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From the Charles A. Dana Research Institute and the Harvard-Thorndike Laboratory, Department of Medicine (Cardiovascular Division) of Beth Israel Hospital (A.C., J.D.G., H.S., S.E.K., J.P.M., P.S.D.), Harvard Medical School, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to either: Antonio Cittadini, M.D., Department of Clinical Medicine and Cardiovascular Sciences, University Federico II, Via Sergio Pansini, 5, 80131 Naples, Italy. E-mail: cittadin{at}unina.it Or Pamela S. Douglas, M.D.,
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Abstract |
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+ 36 ± 5 vs. +19 ± 4;
P < 0.01) and depression of function (%
fractional shortening -12 ± 2 vs. -7 ± 1;
P < 0.01) were both greater in the dwarf group.
Furthermore, dwarf rats failed to develop compensatory hypertrophy of
noninfarcted posterior wall (% posterior wall +5 ± 1
vs. +15 ± 3; P < 0.01).
Therefore, pathologic left ventricular remodeling and functional loss
following myocardial infarction is more marked in conditions of GH
deficiency. An intact GH/IGF-1 axis appears necessary for a normal
response to myocardial infarction injury in the rat. |
Introduction |
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We have recently characterized the cardiac phenotype of a strain of mutant rats with a specific isolated GH deficiency, which resembles human GH deficiency (11). Dwarf rats represent a unique model enabling the study of the physiological role of GH due to the lack of other pituitary abnormalities (12), which has yielded important information concerning the role of GH and IGF-1 on somatic growth (13). Dwarf rats display decreased myocyte size, cardiac atrophy, impaired cardiac contractility, and reduced calcium responsiveness of the myofilaments, which confirm and extend previous observation of cardiac atrophy and impaired cardiac performance observed in childhood onset GH deficiency in man (14, 15, 16).
Therefore, the aim of the current study was to evaluate the pathologic process of cardiac remodeling occurring after myocardial infarction in the absence of GH secretion. We postulated that the lack of a normal hypertrophic response, which is in part mediated by the GH/IGF-1 axis, would worsen postinfarction remodeling and function in dwarf rats.
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Materials and Methods |
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Echocardiography
Transthoracic echocardiograms were performed in all animals
before surgery, and 2 weeks and 6 weeks following myocardial
infarction. Briefly, rats were anesthetized with a combination of
ketamine HCl 50 mg/kg (Parke-Davis, Morris Plains, NJ) and
xylazine 10 mg/kg ip (Lloyd Laboratories, Shenandoa, IA)
and placed on a specially designed apparatus. Echocardiograms were
performed from underneath with a Hewlett-Packard Co. Sonos
1500 (Hewlett-Packard Co., Andover, MA) sector scanner
equipped with a 7.5 MHz phased-array transducer. Two-dimensionally
guided M-mode tracings were recorded with a strip-chart recorder at a
paper speed of 100 mm/sec. Posterior wall thickness and LV internal
dimensions were measured according to the leading edge method of the
American Society of Echocardiography (17). LV outflow
tract diameter was measured on a still-frame two-dimensional image at
the base of the aortic leaflets in a parasternal long-axis view.
Infarct size was measured from the 3-week echocardiogram by observing
the akinetic region in real time and measuring the percentage of the LV
endocardial circumference that was akinetic on a freeze-frame image at
end-diastole, as previously described (5, 18). The
agreement between echocardiographic and histological approaches was
excellent in the previous and in the present study. All measurements,
performed with an off-line analysis system (Cardiac Workstation,
Freeland Systems, Louisville, CO) by one observer who was blinded to
prior results, were based on the average of three consecutive cardiac
cycles.
LV outflow tract velocimetry was recorded from a five-chamber view. Stroke volume was calculated as:
Aortic Velocity Time Integral x [
(left ventricular
outflow tract/2)2],
and multiplied by heart rate to calculate cardiac output. When appropriate, structural and functional indexes were normalized to body weight.
In considerations of the baseline differences between Lewis and dwarf rats, we also calculated the percent change from baseline of all echocardiographic parameters.
Hemodynamic studies
Within 12 h of the final echocardiogram, rats were
anesthetized with ketamine and xylazine at the same doses used for the
echocardiograms. A calibrated 2 French micromanometer-tipped catheter
(Millar Instruments, Houston, TX) was passed via the carotid artery
into the left ventricle under constant pressure monitoring. LV
end-diastolic pressure was recorded with an expanded scale and left
ventricular dP/dt was obtained from a differentiating circuit in the
physiological recorder (model 2400, Gould, Inc., Cleveland, OH).
Because changes in left ventricular shape and uniformity following
myocardial infarction prevent the calculation of true LV wall stress
from monodimensional images, we devised an approximate measure of load
of the noninfarcted myocardium, termed posterior wall load index using
the following formula (5):
0.334 x left ventricular pressure x [LVID/(1 + PWT/LVID)]
where LVID is left ventricular internal diameter (end-systolic or end-diastolic), PWT is posterior wall thickness, and left ventricular pressure is left ventricular peak systolic or end-diastolic pressure. LV measurements were obtained from M-mode recordings, whereas LV pressures were estimated within 12 h of the final echocardiogram under identical anesthetic conditions. Although pressures and dimensions were not measured simultaneously, we believe that additional useful information can be derived from these data.
In summary, the architecture of the left ventricle was assessed by measuring the thicknesses of the LV wall (anterior and posterior wall), the cavity diameters, and the ratio between the cavity diameter and the posterior wall as an index of the extent of LV remodeling. Cardiac function was assessed calculating the endocardial fractional shortening and the cardiac output, whereas the filling pressures and the stress acting on the LV walls during systole and diastole were measured as LV end-diastolic pressure and posterior wall systolic and diastolic load index, respectively.
Postmortem studies
After catheterization, animals were killed and the hearts were
fixed and subsequently analyzed with morphometric histology. Blood
samples and tibial length measurements were obtained from all
animals.
Blood analysis
Blood samples were obtained at the time the rats were killed.
Duplicate hematocrit samples were prepared in microhematocrit tubes.
Serum was prepared from the remainder of the sample and frozen at -20
C for subsequent analysis. Rat GH was measured in rat serum by ELISA,
and total serum IGF-1 by RIA, according to previously described methods
(19, 20).
Histology
Left ventricles were immersion fixed in 10% buffered formalin.
Specimens for histologic examination were obtained from four
cross-sections of the heart, cut from apex to base. The samples were
embedded in paraffin and stained with hematoxylin-eosin for measurement
of muscle fiber diameter and Masson trichrome for assessment of
interstitial fibrosis. The four sections were then projected, and
average infarct size was estimated by measuring the percentage of the
total endocardial circumference replaced by scar tissue. Quantitative
evaluation of myocyte hypertrophy was carried out by morphometry,
according to previously described methods (21), by an
observer blinded to the study protocol, on tissue blocks obtained from
the noninfarcted interventricular septum. Briefly, each section was
projected by using a binocular microscope (Carl Zeiss,
Germany) attached to a video camera at x400 magnification. The system
was interfaced to a personal computer (Apple Computer, Cupertino, CA)
equipped with morphometric software. The circumferences of 100 myocytes
per animal were digitized on each of the four sections, and average
myocyte area calculated. Quantitative assessment of interstitial tissue
as a measure of fibrosis was accomplished with a special grid on which
horizontal and vertical lines provided 100 intersecting points, at
x 400 magnification. Four fields on each of the four sample sites were
examined for each animal, yielding a total of 16 fields in each rat.
Reproducibility studies showed a good correlation between data obtained
from two studies in four rats, for both techniques, with an r value of
0.90.
Northern analysis
Total RNAs, extracted from frozen LV myocardium by the
acid-phenol method as described by Chomczynski and Sacchi
(22), were (20 µg) size fractionated by denaturing gel
electrophoresis and blotted overnight onto Hybond
N+ membranes (Amersham Pharmacia Biotech, Arlington Heights, IL). In addition to the three study
groups, LV myocardium from a group of 4 age and sex matched Lewis rats
was used as control myocardium. The specific IGF-I complementary DNA
(cDNA) probe (5) was radiolabeled with
[
32P]dATP and
[32P]dGTP (Amersham Pharmacia Biotech) by random-priming and used at the specific activity of
at least 2 x 109 cpm/µg. The blots were
prehybridized and hybridized at 65 C for 2 h in RapidHybBuffer
solution (Life Technologies, Inc., Gaithersburg, MD) and
sequentially washed for 30 min with 2 x SSC and 0.1% SDS at room
temperature, followed by washing in 0.5–0.1xSSC and 0.1% SDS at 65 C
until radioactive background was negligible. To normalize for gel
loading variability, all blots were rehybridized to a probe for
glyceraldehyde-3-phosphoate dehydrogenase (GAPDH) (5) as
described before. Thus, arbitrary myocardial IGF-1 messenger RNA (mRNA)
expression was estimated with laser densitometer by normalizing the
autoradiographic density of IGF-1 band to that of corresponding
GAPDH.
Statistical analysis
All values are given as mean ± SEM.
Statistical analysis was performed using a Sun Microsystem Station
equipped with the PROPHET software package. Between-group comparisons
of echocardiographic indexes were performed using a 2-way ANOVA with
repeated measure in one factor (time), followed by Neumann-Keuls test.
One-way ANOVA was employed for the other comparisons, also followed by
Neumann-Keuls test. Percent differences from baseline values of
echocardiographic parameters among the groups were compared using
nonparametric tests. A value of P < 0.05 was
considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Plasma IGF-1 levels, a better index of GH activity, were significantly
decreased in the two groups of dwarf rats compared with the Lewis rats.
IGF-1 transcript levels did not show significant intergroup
differences: (arbitrary units ± SE) controls,
100 ± 4, dwarf-sham, 95 ± 5, dwarf with myocardial
infarction 96 ± 4, Lewis with myocardial infarction 107 ±
10 (Fig. 1). As expected, baseline
evaluation revealed smaller body weight, LV dimensions, and wall
thicknesses in dwarf rats compared with control Lewis (Tables 1 and 2). Moreover, in dwarf rats, cardiac
output was reduced by 41%, whereas fractional shortening was only
slightly lower than normal littermates (Table 3). Over the study protocol, body weight
( + 15%) and LV dimensions showed only little changes from baseline
values in GH-deficient sham rats (Tables 1–3). Despite similar infarct
size at morphometric histology, GH-deficient animals displayed worse
remodeling compared with normal rats. In fact, compared with baseline
values, LV dimensions increased by an average 19% in diastole and 32%
in systole at 6 weeks in infarcted Lewis rats, but by 36% and 62% in
infarcted dwarf rats, respectively (Fig. 2). At 2, and in particular at 6 weeks,
compensatory hypertrophy of the non infarcted posterior wall was
evident in normal infarcted rats, with an average increase of 15%
compared with baseline values. Conversely, GH-deficient rats failed to
develop hypertrophy of the noninfarcted posterior wall. LV systolic
functional loss following myocardial infarction was more marked in
dwarf rats compared with Lewis controls: in fact, at 6 weeks fractional
shortening decreased by 7% in Lewis controls and by an average 12% in
the GH-deficient group (Fig. 2). Morphometric histology of the
noninfarcted myocardium revealed a significant decrease of myocyte area
in both dwarf groups compared with Lewis infarcted rats (Fig. 3). Percent of interstitial tissue as
index of fibrosis was not different among the study groups.
Closed chest LV catheterization with high fidelity recordings showed
elevated values of end- diastolic pressures in both infarcted groups,
without significant differences. The differential effects of myocardial
infarction on LV geometry in the two infarcted groups in presence of
similar LV systolic and diastolic pressures lead to higher values of
diastolic and especially systolic load index of the noninfarcted LV
posterior wall of dwarf infarcted rats compared with controls (Table 3).
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Discussion |
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Although wide interest has grown as to the effects of GH excess in the setting of human and experimental heart failure (3, 4, 5, 6, 7, 8, 9), little is known concerning the role of an intact GH/IGF-1 axis in postinfarction LV remodeling. This could help better delineate the cardiac regulatory role of GH/IGF-1 axis. Considering the postulated trophic role of the GH/IGF-1 axis, it would be reasonable to expect a failure to develop hypertrophy with negative structural and functional reverberations, as our results show.
GH deficiency and postinfarction ventricular remodeling
It has been shown by several investigations in GH excess and
deficiency that GH and IGF-1 contribute to maintaining normal
myocardial mass and function (10). Thus, it is reasonable
to speculate that the worst postinfarction remodeling of dwarf
rats might depend upon their inability to enhance the activity of the
GH/IGF-1 axis. The normal hypertrophic response, which counteracts the
elevated wall stress following myocardial infarction is impaired, and
according to Laplace’s law, the left ventricle dilates more with
negative functional reverberations. This view is supported by the
higher systolic and diastolic stress showed by infarcted dwarf rats
compared with Lewis controls. Moreover, the well known positive
inotropic (23) and vasodilatory (24)
responses mediated by IGF-1, which might play a beneficial role in
setting of myocardial infarction, could also be blunted. To lend
further support to these speculations, the beneficial effects of GH
administration in the setting of experimental myocardial infarction
have been postulated to depend upon GH’s ability potentiate
physiological responses such as the induction of additional hypertrophy
of the noninfarcted myocardium, and enhancement of function of the
surviving myocardium (5). The inability to activate such
mechanisms likely underlies the worse pathologic remodeling of dwarf
rats in the current investigation.
On the other hand, the low activity of the GH/IGF-1 axis consistently documented in human heart failure has been implicated in the pathophysiology of the cardiac failure phenotype and has represented a rationale for the growth factor treatment of this disease condition (3, 4, 5, 6, 7, 8, 9, 10).
Interestingly, although IGF-1 circulating levels were significantly decreased in mutant dwarfs compared with Lewis infarcted rats, myocardial expression of mRNA encoding IGF-1 was not different in the dwarf vs. the Lewis groups. Therefore, at variance with liver and kidney whose local IGF-1 mRNA levels are reduced in dwarf vs. control animals (25, 26), the cardiac muscle behaves consistently with the skeletal muscle that has recently shown to express normal IGF-1 mRNA levels (27). Furthermore, myocardial infarction is not associated with enhanced local production of IGF-1 6 weeks after the injury in both control and dwarf rats. Thus, it appears that the up-regulation of the mRNA encoding IGF-1 and its cognate receptor in the left ventricle of infarcted rats begins only a few hours following coronary ligation (28) but tends to vanish in chronic settings, when the scar and the remodeling process are complete. Congruent with this finding is our previous demonstration of normal myocardial IGF-1 mRNA levels 3 weeks after a large myocardial infarction (5). Future research is needed to verify whether early IGF-1 mRNA up-regulation is attenuated by GH deficiency in this animal model.
Comparison with previous work
Attempts to induce a hypertrophic response in absence of GH
secretion have yielded conflicting results. Our results are congruent
with a series of elegant studies performed during the 1950s, in which
Beznack (29, 30, 31) consistently showed that GH was necessary
for a normal hypertrophic response following aortic banding .
Specifically, experimental aortic constriction caused no cardiac
hypertrophy and hypertension in untreated hypophysectomized rats. On
the other hand, hypophysectomized rats treated with GH showed the same
hypertension and cardiac hypertrophy on aortic constriction as did
normal rats. Conversely, more recently, Lembo et al.
(32) have shown that transgenic mice with low levels of
IGF-1 maintain a normal ability to develop hypertrophy when subjected
to aortic banding. A recent study by Shen et al. also
demonstrated that hypophysectomized infarcted rats exhibit a
hemodynamic profile similar to matched controls (33). It
appears difficult to reconcile such inconsistencies in view of the
different animal models and substitutive therapy employed. However,
some generalizations can be made. In Lembo’s study, although IGF-1
knock-out mice displayed reduced IGF-1 circulating levels, GH levels
were increased by 375% (32). Contractility and systemic
blood pressure were increased at variance with human and other animal
GH-deficient states. Therefore, the transgenic model described does not
appear to be a pure model of GH deficiency, such as the dwarf rat. In
the study by Shen et al., the myocardial infarction was so
small that it did not induce any change of basal hemodynamics in either
hypopituitary or control rats, whereas in our investigation there was a
significant increase of left end-diastolic pressure and of cavity
diameters. It is unlikely that such a limited myocardial injury would
have allowed detection of differences in the remodeling process between
the animal groups. Moreover, the hydrocortisone dosage was far greater
than employed in other investigations, which might have blunted GH’s
actions. At variance with these animal GH-deficient states, the dwarf
rat represents a pure model of GH deficiency, because other pituitary
hormones exhibit normal secretion (12, 13). It closely
mimics childhood onset GH deficiency, because GH is lacking throughout
the entire developmental period, and displays cardiac abnormalities
such as atrophy and impaired function. Moreover, it needs to be
stressed that the mutant dwarf rat derives from a spontaneous single
point mutation and that GH deficiency is the only difference between
normal and dwarf rats (12, 13). Because this is the only
variable, observed differences in remodeling must be a result of the
difference in GH secretion.
Potential study limitations
The ideal way of normalizing physiological parameters is still an
open issue, particularly when dealing with animal models characterized
by abnormal growth patterns. The baseline differences observed in the
current study may indeed confound the data interpretation. In the
current study, however, the interest was mainly focused on the changes
from baseline of LV dimensional and functional parameters between the
two groups of infarcted rats so that each animal served as its own
control. In fact, percent differences do not need normalization to the
different body and heart size. Two considerations further support the
validity of our approach. First, we report measures of LV function that
are independent of the body size, such as fractional shortening, that
show a more marked decrease in the dwarf that in the Lewis rats.
Second, considering the retarded pattern of growth of the dwarf animals
vs. the controls, the differences observed in the remodeling
process between the two infarcted groups are even more significant. As
a prototype, whereas LV end-diastolic diameter in the dwarf rats
increases approximately only 3–4% in 6 weeks, this measures increases
in an age matched control in the same time frame by 7%
(5). Also other measures of LV architecture display a
similar pattern, such as LV end-systolic diameter, or anterior and
posterior wall thicknesses. Consequently, the fact that the same
infarct size leads the left ventricle of the dwarf rats to enlarge more
than the left ventricle of the Lewis control, despite a smaller
relative contribution of the normal growth in the formers, indicates
clearly a worse remodeling process in the dwarf rat.
There are limitations involved in the use of hemodynamic data under anesthesia. However, both animal groups were handled similarly and data should reflect actual intergroup differences.
Conclusions
In a pure model of GH deficiency, myocardial infarction induces
more marked pathologic remodeling characterized by larger cavity size
and lower cardiac function compared with control rats undergoing
similar extent of injury. Therefore, an intact GH/IGF-1 axis appears
necessary for a normal response to myocardial infarction injury in the
rat. These data lend further support to the hypothesis that GH plays a
pivotal role in cardiac physiological and diseased conditions.
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Footnotes |
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Received March 23, 2000.
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