Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 1996;78:536-546

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, W.
Right arrow Articles by Anversa, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, W.
Right arrow Articles by Anversa, P.
(Circulation Research. 1996;78:536-546.)
© 1996 American Heart Association, Inc.

Articles

Aging Does Not Affect the Activation of the Myocyte Insulin-Like Growth Factor-1 Autocrine System After Infarction and Ventricular Failure in Fischer 344 Rats

Wei Cheng, Krzysztof Reiss, Peng Li, Micky J. Chun, Jan Kajstura, Giorgio Olivetti, Piero Anversa

From the Department of Medicine, New York Medical College, Valhalla.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Abstract To determine whether the attenuation in the growth capacity of myocytes in the overloaded aging heart is associated with an impairment in the activation of insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) in the stressed cells, large myocardial infarcts were produced in Fischer 344 rats at 4 and 16 months of age, and the animals were killed 6 hours, 3 days, and 7 days later. After the documentation of cardiac failure, the unaffected myocytes were enzymatically dissociated, and the expression of IGF-1 and IGF-1R was measured at these three time points after surgery. The level of expression of IGF-1R mRNA increased at 3 days and remained elevated at 7 days in both age groups. In addition, an increase in IGF-1R protein in these cells was found, with no apparent difference with age. This phenomenon was coupled with an upregulation of IGF-1 mRNA of comparable magnitude in the younger and older animals. In contrast, the increases in the dimensional properties of myocytes were delayed and of smaller magnitude in the older infarcted rats. Moreover, the expression of atrial natriuretic factor, used as a molecular marker of myocyte cellular hypertrophy, was greater at 3 days in 4-month-old rats and at 7 days in 16-month-old rats. Thus, aging may affect the hypertrophic response of myocytes after infarction but has no impact on the ability of the cells to enhance the expression of IGF-1 and IGF-1R, which may sustain only in part the growth reserve mechanisms of the pathological heart.


Key Words: insulin-like growth factor-1 • insulin-like growth factor-1 receptor • insulin-like growth factor-2 • atrial natriuretic factor • myocyte cell volume


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Coronary heart disease and its complications are major risk factors in the elderly, and myocardial infarction in that group is associated with increased morbidity and mortality.1 2 However, the mechanism responsible for the greater detrimental impact of myocardial infarction on the elderly population is currently unknown. One possibility may be related to the modifications in the mechanical,3 electrical,4 and biochemical5 6 properties of myocytes associated with age that may contribute to the diminished contractile response in the aging heart.1 2 Moreover, a significant loss of myocytes occurs with age,7 8 and this phenomenon limits the growth reserve of the myocardium and its hypertrophic ability.2 In this regard, the expression of the early growth–related genes, c-fos and c-jun, as well as the levels of mRNA encoding sarcoplasmic reticulum Ca2+-ATPase and cardiac calsequestrin are lower in old than in young rats after aortic banding.9 In particular, these defects in the molecular control of myocyte cellular hypertrophy and contractile performance have been considered to play a relevant role in the occurrence of heart failure in aging myocardium.9 However, the bases for the attenuation in the growth capacity of myocytes in the aging heart remain to be identified. In vitro studies have documented that IGF-1 activates DNA synthesis10 and the expression of myosin light chain-2, troponin, and {alpha}-skeletal actin,11 which are consistent with a hyperplastic and hypertrophic response of these cells. Additionally, IGF-1 increases the formation of myofibrils in long-term cultures of adult myocytes.12 An upregulation of IGF-1 mRNA occurs in the myocardium after pressure-overload hypertrophy,13 14 and the density of IGF-1R is increased in the failing human heart.15 Importantly, increases in the expression of IGF-1 and IGF-1R have been shown in the surviving myocytes acutely after infarction, and these changes precede the upregulation in late growth–related genes, DNA replication, and myocyte nuclear mitotic division.16 17 Similar results have also been obtained shortly after nonocclusive coronary artery constriction.18 Therefore, these observations support the notion that the IGF-1–IGF-1R effector pathway may be implicated in the modulation of the reactive growth processes of the pathological heart. On this basis, the hypothesis may be advanced that the mechanism by which the mechanical stimulus on the surface of stressed muscle cells is translated into growth signals at the nucleus is impaired with aging and that this phenomenon involves the IGF-1–IGF-1R system. To analyze this possibility, the consequences of acute myocardial infarction on the expression of IGF-1 and IGF-1R in myocytes were examined in Fischer 344 rats at 4 and 16 months of age. The induction of IGF-2 mRNA was also determined to document potential differences in the activation of the fetal program of these cells. In addition, the expression of ANF was examined to characterize the influence of myocardial infarction on a molecular marker of myocyte hypertrophy. Finally, the changes in myocyte size and shape were measured.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Coronary Artery Occlusion and Ventricular Hemodynamics
Experiments were carried out in male Fischer 344 rats at 4 and 16 months of age (Harlan Sprague-Dawley, Indianapolis, Ind; National Institutes of Health colony). Ligation of the left coronary artery near its origin was performed in animals of both age groups to induce large infarcts of the LV associated with LV failure.19 Sixty rats at 4 months and 76 rats at 16 months were subjected to coronary artery occlusion. Twenty-four rats at 4 months and 42 rats at 16 months died shortly after the operation, mostly because of pulmonary edema. The remaining animals were killed at 6 hours, 3 days, and 7 days after surgery. Twenty-five sham-operated rats, 13 at 4 months and 12 at 16 months, were used as controls. The sham operation was associated with no mortality. Before they were killed, the animals were anesthetized with chloral hydrate (300 mg/kg body wt IP), and the external right carotid artery was cannulated with a microtipped pressure transducer catheter (Millar SPR-249) connected to an electrostatic chart recorder (Gould ES 2000), which was advanced into the LV for the evaluation of LV pressures, +dP/dt, and -dP/dt. Subsequently, a second catheter (Millar SPR-595) was inserted into the right jugular vein and advanced through the superior vena cava and the right atrium into the RV chamber for the measurements of CVP, RV pressures, and dP/dt.19 20

The hemodynamic measurements were used not only for the assessment of global ventricular performance after the induction of coronary artery occlusion but also for an indirect evaluation of infarct size. Previous work in our laboratory has shown that characteristic features of pump function impairment develop in this animal model when the destruction in myocardial mass involves nearly 50% of the myocyte population of the LV inclusive of the septum.19 This correlation has been shown to be present acutely19 and chronically20 after myocardial infarction. On this basis, only rats exhibiting indices of LV failure and extensive myocardial infarction at the time of death were included in the present study.

Myocyte Isolation
At the end of the hemodynamic determinations, hearts were rapidly excised, and myocytes from the LV were enzymatically dissociated.16 17 18 21 22 23 Hearts were placed on a stainless steel cannula for retrograde perfusion through the aorta. The solutions were supplements of modified commercial MEM Eagle Joklik (J.R.H. Biosciences). HEPES-MEM contained (mmol/L) NaCl 117, KCl 5.7, NaHCO3 4.4, KH2PO4 1.5, MgCl2 17, HEPES 21.1, glucose 11.7, L-glutamine 2, and taurine 10, along with 21 mU/mL insulin, amino acids, and vitamins, and was adjusted to pH 7.2 with NaOH. This solution is 292 mOsm, isosmolar with rat serum. Resuspension medium was HEPES-MEM supplemented with 0.5% BSA, 0.3 mmol/L calcium chloride, and 10 mmol/L taurine adjusted to 292 mOsm. The cell isolation procedure consisted of three main steps: (1) Ca2+-free perfusion: Blood washout and collagenase (selected type II, Worthington Biochemical Corp) perfusion of the heart was carried out at 34°C with HEPES-MEM gassed with 85% O2/15% N2. (2) Mechanical tissue dissociation: After removing the heart from the cannula, the LV was separated from the RV free wall and minced. Collagenase-perfused tissue was subsequently shaken in resuspension medium containing collagenase and 0.3 mmol/L calcium chloride. Supernatant cell suspensions were washed and resuspended in resuspension medium. (3) Separation of intact cells: Intact cells were enriched by centrifugation and the supernatant was discarded. This procedure was repeated four or five times in each preparation to remove nonmyocyte cells, cell debris, and the residual collagenase. Each centrifugation was performed at 30g for 3 minutes. Subsequently, {approx}106 cells were suspended in 10 mL isotonic Percoll (final concentration, 41% in resuspension medium) and centrifuged for 10 minutes at 34g. Intact cells were recovered from the pellet and washed, and smears were made to control the purity of the preparation. Rectangular, trypan blue–excluding cells constituted nearly 80% of all myocytes. The number of viable myocytes obtained from the LV decreased as a function of age. At 4 and 16 months, the average numbers of myocytes obtained from the LV of sham-operated control rats were 6x106 and 3.5x106, respectively. Corresponding values in infarcted rats were 3.5x106 and 1.5x106, respectively. The contribution of interstitial cells was assessed by counting 1000 cells in each LV and then computing from the counts the respective fractions of myocytes and nonmyocytes encountered.22 Consistent with previous results,12 20 21 nonmyocytes accounted for <1% of the cell population (Fig 1Down).



View larger version (148K):
[in this window]
[in a new window]
  		Figure 1. Enzymatically dissociated LV myocytes from a Fischer 344 rat at 16 months of age. Original magnification x200, bisbenzimide staining.

RT-PCR
Total RNA was extracted from LV myocytes by following the methodology described by Chomczynski and Sacchi.24 The amount of RNA obtained from 1x106 myocytes was 52±13 µg (n=4). Myocardial infarction affected the yield of LV myocytes but not the quantity of RNA extracted from 1x106 cells. RT-PCR was used to detect IGF-1, IGF-2, and IGF-1R mRNAs in LV myocytes after coronary artery occlusion or sham operation. Details of reactions used in our laboratory are given by Lipson and Baserga.25

Amplimers and probe for IGF-1 were chosen from the sequence for rat IGF-1 ss-mRNA (GenBank, X06043): 5' amplimer, position 134 to 153 bp; 3' amplimer, position 316 to 335 bp, and probe, position 184 to 203 bp. The size of amplification product for rat IGF-1 was 202 bp. Amplimers and probe for IGF-2 were chosen from the sequence for rat IGF-2 ss-mRNA (GenBank, X14834): 5' amplimer, position 1239 to 1258 bp; 3' amplimer, position 1716 to 1735 bp, and probe, position 1475 to 1494 bp. The size of amplification product for rat IGF-2 was 497 bp. Amplimers and probe for IGF-1R were chosen from the sequence of rat IGF-1R ss-mRNA (GenBank, M27293): 5' amplimer, position 165 to 184 bp; 3' amplimer, position 489 to 508 bp, and probe, position 392 to 410 bp. The size of amplification product for rat IGF-1R was 344 bp. For IGF-1, the 5' amplimer is located in exon 3, and the 3' amplimer is located in exon 5. For IGF-2, the 5' amplimer is located in exon 2, and the 3' amplimer is located in exon 6. For IGF-1R, both amplimers are located in exon 2.

Radioactive probes were prepared by oligomer phosphorylation with polynucleotide kinase from Boehringer Mannheim Biochemicals, and [32{gamma}-P]ATP (3000 Ci/mmol) was from Amersham. To ensure that RT-PCR products were not amplified from genomic DNA, all RNA samples were additionally amplified without reverse transcription. In all cases, PCR of RNA samples was negative. To evaluate the quantity of RNA in each lane and the efficiency of the multiple steps involved in the RT-PCR procedure, mRNA for the control gene, human pHE7,26 was amplified in the same reaction mixture in which IGF-1, IGF-2, and IGF-1R mRNA were analyzed. To avoid unspecific annealing between different probes and to minimize the background, hybridization was performed separately for each gene. pHE7 codes for ribosomal protein and is expressed at constant levels throughout the cell cycle.26 The expression of this gene is not affected in growth-inhibited cells.27

RNase Protection Assay
RNase protection assay for IGF-1R was performed by using the methodology described in detail by Sell et al,28 which has previously been used in our laboratory.17 The probe used was transcribed from human IGF-1R cDNA29 cloned into BamHI, Xba I side of the SK polylinker. T7 in vitro transcription generated hot [{alpha}-32P]CTP (800 Ci/mmol) antisense riboprobe from nucleotide 4142 (BamHI) to nucleotide 3800 (Stu I). Subsequently, the in vitro transcript was digested with 2 µL of RNase-free DNase I (2 U/µL) and purified by phenol extraction. After ethanol precipitation, the RNA probe was dissolved in 60 µL of hybridization buffer (80% formamide, 40 mmol/L PIPES, 400 mmol/L NaCl, and 1 mmol/L EDTA), the specific activity of the probe was measured, and aliquots (5x105 cpm) were prepared. Target RNA (5 µg) was added to each RNA-probe aliquot, and the samples were denatured for 5 minutes at 95°C. These samples were then kept at 45°C for overnight hybridization. The next day, hybridization reaction was cooled down to room temperature, and 350 µL of RNase digestion buffer (10 mmol/L Tris-HCl, 5 mmol/L EDTA, and 300 mmol/L sodium acetate), 1 µL of RNase A (7 µg/µL), and 2.5 µL of RNase T1 (10 U/µL) were added; digestion was performed at 30°C for 30 minutes. Subsequently, 10 µL of SDS (20%) and 2.5 µL of proteinase K (20 mg/mL) were added, and samples were incubated for 15 minutes at 37°C. After phenol extraction, hybridization products were coprecipitated with tRNA in the presence of 2 vol ice-cold ethanol. Dried pellets were resuspended in 7 µL of loading buffer (80% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, and 2 mmol/L EDTA), heated at 95°C for 5 minutes, and incubated on ice. Finally, protected hybridization product was size-separated on polyacrylamide (5%)/urea (8 mol/L) sequencing gel and exposed against Kodak x-ray film.

Cross-Linking Assay
This procedure was performed by following the methodology described by Phillips et al.30 Myocyte samples, 3x106 cells each, were washed twice with 5 mL of PBS without Ca2+ and Mg2+ and incubated in 5 mL of serum-free medium (DMEM and 0.1% BSA) for 1 hour at 37°C. Cells were spun down at 500g for 2 minutes and resuspended in 5 mL of serum-free medium. One myocyte sample was pretreated with 50 ng/mL of cold IGF-1 for 5 minutes at 37°C, and then all samples were incubated with [125I]IGF-1 (2x106 cpm/mL) for 3 hours at 4°C. Subsequently, all samples were spun down again at 500g for 2 minutes, washed once with 5 mL of PBS, and incubated with 0.2 mol/L disuccinimidyl subarate for 15 minutes at room temperature. Cells were spun down and suspended in 9 mL of 0.005 mol/L Tris-HCl (pH 7.4) to stop the cross-linking reaction. After 5 minutes of incubation, cells were pelleted at 500g for 2 minutes, lysed with 190 µL of lysis buffer (0.1 mol/L Tris-HCl, 15% glycerol, 2 mmol/L EDTA, 2% SDS, 2% ß-mercaptoethanol, and 0.075% bromophenol blue), and boiled 2 minutes, and proteins were separated on 10% acrylamide gels. The gels were dried after electrophoresis and exposed to x-ray film to visualize radioactive bands. The 135-kD band corresponding to the IGF-1R {alpha}-subunit was quantified with a JAVA image analysis system (Jandel Scientific).

Northern Blot Analysis
This methodology was used to detect the effects of myocardial infarction on a molecular marker of myocyte hypertrophy, ANF. Total RNA (20 µg) was size-separated by electrophoresis in 1.5% agarose-formaldehyde gel, transferred to nitrocellulose membrane, and cross-linked. Prehybridization, hybridization, and washing conditions were used as described by Thomas.31 ANF cDNA (600 bp) was released from pBF/ANF plasmid (obtained from Dr Cricket Seidman) by Pst I digestion. Radioactive probes were prepared by random priming using the multiprime DNA-labeling system and [{alpha}-32P]dCTP (300 Ci/mmol; Amersham). The constancy in amounts of RNA was determined by hybridizing the RNA blots with GAPDH cDNA probe.32

Myocyte Structural Properties
Myocytes were examined with an inverted microscope (Axiovert 10, Zeiss) using a x40 objective, and the images were recorded. Measurements of myocyte dimensions and sarcomere lengths were made in 40 to 50 cells from each LV using a computerized image analysis system. Cells were selected for morphological measurements on the basis of their viability and histological appearance. Specifically, only rod-shaped cells with clear sarcomere striation and absence of membrane blebs were included in the analysis. Cell length and area were measured directly, and average cell width was computed by dividing cell area by cell length. Sarcomere length was measured in three areas within each cell: one in the center and one near each end. Sarcomere length in each cell area was evaluated by measuring groups of 10 sarcomeres, which were combined to compute average sarcomere length in the cell.

Data Analysis
Autoradiograms were analyzed densitometrically by a computerized image analyzer (Jandel Scientific). Signals for IGF-1, IGF-2, IGF-1R, and ANF were divided by the signals for the internal controls for all samples, and quantitative data were expressed in this manner. All results are presented as mean±SD. An identical approach was used for functional data and myocyte dimensional properties. Statistical significance for comparison among groups was determined using ANOVA and Bonferroni's method.33 Values of P<.05 were considered to be significant. Because all measurements could not be collected in every animal, the n values for each determination are listed in the text or the legend for each figure.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
Ventricular Hemodynamics
Measurements of body weight demonstrated that sham operation and myocardial infarction did not affect this parameter in rats at 4 and 16 months of age (TableDown). The molecular studies performed in this investigation required the enzymatic dissociation of myocytes, which precluded the separation of the LV from the RV and the estimation of their individual weights. This is because the dissection of the two ventricles before coronary artery perfusion with collagenase makes this procedure impossible. The two ventricles were cut free only after nearly 2 hours of collagenase perfusion, when the tissue had already markedly changed its characteristics. However, total heart weights were obtained, and these values are listed in the TableDown. None of the small changes in heart weight with infarction reached statistical significance.


View this table:
[in this window]
[in a new window]
  		Table 1. Gross Cardiac Characteristics

Acute myocardial infarction resulted in an {approx}3.0-fold increase in LVEDP in 4- and 6-month-old rats at 6 hours, 3 days, and 7 days after coronary artery occlusion (Fig 2ADown). In contrast, in both age groups, LVPSP decreased by an average of 34% (Fig 2BDown), and LVDP decreased by 49% (Fig 2CDown). Positive dP/dt was also reduced by nearly 46% (Fig 2DDown). A multiple comparison analysis indicated that the alterations in these hemodynamic parameters were comparable at the three time intervals examined in each of the two age groups. However, myocardial infarction in the older animals produced a 10% (P<.05) and a 12% (P<.05) greater reduction in LVPSP and LVDP at 6 hours. Similar differences were noted at 3 days (-8% LVPSP [P<.05] and -13% LVDP [P<.05]) and at 7 days (-9% LVPSP [P<.01] and -11% LVDP [P<.05]).



View larger version (39K):
[in this window]
[in a new window]
  		Figure 2. Effects of myocardial infarction on LVEDP, LVPSP, LVDP, and LV +dP/dt in rats at 4 and 16 months of age at 6 hours (6H), 3 days (3D), and 7 days (7D) after surgery. SO indicates sham-operated control rats. Results are presented as mean±SD. *P<.05 vs control value in the same age group. {dagger}P<.05 vs corresponding value in the younger animal group. For 4-month-old rats, n values are as follows: SO rats, n=10; infarcted rats at 6H, n=6; infarcted rats at 3D, n=14; and infarcted rats at 7D, n=11. For 16-month-old rats, n values are as follows: SO rats, n=9; infarcted rats at 6H, n=6; infarcted rats at 3D, n=14; and infarcted rats at 7D, n=11.

Fig 3Down illustrates that the changes in RVEDP were comparable in the two animal groups at the three time points after infarction. However, RVPSP did not increase in the younger animals, whereas increases of 19% (P<.05) and 23% (P<.01) were noted in the older rats at 3 and 7 days after coronary artery occlusion. The alterations in RV +dP/dt were not statistically significant in either group. Finally, CVP markedly increased in the infarcted rats at both ages, at all intervals.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 3. Effects of myocardial infarction on RVEDP, RVPSP, RV +dP/dt, and CVP in rats at 4 and 16 months of age at 6 hours (6H), 3 days (3D), and 7 days (7D) after surgery. SO indicates sham-operated control rats. Results are presented as mean±SD. *P<.05 vs control value in the same age group. {dagger}P<.05 vs corresponding value in the younger animal group. See Fig 2Up for n values.

When animals at 4 and 16 months were compared, RVEDP was found to be 12% (P=NS), 43% (P<.005), and 47% (P<.005) higher in the older group at 6 hours, 3 days, and 7 days, respectively, after infarction. Moreover, RVPSP was 9% (P<.05) greater at the later time interval in the 16-month-old infarcted rats. Similarly, in this group, CVP values were 36% (P<.01), 38% (P<.05), and 42% (P<.01) higher at 6 hours, 3 days, and 7 days, respectively. In summary, acute myocardial infarction resulted in a significant impairment in ventricular performance that was more severe in the older animal group.

Myocyte Dimensional Properties
These determinations in rats at 4 months of age included eight sham-operated animals, and six, eight, and eight infarcted rats at 6 hours, 3 days, and 7 days, respectively, after surgery. Corresponding numbers of animals at 16 months of age were eight, six, five, and nine. Fig 4Down illustrates the changes in average myocyte size produced by myocardial infarction. Since this cellular parameter was practically identical in myocytes isolated from sham-operated control rats at 6 hours, 3 days, and 7 days after surgery, the collected results were combined in a single group. However, measurements in infarcted rats were kept separate, because variations in cellular dimensions were noted shortly after coronary artery occlusion. In 4-month-old animals, myocardial infarction was associated with a progressive increase in the length of the remaining viable cells of the LV. Although no change was detected at 6 hours, this structural parameter increased 11% (P<.05) and 18% (P<.001) at 3 days and 7 days, respectively, after infarction. In contrast, in the 16-month-old rats, myocyte length did not increase at 3 days, but an 8% (P<.001) increase was noted at 7 days. In both cases, myocyte lengthening occurred in the absence of variations in sarcomere length. Finally, myocyte area increased by 18% (P<.05) and 29% (P<.001) at 3 and 7 days, respectively, after infarction in younger animals and by 17% (P<.01) in the older rats at 7 days only. In summary, myocyte cellular hypertrophy after infarction was delayed and of smaller magnitude in the older animal group.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 4. Effects of myocardial infarction on myocyte dimensional properties in rats at 4 and 16 months of age at 6 hours (6H), 3 days (3D), and 7 days (7D) after surgery. SO indicates sham-operated control rats. Results are presented as mean±SD. *P<.05 vs control value in the same age group. **P<.05 vs the corresponding value in the same age group at 6H. For 4-month-old rats, n values are as follows: SO, n=8; infarcted rats at 6H, n=6; infarcted rats at 3D, n=8; and infarcted rats at 7D, n=8. For 16-month-old rats, n values are as follows: SO, n=8; infarcted rats at 6H, n=6; infarcted rats at 3D, n=5; and infarcted rats at 7D, n=9.

Expression of ANF
The quantity of ANF mRNA in myocytes was measured by Northern blot analysis at 3 and 7 days after infarction in both age groups (Fig 5Down). The 6-hour interval was not included in this evaluation because no indication of myocyte hypertrophy was noted at this time (Fig 4Up). Densitometric data were obtained by dividing the signals for ANF mRNA by the signals for GAPDH to correct for differences in loading. In 16-month-old rats, the level of ANF mRNA was low in LV myocytes of the sham-operated rats. However, the expression of ANF increased 3.0-fold (P<.002) and 4.2-fold (P<.001) at 3 and 7 days, respectively, after infarction. An identical analysis performed in rats at 4 months of age documented that myocardial infarction increased the expression of ANF in the viable LV myocytes by 6.3-fold (P<.001) and 4.7-fold (P<.001) at 3 and 7 days, respectively, after surgery. In addition, there was a 1.48-fold (P<.05) greater ANF mRNA level in 4-month-old rats compared with 16-month-old rats at 3 days after infarction. Conversely, there was a 1.3-fold (P<.001) higher ANF expression in the older animal group at 7 days after infarction.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 5. Top, Detection of ANF mRNA by Northern blot analysis in LV myocytes isolated from sham-operated control (SO) rats and infarcted rats at 3 days (3D) and 7 days (7D) after coronary artery occlusion. Bottom, Quantitative analysis of densitometric data. Results were computed as the optical density ratio of ANF to GAPDH (GAPD). Bars show mean±SD. Individual data points are also presented. *P<.05 vs control value in the same age group. The signal for ANF mRNA increased markedly after infarction. GAPD was used as an internal reference band.

In summary, ANF expression in LV myocytes after infarction was greater at 3 days in 4-month-old rats and at 7 days in 16-month-old rats.

Expression of IGF-1, IGF-2, and IGF-1R
The results illustrated below in Figs 6 through 8DownDownDown concerning RT-PCR data were all obtained by using 1.0 µg of myocyte RNA and 25 cycles of amplification in each determination. The rationale for selecting these conditions is indicated at the end of the description of Fig 9Down. The expression of IGF-1 mRNA in myocytes isolated from the LV of control and experimental animals was determined by RT-PCR (Fig 6Down). The values obtained in each animal are illustrated. IGF-1 mRNA was detected in myocytes from sham-operated control rats, and coronary artery occlusion was associated with enhanced expression of this gene in the remaining viable muscle cells of 4- and 16-month-old rats. This phenomenon was observed at 3 and 7 days after surgery, whereas no changes were apparent at the 6-hour interval. Myocardial infarction produced a greater effect on the older animals, because the lower level of expression of IGF-1 mRNA at baseline in this group was upregulated in a manner comparable to that of younger rats. In this regard, the difference noted in control myocytes between the two age groups was no longer present after coronary artery occlusion.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 6. Top, Detection of IGF-1 mRNA by RT-PCR in LV myocytes isolated from sham-operated control (SO) rats and infarcted rats at 6 hours (6H), 3 days (3D), and 7 days (7D) after coronary artery occlusion. Bottom, Quantitative analysis of densitometric data. Results were computed as the optical density ratio of IGF-1 to pHE7. Bars show mean±SD. Individual data points are also presented. *P<.05 vs control value in the same age group. **P<.05 vs 6H value in the same age group. ***P<.05 vs 3D value in the same age group.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 7. Top, Detection of IGF-2 mRNA by RT-PCR in LV myocytes isolated from sham-operated control (SO) rats and infarcted rats at 6 hours (6H), 3 days (3D), and 7 days (7D) after coronary artery occlusion. Bottom, Quantitative analysis of densitometric data. Results were computed as the optical density ratio of IGF-2 to pHE7. Bars show mean±SD. Individual data points are also presented. *P<.05 vs control value in the same age group. **P<.05 vs 6H value in the same age group. ***P<.05 vs 3D value in the same age group.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 8. Left, Detection of IGF-1R mRNA by RT-PCR in LV myocytes isolated from sham-operated control (SO) rats and infarcted rats at 6 hours (6H), 3 days (3D), and 7 days (7D) after coronary artery occlusion (top) and the quantitative analysis of densitometric data (bottom). Results were computed as the optical density ratio of IGF-1R to pHE7. Bars show mean±SD. Individual data points are also presented. *P<.05 vs control value in the same age group. **P<.05 vs 6H value in the same age group. Top right, Detection of IGF-1R mRNA by the RNase protection assay in LV myocytes isolated from 16-month-old (16 MO) and 4-month-old (4 MO) SO and infarcted rats at 7 days (7D) after surgery. P corresponds to the undigested probe. A marked upregulation of IGF-1R mRNA was noted after infarction. Bottom right, [125I]IGF-1 cross-linked to LV myocytes obtained from SO and infarcted rats at 3 days (3d) and 7 days (7d) after coronary occlusion. Ligand binding was higher after infarction at 3 days (3d) and 7 days (7d) in both groups of animals.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 9. Left, Detection of IGF-1 mRNA by RT-PCR (20 cycles) using increasing quantities of RNA extracted from LV myocytes of sham-operated control (SO) rats and infarcted (MI) rats at 4 months of age (top). This is repeated at 25 cycles for SO and MI rats. The concentrations of RNA were 0.5, 0.75, 1.0, 1.5, and 2.0 µg for each set of five lanes. The level of expression of IGF-1 mRNA in myocytes increased linearly with increasing amounts of RNA. This was apparent at both 20 cycles ({circ}, SO rats, r=.998; {bullet}, MI rats, r=.987) and 25 cycles ({square}, SO rats, r=.992; {blacksquare}, MI rats, r=.944) (bottom). Right, Detection of IGF-2 mRNA by RT-PCR (20 cycles) using increasing quantities of RNA extracted from LV myocytes of SO and MI rats at 4 months of age (top). This is repeated at 25 cycles for SO and MI rats. The concentrations of RNA were 0.5, 0.75, 1.0, 1.5, and 2.0 µg for each set of five lanes. The level of expression of IGF-2 mRNA in myocytes increased linearly with increasing amounts of RNA. This was apparent at both 20 cycles ({circ}, SO rats, r=.994; {bullet}, MI rats, r=.992) and 25 cycles ({square}, SO rats, r=.979; {blacksquare}, MI rats, r=.971) (bottom).

Fig 7Up shows the expression of IGF-2 mRNA by RT-PCR in myocytes from control and infarcted hearts. Individual values are depicted. IGF-2 mRNA levels were barely detectable in 16-month-old rats, whereas the expression of this gene was evident in younger animals. In addition, the amount of IGF-2 mRNA increased at 3 and 7 days after myocardial infarction in rats at 4 months, but only at the later period in the older animal group. On the other hand, coronary artery occlusion was associated with an upregulation of IGF-1R mRNA in the spared myocytes of the LV at 3 and 7 days after surgery, and this adaptation was similar in both age groups (Fig 8Up, left). Moreover, consistent with the expression of IGF-1 and IGF-2, IGF-1R mRNA levels at baseline were lower in rats at 16 months than at 4 months, but this difference did not persist after infarction.

The increase in IGF-1R mRNA detected by RT-PCR at 7 days after infarction was confirmed by the RNase protection assay (Fig 8Up, top right), which further demonstrated an enhanced expression of IGF-1R in myocytes after coronary artery occlusion. The additional band corresponds to an IGF-1R mRNA fragment protected by a shorter riboprobe. A full-length (408-bp) and incomplete IGF-1R RNA transcripts were generated by in vitro transcription. These two fragments were subsequently protected by IGF-1R mRNA in the RNase protection assay and appear as two distinct bands on the gels. Densitometric analysis indicated that the quantity of IGF-1R mRNA in control animals at 4 months increased markedly at 7 days after infarction (densitometric units, 32±6 for control rats [n=4] and 97±7 for rats with myocardial infarction [n=4]; P<.001). A similar upregulation was noted in 16-month-old rats (densitometric units, 23±5 for control rats [n=4] and 65±6 for rats with myocardial infarction [n=4]; P<.001). Cross-linking of [125I]IGF-1 to myocytes at 3 and 7 days showed that more ligand was cross-linked to the 135-kD {alpha}-subunit of the receptor after infarction in 4-month-old rats (densitometric units, 48±2 for control rats [n=3], 96±6 for rats with myocardial infarction 3 days after surgery [n=3], and 80±5 for rats with myocardial infarction 7 days after surgery [n=3]; P<.001) and 16-month-old rats (densitometric units, 18±5 for control rats [n=3], 69±7 for rats with myocardial infarction 3 days after surgery [n=3], and 77±10 for rats with myocardial infarction 7 days after surgery [n=3]; P<.001) (Fig 8Up, bottom right). In summary, aging decreased the expression of IGF-1, IGF-2, and IGF-1R in ventricular myocytes but did not alter the ability to upregulate these genes after myocardial infarction.

Quantification of RT-PCR
Additional experiments were conducted to validate the RT-PCR data, concerning the expression of IGF-1 and IGF-2 in myocytes under control conditions and after myocardial infarction. This was felt necessary because Northern blot analysis for IGF-1 mRNA failed to generate a detectable signal. Moreover, the cDNA probe for rat IGF-2 was not available to us. Therefore, for IGF-1, increasing amounts of myocyte RNA (0.5, 0.75, 1.0, 1.5, and 2.0 µg) were amplified by 20 and 25 cycles. By this approach, it was possible to document that the difference in the expression of IGF-1 mRNA in cells from sham-operated and infarcted rats persisted in each sample (Fig 9Up, left). However, RT-PCR products and quantities of RNA were linearly related but did not increase in absolute terms with the corresponding amounts of RNA. Specifically, the changes in the hybridization signal obtained with 0.5 and 1.0 µg of input RNA increased more than twofold at 20 cycles in both sham-operated and infarcted animals. The lack of an absolute correspondence between quantities of RNA and densitometric values was also noted at 25 cycles. A similar phenomenon was observed for IGF-2, in which the same amounts of RNA and number of amplification cycles were used (Fig 9Up, right). The linear relationships in Fig 9Up were obtained by the least-squares linear curve-fitting method.

The data illustrated in Fig 9Up correspond to 4-month-old rats after sham operation and rats with myocardial infarction at the 7-day time interval. In view of these observations, RT-PCR may be considered quantitative in the detection of relative changes but semiquantitative in the evaluation of absolute changes. Although this is an important limitation, the conclusion can be reached that myocardial infarction produced a significant increase in the expression of IGF-1 and IGF-2 in the viable myocytes of rats at 4 months. Moreover, it is reasonable to infer that the same conclusion may apply to rats at 16 months. For both these genes, 1.0 µg of myocyte RNA and 25 cycles of amplification provided clear hybridization signals in control and infarcted rats. In addition, saturation of the hybridization product was not apparent under these conditions. On this basis, 1.0 µg of myocyte RNA and 25 cycles of amplification were used for the collection of the data illustrated in Figs 6 through 8UpUpUp. In summary, the changes in gene expression detected by RT-PCR after infarction were valid on a semiquantitative basis.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
The results of the present study indicate that aging did not affect the ability of myocytes to respond to the overload associated with acute myocardial infarction by enhancing the expression of IGF-1 and IGF-1R mRNAs in the remaining viable muscle cells of the LV. Similarly, the quantity of IGF-1R protein increased in a comparable manner in the stressed myocytes of young adult and aging infarcted rats. However, the myocyte cellular hypertrophic reaction was retarded and attenuated in the older animals, as documented structurally, by minimal changes in myocyte dimensions, and molecularly, by a delay in the maximal induction of ANF mRNA in the cells. Finally, the expression of IGF-2 mRNA appeared later in aged rats, suggesting that the recapitulation of the fetal phenotype may be required for the initiation of the reactive growth processes of myocytes. Thus, aging impairs the hypertrophic growth capacity of ventricular myocytes but has no influence on the ability of these cells to upregulate the IGF-1–IGF-1R autocrine system. The limitation in the growth reserve of myocytes with age may be responsible for the greater deleterious effects of ischemic injury in older individuals.

Aging, Myocardial Infarction, and Ventricular Failure
One of the major difficulties encountered in the study of the impact of aging on the response of the cardiovascular system is the identification of its initial detrimental impact on the heart.1 2 This is because there is no temporal reference point that can be used to separate changes beyond sexual maturity, which are part of postnatal myocardial growth, from aging changes per se, since they are both controlled by time as a critical factor. However, myocyte cell loss characterizes the aging process in animals7 21 and humans.8 In the Fischer 344 rat, myocardial damage and cell loss are first measurable at 12 months and increase from 12 to 20 months.21 In contrast, no alterations in myocardial structure have been found in 4-month-old rats. Of relevance, ventricular performance is preserved up to 20 to 24 months,9 21 34 35 although defects in myocardial3 and single myocyte36 mechanics appear earlier in life. These were the bases for the selection of the two age intervals in the present study and for the assumption that aging effects were not present in rats at 4 months, whereas their influence was already apparent at 16 months in this model.

Results in this investigation demonstrate that occlusion of the left coronary artery was associated at 6 hours, 3 days, and 7 days with severe impairment of cardiac performance in animals of both age groups. These observations are consistent with previous findings in the same animal model in which ventricular failure has been documented acutely19 and chronically20 37 38 after coronary artery ligation. Although the mortality rate was not analyzed in a statistical manner, in view of the relatively small number of animals included at each time point, this phenomenon appeared to be higher in the older animals. Moreover, the abnormalities in LVEDP, LVPSP, and dP/dt were consistently greater in the 16-month-old than in the 4-month-old infarcted rats. Similarly, RV function was affected more in the older than younger animal group. In this regard, modifications in muscle contractile behavior3 and in the biochemical5 6 and electrophysiological4 properties of the myocardium have been demonstrated with age, in combination with a reduced ability to respond to a prolonged systolic34 and diastolic35 overload. In addition, coronary vascular reserve and resistance become impaired in the aging myocardium,39 and these multiple factors may explain the greater deleterious impact of coronary artery occlusion in the older rats.

Aging, Myocardial Infarction, and Myocyte Cellular Hypertrophy
Several studies in recent years have shown that myocyte cellular hypertrophy occurs after infarction.40 Myocyte enlargement is an early event that progresses throughout the healing process, leading to the reconstitution of a large portion of necrotic myocardium. However, in the presence of extensive infarcts, the addition of contractile mass by cellular hypertrophy is inadequate, and a deficit in functioning tissue characterizes the chronic evolution of the cardiomyopathic heart.20 As indicated by the results of the present study, aging affected the early hypertrophic reaction of the remaining viable cells in rats at 16 months, whereas such an effect was not noted in young animals at 4 months. This phenomenon may have contributed significantly to the greater impairment in cardiac performance observed in the older animal group after infarction.

The attenuation of the hypertrophic response of myocytes in aged rats after coronary artery occlusion was also documented in the present study by a delay in the maximal increase in the expression of ANF in these cells. ANF has repeatedly been considered a reasonable molecular marker of myocyte growth.9 Consistent with this observation, the induction of early growth–related genes is enhanced in young rats subjected to myocardial infarction41 and aortic banding.42 Conversely, the expression of c-fos and c-jun proto-oncogenes is markedly reduced in the overloaded aged heart.9 In addition, the message for sarcoplasmic reticulum Ca2+-ATPase is decreased under these conditions.9

On a cellular basis, myocyte hypertrophy may be accomplished by an increase in myocyte diameter, length, or both. When lateral expansion rather than increased cell length becomes the major growth mechanism, wall thickness increases with no changes in chamber volume.43 In contrast, myocyte lengthening results in a larger cavity volume without affecting wall thickness. A disproportionate increase in cell length with respect to its diameter will generate chamber dilation with moderate thickening of the wall.19 20 23 This latter situation corresponds to the modifications in cell shape encountered in the present study at 3 and 7 days after myocardial infarction in the younger group of rats. On the other hand, such an adaptation was delayed and of smaller magnitude in the older animal group. Since sarcomere length was similar in the surviving myocytes after infarction, the difference in cell lengthening between the two age groups at the 7-day time point was the consequence of the variation in the number of sarcomeres added in series in the 4- and 16-month-old animals. There was an addition of 12 new sarcomeres in myocytes of younger rats and only 5 in the cells of older animals.

Aging, Myocardial Infarction, and IGF-1, IGF-2, and IGF-1R
Recent observations have indicated that myocyte growth may be modulated locally by an IGF-1–IGF-1R autocrine system that becomes activated after myocardial infarction16 17 or coronary artery constriction.18 Although this effector pathway has rarely been claimed to be implicated in the growth reaction of the myocardium in normal44 45 and pathological13 states, its crucial role in the regulation of cell growth in vitro46 and in vivo47 has been well documented. The present experiments are consistent with the notion that alterations in cardiac loading upregulate the IGF-1–IGF-1R autocrine system of myocytes, which may initiate reactive growth adaptations in the stressed cells. Aging did not appear to attenuate this adaptive response of myocytes. However, the lack of synchrony between myocyte hypertrophy and the enhanced expression of IGF-1 and IGF-1R in the cells of older animals after infarction raises the possibility that this signaling mechanism may be implicated more in the regulation of the replicatory machinery of myocytes than in cellular hypertrophy.

Although the claim has been made that ligand activation of IGF-1R on myocytes may be involved exclusively in cellular hypertrophy11 13 14 44 48 or hyperplasia,10 16 17 18 45 49 a cause-and-effect relationship between these two variables remains to be obtained. Myocyte cellular hypertrophy and cell proliferation occur concurrently during physiological7 and pathological21 22 23 myocardial growth, making the separation of these two cellular processes at the molecular level very complex in an in vivo system. Moreover, with the exception of a few instances,16 17 18 the majority of in vivo studies concerning the role of IGF-1 and IGF-1R in myocardial growth used cardiac tissue and not pure preparations of ventricular myocytes.13 14 15 44 By this approach, the adaptation of fibroblasts and endothelial cells cannot be distinguished from that of the muscle compartment of the myocardium. However, experimentations in vitro49 and in vivo16 17 18 with the use of myocytes only tend to support the contention that the IGF-1–IGF-1R system modulates most of the proliferative capacity of these cells. It should be recognized that the present findings, particularly in the younger animals, do not demonstrate that stimulation of the IGF-1–IGF-1R autocrine system was implicated exclusively in one form of myocyte growth. However, similar changes in the expression of IGF-1 and IGF-1R in myocytes were found in the older animal group, in which cellular hypertrophy was depressed and delayed. On the other hand, DNA synthesis and nuclear mitotic division have been documented in young animals acutely after myocardial infarction and severe impairment in ventricular function.17 Thus, the possibility may be advanced that the IGF-1–IGF-1R signaling mechanism may control mostly myocyte proliferation, whereas myocyte cellular hypertrophy may be regulated prevalently by an alternate effector pathway.50 51 52 53 54

The observation made in the present study in terms of the ability of myocytes to enhance their IGF-1–IGF-1R autocrine system after myocardial infarction at two distinct age intervals does not diminish the importance of the fact that the circulating level of IGF-1 and its binding protein, IGFBP-3, decreases from adulthood to senescence in Fischer 344 rats.55 However, IGFBP-3 is reduced more than the corresponding ligand. In healthy individuals, the serum level of IGF-1 and IGFBP-3 declines with aging, but IGFBP-3 decreases less than the ligand, resulting in a reduction in the molar ratio of IGF-1 to IGFBP-3.56 Thus, aging affects the systemic influence of IGF-1 in mammals, although the local IGF-1–IGF-1R effector pathway seems to be preserved in the aging myocardium.


*    Selected Abbreviations and Acronyms
 
ANF = atrial natriuretic factor
CVP = central venous pressure
IGF = insulin-like growth factor
IGF-1R = IGF-1 receptor
IGFBP = IGF binding protein
LV = left ventricle (ventricular)
LVDP = LV developed pressure
LVEDP = LV end-diastolic pressure
LVPSP = LV peak systolic pressure
PCR = polymerase chain reaction
RT = reverse transcriptase
RV = right ventricle (ventricular)
RVEDP = RV end-diastolic pressure
RVPSP = RV peak systolic pressure


*    Acknowledgments
 
This study was supported by grants HL-38132, HL-39902, and HL-40561 from the National Heart, Lung, and Blood Institute. The expert technical assistance of Malgorzata Bakowska and Maria Feliciano is greatly appreciated.


*    Footnotes
 
Reprint requests to Piero Anversa, MD, Department of Medicine, Vosburgh Pavilion–Room 302, New York Medical College, Valhalla, NY 10595.

Received July 19, 1995; accepted December 19, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 

  1. Lakatta EG, Mitchell JH, Pomerance A, Rowe GG. Human aging: changes in structure and function. J Am Coll Cardiol. 1987;10:42A-47A.
  2. Wei JY. Age and the cardiovascular system. N Engl J Med. 1992;327:1735-1739. [Medline] [Order article via Infotrieve]
  3. Anversa P, Puntillo E, Olivetti G, Capasso JM, Sonnenblick EH. Effects of age on mechanical and structural properties of left and right ventricular myocardium of Fischer 344 rats. Am J Physiol. 1989;256:H1440-H1449. [Abstract/Free Full Text]
  4. Wei JY, Spurgeon HA, Lakatta EG. Excitation-contraction in rat myocardium: alterations with adult aging. Am J Physiol. 1984;246:H784-H791.
  5. Capasso JM, Malhotra A, Scheuer J, Sonnenblick EH. Myocardial biochemical, contractile, and electrical performance after imposition of hypertension in young and old rats. Circ Res. 1986;58:445-460. [Abstract/Free Full Text]
  6. Jiang MT, Moffat MP, Narayanan N. Age-related alterations in the phosphorylation of sarcoplasmic reticulum and myofibrillar proteins and diminished contractile response to isoproterenol in intact rat ventricle. Circ Res. 1993;72:102-111. [Abstract/Free Full Text]
  7. Anversa P, Hiler B, Ricci R, Guideri G, Olivetti G. Myocyte cell loss and myocyte hypertrophy in the aging rat heart. J Am Coll Cardiol. 1086;8:1441-1448. [Abstract]
  8. Olivetti G, Melissari M, Capasso JM, Anversa P. Cardiomyopathy of the aging human heart: myocyte loss and reactive cellular hypertrophy. Circ Res. 1991;68:1560-1568. [Abstract/Free Full Text]
  9. Takahashi T, Schunkert H, Isoyama S, Wei JY, Nadal-Ginard B, Grossman W, Izumo S. Age related differences in the expression of proto-oncogene and contractile protein genes in response to pressure overload in the rat myocardium. J Clin Invest. 1992;89:939-946.
  10. Kardami E. Stimulation and inhibition of cardiac myocyte proliferation in vitro. Mol Cell Biochem. 1990;92:129-135. [Medline] [Order article via Infotrieve]
  11. Ito H, Hiroe M, Hirata Y, Tsujino M, Adachi S, Schichiri M, Koike A, Nogami A, Marumo F. Insulin-like growth factor 1 induces hypertrophy with enhanced expression of muscle specific genes in cultured rat cardiomyocytes. Circulation. 1993;87:1715-1721. [Abstract/Free Full Text]
  12. Donath MY, Azpf J, Eppenberger-Eberhardt M, Froesch EF, Eppenberger HM. Insulin-like growth factor 1 stimulated myofibril development and decreases smooth muscle {alpha}-actin of adult cardiomyocytes. Proc Natl Acad Sci U S A. 1994;19:1686-1690.
  13. Hanson MC, Fath KA, Alexander RW, Delafontaine P. Induction of cardiac insulin-like growth factor 1 gene expression in pressure overload hypertrophy. Am J Med Sci. 1993;306:69-74. [Medline] [Order article via Infotrieve]
  14. Donohue TJ, Dworkin LD, Lango MN, Fliegner K, Lango RP, Benstein JA, Slater WR, Catanese VM. Induction of myocardial insulin-like growth factor-I gene expression in left ventricular hypertrophy. Circulation. 1994;89:799-809. [Abstract/Free Full Text]
  15. Toyozaki T, Hiroe M, Hasumi M, Horie T, Hosoda S, Tsushima T, Sekiguchi M. Insulin-like growth factor 1 receptors in human cardiac myocytes and their relation to myocardial hypertrophy. Jpn Circ J. 1993;57:1120-1127. [Medline] [Order article via Infotrieve]
  16. Reiss K, Meggs LG, Li P, Olivetti G, Capasso JM, Anversa P. Upregulation of IGF-1, IGF-1-receptor and late growth related genes in ventricular myocytes acutely after infarction in rats. J Cell Physiol. 1994;158:160-168. [Medline] [Order article via Infotrieve]
  17. Reiss K, Kajstura J, Zhang X, Li P, Szoke E, Olivetti G, Anversa P. Acute myocardial infarction leads to upregulation of the IGF-1 autocrine system, DNA replication and nuclear mitotic division in the remaining viable cardiac myocytes. Exp Cell Res. 1994;213:463-472. [Medline] [Order article via Infotrieve]
  18. Reiss K, Kajstura J, Capasso JM, Marino TA, Anversa P. Impairment of myocyte contractility following coronary artery narrowing is associated with activation of the myocyte IGF1 autocrine system, enhanced expression of late growth related genes, DNA-synthesis and myocyte nuclear mitotic division in rats. Exp Cell Res. 1993;207:348-360. [Medline] [Order article via Infotrieve]
  19. Olivetti G, Capasso JM, Sonnenblick EH, Anversa P. Side-to-side slippage of myocytes participates in ventricular wall remodeling acutely after myocardial infarction in rats. Circ Res. 1990;67:23-34. [Abstract/Free Full Text]
  20. Olivetti G, Capasso JM, Meggs LG, Sonnenblick EH, Anversa P. Cellular basis of chronic ventricular remodeling after myocardial infarction in rats. Circ Res. 1991;68:856-869. [Abstract/Free Full Text]
  21. Anversa P, Palackal T, Sonnenblick EH, Olivetti G, Meggs LG, Capasso JM. Myocyte cell loss and myocyte cellular hyperplasia in the hypertrophied aging rat heart. Circ Res. 1990;67:871-885. [Abstract/Free Full Text]
  22. Anversa P, Fitzpatrick D, Argani S, Capasso JM. Myocyte mitotic division in the aging mammalian rat heart. Circ Res. 1991;69:1159-1164. [Abstract/Free Full Text]
  23. Kajstura J, Zhang X, Reiss K, Szoke E, Li P, Lagrasta C, Cheng W, Darzynkiewicz Z, Olivetti G, Anversa P. Myocyte cellular hyperplasia and myocyte cellular hypertrophy contribute to chronic ventricular remodeling in coronary artery narrowing–induced cardiomyopathy in rats. Circ Res. 1994;74:383-400. [Abstract/Free Full Text]
  24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]
  25. Lipson KE, Baserga R. Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe. Proc Natl Acad Sci U S A. 1989;86:9774-9777. [Abstract/Free Full Text]
  26. Kao HT, Nevins JR. Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection. Mol Cell Biol. 1983;3:2058-2065. [Abstract/Free Full Text]
  27. Surmacz E, Reiss K, Sell C, Baserga R. Cyclin D1 messenger RNA is inducible by platelet-derived growth factor in cultured fibroblasts. Cancer Res. 1992;52:4522-4525. [Abstract/Free Full Text]
  28. Sell C, Chang CD, Koniecki J, Chen HM, Baserga R. A cryptopromoter is activated in the proliferating cell nuclear antigen gene of growth arrested cells. J Cell Physiol. 1992;152:177-184. [Medline] [Order article via Infotrieve]
  29. Ullrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, LeBon T, Tathuria S, Chen E, Jacobs S, Francke U, Ramachandran J, Fujita-Yamaguchi Y. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J. 1986;5:2503-2512. [Medline] [Order article via Infotrieve]
  30. Phillips PD, Pignolo RJ, Cristofalo VJ. Insulin-like growth factor-1: specific binding to high and low affinity sites and mitogenic action throughout the life span of WI-38 cells. J Cell Physiol. 1987;133:135-143. [Medline] [Order article via Infotrieve]
  31. Thomas PS. Hybridization of denatured RNA transferred on dotted nitrocellulose paper. Methods Enzymol. 1982;100:255-266.
  32. Marino TA, Halder S, Williamson EC, Beaverson K, Walter RA, Marino DR, Beatty C, Lipson KE. Proliferating cell nuclear antigen in developing and adult rat cardiac muscle cells. Circ Res. 1991;69:1353-1360. [Abstract/Free Full Text]
  33. Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res. 1980;47:1-9. [Abstract/Free Full Text]
  34. Isoyama S, Wei JY, Izumo S, Fort P, Schoen FJ, Grossman W. Effects of age on the development of cardiac hypertrophy produced by aortic constriction in the rat. Circ Res. 1987;61:337-345. [Abstract/Free Full Text]
  35. Isoyama S, Grossman W, Wei JY. Effect of age on myocardial adaptations to volume overload in the rat. J Clin Invest. 1988;81:1850-1857.
  36. Capasso JM, Fitzpatrick D, Anversa P. Cellular mechanisms of ventricular failure: myocyte kinetics and geometry with age. Am J Physiol. 1992;262:H1770-H1781. [Abstract/Free Full Text]
  37. Pfeffer JM, Pfeffer MA, Braunwald E. Influence of chronic captopril therapy on the infarcted left ventricle of the rat. Circ Res. 1985;57:84-95. [Abstract/Free Full Text]
  38. Pfeffer JM, Pfeffer MA, Fletcher PJ, Braunwald E. Progressive ventricular remodeling in rat with myocardial infarction. Am J Physiol. 1991;260:H1406-H1414. [Abstract/Free Full Text]
  39. Hachamovitch R, Wicker P, Capasso JM, Anversa P. Alterations of coronary blood flow and reserve with aging in Fischer 344 rats. Am J Physiol. 1989;256:H66-H72. [Abstract/Free Full Text]
  40. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Circulation. 1990;81:1161-1172. [Abstract/Free Full Text]
  41. Reiss K, Capasso JM, Huang HE, Meggs LG, Li P, Anversa P. ANG II receptors, c-myc, and c-jun in myocytes after myocardial infarction and ventricular failure. Am J Physiol. 1993;264:H760-H769. [Abstract/Free Full Text]
  42. Komuro I, Kurabayashi M, Takaku F, Yazaki Y. Expression of cellular oncogenes in the myocardium during the developmental stage and pressure-overloaded hypertrophy of the rat heart. Circ Res. 1988;62:1075-1079. [Abstract/Free Full Text]
  43. Grossman W, Jones D, McLaurin LP. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest. 1975;56:56-64.
  44. Engelmann GL, Boehm KD, Haskell JF. Insulin-like growth factors and neonatal cardiomyocyte development: ventricular gene expression and membrane receptor variations in normotensive and hypertensive rats. Mol Cell Endocrinol. 1989;63:1-14. [Medline] [Order article via Infotrieve]
  45. Cheng W, Reiss K, Kajstura J, Kowal K, Quaini F, Anversa P. Downregulation of the IGF-1 system parallels the attenuation in the proliferative capacity of rat ventricular myocytes during postnatal development. Lab Invest. 1995;72:646-655. [Medline] [Order article via Infotrieve]
  46. Pietrzkowski Z, Lammers R, Carpenter AM, Soderquist M, Limardo PD, Phillips A, Ullrich A, Baserga R. Constitutive expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor abrogates all requirements for exogenous growth factors. Cell Growth Differ. 1992;3:199-205. [Abstract]
  47. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstradiatis A. Mice carrying null mutations of the genes encoding insulin-like growth factor 1 (IGF-1) and type 1 IGF receptor (IGF-1R). Cell. 1993;75:73-82. [Medline] [Order article via Infotrieve]
  48. Duerr RL, Huang S, Miraliakbar HR, Clark R, Chien KR, Ross J Jr. Insulin-like growth factor-1 enhances ventricular hypertrophy and function during the onset of experimental cardiac failure. J Clin Invest. 1995;95:619-627.
  49. Kajstura J, Cheng W, Reiss K, Anversa P. The IGF-1-IGF-1 receptor system modulates myocyte proliferation but not myocyte cellular hypertrophy in vitro. Exp Cell Res. 1994;215:273-283. [Medline] [Order article via Infotrieve]
  50. Meggs LG, Tillotson J, Huang H, Sonnenblick EH, Capasso JM, Anversa P. Noncoordinate regulation of alpha-1 adrenoreceptor coupling and reexpression of alpha skeletal actin in myocardial infarction-induced left ventricular failure in rats. J Clin Invest. 1990;86:1451-1458.
  51. Meggs LG, Coupet J, Huang H, Cheng W, Li P, Capasso JM, Homcy CJ, Anversa P. Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats. Circ Res. 1993;72:1149-1162. [Abstract/Free Full Text]
  52. Dostal DE, Rothblum KN, Chernin MI, Cooper GR, Baker KM. Intracardiac detection of angiotensinogen and renin: a localized renin-angiotensin system in neonatal rat heart. Am J Physiol. 1992;263:C838-C850. [Abstract/Free Full Text]
  53. Sodoshima J, Xu J, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell. 1993;75:977-984. [Medline] [Order article via Infotrieve]
  54. Zhang X, Dostal DE, Reiss K, Cheng W, Kajstura J, Li P, Huang H, Sonnenblick EH, Meggs LG, Baker KM, Anversa P. Identification and activation of the autocrine renin-angiotensin system in adult ventricular myocytes in vivo. Am J Physiol. 1995;269:H1791-H1802. [Abstract/Free Full Text]
  55. Benedict MR, Lu MJ, Florini JR, Woo J, Richman RA. The differential regulation of insulin-like growth factor (IGF) binding proteins by IGF-1 during the life span of the rat. J Gerontol. 1994;49:215-223.
  56. Juul A, Main K, Blum WF, Lindholm J, Ranke MB, Skakkebaek NE. The ratio between serum levels of insulin-like growth factor (IGF)-I and the IGF binding proteins (IGFBP-