ARTICLE |
Correspondence to: Louise M. Burrell, Dept. of Medicine, U. of Melbourne, Austin and Repatriation Medical Centre, Studley Road, Heidelberg 3084, Victoria, Australia. E-mail: burrell@austin.unimelb.edu.au
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Summary |
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This study evaluated the effects of angiotensin-converting enzyme (ACE) inhibition after myocardial infarction (MI) on cardiac remodeling and gene expression and localization of components (ligands, receptors, and binding proteins) of the cardiac insulin-like growth factor (IGF) system. After ligation of the coronary artery, rats were randomized to vehicle or ACE inhibitor (Captopril, 50 mg/kg/day) for 4 weeks. Blood pressure, cardiac remodeling, and components of the IGF system were localized in the heart using in situ hybridization (ISH) and immunohistochemistry (IHC). The average infarct size was 42%. There were regional differences in the expression of the IGF system after MI, with increased IGF-I mRNA abundance in the border (24-fold) and infarct (12-fold) and increased IGF-binding protein (IGFBP)-3 mRNA in all areas of the failing left ventricle (threefold). Captopril reduced blood pressure, attenuated cardiac remodeling, and caused a threefold increase in IGF-I receptor mRNA and protein in infarct, border zone, and viable myocardium (p<0.01). Captopril had no effect on IGF-I mRNA but further increased IGFBP-3 mRNA and protein in the border zone, (p<0.05). The changes in the cardiac IGF system following MI are highly localized, persist for at least 4 weeks, and can be modulated by ACE inhibition. These data suggest that the benefits of ACE inhibitors in attenuation of cardiac remodeling may be mediated in part through increased expression of the IGF-I receptor sensitizing the myocardium to the positive effects of endogenous IGF-I. (J Histochem Cytochem 51:831839, 2003)
Key Words: experimental, heart, pathophysiology, cellular, ACE inhibitors, infarction, gene expression, growth factors, hormones
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Introduction |
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MYOCARDIAL INFARCTION (MI) causes activation of neurohormonal systems that preserve circulatory homeostasis but also play a role in the development and progression of congestive heart failure (CHF) through cytokine activation and cardiac remodeling (
A number of studies have assessed the role of the cytokine insulin-like growth factor (IGF)-I and growth hormone (GH) on myocyte injury and myocardial function after MI, and both IGF-I and GH have emerged as potential agents in the therapy of CHF (
Both the reninangiotensin system (RAS) and the IGF system are activated after MI, with increases in IGF-I mRNA (
Although the data suggest a potential therapeutic role for IGF-I/GH after MI, the effects of standard treatment with ACE inhibition after MI on the cardiac IGF system have not been assessed. Such information is useful in determining the mechanism of benefit of GH/IGF-I treatment, particularly because if it is to enter the therapeutic arena, it will be most likely used as an adjunct to ACE inhibition. The aim of this work was therefore to assess the effect of ACE inhibition with captopril on the cardiac IGF system after coronary artery ligation in the rat. The study included evaluation of cardiac remodeling and gene expression and localization of all components (ligands, receptors, and binding proteins) of the IGF system in the heart. Although most physiological actions of IGF-I and IGF-II are mediated via the IGF-I receptor, this interaction is modulated by at least six structurally related IGF binding proteins (IGFBP-1 to 6) that bind to IGF-I to inhibit or potentiate its activity (
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Materials and Methods |
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Experimental procedures were performed according to the National Health and Medical Research Council of Australia Guidelines for Animal Experimentation. Rats were housed at 2325C in a 12:12 light:dark cycle, with ad libitum food containing 0.40.6% NaCl and water.
Experimental Design
LV free-wall MI was induced in female SpragueDawley rats (200250 g) by ligation of the proximal anterior descending artery as described previously (
In situ hybridization (ISH) was used to localize mRNA for the IGF system (IGF-I, IGF-II, IGF-I receptor and IGFBPs 16) in Control and MI LV and to examine changes in this system after captopril (
ISH Histochemistry
ISH was performed on 4-µm sections from four levels of LV from MI (vehicle n=5, captopril n=5) and Control rats (vehicle n=5, captopril n=5) to localize the IGF system mRNA (IGF-I, IGF-II, IGF-I receptor, and IGFBPs 16).
Labeling of RNA Probes and Hybridization.
Complementary (antisense) or noncomplementary (sense) RNA probes were synthesized for rat IGFBP-16 (kindly provided by Dr. S. Shimasaki; Whittier Institute, La Jolla, CA), IGF-II (provided by Dr. P.K. Lund; University of North Carolina, Chapel Hill, NC), and IGF-I receptor and IGF-I (provided by Dr. C. J. Roberts, Jr and Dr. D. Le Roith, NIH, Bethesda, MD) and hybridized as previously described (
Quantitation of Macroscopic Autoradiographs.
Quantitation was performed using a microcomputer imaging device (Imaging Research; St. Catharines, Ontario, Canada) run by an IBM PC. Sections from four levels of the LV from each animal were used for quantitation. In MI hearts, the viable myocardium, infarct, and border zone were quantitated separately. The border zone is the area of high cellular infiltrate at the border zone of the fibrotic scar tissue of the infarct. The optical densities of the autoradiographs were calibrated in terms of radioactivity density as dpm/mm2 by reference to radioactive standards (Amersham) carried through the procedures (
Immunohistochemistry
Immunohistochemistry was carried out on 4-µm sections from four levels of LV from MI (vehicle n=10, captopril n=10) and Control rats (vehicle n=10, captopril n=5). A rabbit anti-mouse IGFBP-3 polyclonal antiserum (GroPep; Adelaide, Australia), which crossreacts with rat IGFBP-3, at a dilution of 1:150 was employed. A biotinylated goat anti-rabbit secondary antibody at a dilution of 1:400 (Vector Laboratories; Burlingame, CA) and the Elite Vectastain ABC kit (Vector Laboratories), followed by diaminobenzidine (Sigma; St Louis, MO) were used to visualize antibody binding. Negative control slides were incubated with normal goat serum; the primary antibody was excluded. For detection of IGF-I receptor protein, a mouse MAb to the IGF-I receptor (ß-subunit) (Neomarkers; Union City, CA), which crossreacts with the rat IGF-I receptor, was used at a dilution of 1:100. This antibody was used in conjunction with a Catalyzed Signal Amplification System (DAKO, Carpinteria, CA) and the peroxidase method of labeling according to the manufacturer's instructions.
Quantitation of IHC Staining.
Immunohistochemical staining for IGFBP-3 and IGF-I receptor protein was quantitated (n=510/group) using computerized image analysis (AIS Imaging; St. Catharines, Ontario, Canada). All sections used for quantitation were fixed, processed, sectioned, and immunolabeled at the same time and under the same conditions to limit variability. Sections from four levels of the LV from each animal were used for quantitation. In MI, the viable myocardium, infarct and border zone were quantitated separately. Twelve fields (x20 objective) from each region of the heart (viable myocardium, infarct, and border) were selected for assessment according to a predefined grid pattern. Images were imported into the AIS imaging program using a color video camera and a standard light microscope. The detection level threshold for positively stained areas (brown for DAB staining) was set so that the processed image accurately reflected the positively stained areas as visualized by light microscopy and on the unprocessed digital image. An average intensity for the selected area was then calculated. The percentage area of chromogen staining was determined by calculating the number of selected pixels (positively stained areas) in a given area and was expressed as a percentage of the entire image. The average intensity and area of staining were then multiplied to give the final figure (arbitrary units) (
Statistics
Results are presented as mean ± SEM. Before analysis of ISH histochemistry quantitation, results were log-transformed to stabilize variance where appropriate. Differences between values for both ISH and IHC were assessed using two-factor ANOVA, one factor being the presence of MI and the other being treatment (vehicle or captopril), followed by post-hoc analysis using the Fisher LSD test.
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Results |
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All sham-operated rats (Control, n=32) survived to 24 hr and were randomized to vehicle (n=17) or captopril (n=15). Of the rats operated on to produce an MI, 80% (n=20) were alive at 24 hr and were randomized to vehicle (n=10) or captopril (n=10).
Infarct Size, Body Weight, Blood Pressure, Cardiac Mass, and Hormones
The average infarct size was 42% and was similar in vehicle and captopril-treated MI (Table 1). No Control animal had evidence of cardiac damage. There was no pretreatment difference in body weight or systolic blood pressure between Control and MI rats (not shown). Control rats gained weight throughout the duration of the study with no treatment effect, whereas MI rats treated with captopril gained less weight than vehicle-treated rats (p<0.01). Captopril reduced SBP in both Control and MI (p<0.01). Results after 4 weeks of treatment are shown in Table 1. MI rats had CHF with increased LV and lung mass (p<0.05) compared to Control rats (Table 1). Captopril reduced LV mass in both MI and Control, whereas lung mass was reduced by captopril in MI only (p<0.05). Captopril increased PRA in all rats (p<0.01). Plasma ANP concentrations were elevated in MI rats compared to Control (p<0.01) and were reduced by captopril (p<0.05) (Table 1).
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Expression of the Cardiac IGF System After Infarction and Effects of ACE Inhibition
The cellular distribution of components of the IGF system 4 weeks after MI was similar to that previously published 24 weeks after MI (
IGF-I and IGF-II mRNA. As we have shown previously, after MI IGF-I mRNA levels are elevated with 24- and 12-fold increases in the border and infarct zones, respectively, compared to the viable myocardium (p<0.05) and twofold increases in the border compared to infarct (p<0.05) (Fig 1). ACE inhibition had no effect on IGF-I mRNA or on IGF-II mRNA.
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IGF-I Receptor mRNA and Protein. IGF-I receptor mRNA levels were similar in the viable myocardium of MI compared to Control rats, and were increased three- to fourfold in all areas of the infarcted heart (viable myocardium, border, infarct) by ACE inhibition (p<0.01; Fig 2A). ACE inhibition also increased IGF-I receptor protein levels (Fig 2B) in viable myocardium (p<0.05), border, and infarct (p<0.01). IGF-I receptor protein was localized to the cell membrane of myocytes in the Control (data not shown) and MI rats (Fig 3A and Fig 3B). An increase in IGF-I receptor protein levels was also seen in the infarct and border zone (Fig 2B; p<0.01), mainly in the endothelium of vessels within the infarct (Fig 3C and Fig 3D) and border (Fig 3E and Fig 3F).
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IGF Binding Proteins. IGFBP-3 mRNA levels were increased 2.4-fold in the viable myocardium of MI compared to Control rats (p<0.05) and were further increased in the border (3.5-fold) and infarct (three-fold) (p<0.01) (Fig 4A). ACE inhibition increased IGFBP-3 mRNA by 50% in the infarct and border (p<0.05; Fig 4A) but had no effect in the viable myocardium. This was associated with changes at the protein level, with ACE inhibition increasing IGFBP-3 protein significantly in the border (p<0.05; Fig 4B) and approaching significance in the infarct area (p=0.07; Fig 4B).
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IGFBP-3 protein was localized to myocardial cells (Fig 5A and Fig 5B) and to infiltrating cells and vessels of the infarct (Fig 5C and Fig 5D) and border (Fig 5E and Fig 5F), correlating with the distribution of mRNA in these areas. The distribution of IGFBP-3 protein was more widespread in the myocardial cells than that of the mRNA, which may reflect the fact that IGFBP-3 is a secreted protein and is therefore not confined to sites of synthesis (
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Expression of IGFBP-1 and IGFBP-2 mRNA was minimal in normal and infarcted hearts and was unchanged with ACE inhibition (data not shown). IGFBP-4 mRNA levels were 2.8-fold higher in viable myocardium of MI than Control (p<0.05) (Table 2) and were further increased in the border (6.6-fold) and infarct (4.6-fold) (p<0.01). IGFBP-6 mRNA expression was increased 5.5-fold in the border (p<0.01) (Table 2). Expression of IGFBP-5 mRNA was not significantly different between Control and CHF. There was no effect of ACE inhibition on IGFBP-4, -5, or -6 mRNA levels (Table 2).
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Discussion |
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The main findings of this study are as follows: (a) the changes in the cardiac IGF system after MI are highly localized and persist for at least 4 weeks; (b) ACE inhibition attenuates cardiac remodeling and pulmonary congestion; and (c) ACE inhibition enhances expression of IGF-I receptor gene and protein in the failing heart and causes further increases in IGFBP-3 gene and protein in the border zone. These data suggest a significant interaction between the cardiac IGF system and the RAS in the remodeling heart after MI. The benefits of ACE inhibitors in attenuation cardiac remodeling after MI may be mediated in part through increased expression of the IGF-I receptor sensitizing the myocardium to the positive effects of endogenous IGF-I.
This model of MI-induced CHF causes hemodynamic alterations and neurohormonal changes (
The results of this study agree with our previous findings, in that MI increased expression of IGF-I and IGFBP-3, -4, and -6 mRNA in the border and infarct and increased IGFBP-3 and -4 mRNA in the viable myocardium, with no change in the gene expression of IGF-II or the IGF-I receptor (
Many studies support a protective and anti-apoptotic role for GH and/or IGF-I in myocardial ischemia and MI (
This study assessed whether the cardiac benefits of ACE inhibition may be mediated in part through changes in the cardiac IGF system. As we have shown previously (
We found that the increase in IGF-I receptor expression was not associated with any change in expression of the endogenous ligand IGF-I. Gene expression profiling has produced similar results in the same model; captopril increased IGF-I receptor expression in the absence of changes in IGF-I expression (
The IGFBPs, which are potent modulators of the biological actions of IGF-I, were also assessed to more fully understand the role of the local IGF system in CHF. IGFBP-3 mRNA is present in the human heart and is more abundant in ventricles from patients with ischemic heart disease and hypertrophic cardiomyopathy than in controls (
Experimental studies have clearly demonstrated the benefits of GH and/or IGF-I after myocardial ischemia or infarction. However, because randomized clinical trials have also established ACE inhibitors as standard therapy following MI (
Our present study uses a histological approach to allow better localization of mRNA and protein, an approach that is not as sensitive as Northern and Western blotting, but that clearly demonstrates significant, highly localized changes in expression of the cardiac IGF system with MI-induced CHF in the rat that can be modulated by ACE inhibition. The benefits of ACE inhibition in attenuation of cardiac remodeling may be mediated in part through the upregulation of the cardiac IGF-I receptor, sensitizing the myocardium to the positive effects of endogenous IGF-I. These results are of functional significance if a therapeutic strategy is directed at the potentiation of a specific IGF-I/IGF-I receptor interaction. Because we found no change in expression of endogenous cardiac IGF-I with ACE inhibition, this study provides a rationale for the addition of GH and/or IGF-I to ACE inhibition in the management of ischemic heart failure.
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Acknowledgments |
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Supported by the National Heart Foundation of Australia, the Austin Hospital Medical Research Foundation, and the Sir Edward Dunlop Medical Research Foundation.
Received for publication August 8, 2002; accepted December 6, 2002.
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