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Wallenberg (E.O., E.B., R.M., V.K., M.F., Å.H., F.W.) and Lundberg (B.M., B.S.) Laboratories, Research Center for Endocrinology and Metabolism (J.I.), Sahlgrenska University Hospital, SE 413 45 Gothenburg, Sweden
Address all correspondence and requests for reprints to: Jörgen Isgaard, M.D., Ph.D., Research Center for Endocrinology and Metabolism, Sahlgrenska University Hospital, SE 413 45 Gothenburg, Sweden. E-mail: jorgen.isgaard{at}medic.gu.se
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Abstract |
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Myocardial infarction (MI) was induced in male Sprague Dawley rats. Three different groups were studied: MI rats treated with saline (n = 7), MI rats treated with GH (MI + GH; n = 11; 3 mg/kg·day), and sham-operated rats (sham; n = 8). All rats were investigated with 31P magnetic resonance spectroscopy and echocardiography at 3 days after MI and 3 weeks later. After 3 weeks treatment with GH, the phosphocreatine/ATP ratio increased significantly, compared with the control group (MI = 1.69 ± 0.09 vs. MI + GH = 2.42 ± 0.05, P < 0.001; sham = 2.34 ± 0.08). Treatment with GH significantly attenuated an increase in left ventricular end systolic volume and end diastolic volume. A decrease in ejection fraction was prevented in GH-treated rats (P < 0.05 vs. MI). Myocardial and plasma noradrenaline levels were significantly lower in MI rats treated with GH. These effects were accompanied by normalization of plasma brain natriuretic peptide levels (sham = 124.1 ± 8.4; MI = 203.9 ± 34.7; MI + GH = 118.3 ± 8.4 ng/ml; P < 0.05 vs. MI).
In conclusion, GH improves myocardial energy reserve, preserves left ventricular function, and attenuates pathologic postinfarct remodeling in the absence of induction of left ventricular hypertrophy in postinfarct rats. The marked decrease in myocardial content of noradrenaline, after GH treatment, may protect myocardium from adverse effects of catecholamines during postinfarct remodeling.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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31P Magnetic resonance spectroscopy (31P MRS) has been proven to be a powerful tool in the studies of cardiac energy metabolism because it allows unique noninvasive and nondestructive investigation of the heart (6). The technique has been successfully used in vitro (7) and in vivo (8, 9) in different animal models, as well as in humans, for evaluation of cardiac energy metabolism during infarction (10), cardiomyopathy (11), and heart failure (12).
Activation of regional sympathetic systems in the heart and in other organs is a known phenomenon in heart failure (13, 14). Overwhelming evidence supports the concept that overactivation of this system significantly contributes to the progression of CHF (13, 15). Furthermore, a marked activation of the sympathetic nervous system has also been reported in GH-deficient patients (16). However, the relationship between the GH-insulin-like growth factor I (GH-IGF-I) axis and sympathetic system has not been fully clarified at the present time.
The aims of this study were: 1) to examine, in vivo, the effects of GH on myocardial energy status (using volume-selective 31P MRS) and function (using echocardiography) in rats during early postinfarct remodeling phase; and 2) to evaluate the effect of GH treatment on myocardial and plasma catecholamines, myocardial ß-adrenoreceptors, and plasma brain natriuretic peptide (BNP) content.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Echocardiography and hemodynamics
Echocardiography was used to assess left ventricular function
and geometry using previously validated two-dimensional, M-mode and
Doppler techniques (18). Left ventricular meridional wall
stress was calculated as previously described (9, 19).
Examinations were performed 3 days post infarct and 3 weeks later. The
size of MI was estimated according to the score system
(20). Although the score system used for estimation of
infarct size provides a reasonable estimate, the low number of animals
may have been a limitation.
Only the rats with large infarcts and ejection fraction (EF) less than 45% were selected. Animals that did not shown signs of MI were defined as sham-operated (sham, n = 8). Rats with MI were randomized into two groups [control group (MI, n = 7) and GH group (MI + GH, n = 11)]. The MI + GH group received recombinant human GH (rhGH) continuously (3 mg/kg·day for 3 weeks), delivered by implanted osmotic pumps (Alzet, 2ML4, Alza Corp., Palo Alto, CA), starting at 3 days post infarct. The control group (MI) received saline. Systolic blood pressure was measured by the indirect cuff-tail method (9) (RTBP Monitor, Harvard Apparatus, Inc., South Natik, MA).
In vivo 31P MRS of the rat heart
Volume-selective, cardiac 31P MRS
experiments were performed on a 2.35 Tesla horizontal magnet with a
20-cm bore (BioSpec 24/30, Bruker Medical GmbH,
Rheinstetten, Germany) according to the method previously described by
our laboratory (9). The myocardial PCr/ATP ratio was
corrected for partial saturation and for blood contamination (6, 21). After completion of the second 31P
MRS examination, the rats were killed by rapid excision of the
heart.
Radioligand binding for ß-adrenoceptor assessment
Cardiac tissue, without macroscopic adipose or connective
tissue, was minced and homogenized in buffer consisting of 50
mM Tris HCl and 10 mM
MgCl2. The protein content of homogenate was
measured according to Lowry. ß-adrenoceptors were determined by use
of (125I) iodocyanopindolol using standard
binding assay.
Serum levels of rhGH and IGF-I
The serum concentration of IGF-I was determined by a
hydrochloric acid-ethanol extraction RIA using human IGF-I for labeling
(Nichols Institute Diagnostics, San Juan Capistrano, CA).
The assay was performed according to the manufacturer’s protocol. The
levels of rhGH in the plasma from rats treated with rhGH were
determined by a polyclonal antibody-based immunoradiometric assay
(Pharmacia, Uppsala, Sweden).
Biochemical analysis of catecholamines in plasma and
myocardium
Myocardial and plasma levels of catecholamines, noradrenaline
(NA), adrenaline (A), and dopamine (DA) were measured by means of HPLC
with electrochemical detection (Gynkotek HPLC, Germering bei
München, Germany) according to the standard techniques using a
modified procedure (22).
BNP
The blood sample was drawn from the right ventricle (RV) and was
transferred to ice-cold tubes containing aprotinin (1000 kIU/ml) and
Na2EDTA (1 mg/ml) and was centrifuged at 4 C.
Plasma samples were stored at 20 C until assay. The plasma
concentration of BNP was measured with 250 µl plasma using RIA
according to the manufacturer’s protocol (Peninsula Laboratories, Inc., San Carlos, CA).
Hypophysectomy
To evaluate whether GH has a direct effect on myocardial
bioenergetics, cardiac 31P MRS was performed in
hypophysectomized male Sprague Dawley rats (Möllegård,
Breeding Centre, Ltd., Ejby, Denmark). Two different groups of rats
were used: hypophysectomized rats replaced only with hydrocortisone
(0.25 mg/kg·day) and L-T4 (20
µg/kg·day) (n = 5); and hypophysectomized rats replaced
with hydrocortisone, L-T4 in the same
doses, and GH (1 mg/kg·day) (n = 6). Replacement with
hydrocortisone and L-T4 was started
immediately after hypophysectomy, whereas GH was given during
weeks 5–6 after hypophysectomy. Hydrocortisone and
L-T4 were delivered by implanted
osmotic pumps, whereas GH was given by sc injection.
31P MRS examination of the heart was performed 6
weeks after hypophysectomy.
Statistics
One-way ANOVA (of single measurements and change from baseline/3
days post infarct), followed by Fisher PLSD post hoc test,
was applied to detect significant differences. Normal distribution of
the data was assessed using a Kolmogorov-Smirnov test. When data were
not normally distributed, a nonparametric test was applied. The value
P < 0.05 was considered as statistically significant.
All data are presented as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). During the treatment period of 3
weeks, there was a significant increase in body weight in all
experimental groups. All three groups have shown similar increase in
body weight during the experimental time. However, body weight was
significantly lower in MI rats, compared with the sham group, also at
the end of the study. Treatment with rhGH did not result in further
increase in body weight, compared with the controls. There was no
significant difference in LV or RV weight after rhGH treatment,
compared with the control group.
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), heart rate (HR), EF, and left ventricular
dimensions (Tables 2 and 3). At the same time, fractional
shortening (FS) was significantly lower, whereas left ventricular
volumes in systole and diastole were significantly higher in the GH
group. Rats with MI had significantly lower EF, FS, and higher LV
volumes and dimensions, compared with sham rats, indicating early
systolic dysfunction and rapid dilatation of infarcted LV.
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The decceleration slope of the mitral E-wave was significantly higher in MI rats, compared with sham, at base line and was not affected 3 weeks later, after the treatment with GH. There was a tendency toward decreased E/A wave ratio in the GH-treated group. Systolic blood pressure was not different between the groups either 3 days or 3 weeks post infarct. Compared with base line, WS decreased only in the GH group, although this difference was not statistically significant.
Left ventricular energy status assessed by volume-selective
31P MRS
The PCr/ATP ratio was not significantly different between control
and rhGH groups at the base line (MI = 1.45 ± 0.07; MI +
GH = 1.58 ± 0.06; sham = 2.42 ± 0.3), whereas
both groups had significantly lower PCr/ATP ratio, compared with
the sham group (P < 0.0001) (Fig. 1, A and B). After 3 weeks treatment with
rhGH, the PCr/ATP ratio increased significantly, compared with the
control group, and reached the same value as in the sham group,
suggesting normalization of cardiac energy status (MI + GH =
2.42 ± 0.05 vs. MI = 1.69 ± 0.09,
P < 0.001; sham = 2.34 ± 0.08). In
hypophysectomized rats without GH substitution, the PCr/ATP ratio was
significantly decreased at 6 weeks after hypophysectomy, whereas 2
weeks of GH substitution normalized the PCr/ATP ratio (1.63 ±
0.12 vs. 2.45 ± 0.05; P < 0.0001)
(Fig. 2).
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).
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and Fig. 3A). Myocardial DA was
significantly lower in the GH group, compared with the sham group,
whereas the difference was not significant, compared with controls.
Neither myocardial nor plasma concentration of A was different between
the groups. The rhGH-treated rats had significantly lower plasma NA
levels, compared with the control group (Fig 3B). There was no
difference in plasma levels of DA and A and in myocardial content of
ß-adrenoceptors between the groups.
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). No difference was found between sham
and rhGH-treated rats.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects of early treatment with rhGH on cardiac
bioenergetics
Accumulating evidence suggests that the failing heart is an
energy-depleted organ (23). Our laboratory has previously
reported that disturbances in myocardial energy metabolism ensue early
in the postinfarct period (9) and that lowering of
myocardial energy reserve correlates with parameters of LV systolic and
diastolic dysfunction, as well as with LV wall stress
(24). Failing and hypertrophied myocardium is
characterized by several consistent changes in the cellular energetic
system, regardless of species (25, 26). The PCr-to-ATP
ratio is a commonly used index of cellular energy status, because it
reflects the equations of cellular phosphorylation potential [a
driving chemical force for energy-dependent intracellular events
(12, 27)].
It is well known that GH has profound influence on the regulation of carbohydrate, protein, and fatty acid metabolism. However, little is known about the specific metabolic effects of GH at the level of the heart and whether GH directly affects cellular energy metabolism. Initial experimental and clinical studies in which GH was used as treatment for heart failure suggested that GH might lower energetic demand of the failing heart by reducing LV wall stress and afterload. Other reports suggested that GH might positively affect the economy of energy utilization at cross-bridge reactions in the myocytes (28). However, the hypothesis that GH could improve cardiac energy status was never tested adequately and directly in vivo. In this study, the effect of GH treatment on myocardial bioenergetics was assessed in vivo and noninvasively using volume-selective cardiac 31P MRS. The study demonstrates that short-term treatment with rhGH in the early postinfarct phase normalizes the myocardial PCr/ATP ratio. Because the treatment with rhGH normalized the PCr/ATP ratio without induction of either LV hypertrophy or significant change in WS, antiremodeling effect cannot entirely explain this result. It is more likely that the improvement in myocardial bioenergetics is largely independent of GH’s antiremodeling effect. This hypothesis is supported by our observation that hypophysectomized rats without GH substitution have shown a decreased myocardial PCr/ATP ratio, which was normalized after 2 weeks of GH substitution. The underlying mechanism for this is unknown, but previous studies have shown that GH is involved in the regulation of cellular enzymes important for creatine synthesis (29). Further experiments are needed to evaluate whether insufficient creatine production may be the reason for decreased PCr/ATP in GH-deficient rats. It has also been shown that IGF-I stimulates uptake of both creatine and inorganic phosphate by muscle cells under in vitro conditions (30, 31). It is tempting to speculate that treatment with GH could increase uptake of creatine and Pi by local stimulation of IGF-I production. However, because the plasma levels of IGF-I were not different between rhGH-treated and control groups, and local levels of IGF-I were not measured, this assumption remains to be investigated in the future. Furthermore, it is also important to investigate whether GH directly affects these transport mechanisms mediated through GH receptors in the myocardium.
Effects of GH on left ventricular function and morphology
Previous studies have demonstrated beneficial effects of GH on
systolic function in rats after experimental MI (1, 32).
In the present study, a moderate effect of rhGH on cardiac performance
was observed, manifested as a preservation of function rather than
improvement. This may be attributed to the fact that rhGH was added in
the early post-MI phase rather than in a chronic setting, as previously
reported (1). However, normalization of plasma BNP after
rhGH treatment strongly suggests early beneficial effects of rhGH on
hemodynamics after MI. To our knowledge, this is the first time that
rhGH effects on BNP levels have been reported.
Treatment with rhGH attenuated pathologic remodeling of LV in the rats with MI. This is in accordance with previous studies (32), although these findings are not always consistent (33, 34). In the study by Shen et al. (34), GH treatment did not affect cardiac function and remodeling either in normal or hypophysectomized female rats with MI. There are several possible reasons for the divergence in the results. In this study, male rats, instead of female, were used. Previous studies have clearly demonstrated that gender differences exist regarding response to the treatment with GH. Female rats have slower spontaneous growth rate and show a less pronounced response to the exogenously administered GH (35). The reason for this is unknown, but multiple studies have indicated a lower number of GH receptors and higher concentration of GH-binding proteins as possible contributing factors (36). Additionally, the use of one-time observations (34) may preclude detection of small, but significant, beneficial effects of GH treatment. Although attenuation of increase in LV volumes was only modest, this effect, together with no significant increase in the LV weight, suggests an antiremodeling effect of rhGH treatment. These findings prompt the tempting hypothesis that patients with postinfarct remodeling ,and possibly CHF, may benefit from the rhGH treatment without need for induction of LV hypertrophy, as previously proposed (2). This is an important issue because it is generally accepted that LV hypertrophy is an independent risk for mortality in the patients with CHF in progress.
Effects of GH on catecholamines and ß-adrenoceptors
One rather unexpected result was that of markedly decreased
myocardial content of NA in animals treated with rhGH. Not only
myocardial content of NA but also plasma NA were significantly lower in
rats receiving rhGH. The interaction between GH and sympathetic system
is probably complex and is not completely understood at the present
time. Previous clinical studies indicate that GH may have pronounced
effects in the regulation of sympathetic function, which is based on
the fact that patients with GH deficiency have markedly increased
activation of the sympathetic nerve fibers firing in skeletal muscle
(16). Furthermore, GH treatment of patients with dilated
cardiomyopathy results in attenuation of cardiac sympathetic
activation under stress conditions (37). Our findings
are congruent with these observations. The marked lowering of
myocardial catecholamine content, after rhGH treatment, suggests an
important role of rhGH in regulation of the cardiac sympathetic system
and catecholamines. The present study does not allow any conclusions
regarding the mechanisms and pathophysiological importance for these
findings, and further studies are necessary. However, it is unlikely
that diminished stores of myocardial NA are a result of increased
release and/or decreased uptake. This assumption is supported by the
fact that neither HR nor myocardial content of ß-adrenoceptors was
different between the groups. It has been established that
down-regulation of ß-adrenoceptors is one consequence of cellular
exposure to high NA levels (38). In the early phase of
heart failure, there is an organ-selective activation of the cardiac
sympathetic system, and the catecholamine spillover from the heart is
3–4 times higher than normal (14). If GH specifically
increases release and/or decreases reuptake of NA, one would expect
depletion of myocardial stores to be associated with increased plasma
NA levels. Even if the long-term consequences of this effect are not
known, lowering of tissue NA content in the early postinfarct phase,
together with low plasma NA concentration, could have protective
effects on damaged and remodeling myocardium. Experimental studies have
shown that NA, in high concentration (similar to that known to occur in
the neuromuscular synapses of failing myocardium), exerts direct
pathological effects on cardiomyocytes. These effects include cell
necrosis, stimulation of apoptosis, increase in interstitial fibrosis,
arrhythmias, and others (39, 40). Furthermore, increased
adrenergic drive can decrease the contents of creatine and CK in the
heart, indicating adverse effects on cellular energetic homeostasis
(41). While improving LV function by other mechanisms
independent of the sympathetic system, addition of rhGH could, at the
same time, protect myocardium from the side effects of sympathetic
overactivation. This hypothesis, however, has to be taken with caution
and proven in future experiments. One has to keep in mind that
depletion of myocardial catecholamines is a consistent finding in
patients with advanced heart failure. Exhaustion of catecholamine
stores in cardiac neurons is proposed to be involved in mechanisms
behind LV dysfunction.
In summary, 3 weeks of treatment with rhGH, in the early postinfarct phase, improved myocardial energy status, attenuated postinfarct pathologic remodeling of LV, and decreased myocardial and plasma catecholamine levels in rats with large MI. These effects were accompanied by preservation of left ventricle function and normalization of BNP levels in plasma. These findings provide novel evidence in favor of the concept that GH may have a place in the treatment of postinfarct remodeling.
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Acknowledgments |
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Footnotes |
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Received May 8, 2000.
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References |
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, P <
0.0001 vs. Sham; #, P < 0.0001
vs. base line; *, P < 0.0001
vs. MI; ¤, P < 0.001 effect of
treatment with rhGH (MI-rhGH) vs. control (MI).
