In the brain, ouabain-like compounds (OLC) and the reninangiotensin system (RAS) contribute to sympathetic hyperactivity in rats after myocardial infarction (MI). This study aimed to evaluate changes in components of the central vs. the peripheral RAS. Angiotensin-converting enzyme (ACE) and angiotensin type 1 (AT1) receptor binding densities were determined by measuring 125I-labeled 351A and 125I-labeled ANG II binding 4 and 8 wk after MI. In the brain, ACE and AT1 receptor binding increased 8–15% in the subfornical organ, 14–22% in the organum vasculosum laminae terminalis, 20–34% in the paraventricular nucleus, and 13–15% in the median preoptic nucleus. In the heart, the greatest increase in ACE and AT1 receptor binding occurred at the infarct scar (∼10-fold) and the least in the right ventricle (2-fold). In kidneys, ACE and AT1 receptor binding decreased 10–15%. After intracerebroventricular infusion of Fab fragments to block brain OLC from 0.5 to 4 wk after MI, increases in ACE and AT1 receptors in the subfornical organ, organum vasculosum laminae terminalis, paraventricular nucleus, and medial preoptic nucleus were markedly inhibited, and ACE and AT1 receptor densities in the heart increased less (6-fold in the infarct scar). In kidneys, decreases in ACE and AT1 receptor binding were absent after treatment with Fab fragments. These results demonstrate that ACE and AT1 receptor binding densities increase not only in the heart but also in relevant areas of the brain of rats after MI. Brain OLC appears to play a major role in activation of brain RAS in rats after MI and, to a modest degree, in activation of the cardiac RAS.
- renin-angiotensin system
- heart failure
components of the brain renin-angiotensin system (RAS) are present in circumventricular organs, such as the organum vasculosum laminae terminalis (OVLT) and subfornical organ (SFO), which are exposed to blood-borne angiotensins, and many regions within the blood-brain barrier (BBB), such as the paraventricular nucleus (PVN), median preoptic nucleus (MnPO), and suprachiasmatic nucleus (1, 3). Central angiotensin type 1 (AT1) receptor stimulation plays a critical role in determination of sympathetic hyperactivity and progression to congestive heart failure (CHF) in rats after myocardial infarction (MI). Thus intracerebroventricular infusions of the AT1 receptor blocker losartan normalize sympathetic hyperactivity and impairment of baroreflex function (31) and prevent a significant component of left ventricular (LV) remodeling and impairment of LV pump function after MI (14). Intracarotid injections of the angiotensin-converting enzyme (ACE) inhibitor captopril or losartan normalize increased neuronal firing in the PVN of rats with CHF after MI (32). Functional studies provide evidence for involvement of the brain RAS. Whether, where, and which components of the RAS in rats with CHF after MI actually increase have not been explored. Studies by our group have shown that, in the brain, ouabain-like compounds (OLC) also play a critical role in the central pathways leading to sympathetic hyperactivity after MI (11). Moreover, effects of OLC in the central nervous system (CNS) appear to lead to activation of the brain RAS and, thereby, sympathetic hyperactivity (9). Whether a chronic increase in OLC release increases the activity of the brain RAS through increased angiotensin II (ANG II) release and/or increases other components of the RAS, such as ACE and AT1 receptor densities, has not been studied.
Therefore, the present study had two objectives: 1) to assess AT1 receptor and ACE binding densities by in vitro autoradiography in relevant brain nuclei in rats with mild-to-moderate CHF after MI relative to changes in the heart (24, 25) and kidneys and 2) to assess the role of brain OLC in the mechanisms leading to changes in AT1 receptor and ACE densities by chronic blockade of brain OLC using intracerebroventricular infusion of antibody Fab fragments binding ouabain and OLC with high affinity (11).
MATERIALS AND METHODS
Male Wistar Rats (Charles River Breeding Laboratories, Montreal, QC, Canada; 200–250 g body wt) were housed at 24°C on a 12:12-h light-dark cycle and given regular rat chow and tap water. All experimental procedures were carried out in accordance with the guidelines of the University of Ottawa Animal Care Committee for the Use and Care of Laboratory Animals. After a 5-day acclimatization, open-chest surgery was performed for a one-stage (22) or a two-stage MI model (13). Briefly, under halothane inhalation anesthesia, the thorax was opened at the fourth or fifth left intercostal space. The left coronary artery was ligated 2–3 mm from its origin with a 6-0 silk suture attached to an atraumatic needle (model K801H, Ethicon). For two-stage ligation, a loose loop of suture was placed around the left coronary artery without ligating the artery, and the ends of the suture were pulled to the back of the neck with a trocar. At 1 wk after recovery from the surgery, in conscious rats, the occluder was carefully pulled until it was no longer possible to move the occluder in relation to the outer guide. Sham rats underwent the same surgical procedure without ligation. Buprenorphine (0.03 mg/ml, 0.1 ml/rat, twice daily for 3 days) was used to relieve pain.
Experiment 1. At 1 and 2 mo after MI, four experimental groups were studied: one-stage MI (sham and MI) and two-stage MI (sham and MI). Under halothane anesthesia, a polyethylene (PE)-50 catheter was inserted in the LV through the right carotid artery. At 4 h after recovery from the anesthesia, the rat was placed in a cage without restraint for continuous recording of LV end-diastolic pressure (LVEDP), LV peak systolic pressure (LVPSP), and heart rate after 30 min of rest (11).
Experiment 2. At 3–5 days after the ligation, under halothane anesthesia, an L-shaped, 23-gauge stainless steel cannula was placed into the left lateral ventricle of the brain (9, 31). The longer arm of the cannula was connected via a PE catheter to an osmotic minipump (model 2002, Alza, Palo Alto, CA; 12 μl/day), which was filled with antibody Fab fragments (Digibind, Glaxo Wellcome, Toronto, ON, Canada) or γ-globulins (Sigma, St. Louis, MO) as a control (200 μg/day for both). Sham-operated rats were treated with γ-globulin (sham + IgG), and rats with CHF after MI were randomly divided into intracerebroventricular γ-globulin (CHF + IgG) and intracerebroventricular Fab fragment (CHF + Fab) groups. All pumps were implanted subcutaneously on the back. At 2 wk after the intracerebroventricular surgery, under halothane anesthesia, the pumps were replaced with new pumps filled with the same compounds. At the end of the 4-wk infusion, LVEDP, LVPSP, and heart rate were measured as described in Experiment 1.
Assessment of Infarct Size
Because the hearts were needed for autoradiography, MI sizes were determined by visual inspection and categorized as large (>30% of LV) or small (<30% of LV) for inclusion in the large-MI or the small-MI group.
Quantitative In Vitro AT1 Receptor and ACE Autoradiography
After the animals were decapitated, the brain, heart, and kidneys were removed and quickly frozen in 2-methylbutane at –40°C and stored at –80°C. The standard autoradiography protocol was performed (28). Briefly, cryostat serial 20-μm sections were mounted onto Superfrost Plus microscope slides (VWR, West Chester, PA) and stored at –80°C. To investigate AT1 receptor binding, sections were preincubated in 5 mM Na2EDTA, 0.2% BSA, and 0.4 mM bacitracin (Sigma) at room temperature for 15 min and then incubated in the same buffer with 0.3 μCi/ml 125I-Sar1,Ile8-ANG II (2,176 Ci/mmol; Washington State University Peptide Radioiodination Service Center, Pullman, WA) plus PD-123319 (10–5 M; Sigma), an AT2 receptor antagonist, for 1 h at room temperature. Nonspecific binding was determined in the presence of 1 μM unlabeled ANG II (Sigma). For ACE autoradiography, the 10 mM phosphate incubation buffer (pH 7.4) contained 0.3 μCi/ml (30 pM) of 125I-labeled 351A (125I-351A) and 0.2% BSA. Nonspecific binding was determined in the presence of 100 mM EDTA, which completely abolished the 125I-351A binding signal. The concentrations of the two ligands are based on previous studies (24, 25) and cause maximal binding for measurement of actual densities.
After four washes, the slides were dried and then exposed to Kodak Biomax MR film (Eastman Kodak, Rochester, NY) for 48 h, along with a set of methylacrylate 125I standards (Washington State University Peptide Radioiodination Service Center). The film was processed in a Kodak X-OMAT automatic developer. 125I-labeled ANG II (125I-ANG II) binding and ACE binding densities were quantified using a computer-assisted image analysis system (AIS/C, Imaging Research, St. Catharines, ON, Canada) and converted to femtomoles per milligram and femtomoles per gram, respectively, by comparison with the calibrated relative optical density of the 125I standards. Specific binding density was calculated as total binding minus nonspecific binding, which was <2–5% in control and experimental rats.
The brain nuclei localization in the cryostat sections was defined according to the rat brain atlas of Paxinos and Watson (20). AT1 receptors and ACE in cardiovascular regulatory nuclei outside the BBB (OVLT and SFO) as well as nuclei inside the BBB (PVN and MnPO) were examined. As peripheral tissues, the heart and the renal medulla and cortex were included.
Values are means ± SE. One-way ANOVA was used to determine the effects of treatments and MI on the various parameters. Statistical significance was defined as P < 0.05. In all instances, groups undergoing the one- and two-stage MI surgery followed the same pattern, and results were therefore combined for the one- and two-stage groups into one group for the small MI and one group for the large MI.
To calculate percent changes in densities in the MI groups, the average of the control group was considered 100%, and the values in the MI groups are expressed as a percentage of this average.
In the large-MI groups, resting LVPSP was significantly decreased and LVEDP increased at 1 and 2 mo. In the small-MI groups, these changes were significant only at 2 mo (Table 1). Heart rate was similar in all groups.
After blockade of brain “ouabain,” LVEDP increases in MI + Fab groups were significantly less than in the MI control groups, and LVPSP showed only a minor [nonsignificant (NS)] decrease.
Changes in AT1 Receptor Density
Brain. Tables 2 and 3 show absolute densities and Fig. 1 shows an actual autoradiograph. Figures 2 and 3 show relative changes in densities. At 4 wk after MI, in rats with large MI, AT1 receptor binding was clearly increased in the OVLT, SFO, and PVN, but only modestly in the MnPO. At 8 wk after MI, AT1 receptor binding remained elevated in the OVLT, SFO, and PVN and was also increased in the MnPO. Rats with small MI showed minor (5–10%) increases (Table 2 and Fig. 2).
In the central blockade experiment, AT1 receptor binding was significantly reduced in the SFO, OVLT, PVN, and MnPO in Fab-treated MI rats compared with MI controls. Densities remained modestly elevated in rats with large MI, but not in rats with small MI (Table 3 and Fig. 3).
Heart. AT1 receptors are normally expressed in low numbers in the rat heart (Table 4). After MI, AT1 receptor binding was clearly increased at the site of the infarct, increased somewhat less in the peri-infarct zone and noninfarct part of the LV, and increased only modestly in the right ventricle (RV) at 4 and 8 wk (Table 4). Rats with large MI showed only minor increases relative to rats with small MI.
In MI rats treated with Fab fragments, AT1 receptor binding was significantly reduced at the site of the infarct and in the peri-infarct zone of the LV, but densities remained significantly increased compared with sham groups (Table 5). Fab treatment tended (NS) to decrease AT1 receptor binding in the noninfarct zone of the LV and RV.
Kidney. High AT1 receptor densities were found in the medulla and cortex (Table 4). After MI, AT1 receptor binding was significantly decreased in the medulla of rats with large MI at 4 and 8 wk and showed minor decreases in the cortex. In the Fab-treated MI group, the decrease in the medulla was less and not statistically different from the sham group (Table 5).
Changes in ACE Density
Brain. At 4 and 8 wk after MI, ACE binding was significantly increased in the SFO, OVLT, and PVN, but only modestly in the MnPO. Increases were less in rats with small MI than in rats with large MI (Table 2 and Fig. 2).
Treatment with Fab fragments clearly reduced ACE binding in the SFO, OVLT, PVN, and MnPO, but the ACE density remained increased in the OVLT and PVN of rats with large MI compared with the sham group (Table 3 and Fig. 3).
Heart. After MI, changes in ACE binding were similar to those in AT1 receptor binding. At 4 and 8 wk after MI, ACE binding was obviously increased at the site of the infarct, somewhat less in the peri-infarct zone and noninfarct part of the LV, and more modestly in the RV. Rats with large MI showed only minor further increases relative to rats with small MI (Table 4).
In MI rats treated with Fab fragments, ACE binding was significantly decreased at the site of the infarct, in the peri-infarct zone, and in the RV, but densities remained significantly increased compared with sham groups (Table 5). Fab treatment tended (NS) to decrease ACE binding in the noninfarct zone of the LV.
The present study demonstrates tissue-specific regulation of two important components of tissue RASs, i.e., AT1 receptor and ACE densities after MI: 1) marked upregulation in the heart (>4-fold in the LV), 2) modest (15–30%) increases in brain nuclei involved in regulation of sympathetic tone and cardiovascular homeostasis, and 3) significant decreases (10–20%) in the kidney. Chronic blockade of brain OLC after MI, to a large extent, prevented the increases in AT1 receptor and ACE densities in the brain nuclei studied, attenuated the increases in the heart, and largely prevented the decreases in the kidney.
In rats after MI, functional studies clearly established that the brain RAS through AT1 stimulation plays an essential role in the sympathoexcitation (31) and contributes to the progression of LV dysfunction (14). The present study demonstrates that this functional dependency is associated with increased densities of two components of the brain RAS: ACE and AT1 receptor densities increased in parallel in brain nuclei outside (OVLT and SFO) and inside (MnPO and PVN) the BBB involved in regulation of sympathetic tone. Interestingly, even rats with small MI and only small increases in LVEDP showed some increases in densities, suggesting that modest LV dysfunction is already sufficient to activate central pathways. This finding is consistent with our previous study, in which Fra-like immunoreactivity was a marker of long-term neuronal activation, which also showed significant increases in the PVN and supraoptic nucleus of rats with small MI (27). Modest LV dysfunction also leads to impairment of arterial baroreflex function (5, 6). LV dysfunction may activate the CNS through a variety of pathways, e.g., activation of the cardiac sympathetic afferent reflex (17) and the circulatory renin-angiotensin-aldosterone system (12). We previously showed a 50–100% increase in plasma ANG II up to 2 mo after MI (12). One may speculate that circulating ANG II and/or aldosterone (4, 7, 19, 23, 30) increases AT1 receptor and ACE densities and neuronal activity in the SFO and OVLT and then the intrinsic RAS is activated in nuclei inside the BBB, such as the MnPO and PVN. Consistent with this concept, intracarotid injections of an AT1 receptor blocker, ACE inhibitor, or aldosterone receptor antagonist normalize increased neuronal activity in the PVN of rats with CHF after MI (32).
On the other hand, chronic blockade of brain OLC fully prevented the increases in brain AT1 receptor and ACE densities in rats with small MI inside and outside the BBB and a major part of the increases in rats with large MI. In addition, in rats with small MI, blockade of brain OLC prevented the modest increase in LVEDP. In these rats, blockade of brain OLC may prevent sympathetic hyperactivity and, thereby, the development of modest LV dysfunction and increases in plasma ANG II and aldosterone and their central effects. In rats with large MI, chronic blockade of brain OLC prevented part of the increase in LVEDP and LV dilation (similar to our previous study) (14). The component prevented by blockade of brain OLC may reflect progression of LV dysfunction due to sympathetic hyperactivity, whereas the component remaining may reflect the loss of functional myocardium per se and the associated increase in end-diastolic wall stress. In this case, some activation of, e.g., the cardiac sympathetic afferent reflex and plasma ANG II and aldosterone may still occur and, therefore, some increase in brain AT1 receptor and ACE densities but, in the presence of blockade of brain OLC, does not lead to sympathetic hyperactivity. How and where in the CNS OLC and the RAS interact have not been elucidated. In rats with salt-sensitive hypertension, OLC and AT1 receptors in the MnPO appear to play a major role (2).
The heart showed clear increases in AT1 receptor and ACE densities that were fairly similar at 4 and 8 wk after MI: up to 10-fold at the site of the infarct and 4- to 5-fold in the noninfarcted LV. The pattern of increase and the extent of the increases in densities are similar to those reported in previous studies (24–26). These studies used large MI and did not provide data for rats with small MI. Severalfold increases in cardiac AT1 receptor and ACE densities in the noninfarcted LV of rats with small MI and only modest increases in LVEDP, and not much greater increases in rats with large MI, despite more clear increases in LVEDP, were somewhat unexpected. Moreover, blockade of brain OLC normalized LVEDP in rats with small MI and blunted the increase (18 ± 3 vs. 6 ± 1) in rats with large MI, but 60–70% of the increases in densities persisted in rats with small or large MI. For the whole LV, stretch appeared therefore to play only a modest role in the increased densities. However, one has to consider that autoradiography does not separate densities on myocytes vs. fibroblasts. Myocytes, but not fibroblasts, respond to stretch with increased expression of RAS genes and proteins (16, 18). Thus it is possible that myocyte densities differed more substantially in rats with small MI than in those with large MI and after blockade of brain OLC. Besides stretch, cardiac sympathetic hyperactivity, ANG II, and aldosterone may contribute to the difference between blockade of brain OLC and no blockade.
In the kidneys, AT1 receptor and ACE densities showed modest decreases that were significant only in rats with large MI. Only minor (NS) decreases persisted after blockade of brain OLC. Chronic ANG II infusions significantly decreased AT1 receptor binding but not total AT1 receptor-protein abundance, suggestive of increased internalization of the receptor-agonist complex without an increase in expression. In contrast, the ANG II infusion significantly increased ACE densities in the proximal convoluted tubules (8). It appears therefore that an increase in ANG II alone cannot explain the decreases in AT1 receptor and ACE densities after MI. The authors are aware of one previous study showing no changes in renal AT1 receptor mRNA 30 days after MI, as assessed by Northern blotting (10).
Limitations of the Present Study
In vitro autoradiography evaluates the extent of membrane-bound receptors (AT1) or enzyme (ACE). Changes in these densities cannot be directly related to changes in function of the local RAS. In particular, changes in internalization or turnover may affect the membrane-bound component inversely. Other techniques (e.g., measurements of mRNA or protein) or approaches (e.g., ACE inhibitor to block ANG II formation and, therefore, AT1 receptor endocytosis) (29) are needed to complement our findings. In addition, other techniques are needed to address which specific types of cells show the changes in densities.
In conclusion, the present study demonstrates that, in the chronic phase after MI, rats exhibit significant increases in AT1 receptor and ACE densities not only in the heart, but also in brain nuclei involved in regulation of sympathetic tone and cardiovascular homeostasis. Chronic blockade of brain OLC attenuates LV dysfunction after MI and the increases in densities, to a large extent in the brain and to a modest degree in the heart. These findings support functional studies showing a major role for brain OLC in development of sympathetic hyperactivity after MI involving the brain RAS.
The derivate of lisinopril, 351A, was kindly donated by Dr. Sun, Division of Cardiovascular Diseases, Department of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163.
Present address of H. Wang: Laboratory of Cardiac Growth and Differentiation, Institut de Recherches Cliniques de Montreal, 110 des Pins Ouest, Montreal, QC, Canada H2W 1R7.
This study was supported by Canadian Institutes of Health Research Operating Grant MOP 13182 and Heart and Stroke Foundation of Ontario Grant T4716. H. Wang was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research/Canadian Hypertension Society. F. H. H. Leenen holds the Pfizer Chair in Hypertension Research, an endowed chair supported by Pfizer Canada, the University of Ottawa Heart Institute Foundation, and the Canadian Institutes of Health Research.
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- Copyright © 2004 by the American Physiological Society