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1Departments of Medicine, 2Radiology, 3Biomedical Engineering, and 4Health Evaluation Sciences, University of Virginia Health System, Charlottesville, Virginia; 5Department of Pathology, University of Alabama, Birmingham, Alabama; and 6Department of Cardiovascular Medicine, Kyoto Prefectural University School of Medicine, Kyoto, Japan
Submitted 18 August 2005 ; accepted in final form 5 October 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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magnetic resonance imaging; angiotensin; myocardial infarction; receptors
The latter notion is supported by two major lines of investigation. First, it has been shown in animal models that the lack of AT2-R worsens outcomes after experimental MI (1, 5, 18). In mice that were not able to mount a profibrotic and hypertrophic response, cardiac rupture was significantly increased post-MI (1, 5). Second, it has been shown that the protective effect of AT1-R blockade by ARBs is attenuated in the presence of AT2-R inhibition (7, 13, 27). Data suggest that the AT2-R has a minimal role in blood pressure lowering in the setting of AT1-R blockade, but it is thought to reduce fibrosis and myocyte hypertrophy after MI (27). Our group has demonstrated that AT2-R overexpression is associated with improved baseline LV function and attenuates post-MI remodeling and that nitric oxide is part of the downstream signaling pathway (2, 30)
We hypothesized that the AT2-R mediates much of the benefit of AT1-R blockade in the myocardium, and therefore we studied the AT1-R/AT2-R interplay by pharmacological and genetic inhibition of the AT1-R in AT2-R cardiac transgenic (TG) mice compared with appropriate controls. We hypothesized that if AT2-R plays a major role in both left ventricular (LV) remodeling and modulating the benefits of AT1-R blockade, then losartan (Los) therapy and knocking out the AT1-R may not be additive to AT2-R overexpression.
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METHODS |
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Animal protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and were approved by the University of Virginia Animal Care and Use Committee. All five arms of the present study were performed in mice of C57Bl/6 background; in three arms with cardiac overexpression of the AT2-R, as described previously (2, 30). This strain was originally created by Dr. H. Matsubara (Kyoto Prefectural University School of Medicine, Kyoto, Japan) (16).
For one arm of the study, AT2-R TG mice and mice with systemic knockout (KO) of the AT1a-R were cross-bred. Heterozygote AT1a-R deficient mice, originally derived from a line previously described (6) and backcrossed more than five generations into the C57Bl/6 strain were bred in the laboratory of Dr. Amy Mangrum to generate wild-type (WT; +/+), heterozygous (+/–), and homozygous null (–/–) littermates. After the AT2-R TG mice were cross-bred with homozygous AT1a-R null mice, subsequent generations yielded mice with both cardiac overexpression of the AT2-R and systemic knockout of the AT1a-R. Transgene expression and knockout were confirmed by Southern blot analysis of genomic DNA from mouse tails.
Study Design
This study consisted of five arms with a total of 59 mice: 1) WT, untreated (n = 12); 2) WT, treated with the AT1-R blocker losartan (10–20 mg·kg–1·day–1 in drinking water) from day 1 to day 28 post-MI (WT-Los; n = 10); 3) cardiac overexpression of the AT2-R (AT2-TG; n = 14); 4) cardiac overexpression of the AT2-R, treated with losartan (AT2-TG+Los; n = 13); and 5) cardiac overexpression of the AT2-R and null for the AT1a-R (AT2-TG/AT1KO; n = 10). All mice were 8–14 wk of age at the start of the study period. We measured daily water intake in all losartan-treated animals to assure that mice were receiving the prescribed doses of losartan. MI was created on day 0. Noninvasive measurements of heart rate and systolic blood pressure were performed weekly postinfarction with the use of a Visitec-2000 tail-cuff apparatus. Cardiac magnetic resonance imaging (CMR) was performed at baseline and on days 1, 7, and 28 post-MI. Invasive hemodynamic measurements were performed before mice were euthanized and cardiac tissue was sampled for histological analysis.
Infarct Surgery
Myocardial infarction was created as described previously (30). Briefly, after mice were anesthetized with pentobarbital sodium at a dose of 80–100 mg/kg ip, the chest was opened and the left anterior descending artery was ligated with a 7-0 silk suture for 60 min over a short PE-50 tube; this was followed by reperfusion. Occlusion was verified by visual confirmation of color changes in the ischemic regions of the LV and by widened QRS complex on ECG.
Cardiac Magnetic Resonance Imaging
Murine CMR was performed as previously described (30). Briefly, a 4.7-T MRI system (Varian 200/400, Inova) with Magnex gradients (80 G/cm maximum strength) was used with a custom-built Litz radiofrequency coil (Doty Scientific, Columbia, SC). Anesthesia was induced with 3% isoflurane and was maintained with a 1% isoflurane-oxygen mixture throughout the study. Core body temperature was maintained at 37.0 ± 0.1°C by using an external water bath. ECG triggering was achieved with a gating/monitoring system (SA Instruments, Stony Brook, NY). After localizer images, contiguous short-axis bright blood cine images of the LV were obtained with a 2D-FLASH gradient echo sequence (Fig. 1). Cine-imaging parameters were as follows: repetition time, 8–10 ms; time to echo, 3.1–3.9 ms; flip angle, 20°; field of view, 2.56 cm; matrix, 128 x 128; slice thickness, 1 mm; voxel size, 100 x 100 x 1,000 µm3; and number of excitations, three. We obtained 12–14 phases for each of the six to eight contiguous short-axis slices. Infarct imaging was done by using the same sequence on day 1 post-MI, after the intraperitoneal injection of 0.3 mM/kg Gd-diethylenetriamine pentaacetic acid, by using a flip angle of 60° to increase postcontrast T1 weighting. This technique has been carefully validated against pathological measures of infarct size by our group (29).
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Short-axis cine images were analyzed by using ARGUS image analysis software (Siemens Medical Solutions, Princeton, NJ). Epi- and endocardial borders were planimetered to determine end-diastolic volume (EDV), end-systolic volume (ESV), LV mass (LVM), and ejection fraction (EF). Volumes and mass were indexed to body weight (EDVI, ESVI, and LVMI). Infarct size was measured on day 1 postcontrast cine images by planimetry of the area of late gadolinium enhancement with signal intensity >2 SD above the signal intensity of remote myocardium (29).
Hemodynamic Measurements
Invasive hemodynamic measurements were performed after the day 28 CMR examination. Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 µg/g body wt), enhanced by local subcutaneous injection of 0.5% bupivicaine (100 µl). Peripheral arterial pressure was measured by cannulating the right common carotid artery with PE-10 tubing, and LV pressure was measured after direct puncturing of the LV with a 27-gauge needle in the fifth intercostal space, connected to PE-50 tubing. Blood pressure, LV pressure, heart rate, and developed pressure (dP/dt) were recorded with a MacLab recording system.
Collagen Analysis
We performed collagen analysis as previously described (2). Briefly, hearts were removed after mice were euthanized, fixed in formaldehyde, and embedded in paraffin. Six-micrometer slices were prepared and stained with picric acid Sirius red. Morphometric analysis was done in the infarct border zone (adjacent) and in remote regions with the use of an Olympus microscope with a green 540-nm filter, interfaced with a CCD72 videocamera. Analysis was done with a Universal Imaging Image 1/AT morphometry system (West Chester, PA). Volume percent collagen was determined by measuring three to four sections from each region by calculating the mean from all fields in each region in each animal.
Myocyte Analysis
Three to five tissue samples from each heart were sectioned at 5-µm thickness, stained with picric acid Sirius red, and examined with an Olympus AH2 research microscope by using rhodamine epifluorescence. With the use of a x40 objective (x660 on monitor), the cross-sectional area was determined on a minimum of 60 myocytes from each animal in adjacent and remote noninfarcted regions, selected from areas judged to be within 20° of true cross section, by using a Universal Imaging AT1 image analysis system. The mean area was calculated for each animal.
Statistical Methods
Infarct sizes between groups and noninvasive and invasive hemodynamic parameters were compared by one-way ANOVA with Tukey subtesting. Regional percent collagen and regional myocyte size between groups were compared by two-way ANOVA with Tukey subtesting. Volumetric parameters were compared between all five groups by using F tests in repeated-measures models by using day 0 data as a covariate. Analyses were carried out by using PROC MIXED in SAS 9.1 (SAS Institute, Cary, NC). All values are presented as means ± SE; P < 0.05 was considered significant.
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RESULTS |
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Infarct size, expressed as percent LVM on day 1 post-MI, was similar among groups (42 ± 2% for WT, 39 ± 3% for WT+Los, 41 ± 3% for AT2-TG, 41 ± 2% for AT2-TG+Los, and 39 ± 2% for AT2-TG/AT1KO; P = not significant).
Noninvasive Blood Pressure
When noninvasive systolic blood pressures were averaged over the 4-wk post-MI time course, the AT2-TG/AT1KO group demonstrated lower systolic blood pressures than the other groups (111 ± 3 for WT, 106 ± 2 for WT+Los, 109 ± 3 for AT2-TG, 111 ± 3 for AT2-TG+Los, and 98 ± 3 mmHg for AT2-TG/AT1KO; P < 0.001).
LV Volumetric Parameters
A complete description of parameters is given in Table 1.
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Day 1. Acute LV dilatation and dysfunction on day 1 post-MI was characterized by an increase in ESVI and a fall in EF in all groups. EDVI was smaller in all three AT2-R TG groups than WT at this time point.
Day 7. LV dilatation continued through this time point, although ESVI and EF were best preserved in the AT2-TG/AT1KO and WT+Los groups (Table 1 and Figs. 2 and 3). EDVI was lower in all groups compared with WT.
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Day 28 Hemodynamics
On invasive hemodynamic measurements performed after completion of day 28 post-MI imaging, heart rate was similar in the five groups (Table 2). Invasive systolic, diastolic, and mean arterial pressures were lowest in the WT+Los and AT2-TG/AT1KO groups. (Table 2) LV end-diastolic and end-systolic pressures were also significantly lower in the WT+Los and AT2-TG/AT1KO groups. Positive dP/dt was similar in the five groups (Table 2).
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The fibrosis identified in noninfarcted regions was interstitial fibrosis. Collagen content on day 28 post-MI was not different among the five groups in remote regions, but in adjacent regions it was significantly lower in AT2-TG/AT1KO compared with all other groups (Table 3). For purposes of comparison, collagen content in noninfarcted WT mice is 1.2 ± 0.4%.
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Myocyte hypertrophy occurred in all groups on day 28 post-MI and was greater in adjacent noninfarcted regions than in remote regions (Table 4). For comparison purposes, myocyte size in noninfarcted C57Bl/6 WT controls is 164 ± 31 µm2. However, regional myocyte hypertrophy in both regions was similar among groups (Table 4).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Infarct size was similar among groups as measured on day 1 post-MI. Systolic blood pressure was lower in AT2-TG/AT1KO than in other groups from postinfarction through late in the post-MI course. By day 28 post-MI, when corrected for baseline differences, EDVI and ESVI were higher and EF was lower in WT than in all other groups. EF was highest and EDV and mass indexes were lowest in the AT2-TG/AT1KO group at day 28. The AT2-TG/AT1KO demonstrated less fibrosis in adjacent noninfarcted regions. Regional myocyte hypertrophy was similar in all groups.
Throughout the study, LV size and function were similar among the AT2-TG and AT2-TG+Los groups, suggesting that pharmacological inhibition of the AT1-R pathway provides no additional attenuation of post-MI LV remodeling in the setting of cardiac AT2-R overexpression. We chose a dose of losartan that has been shown to attenuate LV remodeling in the setting of hypertrophic cardiomyopathy in mice, without lowering systolic blood pressure (12). Indeed, LV remodeling was significantly attenuated in the WT mice treated with losartan alone when compared with untreated WT, equivalent to the group with cardiac overexpression of AT2-R.
Systolic blood pressure was, on average, 12–14 mmHg lower in AT2-TG/AT1KO compared with both AT2-TG and AT2-TG+Los throughout the study, consistent with previously reported data (21). This blood pressure lowering may have contributed to the additional benefit of AT1a-R knockout over and above AT2-R overexpression noted in the present study. In addition, AT2-TG/AT1KO showed additional effects on limiting fibrosis in adjacent noninfarcted regions, consistent with prior studies with a AT1a-R knockout model (4). A primary benefit of inhibiting the AT1-R may be due to this effect on collagen in noninfarcted regions. However, collagen levels before MI were not measured in AT2-TG/AT1KO mice and may be different at baseline, although baseline interstitial collagen in WT mice is quite low. These adjacent noninfarcted regions play a crucial role in the remodeling process (11) due to local increases in wall stress, a major stimulus to ANG II release (22). Cellular hypertrophy was similar among groups, but the power of the study to demonstrate differences in myocyte size was limited.
A recent study (10) in a canine model demonstrated that one of the effects of AT1-R blockade is to upregulate the AT2-R. This may explain why WT+Los findings were very similar to those of the AT2-TG group in the present study. The role of the AT2-R post-MI has been previously investigated in knockout models. In mice that were not able to mount a profibrotic and hypertrophic response, cardiac rupture was significantly increased post-MI (1, 5), suggesting a protective effect post-MI to maintain myocardial scar formation. However, the AT2-R null mouse may not be an ideal model of disease states because the AT2-R is expressed at very low levels in normal animals and is only upregulated in disease states. Therefore, some have suggested that models overexpressing the AT2-R may be more appropriate for study (23). For that reason, we have chosen to study AT2-R overexpression. Our previous studies (30) have demonstrated the protective effects of AT2-R overexpression in the post-MI setting. Pharmacological approaches to increase levels of the AT2-R post-MI may indeed be a reasonable goal based on these studies.
A similar interrelationship between the AT1-R and AT2-R has been shown post-MI with pharmacological inhibition of the AT2-R. With the use of a pharmacological AT2-R antagonist, one group demonstrated that the infarct size-reducing effect of AT1-R antagonism was dependent on the AT2-R (7). The beneficial effects of AT1-R blockade in rats with post-MI heart failure, including attenuation of interstitial fibrosis, hypertrophy, and LV volumes, were shown to be antagonized by pharmacological blockade of the AT2-R (14). This is additional evidence that much of the benefit of AT1-R blockade may be mediated by the AT2-R. In this study, treatment was started 2 mo after myocardial infarction, thus representing a more chronic heart failure state rather than an acute post-MI condition. These effects were reduced by bradykinin receptor blockade and eliminated in rats with impaired kininogen transport mechanisms, supporting the hypothesis that kinins, at least in part, mediate the benefits of the AT2-R. Furthermore, the benefits of valsartan postinfarction on LV EF, cardiac output, myocyte size, and fibrosis were significantly reduced in AT2-R knockout mice (27). These data lend further credence to the notion that the AT2-R mediates some of the benefit of AT1-R blockade and support our findings of equivalence of pharmacological blockade of AT1-R and AT2-R overexpression.
The interplay between the AT1-R and AT2-R has been studied in models other than post-MI remodeling. The AT1-R blocker valsartan inhibited both coronary arterial thickening and perivascular fibrosis in aortic-banded mice, but it was more potent in WT mice than in AT2-R null mice, suggesting that the AT2-R is necessary for the benefits of AT1-R blockade to be realized (26). In spontaneously hypertensive rats, AT1-R antagonism reduced blood pressure and myocardial fibrosis, which was in turn partially prevented by AT2-R antagonism (24). In aged rats, AT2-R antagonism reversed the reduction in myocardial hypertrophy and fibrosis afforded by AT1-R blockade with candesartan.(9) Thus data in models of infarction, hypertrophy, and aging show that the AT2-R is essential to mediate the effect of AT1-R blockade.
The downstream signaling pathways responsible for the benefits of AT2-R overexpression remain only partially elucidated. Nitric oxide has been shown to be an important mediator (2). Bradykinin may well be an important intermediate signaling molecule between the AT2-R and nitric oxide as it is in the kidney (3). Bradykinin itself has little direct effect on post-MI LV remodeling as demonstrated by lack of effect with bradykinin B2 receptor knockouts (28). However, the absence of the B2 receptor limited the benefits of ACE inhibitors and AT1-R blockade (28). Further work is necessary to clarify these issues.
Limitations
ANG II levels were not directly measured, and therefore the level of pharmacological inhibition of the AT1-R by losartan was not assessed directly. However, water consumption by the mice was carefully measured to ensure that they received the prescribed doses of losartan. In addition, WT mice treated with the same dose of losartan demonstrated significant attenuation of LV remodeling. Higher, supratherapeutic doses of losartan that significantly lower blood pressure may have had an effect similar to that of AT2-TG/AT1KO. However, the effects of ARBs in postinfarct remodeling have been shown to be independent of blood pressure lowering in animal models (14, 15). Blood pressure lowering was insignificant in clinical trials of ACE inhibition or ARBs post-MI and is unlikely to be the primary mode of benefit (19, 20).
The AT1-R knockout model was specific to the AT1a-R, thereby leaving the AT1b-R intact. However, the predominant effect of angiotensin on the AT1-R is thought to be mediated through the AT1a-R. The study was underpowered to examine some of the secondary end points such as myocyte size, but the most important clinical effect that impacts mortality in a given animal is that on LV size and function post-MI.
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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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  D. C. Isbell, S. Voros, Z. Yang, J. M. DiMaria, S. S. Berr, B. A. French, F. H. Epstein, S. P. Bishop, H. Wang, R. J. Roy, et al. Interaction between bradykinin subtype 2 and angiotensin II type 2 receptors during post-MI left ventricular remodeling Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3372 - H3378. [Abstract] [Full Text] [PDF]
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P < 0.05 vs. WT-Los; 
P < 0.05 vs. AT2-TG.