P < 0.01). Total p70S6K protein levels were also increased (133 ± 8 vs. 100 ± 5%; P < 0.01). Levels of these signaling proteins in TG were intermediate between those in and NTG mice.
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To investigate whether the activation of the growth-signaling pathways observed in 18-wk-old TG mice in vivo is directly linked to AT2 overexpression, levels of these signaling molecules were assessed in LV tissues from 18-day embryos, 3-day-old neonatal mice (an early time point when the AT2 transgene driven by the MLC-2v promoter is activated), and 2-wk-old mice. The protein levels of signaling molecules in TG mice were expressed as a percentage of NTG levels at each time point. In LV tissues of 18-day TG embryos, no differences were observed compared with NTG embryos. In LV tissues of TG mice, levels of phosphorylated PKC- and PKC-II were 154% of NTG in 3-day neonates and 163% of NTG in 2-wk-old animals. Total PKC- and PKC- levels were not increased at either time point. In TG mice, levels of phosphorylated p70S6K (Thr421/Ser424) were 136 and 131% of 3-day- and 2-wk-old NTG mice, respectively. Levels of phosphorylated p85S6K (Thr389) were 162 and 164%, and total p70S6K protein levels were 132 and 138% of 3-day- and 2-wk-old NTG mice, respectively. An increase in the level of phosphorylated ERK1/2, which was observed in 18-wk-old TG mice, was not observed in 3-day- and 2-wk-old TG LV tissue.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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AT2 receptors and growth. In vitro studies of vascular smooth muscle cells (34), endothelial cells (29), cardiac fibroblasts, and neonatal cardiomyocytes (5, 31) suggest that the AT2 receptor inhibits cell growth and proliferation. However, recent reports indicate that AT2 activation may have the potential to stimulate growth during vascular remodeling (16, 22) as well as in cardiac hypertrophy (26). Reports of the effects of AT2 gene deletion on hypertrophic growth in TG mice are conflicting with differing models showing a requirement (12, 28) vs. no effect (3) of the AT2 receptor on the development of pathological hypertrophy. In TG mice with expression of AT2 receptors driven by the -MHC promoter, no difference in cardiac size was observed between the TG and NTG mice, whereas cardiac chronotropic properties were modified, which suggests a potential confounding effect of transgene expression in atria (19). The present study is more precise, and AT2 receptor expression targeted only to the ventricles by the MLC-2v promoter provides a model to elucidate the effects of expression of the AT2 receptor on ventricular myocyte remodeling and function.
AT2 receptors and remodeling. The TG mice, which had severe depression of LV systolic function in vivo, had microscopic findings suggestive of myocyte apoptosis that were accompanied by secondary replacement by fibrillar collagen. Leakage of cytochrome c into the cytoplasm is indicative of cells entering the apoptotic pathway (6). Both immunogold labeling and confocal localization showed the presence of leakage of cytochrome c into the cytoplasm in the TG mice compared with the TG and NTG mice. These observations are consistent with in vitro observations of the proapoptotic effects of excess AT2 receptor expression in smooth muscle cells (34) and cardiomyocytes (9). Morphological as well as hemodynamic abnormalities were less prominent in the TG mice. These observations suggest that the level of AT2 expression influences the magnitude of both morphological and physiological parameters of heart failure.
Possible mechanisms of remodeling in AT2 TG mice. We observed that levels of phosphorylated PKC- and - were increased shortly after birth and continued to be elevated at the 18-wk stage of overt heart failure in TG mice that expressed AT2. These data support the hypothesis that AT2 overexpression in vivo in ventricular myocytes is associated with the activation of PKC- and -, which are implicated in cardiac remodeling and heart failure (24). AT2 overexpression is also associated with the early activation of p70S6K/p85S6K and the increase of total levels of p70S6K protein. These findings are consistent with the observations in one model of AT2 null mice (12, 28) that showed that deletion of the AT2 gene prevented hypertrophy caused by chronic ANG II infusion as well as pressure overload. Those studies showed that the absence of LV hypertrophy in AT2 null mice during pressure overload was related to the reduction of phosphorylated p70S6K as well as total p70S6K levels. Those observations contrast with findings in a separate AT2 null model in which vascular and cardiac hypertrophy was observed to be associated with increased P70S6K (7). In the present study, an increase in phosphorylated ERK1/2 was only observed in 18-wk-old animals, which suggests that late activation of this pathway is not directly linked to AT2 overexpression and is a late secondary response during the progression to heart failure.
In addition, a highly novel mechanism of action of AT2 receptors was recently proposed by AbdAlla et al. (1) who reported evidence of AT2-AT1 receptor heterodimerization such that AT2 inhibits activation of AT1 by direct binding independent of AT2 activation. This mechanism would provide a complex mode of AT2 function distinct from signaling cascades activated by ANG II binding to the AT2 receptor. It is a formal possibility that the FLAG tag itself could cause alterations in protein function; however, the innocuous nature of the tag has been recently documented in a number of contractile protein-based TG mice (14).
Implications for human cardiac disease. It is not known whether the levels of ventricular myocyte AT2 expression observed in this TG model simulate or are in excess of that observed in the ventricle in advanced human heart failure. In human cardiac disease, both the regulation and localization of AT2 are controversial. Regitz-Zagrosek et al. (27) reported no increase in ventricular AT2 receptors in human heart failure. In contrast, in ventricular tissue from patients with severe heart failure, a downregulation of AT1 and marked increase in the proportion of AT2 to AT1 receptors were reported by Asano et al. (4) using ligand-binding studies and by Haywood et al. (11) using RT-PCR without cell-specific localization. Using autoradiography localization of AT1 and AT2 receptors in human heart tissue, both Wharton et al. (33) and Tsutsumi et al. (30) reported a marked upregulation of AT2 in failing ventricular myocardium in both nonmyocyte and myocyte cell types with a preponderance of its localization in regions with fibrosis. In contrast, Matsumoto et al. (21) found no upregulation of AT2 receptors in ventricular tissue from patients undergoing bypass surgery with normal or minimally depressed ventricular function and identified AT2 receptors only in cardiomyocytes but not in fibroblasts.
Summary. Further studies, including in vitro studies in adult myocytes, are required to understand the effects of AT2 receptor expression on growth, apoptotic signaling, and contractility. The present observations raise the formal possibility that the AT2 receptor may be a target for intervention in chronic heart failure.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.01 vs. NTG mice; 
P < 0.05 vs. 
P < 0.01 vs.