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Research Center for Endocrinology and Metabolism, the Department of Internal Medicine (Å.T., B.-Å.B., J.I.), Wallenberg Laboratory (E.B., Å.H.), the Department of Clinical Physiology (K.C., V.K.), and the Department of Clinical Pharmacology (X.Y.S., T.H.), Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden
Address all correspondence and requests for reprints to: Jörgen Isgaard, M.D., Ph.D., Research Center for Endocrinology and Metabolism, Gröna Stråket 8, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. E-mail: jorgen.isgaard{at}ss.gu.se
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Introduction |
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The hexapeptide hexarelin (Hex) is chemically more stable and a more potent GH secretagogue than its analog GHRP-6 (10). Its actions have been investigated in both experimental and clinical studies (11, 12, 13, 14, 15, 16). The results of many studies have shown that Hex increases GH secretion, but there are few studies of the clinical long term effects. However, Hex has been reported to accelerate growth in short children, influence parameters of bone formation and muscle morphology in aged dogs, and restore sodium channel properties in skeletal muscle in aged rats (17, 18, 19).
The myocardium expresss functional receptors for both GH and insulin-like growth factor I (IGF-I), and it is now well known that the GH/IGF-I axis has an important role during cardiac development and for maintaining the structure and function of the heart (20). In GH deficiency one of the clinical signs is an impaired cardiovascular performance, which may be reversed after GH substitution therapy, particularly in terms of increased stroke volume and reduced peripheral resistance (21). GH has also been shown to improve systolic function in normal subjects (22). In recent studies of patients with congestive heart failure due to dilated cardiomyopathy, both acute administration of GH and more chronic treatment have been shown to exert major hemodynamic effects (23, 24). The most important findings in these studies were improved parameters of systolic heart performance, decreased peripheral resistance, and enhanced ventricular work despite a reduction in oxygen consumption and energy production. These promising results could not be confirmed in a small placebo-controlled study despite increased levels of serum IGF-I (25). However, there is also abundant experimental evidence for enhanced cardiac performance by GH, among them several studies using a rat model of experimental myocardial infarction (MI) (26, 27, 28, 29). Besides a pronounced vasodilatation and lowering of total peripheral resistance by GH/IGF-I, enhanced myocardial contractility is likely to contribute (30, 31, 32). Moreover, in animal models of GH excess there is a shift toward a myosin isoform with a lower adenosine triphosphatase activity, which may decrease the energy demand of the contractile process (30).
Recently, two groups have shown cardiac effects of Hex, including protection from ischemia in rats (33, 34) and acute hemodynamic effects in humans (35). In the present study we have characterized, in vivo and noninvasively, the hemodynamic effects of Hex. We have used echocardiography in a rat model 4 weeks after experimental myocardial infarction and compared the effects of Hex with those of GH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Study protocol: animal model of myocardial infarction
During short lasting methohexital sodium (75 mg/kg, ip; Brietal,
Eli Lilly & Co., Indianapolis, IN) anesthesia, the rats
were intubated and artificially ventilated with a respirator with room
air (4 ml/beat·kg; 78 beats/min and 40/60% inspiration/expiration
ratio) using a Carlsson ventilator (no. 8908, Mölndal, Sweden). A
left thoracotomy was performed, exposing the left ventricular
(LV) wall of the rats. The left coronary artery was ligated by
positioning a suture between the pulmonary artery out-flow tract and
the left atrium. The lungs were thereafter hyperinflated, positive
end-expiratory pressure was applied, and the thorax was immediately
closed (36). All rats (n = 56) were operated upon within 3 days.
Ten rats died during surgery or the postoperative phase.
After a recovery period of 4 weeks, the remaining operated rats (n = 46) were examined by transthoracic echocardiography. Again, all rats were examined within 3 days in the corresponding order as they were operated upon 4 weeks previously. The rats were anesthetized using ketamine hydrochloride (Ketalar, Parke-Davis, Barcelona, Spain; 50 mg/kg) and xylazine hydrochloride (Rompun vet, Bayer Corp., Leverkusen, Germany; 10 mg/kg, ip). Nine rats died during anesthesia. The chests were shaved, the rats were placed prone on the left decubitus, and echocardiography was performed (see below), blinded for treatment. According to infarction size, all rats with MI were distributed into five groups based on a score system described in echocardiographic methods section. Animals with no echocardiographic signs of MI were excluded from the study before randomization (n = 3). According to infarction size, the rats were then randomized to the different treatment groups, and treatment was started with recombinant human GH (2.5 mg/kg·day; n = 8), hexarelin [10 µg/kg·day (Hex10; n = 9) and 100 µg/kg·day (Hex100; n = 8)], or 0.9% NaCl (n = 9). GH and hexarelin were injected sc twice daily, and NaCl was injected once daily. Recombinant human GH (Genotropin) and hexarelin (His-D-2-methyl-Trp-Ala-Trp-D-Phe-Lys-NH2) were gifts from Pharmacia & Upjohn, Inc. (Stockholm, Sweden).
After the treatment period the rats were anesthetized and subjected to echocardiography a second time, also blinded for treatment. The examination was finished within 3 days so that the treatment period for all rats was 14 days. No rats died during the second echocardiographic examination. Untreated intact rats (n = 6) were used as normal controls and were examined with echocardiography only at the end of the experiment. After completion of the second echocardiographic examination, blood was sampled from the inferior caval vein, and the rats were killed by rapid excision of the heart. The atria were trimmed free from the ventricles, the right ventricle (RV) was separated from the LV plus septum, and the parts were weighed. Left kidney and liver tissue were collected, and the kidney was weighed. Blood samples were centrifuged, and serum was kept at -20 C until assayed for IGF-I concentration.
Echocardiography
All Doppler echocardiographic recordings were performed by an
investigator blinded to treatment allocation, and interpretations were
performed by two independent observers, also blinded to treatment.
Echocardiographic examination of the rats was performed before
treatment and at the end of the treatment period, as previously
described (28, 37). All rats were evaluated by a commercially available
echocardiographic system equipped with a 10-MHz linear array transducer
(Acuson, Sequoia-512, Mountain View, CA). Electrocardiographic
electrodes were placed for timing of intracardiac events. Optimal image
sequences were recorded on the S-VHS videorecorder and saved as digital
cine loops (three consecutive heart cycles, high resolution zoom with a
high frame rate) on the optical disc for future analysis. Images were
obtained through the left parasternal rib spaces by placing the
transducer against the chest wall from below. A long axis view of the
LV was obtained, and subsequently perpendicular to this, a
two-dimensional short axis view of the LV was acquired at the level
between the papillary muscles and mitral valve. By superior angulation
in the short axis view, the pulmonary artery was visualized and
recorded along the transecting beam with and without color Doppler
display. Pulsed wave Doppler signals and velocity time integrals were
obtained with the sampling site placed in the pulmonary artery well
below the valve level, avoiding turbulent flow or higher velocities
from the valve area. Measurements of main pulmonary artery (MPA)
diameter were performed at the level of the pulsed wave sampling site
from digitized cine loops in three consecutive heartbeats.
The size of the myocardial damage/infarction was estimated visually at the baseline examination from the two-dimensional short axis images as follows: no akinetic or severely hypokinetic segments (no damage), grade 0; akinesis/hypokinesis comprising up to 60° circumference (small damage), grade 1; 61–120° (moderate damage), grade 2; 121–180° (large damage), grade 3; and more than 180° (very large damage), grade 4. Average infarction size grades in the groups were 2.4 ± 0.4 (NaCl), 2.4 ± 0.4 (GH), 2.4 ± 0.3 (Hex10), and 2.5 ± 0.3 (Hex100; mean ± SEM).
Short axis views of the LV were recalled and displayed. Images were selected at end-diastole, end-systole, and short axis LV, and myocardial areas were measured. The mean of three tracings from one to three beats was used depending upon the quality of recorded beats. Area fractional shortening was calculated as (diastolic - systolic)/diastolic LV short axis area.
Stroke volume (SV) was calculated as product of MPA mean velocity time integral (mean of three consecutive pulmonary artery velocity profiles) and corresponding MPA mean area. To minimize measurement errors in longitudinal comparison of SV, we used the baseline estimation of pulmonary artery dimension also at the end of treatment, however adjusted by a coefficient determined by the relative gain in body weight (BW). Multiplying stroke volume by heart rate yielded cardiac output (CO). Total peripheral resistance (TPR) was calculated as mean arterial pressure (MAP)/CO.
Meridional wall stress was determined at 2 weeks according to Mirsky by the formula (38): 1.33 x MAP x (ESA/ESMA) x 103 kdyn/cm2. For the calculation of end-systolic wall stress, we used MAP as an equivalent of end-systolic pressure (39). ESA indicates end-systolic short axis area of the LV cavity, and ESMA indicates LV end-systolic short axis myocardial area.
Blood pressure measurement
An arterial catheter was placed during anesthesia in the aortic
arch via the left carotid artery. MAP was recorded on a Grass model 7D
polygraph (Quincey, MA) via a Statham P 23 DC transducer (Gulton
Statham Transducers, Inc., Costa Mesa, CA). Blood pressure
values were calculated as means of 30 samples taken during 60
sec.
Serum IGF-I
The serum concentration of IGF-I was determined by a
hydrochloric acid-ethanol extraction RIA using authentic IGF-I for
labeling (Nichols Institute Diagnostics, San Juan
Capistrano, CA). The assay was performed according to the
manufacturer’s protocol after centrifugation of precipitated serum
proteins at 4 C followed by neutralization with Tris base and another
centrifugation at 4 C.
Isolation of RNA
RNA was isolated using Trireagent (Sigma, St.
Louis, MO) according to the manufacturer’s protocol. Briefly, tissue
was homogenized in Trireagent using a Polytron (Brinkmann Instruments, Inc., Westbury, NY). After centrifugation,
isopropanol was added to the supernatant, followed by centrifugation.
The RNA pellet was washed with 75% ethanol and then dissolved in
diethylpyrocarbonate water.
IGF-I probe
A sequence corresponding to the base coordinates 267–286 and
366–385 (145 bp) of the IGF-I complementary DNA (40) was subcloned
into the SapI-XhoI sites of pCRII
(Invitrogen, Carlsbad, CA) and subsequently used to
generate sense messenger RNA (mRNA) as synthetic standard and antisense
probe. RNA antisense probes were generated from linearized plasmids
(SapI) using T7 RNA polymerase. Synthetic standard was
generated using XhoI-linearized plasmid and Sp6 RNA
polymerase.
Solution hybridization and ribonuclease (RNase) protection
assays
A solution hybridization assay (41) was used to quantify IGF-I
mRNA in cardiac, liver, and kidney tissue, respectively. The
hybridization was performed at 70 C for 24 h in 0.06 M
NaCl, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, 0.1%
SDS, 0.75 mM dithiothreitol, 25% formamide, and
35S-labeled IGF-I probe. After the addition of
100 µg herring sperm DNA, the samples were treated with 40 µg/ml
RNase A and 2 µg/ml RNase T1
(Sigma). Trichloroacetic acid-precipitated protected
hybrids were then collected on glass-fiber filters (GF/C,
Whatman, Maidstone, UK) and counted in a scintillation
counter. The signal was compared with a standard curve based on known
amounts of synthetic IGF-I mRNA, and results were related to total RNA
content.
To visualize the hybridization of labeled probe to mRNA, a RNase protection assay was performed using RPA II kit 1410 (Ambion, Inc., Austin, TX). Briefly, samples of 40 µg total cellular RNA were hybridized at 45 C overnight with 5 x 105 cpm of the 35S-labeled rat IGF-I mRNA antisense probe, then digested with RNase. The RNA:RNA hybrids were precipitated, resuspended, and separated on a 6% polyacrylamide/8 M urea gel. The signals from protected fragments were visualized on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA), showing a major protected fragment of 145 bp (data not shown).
Statistical analysis
Values are given as the mean ± SEM. Heart and
kidney weights (only when normalized to BW); serum IGF-I; IGF-I mRNA in
liver, kidney, and cardiac tissue; and hemodynamic variables available
only at the end of the experiment were compared using one-way
ANOVA/post-hoc Fisher’s protected least significant
difference test between the MI groups and unpaired t test
between intact controls and MI NaCl rats. When data were available
before and after treatment (i.e. BW and echocardiographic
variables), a one-way ANOVA/post-hoc Fisher’s protected
least significant difference test was performed on the changes in the
four MI groups, but no statistical comparisons were performed with
intact rats.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 1)). Total heart weight/BW was higher in NaCl vs. intact
rats (P < 0.05). There were no significant differences
in kidney weight/BW or LV and RV weight/BW (Table 1). There were no
significant differences in serum IGF-I between NaCl-treated and Hex- or
GH-treated rats, although there was a tendency toward increased values
in the GH group. IGF-I mRNA expression in the liver did not
differ between any of the groups. Cardiac IGF-I mRNA was increased in
NaCl vs. intact rats, but was not further increased by
treatment with GH or Hex (Table 1). Kidney IGF-I mRNA was increased in
the GH group vs. NaCl, whereas IGF-I mRNA expression in both
Hex groups was comparable to expression in the NaCl group and
significantly lower than that in the GH group (Fig. 1B).
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and Table 2)). When
normalized to BW, the effect was still significant for Hex100 with a
borderline P value (P = 0.060) for GH. GH
and Hex100 also increased CO compared with NaCl and Hex10 (Fig. 3). CO/BW was increased by Hex100, with a
borderline significance for GH (P = 0.056). At the end
of the treatment period, TPR was lower in the GH and Hex100 groups
compared with that in NaCl and Hex10 rats (Fig. 4). No improvement of other systolic
parameters by GH/Hex treatment could be detected (Table 2). No
significant effects were seen on diastolic parameters, i.e.
deceleration rate and early vs. late LV filling
velocities ratio (data not shown). Heart rate did not differ
between the groups. MAP was lower in the GH group vs. that
in the NaCl group, but was not significantly changed by Hex treatment,
although there was a tendency toward lower MAP in the Hex100 group.
End-systolic wall stress was increased in all NaCl rats vs.
intact rats, but there were no significant effects of treatment. There
were no effects on LV geometry variables such as ESA and EDA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Only one systolic parameter was significantly affected by Hex and GH treatment, i.e. stroke volume. However, SV as well as its derivatives, CO and TPR, are considered to be important variables in heart failure (42). Moreover, the effects on stroke volume and cardiac output were still present when normalized to BW. The small changes in cardiac dimensions, e.g. ESA and EDA, were not significantly different between treatments and were not sufficient as an explanation of the effect on SV.
The mechanisms behind the hemodynamic effects of GH may be complex. The decrease in MAP/TPR may be a nitric oxide-mediated effect (43), and a reduction of afterload may, in turn, result in an increase in SV. However, there are also studies providing evidence of an effect on cardiac contractility by GH (30, 31); recent findings by Tajima et al. suggest an up-regulation of the sarcoplasmatic Ca2+-adenosine triphosphatase and enhancement of the contractile reserve (32).
Previous studies have shown that Hex has a stimulatory effect on GH secretion (15), and the similarities in hemodynamic profile would indicate that its actions may be mediated through GH. However, no other signs of increased GH activity by Hex was detected in our study, including BW gain and kidney IGF-I mRNA levels, where GH had clear-cut effects. Notably, the GH-releasing effect of Hex has not been studied in this rat MI model, and serum levels of GH in Hex animals are unknown. Thus, Hex may exert its hemodynamic effects through small increases in GH, not sufficient for increasing BW gain and kidney IGF-I mRNA. Moreover, different secretion patterns may evoke differential effects at the organ level.
Recent studies have given support for a direct, GH-independent action of Hex on the myocardium. Locatelli et al. studied the effects of ischemia and reperfusion on cardiac function in hypophysectomized rats and found that Hex administrated before ischemia preserves cardiac performance (34). Bisi et al. reported an increased LV ejection fraction, peaking 30 min after a single iv injection of Hex in normal male subjects, not seen after GH administration despite similar peak values of GH after the two substances (35). Expression of mRNA for the cloned GHS receptor in rat heart has been reported (44), although this has not been found by other groups (45, 46). Ong et al. reported Hex binding to rat cardiac membranes and suggest the existence of a different GHS-R subtype in the myocardium (3, 47). These data on direct cardiac effects are in alignment with our results of systolic effects of Hex without other signs of increased GH activity. Moreover, the existence of GHS/Hex receptors in the vascular endothelium has been suggested, as Hex treatment seems to alter the reactiveness of the aortic ring in hypophysectomized male rats (48). These new findings could also be in accordance with the decrease in TPR by Hex100 treatment in our study.
Our finding of increased BW gain in GH-treated rats is in line with
previous observations (26, 27), and increased IGF-I mRNA expression in
kidney gives further support for peripheral effects by GH. As the liver
is the major source of circulating IGF-I (49), the effects on liver
IGF-I mRNA are closely linked to serum IGF-I. We found no significant
effect of GH (or Hex) on serum or liver IGF-I in MI rats. A possible
explanation may be increased cytokine activity, e.g. tumor
necrosis factor-
, observed in heart failure (50), which may cause a
reduction in the liver production of IGF-I (51). Local expression of
IGF-I mRNA in the heart was increased after MI as previously
demonstrated (28), but GH (and Hex) failed to increase it further when
studied at this single time point 6 weeks after MI, which is consistent
with previous results from our laboratory (28). Thus, although the
activation of specific transcripts or regional differences in IGF-I
expression not have been excluded here, activation of cardiac IGF-I
expression appears to be of minor importance for both GH and Hex
effects on hemodynamics after MI.
In contrast to GH, Hex-treated animals did not gain more weight than controls, suggesting the absence of anabolic effects mediated by GH. Other groups have also reported no effect of Hex on BW gain in male rats (33). The lack of Hex effect on serum IGF-I is in accordance with several previous studies of administration of Hex or GHRP-6 to animals and humans, with different routes of administration (18, 33, 52, 53, 54). However, data are conflicting, and increased levels of serum IGF-I after Hex/GHRP-6 treatment have also been reported in some studies (15, 17, 55, 56, 57).
There are now several studies of the cardiac and peripheral hemodynamic effects of GH and/or IGF-I, and among them, promising clinical data pointing to a future role of GH/IGF-I in cardiovascular therapy (24). GHS have the ability to amplify the physiological pulsatile GH secretion, and several of the GHS are orally bioavailable, in contrast to GH (13). In the current study we report that Hex improves variables of in vivo cardiac function, comparable to GH. Further investigation of the cardiovascular effects of Hex and other GHS is mandatory and should include studies of the direct action of GHS on the myocardium.
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Acknowledgments |
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Footnotes |
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Received July 21, 1999.
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