ARTICLE |
Correspondence to: Rachael Dean, Dept. of Medicine, University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg 3084, Victoria, Australia.
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Summary |
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Although cardiac effects of growth hormone (GH) and insulin-like growth factor (IGF)-I have been reported in experimental models of heart failure and in human dilated cardiomyopathy, the IGF system has not been comprehensively assessed in the failing heart. We therefore localized the IGF system in the left ventricle during congestive heart failure after myocardial infarction (MI) in the rat. The left anterior descending coronary artery was ligated in adult female SpragueDawley rats and hearts were examined after 6 months when congestive heart failure had developed. In situ hybridization histochemistry was used to localize mRNA for the components of the IGF system in the left ventricle of sham and congestive heart failure animals. We were able to detect changes in the spatial distribution of mRNA for IGF-I and IGF binding proteins 3, 4, 5, and 6 in the left ventricle during congestive heart failure after MI. IGF-I and the binding proteins were predominantly increased in the infarct/peri-infarct area of the left ventricle. Other components of the IGF system were indistinguishable from the low to undetectable levels in sham-operated rats. These results demonstrate that the IGF system is altered in the failing heart and suggest that the IGF system plays an important role in the response of the heart to MI and consequent failure. (J Histochem Cytochem 47:649659, 1999)
Key Words: heart failure, binding protein, in situ hybridization, left ventricle, myocardial infarction, insulin-like growth factor, rat
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Introduction |
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Insulin-like growth factor (IGF)-I stimulates tissue growth and differentiation in a paracrine, autocrine, and/or endocrine fashion (
Congestive heart failure (CHF) is the final common pathway of many cardiovascular diseases, such as coronary artery disease and hypertension. CHF is considered to be an irreversible and progressive process characterized by ventricular dilatation and hypertrophy, a process known as remodeling, and by diminished pump performance.
Changes in expression of components of the cardiac IGF system have been reported in different models of cardiac ischemia, including acute myocardial infarction (MI) (
This is the first study to assess all components of the cardiac IGF system (ligands, receptors, and IGFBPs) at the cellular level in normal or abnormal left ventricle (LV). Furthermore, we describe in detail the endogenous responses of the IGF system in the rat LV 6 months after MI, when CHF is firmly established. This is a clinically relevant time point because CHF is a progressive disease. Furthermore, it is pathophysiologically relevant because remodeling continues even though the scar is mature.
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Materials and Methods |
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Animals
Experimental procedures were approved by the Austin Hospital Animal Research Ethics Committee and performed according to the National Health and Medical Research Council of Australia guidelines for animal experimentation. Female SpragueDawley rats (200250 g) were obtained from the Biological Research Laboratory, Austin and Repatriation Medical Centre, Heidelberg, Australia. Rats were housed at 2325C in a 12:12 light:dark cycle, with ad libitum food containing 0.40.6% NaCl (Norco) and water.
Surgical Production of MI
The rat model of MI resulting in CHF has been extensively used to examine the efficacy of therapeutic interventions in cardiac remodeling and survival (
Biochemical, Hormonal, and Cardiovascular Structural Data
Six months after operation, rats were sacrificed and trunk blood was collected into EDTA tubes for measurement of atrial natriuretic peptide (ANP) and plasma renin activity (
Infarct Size
The LV/interventricular septum was fixed in 10% buffered formalin. Each LV was sectioned at four levels from the base to the apex and paraffin-embedded. Sections were cut and stained with Masson's trichrome, and hematoxylin and eosin. The mean epicardial and endocardial scar circumference was compared to total LV circumference to calculate total infarct size (
In Situ Hybridization Histochemistry
Four-µm sections were cut from each level from CHF and sham-operated (n = 4/group) hearts for in situ hybridization histochemistry, as previously described (
Labeling of RNA Probes
Complementary (anti-sense) or noncomplementary (sense) RNA probes were synthesized for rat IGFBP-1 to -6 (kindly provided by Dr. S. Shimasaki; Whittier Institute, La Jolla, CA), IGF-II (kindly provided by Dr. P.K. Lund; University of North Carolina, Chapel Hill, NC), and IGF-I receptor and IGF-I (kindly provided by Dr. C. J. Roberts, Jr., and Dr. D. Le Roith; National Institutes of Health, Bethesda, MD), as previously described (
Hybridization
Before hybridization, sections were dewaxed, hydrated in graded ethanol and ultrapure water (MilliQ Purification Systems; Millipore Corporation, Bedford, MA), treated with Pronase E (Sigma Chemical; St. Louis, MO) (125 mg/ml), dehydrated in 70% ethanol, and air-dried. The 35S-labeled RNA probes (5 x 105 cpm/25 µl hybridization buffer) were added to a hybridization buffer consisting of 300 mmol/liter NaCl, 10 mmol/liter Tris-HCl, pH 7.5, 10 mmol/liter Na2HPO4, pH 6.8, 5 mmol/liter EDTA, pH 8.0, 1 x Denhardt's solution, 50 mg/ml yeast RNA, 50% deionized formamide, and 10% (w/v) dextran sulfate. The hybridization buffer containing labeled probe was preheated to 85C (5 min). Appropriate volumes were placed on the sections, which were then coverslipped and placed in humidified (50% formamide) chambers for 1416 hr at 60C.
After hybridization, coverslips were removed and slides were washed in 2 x standard saline citrate (2 x SSC: 0.3 mol/liter NaCl, 0.33 mol/liter Na3C6H5O7.2H2O) containing 50% formamide preheated to 55C, and then washed in 2 x SSC/50% formamide at 55C for 1.5 hr. Sections were then washed in several changes of RNase A buffer (10 mmol/liter Tris-HCl, pH 7.5, 1 mmol/liter EDTA, pH 8.0, 0.5 mol/liter NaCl) preheated to 37C and then treated with 150 µg/ml RNase A in RNase A buffer for 1 hr at 37C, followed by a wash in 2 x SSC at 55C for 45 min. After final dehydration through graded ethanol, slides were air-dried and autoradiographed with Kodak XAR (Eastman Kodak; Rochester, NY) film for 13 days at room temperature. Slides were then dipped in photographic emulsion (Amersham; Poole, UK), stored with desiccant at 4C for 1421 days, developed in Kodak D19, fixed in Ilford Hypam, and stained with hematoxylin for cellular localization.
Quantitation of Macroscopic Autoradiographs
Quantitation was carried out using a microcomputer imaging device (MCID) (Imaging Research; St Catherines, Ont, Canada) run by an IBM PC. Four sense and four antisense sections from each animal were used for quantitation. In CHF hearts, the hypertrophied LV and infarct/peri-infarct regions were quantitated separately. The optical density of the autoradiographs was calibrated in terms of radioactivity density by reference to 14C radioactive standards (Amersham) carried through the procedures. 14C radioactive standards can be used to calibrate 35S signal in tissue (
Immunohistochemistry
Immunohistochemistry for macrophages was carried out on protease-digested 4-µm paraffin sections of formalin-fixed LV. Standard techniques using a rat macrophage antibody, AIAD51240 (Accurate Chemicals; Westbury, NY) at a dilution of 1:200 and the Elite Vectastain ABC kit (Vector Laboratories; Burlingame, CA) were employed.
Statistics
Results are presented as mean ± SEM. Comparisons of tissue weights and vasoactive hormone levels were made by unpaired t-tests. Quantitative in situ hybridization variables were log-transformed before statistical analysis to stabilize variance. Comparisons were made using one-way ANOVA followed by a Scheffe F-test. Results were considered significant when p<0.05.
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Results |
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Coronary Artery Ligation in the Rat Results in CHF After 6 Months
The average size of MI in CHF rats was 39 ± 2%. No sham-operated animal had evidence of cardiac damage. All rats grew normally over the study period, and body weights in sham and CHF rats were not different (Table 1). CHF rats were characterized by increased relative LV, RV, LA, RA, and lung masses (Table 1), consistent with the presence of CHF (
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IGF-I mRNA Is Increased in the Peri-infarct and Infarct Zones
After MI, there was a significant increase in IGF-I mRNA expression in the peri-infarct/infarct area (p= 0.0016; Figure 1; Table 2). This was particularly evident in the cellular infiltrate (Figure 2A and Figure 2B), endocardium (Table 3), and blood vessels within the infarct/peri-infarct zone. However, there was little expression of IGF-I mRNA in the hypertrophied LV away from the infarct. No detectable IGF-I mRNA signal was observed in the LV of sham-operated rats (Figure 1; Table 3). In contrast, arterial endothelium was the sole site of expression of IGF-II mRNA in sham LV and was unchanged in the hypertro-phying LV or infarct/peri-infarct regions of CHF LV (Table 3).
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IGF-I receptor mRNA was moderately expressed in myocardial cells of sham and CHF LV (Table 3). A similar level of signal representing IGF-I receptor mRNA was also seen in the infarct and peri-infarct cellular infiltrate (Table 2 and Table 3).
Cell-specific Localization of mRNA for IGFBPs in Normal LV
In sham-operated rats with normal LV, IGFBP-3 mRNA signal was observed over individual cells of the endocardium, pericardium, over the conducting system, and over cells on the aortic side of the aortic valve (Figure 3A and Figure 3B, Table 4).
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Signal representing IGFBP-4 mRNA was abundant over endothelial cells of blood vessels (Figure 3C and Figure 3D) and, to a lesser extent, over individual cells of the valve leaflet (Table 4).
The conducting system and blood vessels within the pericardium showed high levels of IGFBP-5 mRNA hybridization in sham LV (Figure 3E and Figure 3F; Table 4). In contrast, vessels within the myocardium were not labeled, but groups of cells surrounding the vessels were (Figure 3G and Figure 3H; Table 4); these may represent bundles of conducting fibers, infiltrating cells, or nerves. Individual cells throughout the myocardium and within the endocardium were also labeled (Figure 3E and Figure 3F; Table 4).
In sham LV, low-abundance signal representing IGFBP-6 mRNA was observed at the junction of valve leaflets and the myocardium in an area that may represent part of the A-V node, as well as over cells surrounding arteries (Table 4). Signal representing IGFBP-1 and IGFBP-2 was very low over the myocardium in sham LV (Table 4).
Expression of IGFBPs Is Elevated over the Scar and Peri-infarct Zone of Infarcted LV
Expression of IGFBPs 1 and 36 was significantly elevated in the infarct and peri-infarct regions (Figure 1; Table 2) of CHF LV (IGFBP-1, p=0.01; IGFBP-3, p=0.0154; IGFBP-4, p<0.0001; IGFBP-5, p=0.02; and IGFBP-6, p=0.0016). IGFBP-4 mRNA was also increased in the hypertrophied LV of CHF rats (p= 0.0002).
At the microscopic level, IGFBP-3 (Figure 4A and Figure 4B) and IGFBP-4 (Figure 4C and Figure 4D) mRNA signals were detectable over the infiltrating cells and throughout the cells of the connective tissue of the peri-infarct area (Table 4). Cells of the myocardium away from the infarct in the hypertrophying LV were also labeled for IGFBP-4 mRNA. IGFBP-5 mRNA labeling of infarct and peri-infarct infiltrating cells was not as intense as those of IGFBP-3 and IGFBP-4 but was largely confined to cells lining the blood vessels in these regions (Figure 4E and Figure 4F; Table 4). This was in contrast to the lack of IGFBP-5 mRNA labeling over blood vessels throughout the hypertrophied LV.
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IGFBP-6 mRNA was moderately expressed over the entire infarct and peri-infarct area, including infiltrating cells and cells of the mature scar, but was not increased over vessels (Figure 4G and Figure 4H; Table 4). Signal representing IGFBP-1 or IGFBP-2 mRNA was very low over the entire myocardium in both sham and CHF left ventricles, including the scar (Table 4). Although the increase in IGFBP-1 mRNA in the infarct/peri-infarct area was statistically significant, levels of signal were very low and, given the lack of increase in signal over specific cells at the microscopic level, the biological significance of this result is questionable. Furthermore, the rise in IGFBP-1 mRNA was inconsistent because it was seen in only two of the four animals.
Pericardial Expression of IGF-I and IGFBP 36 mRNA Is Elevated in Infarcted LV
Infiltrating cells of the pericardium of CHF LV were abundantly labeled for IGFBP-3 (Figure 5A and Figure 5B), IGFBP-4 (Figure 5C and Figure 5D), IGFBP-5 (Figure 5E and Figure 5F), IGFBP-6 (Figure 5G and Figure 5H), and IGF-I (Figure 5I and Figure 5J) mRNAs (Table 3 and Table 4).
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Macrophages Were Not the Sole Type of Infiltrating Cell to Express Components of the IGF System
Immunohistochemistry for macrophages was carried out on serial sections of LV. The sections used for immunohistochemistry were adjacent to those used for in situ hybridization. This enabled us to determine whether labeling of cells in the infarct/peri-infarct area and pericardium with in situ hybridization was confined to macrophages. We found that signal for IGF-I and IGFBP-3, -4, -5, and -6 mRNA co-localized with macrophage staining; however this was not the only cell type to express the mRNA (not shown).
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Discussion |
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This is the first study to localize the components of the IGF system at the cellular level in the LV of normal and infarction-induced failing rat hearts. Coronary ligation in the rat produces a uniform model of CHF characterized by increased cardiac and lung weight and increased plasma atrial natriuretic peptide levels.
In this model, mRNA for IGF-I and IGFBP-36 was significantly increased in the infarct/peri-infarct area of the failing LV. However, there was little change in the hypertrophying myocardium, with only IGFBP-4 showing a significant increase. The components of the IGF system each had unique patterns of cellular distribution, with different cells of the LV, including infiltrating cells, vessels, valve leaflet cells, and myocytes, being labeled.
The heart is a GH-dependent organ (
Our study demonstrates that IGF-I mRNA remained significantly elevated in the peri-infarct and infarct areas but not in the hypertrophied LV 6 months after MI. Previous studies using a rat model of MI have shown that IGF-I immunoreactivity is increased in the viable myocardium immediately adjacent to the infarct and, to a lesser extent, over myocardial tissue distant from the infarct (24 hr and 20 days after infarct) (
The role of the IGFBPs in the setting of CHF is complex because they may stimulate or inhibit IGF actions (Bach et al. 1995). In the first 3 days after myocardial microembolization in porcine heart, there are elevations in IGFBP-3 and -6 mRNA and a decline in IGFBP-5 mRNA in the ischemic tissue (
Although the infiltrating inflammatory cells of the infarct and peri-infarct region were the major cell types to show an increase in IGFBP mRNA, they were not the only cell types to do so. For example, IGFBP-3 and -4 mRNA were increased in connective tissue cells, such as fibroblasts, and IGFBP-5 mRNA was increased over vascular endothelial cells in the infarcted region. This lends support to the contention that the response is not purely confined to infiltrating inflammatory cells but also involves the resident cells of the myocardium. Furthermore, resident fibroblasts and macrophages distant from the infarct did not have high levels of expression, indicating that the increased expression in the peri-infarct/infarct region was not solely due to the infiltration of cells with high levels of basal expression.
This study has demonstrated significant changes in the expression of components of the IGF system in failing myocardium 6 months after infarction. There was a dramatic upregulation of the IGF system in the infarct/peri-infarct area, an area of repair and remodeling, suggesting that the IGF system plays an important role in the response of the heart to ischemic injury and consequent cardiac failure. Because the results of the present study at 6 months after myocardial infarction differ from those of previous studies examining the IGF system soon after myocardial infarction (
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Acknowledgments |
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Supported by grants from the National Heart Foundation and the Austin Hospital Medical Research Foundation.
Many thanks to Mr John Risvanis for carrying out the immunohistochemistry.
Received for publication September 21, 1998; accepted December 1, 1998.
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