© 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
From the Department of Medicine, New York Medical College, Valhalla.
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Abstract |
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 Top Abstract IntroductionMaterials and Methods Results Discussion References |
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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
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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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
-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 |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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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, 
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 1
).
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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
-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 [-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 -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
[-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.
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Results |
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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 (Table
). 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 Table. None of the
small changes in heart weight with infarction reached statistical
significance.
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Acute myocardial infarction resulted in an 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 2A). In
contrast, in both age groups, LVPSP decreased by an average of 34%
(Fig 2B), and LVDP decreased by 49% (Fig 2C).
Positive dP/dt was also
reduced by nearly 46% (Fig 2D). 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]).
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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 3 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.
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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 4 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.
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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 5). The 6-hour interval was not included in this
evaluation because no indication of myocyte hypertrophy was
noted at this time (Fig 4
). 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.
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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
8 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 9. The expression of IGF-1 mRNA in myocytes isolated
from the LV
of control and experimental animals was determined by RT-PCR (Fig
6). 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.
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, SO rats, r=.998; 
, MI rats,
r=.987) and 25 cycles (
, SO rats,
r=.992;
, 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 (
Fig 7 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
8,
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 8,
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
-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 8, 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
9, 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 9,
right). The
linear relationships in Fig 9 were obtained by the
least-squares
linear curve-fitting method.
The data illustrated in Fig
9 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 8.
In summary, the changes in gene
expression detected by RT-PCR after infarction were valid on a
semiquantitative basis.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
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 |
|---|
|
|
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.
|
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P. Anversa and J. Kajstura Ventricular Myocytes Are Not Terminally Differentiated in the Adult Mammalian Heart Circ. Res., July 13, 1998; 83(1): 1 - 14. [Full Text] [PDF]
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G. Redaelli, A. Malhotra, B. Li, P. Li, E. H. Sonnenblick, P. A. Hofmann, and P. Anversa Effects of Constitutive Overexpression of Insulin-Like Growth Factor-1 on the Mechanical Characteristics and Molecular Properties of Ventricular Myocytes Circ. Res., March 23, 1998; 82(5): 594 - 603. [Abstract] [Full Text] [PDF]
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