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Endocrinology Vol. 141, No. 11 3993-3999
Copyright © 2000 by The Endocrine Society

ARTICLES

Pretreatment with Growth Hormone-Releasing Peptide-2 Directly Protects against the Diastolic Dysfunction of Myocardial Stunning in an Isolated, Blood-Perfused Rabbit Heart Model1

Frank Weekers, Eric Van Herck, Jörgen Isgaard and Greet Van den Berghe

Department of Intensive Care Medicine and Center for Experimental Surgery & Anaesthesiology (F.W., G.V.d.B.) and Laboratory for Experimental Medicine and Endocrinology (E.V.H.), Catholic University of Leuven, Leuven, Belgium; Research Center for Endocrinology and Metabolism, Sahlgrenska University Hospital (J.I.), Göteborg, Sweden

Address all correspondence and requests for reprints to: Frank Weekers, M.D., Department of Intensive Care Medicine, University Hospital Gasthuisberg, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium. E-mail: Frank.Weekers{at}uz.kuleuven.ac.be


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Brief coronary occlusion followed by reperfusion leads to reversible myocardial dysfunction (stunning) which can induce irreversible damage of other organ systems. We studied the effects of pretreatment with recombinant human GH (rhGH) and the GH-secretagogue GHRP-2 on myocardial stunning in a blood-perfused isolated rabbit heart model.

In a first set of experiments, effects of bolus rhGH administration (3.5 mg/kg) (n = 5) into the aortic root of unpretreated animals were compared with those of saline (n = 6). In a second set, animals were pretreated for 14 days with SC rhGH 3.5 mg/kg·day (n = 9) or 160 µg/kg·day GHRP-2 (n = 8) in two divided doses. Body weight and plasma concentrations of rhGH, rabbit GH (rGH) and IGF-I were determined before and at the end of 14 days pretreatment. Hearts were excised and submitted to 15 min ischemia followed by 80 min reperfusion, after which postischemic recovery was compared with nonischemic hearts mounted into the same system. At study end, all hearts were snap-frozen to examine markers of apoptosis.

Circulating levels of rabbit GH (rGH) remained identical in all animals. Pretreatment with rhGH for 14 days induced a 142 ± 116% rise of serum IGF-I vs. 8 ± 15% with GHRP-2 (P < 0.001) and increased body weight with 6.8 ± 2.5% vs. 3.4 ± 3.3% with GHRP-2 (P = 0.01).

A bolus injection of rhGH did not alter myocardial function compared with saline allowing data from these experiments to be pooled into one ischemic control group for further analysis of the effect of pretreatment. No difference in postischemic recovery of left ventricular systolic function among the unpretreated, rhGH pretreated and GHRP-2 pretreated hearts was apparent. At the end of reperfusion, a 3-fold higher end-diastolic pressure (EDP) persisted in the unpretreated and rhGH pretreated hearts compared with the nonischemic hearts. In the GHRP-2 pretreated hearts, EDP decreased to half the pressure observed in unpretreated and rhGH pretreated hearts (all P <= 0.02), a level which was indistinguishable from that in the nonischemic hearts, suggesting full postischemic recovery of diastolic function. There were no signs of increased apoptosis in the experimental groups.

In conclusion, 14 days pretreatment with GHRP-2, but not rhGH, protected selectively against the diastolic dysfunction of myocardial stunning in this model. This observation may open perspectives for GH-secretagogues as cardioprotective agents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TEMPORARY MYOCARDIAL ISCHEMIA followed by reperfusion causes reversible cardiac dysfunction known as myocardial stunning (1). This functional impairment in the absence of necrosis occurs after emergency revascularisation (medical or endovascular) as well as in the setting of low flow coronary bypass surgery. Extracorporeal bypass and intermittent aortic cross clamping for coronary surgery inevitably causes temporary coronary hypoperfusion. The ensuing myocardial dysfunction consists of impaired systolic and diastolic function, as evidenced by reduced contractility and relaxation of the heart. In patients with limited myocardial reserve, the myocardial dysfunction—in itself reversible—may lead to shock and multiple organ failure which can be irreversible and potentially lethal. The mechanisms underlying myocardial stunning remain incompletely understood but likely involve oxidative stress, impaired Ca2+-responsiveness of the myofibrils and cytosolic Ca2+ overload, the latter due to impaired uptake of Ca2+ by the sarco-endoplasmatic reticulum (SR) and to reduced Ca2+ Mg2+ATPase (2). Adequate prevention and targeted treatment are still lacking.

There is increasing evidence for an important role of the mediators of the somototrophic axis in regulating myocardial growth and performance. Evidence by and large originates from the now extensive experience with GH replacement in patients with GH deficiency. GH substitution to these patients results in improved systolic function and normalization of the reduced left ventricular mass (3, 4, 5). A number of experimental studies, both in intact rats (6) and in rats with surgically induced myocardial infarction (7, 8, 9, 10), showed that GH and IGF-I, alone or in combination, improve systolic function, even in combination with ACE inhibitors (11). In addition, GH has shown to attenuate remodeling of the left ventricle (LV) in rats after myocardial infarction, by reducing LV dilation and stimulating hypertrophy of the noninfarcted myocardium (12, 13). However, clinical data on beneficial effects of GH treatment in non-GH-deficient patients with congestive heart failure (CHF) remain controversial (14, 15, 16).

GH-releasing hormone (GHRH) of hypothalamic origin is currently considered to be the major endogenous and specific secretagogue for GH (17). Since the early 1980s (18), a series of synthetic peptides (GH-releasing peptides or GHRPs) and nonpeptide agents have been discovered that potently release GH from the pituitary, through a distinct G protein-coupled receptor located in the hypothalamus and the pituitary (19). Ong et al. (20) identified a specific GHRP receptor in the myocardium. In healthy volunteers, bolus administration of hexarelin, a member of the GHRP family, was found to increase left ventricular ejection fraction (LVEF) (21). Moreover, 3 weeks treatment of senescent rats with hexarelin and, to a lesser extent, with GH appeared to protect against the myocardial dysfunction induced by ischemic necrosis (22). Two more recent experimental studies in rats reported beneficial cardiac effects of hexarelin, including protection from ischemia (23) and improved systolic function after myocardial infarction (24). Only very recently, an endogenous ligand (labeled ghrelin) for the GHRP receptor has been isolated from the rat stomach and ghrelin immunoreactive neurons were found to be present in the hypothalamic arcuate nucleus. Furthermore ghrelin was found to circulate in the blood and since receptors for GHRP are ubiquitously present throughout the body, this suggested a systemic role for this peptide (25).

In view of the presence of GHRP receptors in the myocardium and the postreceptor effects of GHRPs on the pituitary GHRP receptor, which involve Ca2+ handling (26), we hypothesized that pretreatment with GHRP-2 might exert a protective effect on myocardial stunning. We therefore investigated the effects of 2 weeks pretreatment with either recombinant human GH (rhGH) or GHRP-2 on true myocardial stunning (reversible dysfunction in the absence of myocardial necrosis) in an isolated blood-perfused rabbit heart model (27).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
We used the isolated, blood-perfused rabbit heart model that has been extensively described elsewhere (27). New Zealand white rabbits were purchased from a local rabbitry and were housed individually for at least 14 days before the experiments. The animals were fed once daily and exposed to 14-h day and 10-h night cycle. All animals were treated according to the "Principles of Laboratory Animal Care" formulated by the U.S. National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the U.S. National Institute of Health. The study protocol was approved by the University of Leuven ethical review board for animal research.

Isolated heart preparation
The hearts were excised through a median sternotomy under general anesthesia with 5 mg/kg piritramide intramuscularly (Dipidolor, 10 mg/ml; Janssen Pharmaceutica, Beerse, Belgium), sodium pentobarbital 20–25 mg/kg (Nembutal 60 mg/ml Sanofi Pharmaceuticals, Inc., Winthrop, NY) and pancuronium (Pavulon 4 mg/2 ml Organon-Technica, Boxtel, The Netherlands) iv. The hearts were immediately immersed into ice cold Ringers (0-4 C) and the aortic root was cannulated. Within 1 min after excision, they were mounted into the perfusing system (modified Langendorff system) as described below and the perfusion was started. The hearts almost immediately resumed active contractions. The left ventricle was vented through a 20G catheter placed through the apical wall. The pulmonary artery was cannulated to drain blood from the sinus coronarius and the right atrial cuff was ligated. Then a fluid-filled latex balloon connected to a pressure transducer (Model 1280C, Hewlett-Packard Co., Waltham, MA) was placed in the left ventricle through the left atrium and mitral valve for pressure recordings. Finally, the heart was paced at 3 Hertz and temperature was monitored by a temperature probe inserted in the right ventricle muscular wall. The latex balloon was filled to a left ventricle end diastolic pressure (LVEDP) of 10 mmHg. Left ventricular systolic pressure (LVSP) and LVEDP were recorded. Coronary flow was continuously recorded (flow probe T206 small animal flowmeter; Transonic Systems Inc., Ithaca, NY) and perfusion pressure was kept constant by assuring constant spilling of the reservoir. The perfusion pressure was also monitored through a side arm of the aortic cannula connected to a pressure transducer.

Perfusing system
Isolated hearts were placed in a blood perfused, support rabbit driven system as described in detail by Chen et al. (27). This system induces a more physiological coronary perfusion compared with other models because it provides whole blood. In brief, the New-Zealand white support rabbits were premedicated with 5 mg/kg piritramide intramuscular. Both ear veins were cannulated. Anesthesia was induced by iv injection of sodium pentobarbital (Nembutal, 20–25 mg/kg). A tracheostomy was performed and the animals were ventilated with a Mark 7 Bird ventilator. Ventilator settings were adapted to keep blood gasses within the physiological range. Continuous iv anesthesia was delivered by piritramide (2 mg/kg·h) and sodium pentobarbital (4 mg/kg·h) infusions via both ear veins. The animal was heparinized by a continuous infusion of 300 IU/kg·h heparin. Then, the femoral artery was cannulated with a 20G catheter for blood pressure monitoring and blood gas analysis. The left carotid artery was cannulated with a 16G catheter for withdrawal of arterial blood. The right internal jugular vein was cannulated with a 14G catheter for return of venous blood from the perfusing system.

Blood was withdrawn from the carotid artery by a roller pump into a overflow reservoir located 85 cm above the isolated heart, thus generating a 70 mmHg perfusion pressure through the coronary arteries. The heart chamber around the isolated heart contained blood drained from the overflow reservoir and blood drained from the sinus coronarius and Thebesian veins of the isolated, contracting heart. From this heart chamber, blood was returned to the support animal over a filter (200- micron blood filter, Baxter SA, Lessines, Belgium). To maintain a myocardial temperature of 37 ± 0.5 C, the overflow reservoir, the heart chamber and all bloodlines were warmed with a water heating system. All tubing consisted of silicon. The heart chamber and the overflow reservoir were of polyethylene. The perfusing system was primed with 60 ml of Ringer’s solution, 40 ml Plasmasterile (Fresenius AG, Bad Homburg, Germany), 3 ml sodium bicarbonate 0.8 M and 1500 IU heparin.

Treatment groups
Thirty three animals were randomly allocated to one of five study groups.

Acute GH effect experiments
Group 1 (rhGH treatment group; n = 6) and group 2 (saline group; n = 5): after isolation of the heart and after mounting it into the perfusion system allowing for 15 min of stabilization, a bolus of 3.5 mg/kg rhGH (Genotropin, Pharmacia & Upjohn, Inc., Stockholm, Sweden) or saline, respectively, was infused intracoronary over 10 min with an infusion pump followed by a restabilization period of 15 min. Then, the flow to the heart was interrupted for 15 min. After 15 min of global ischemia, reperfusion was established and recovery followed for 80 min.

Pretreatment experiments
Group 3 (rhGH pretreated group; n = 9) and group 4 (GHRP-2 pretreated group; n = 8): animals received sc 3.5 mg/kg·day rhGH or 160 µg/kg·day GHRP-2 (Kaken Pharmaceutical Co., Ltd., Tokyo, Japan), respectively, in two divided doses for 14 consecutive days (last dose given on the morning of the experiment) after which they were killed and the hearts isolated and submitted to the same ischemia/reperfusion procedure as described above and observed over the same time window. The dose of 80 µg/kg GHRP-2 was chosen after a dose-response study that revealed the this dose is above the threshold necessary to evoke a significant GH response in healthy rabbits (unpublished data).

No ischemia experiments
Group 5 (nonischemic group; n = 5): untreated animals of which hearts were isolated and mounted into the system without a subsequent ischemic insult, to evaluate the stability of the isolation/perfusion model over the studied time window.

Measurements
The change in body weight over 14 days of treatment was determined in each animal.

Blood was sampled before treatment initiation and after extirpation of the heart (2–6 h after last injection) for determination of serum levels of rabbit GH (rGH), rhGH, and IGF-I. All samples were analyzed within a single assay run. A specific RIA for determination of rGH levels was used (chemicals kindly provided by Dr. A. Parlow, NIH). The detection limit was 1 µg/liter and the within-assay CV was 2.3%. Serum levels of rhGH were determined by RIA as previously described (28). IGF-I was determined by RIA in acid ethanol-extracted plasma using a guinea pig antiserum against human recombinant IGF-I (within assay coefficients of variation was 7.4%).

In the isolated hearts, following hemodynamic parameters were recorded: LVSP, LVEDP, coronary flow and blood pressure of the support animal. All these parameters were recorded on a heatwriting chart recorder (WT-655G; Nihon Kohden Corp., Tokyo, Japan).

At the end of the experiments, all hearts were removed from the Langendorff system and snap-frozen in liquid nitrogen and stored in -70 C for determination of markers of apoptosis.

TUNEL staining
From randomly selected hearts, 10-mm thick sections were cut on cryostat and adhered to microscopic slides. The sections were then fixed in 4% paraformaldehyde in PBS, pH 7.4. Endogenous peroxidase was quenched with preincubation of the sections in 3% H2O2 in PBS for 5 min. The sections were then stained according to the TUNEL method using ApoTag plus peroxidase kit (Oncor) according to the manufacturer’s instructions. Thereafter the sections were counterstained with methyl green and mounted. Sections preincubated with DNase I and sections from ovary of 21-day-old rats served as positive controls (29).

DNA gel electrophoresis
From randomly selected hearts, tissue (4–5 mg) was added to 525 µl lysis buffer (475 ml of TEN: 50 mM Tris pH 8.0, 100 mM EDTA, pH 8.0, 100 mM NaCl pH 8.0, 25 µl 20% SDS, and 25 µl proteinase K 10 mg/ml) and incubated over night at 56 C. The DNA was extracted twice with 525 µl phenol/chloroform. After each extraction, the samples were centrifuged for 10 min at 13,000 rpm. The samples were thereafter precipitated with 2 volumes of 99.5% ethanol and centrifuged for 1 min at 13,000 rpm. The nest of DNA was transferred to 70% ethanol and washed for 1 h. Thereafter, the samples were again centrifuged for 1 min at 13,000 rpm and the ethanol was removed, 15 ml DEPC water was added and the pellet was left to dissolve over night. Ten micrograms of DNA was then run on a 1.8% agarose gel and stained in an ethidium bromide bath for 1 h.

Data analysis
Results are presented as mean ± SD unless indicated otherwise. Hemodynamic changes were always considered relative to the reference value obtained at the end of the preischemic stabilization period or its equivalent time point in the nonischemic controls (set at 100%). Data analysis was performed using one way ANOVA with Fisher’s PLSD post hoc testing for multiple comparisons, by paired and unpaired Student’s t test and Mann Whitney-U test when appropriate. A P value of <0.05 was construed as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Blood pressure recorded in the support animals was identical in all study groups (data not shown).

1. Acute effects of rhGH vs. saline administration
Administration of 3.5 mg/kg rhGH over 15 min directly into the aortic root did not alter any of the measured hemodynamic parameters compared with saline (data not shown). Therefore, the hemodynamic data of both groups were pooled as ischemic controls for the pretreatment experiments.

2. Effects of pretreatment with rhGH and GHRP-2
2.1. Body weight

Pretreatment with rhGH for 14 days induced a mean 6.8 ± 2.5% increase in body weight vs. 3.4 ± 3.3% in the GHRP-2 pretreated group (P = 0.01).

2.2. GH and IGF-I levels (Table 1Go)
After 14 days of rhGH pretreatment, rhGH serum concentration was 2120 ± 1519 µg/liter (range 9–4100 µg/liter), whereas no rhGH was detectable in the GHRP-2 pretreated group. The levels of rGH were statistically identical in both groups (Table 1Go). The incremental change in IGF-I after 14 day treatment was 142 ± 116% in the rhGH pretreated group vs. 8 ± 15% in the GHRP-2 pretreated group (P < 0.001).


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  		Table 1. Serum concentrations of rhGH, rGH, and IGF-I before and after treatment

 
2.3. Hemodynamic measurements (Table 2Go and Fig. 1Go)
2.3.1. Left ventricle systolic pressure (LVSP) (Fig. 1Go, upper panel)


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  		Table 2. Baseline hemodynamic measures in the four experimental groups

 


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  		Figure 1. Recovery of left ventricular systolic pressure (upper panel), left ventricular end diastolic pressure (middle panel), and coronary flow (lower panel) after 15 min ischemia followed by 80 min reperfusion in the four groups. Baseline value is set at 100% and recovery is expressed as percent recovery from baseline. Open circles represent data from the nonischemic group, filled squares from the unpretreated ischemic group, filled triangles from the rhGH pretreated group, and filled diamonds from GHRP-2 pretreated group. All data are expressed as mean ± SEM.

 
In the nonischemic hearts (n = 5), a slight decrease in LVSP to 89 ± 5% occurred over the relevant 95 min after the baseline reference point. In the three ischemic groups, systolic function ceased following onset of ischemia with no developed pressure. Upon reperfusion, and after a short period of fibrillation, all hearts resumed contraction. At the end of the reperfusion period, there was an incomplete recovery of systolic pressure up to 64 ± 9% in the unpretreated hearts (n = 11), to 69 ± 10% in the rhGH pretreated hearts (n = 9) and to 57 ± 7% in the GHRP-2 pretreated hearts (n = 8). Both pretreatment groups behaved no different from the unpretreated group, although at 80 min reperfusion, the difference between the rhGH and GHRP-2 pretreated groups reached statistical significance (P = 0.01).

2.3.2. Left ventricle end-diastolic pressure (LVEDP) (Fig. 1Go, middle panel)
With the onset of ischemia, ischemic contracture developed as indicated by an increase in LVEDP in this isovolumic model. After 15 min of ischemia, LVEDP reached 158 ± 36% of the preischemic value in the unpretreated hearts, 135 ± 22% in the rhGH pretreated hearts and 145 ± 30% in the GHRP-2 pretreated hearts (no difference among the three groups). In the nonischemic hearts, LVEDP was 89 ± 11% at this time point, which was lower than in all three ischemic groups (all P < 0.008).

Upon reperfusion, LVEDP gradually decreased again in all three ischemic groups. At 80 min of reperfusion, LVEDP declined from 158 ± 36% at 15 min of ischemia to 96 ± 28% (P < 0.001) in the unpretreated hearts, from 135 ± 22% to 100 ± 43% (P < 0.0001) in the rhGH pretreated hearts and from 145 ± 30% to 58 ± 33% in the GHRP-2 pretreated hearts (P < 0.0001). One-way ANOVA with Fisher’s PLSD revealed a higher LVEDP in the unpretreated and in the rhGH pretreated hearts than in the nonischemic hearts (P = 0.0016 and 0.0014, respectively) indicating incomplete recovery, whereas in the GHRP-2 pretreated hearts, LVEDP declined further than in the unpretreated (P = 0.02) and in the rhGH pretreatment hearts (P = 0.017) but became indistinguishable from that observed in the nonischemic group, suggesting full recovery of diastolic function exclusively in the GHRP-2 pretreated hearts.

2.3.3. Coronary flow (Fig. 1Go, lower panel)
In the nonischemic hearts there was no significant change in coronary flow over the relevant 95 min. Coronary flow was reduced to zero during ischemia. Upon reperfusion, an initial hyperemia occurred, maximal after 5 min of reperfusion, immediately followed by a decrease in flow toward baseline. Coronary flow at 5' reperfusion was 170 ± 60% in the unpretreated hearts, 177 ± 25% in the rhGH pretreated hearts and 157 ± 41% in the GHRP-2 pretreated hearts (NS as assessed by ANOVA). Coronary flow after 80 min reperfusion was significantly lower in the GHRP-2 pretreated hearts (71 ± 15%) compared with unpretreated hearts (92 ± 17%, P = 0.04) and to the rhGH pretreated hearts (92 ± 30%, P = 0.04).

3. Effects on apoptosis
To determine the presence of apoptosis, low molecular weight DNA was extracted from the hearts and analyzed by agarose gel electrophoresis. Cardiac tissue from unpretreated, rhGH pretreated and GHRP-2 pretreated animals did not show DNA laddering (data not shown). In contrast, ovaries from 21-day-old rats displayed a clear laddering pattern. TUNEL staining of the ventricles showed only occasionally labeled cells, and there was no difference between the different treatment groups (data not shown). In conclusion, there were no signs of increased apoptosis in the different experimental groups.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this isolated, blood-perfused rabbit heart model of stunning, in which brief ischemia and reperfusion induced functional impairment without detectable necrosis or apoptosis, we found 2 weeks pretreatment with the GH-secretagogue GHRP-2, but not GH itself, to be protective selectively against the diastolic component of postischemia-reperfusion myocardial dysfunction. This improvement of diastolic stiffness was independent of coronary blood flow and of serum levels of GH and IGF-I, pointing to a possible direct effect of the GH-secretagogue on cardiomyocytes comparable to that of ischemic preconditioning.

Finding the effect of GHRP-2 on diastolic function to be independent of GH/IGF-I was striking. Indeed, after 2 weeks pretreatment with two daily doses of GHRP-2, serum rGH and IGF-I concentrations were unaltered, which can be explained by intact feedback inhibition loops or desensitization of the somatotropes. Similar observations were previously made with hexarelin treatment in rats (24). However a minor effect on pulsatile GH secretion—which did not evoke a rise in serum IGF-I—could easily have been missed by single sample assessment, a methodological aspect that also explains the large variability in serum rGH before treatment initiation. More importantly, neither acute administration of rGH nor pretreatment with two daily doses of rhGH, which substantially elevated circulating IGF-I, had an effect comparable to that of GHRP-2. If anything, GH pretreatment induced a minor positive effect on systolic function after ischemia and reperfusion. It is long known that GH is necessary to maintain cardiac weight and that GH excess produces myocardial hypertrophy (30). Moreover, GH treatment in healthy volunteers has been shown to increase cardiac output, contractility and fractional shortening without generating an effect on diastolic parameters (31). Patients with GH excess due to acromegaly have an increased myocardial contractility and a low systemic vascular resistance with no change in morphology early in the disease process (32). These effects of GH may be mediated by circulating and/or locally produced IGF-I (33, 34). The weight gain as well as the substantial IGF-I rise selectively observed in the GH-pretreated animals may indicate acromegaly-like effects underlying the observed minor positive effect on systolic function after ischemia and reperfusion in this particular group.

The protection observed with GHRP-2 pretreatment, but not with GH pretreatment, on postischemic diastolic dysfunction points to a direct effect of the GH-secretagogue, as suggested by other authors (22). A specific GHRP receptor, with binding characteristics distinct from those of the pituitary GHRP receptor, has been identified on myocardial membranes (20). Furthermore, an endogenous ligand for the GH secretagogue receptor has been isolated and was found to circulate in human blood (25). This strengthens the hypothesis that GH secretagogues may have a direct effect on the cardiovascular system. The signal transduction systems involved in GHRP-induced activation of the cardiac GHRP receptor have not yet been studied in detail. In the somatotropes, GHRPs first release Ca2+ from its intracellular stores and then cause Ca2+ influx by an increase in membrane Ca2+ permeability, the latter due to depolarization of the somatotrope membrane and to the action of second messengers on Ca2+ channel proteins. The second messenger pathways involved in the pituitary GHRP actions are the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, the protein kinase C (PKC) and the inositol triphosphate (InsP3) pathway (35). It is conceivable that activation of the cardiac GHRP receptor evokes comparable changes in intracellular Ca2+. Bolus administration of hexarelin, another member of the GHRP family, has been found to acutely increase left ventricle ejection fraction in healthy human volunteers (36). This seems to be primarily a systolic effect that has been attributed to an acute release of intracellular Ca2+. In the nonischemic condition, reuptake of intracellular Ca2+ is not impaired and is stimulated by increases in free intracellular Ca2+. Prolonged repetitive GHRP boluses in healthy animals—as in the GHRP-2 pretreatment regimen—conceivably induced adaptive activation of Ca2+ re-uptake pathways which, as in ischemic preconditioning (37), may prevent cytosolic Ca2+ overload in case of subsequent ischemia and reperfusion. Indeed, (late) ischemic preconditioning also selectively improves diastolic stiffness in stunning, an effect which is thought to involve up-regulation of protein kinase C activity (38, 39). A GHRP-2 induced "preconditioning" and modulation of postreceptor pathways could explain the improved recovery of diastolic function in the GHRP-2 pretreated hearts. An alternative explanation for this observation could be activation of glycolyis in cardiomyocytes by GHRP-2 pretreatment, either directly or indirectly, which could limit Ca2+ overload during reperfusion (40).

Both limiting the early reperfusion hyperemia and increasing coronary bloodflow after ischemia have been suggested to protect against postischemic myocardial dysfunction (41, 42, 43, 44). We noted a reduction in coronary bloodflow with GHRP-2 pretreatment exclusively during the late reperfusion phase. It appears unlikely, however, that changes in coronary flow explain our current findings. Indeed, no effect of GHRP-2 pretreatment on the initial peak hyperemia was detected and the GHRP-2 induced improvement of diastolic function preceded the decrease of coronary flow. As observed with ischemic preconditioning, GHRP-2 pretreatment may have improved myocardial efficiency and reduced myocardial hypermetabolism after reperfusion, which in turn could explain the somewhat lower coronary flow in the GHRP-2 pretreated hearts.

In contrast to Rossioni et al. (22), who found a significant protective effect of 3 weeks pretreatment with hexarelin on systolic function, systolic performance was unaltered in our experiments. Differences in age of the animals, species, duration of pretreatment and differences between GHRP compounds used could account for the different outcomes. Furthermore, the absence of apoptosis in our model confirms the findings of Gottlieb et al. (45) that brief ischemia induces no apoptosis and indicates that our investigation took place in a true model of myocardial stunning, which is postischemic dysfunction without concomitant necrosis.

In conclusion, GHRP-2 pretreatment protected selectively against diastolic myocardial dysfunction induced by temporary ischemia and reperfusion in an isolated blood-perfused rabbit heart model of stunning, an effect which was independent of GH and IGF-I. If this novel finding can be confirmed in a clinical study, it may have important implications for GH-secretagogues as future cardioprotective agents for use in low flow bypass surgery.


   Acknowledgments
 
Mr. Dirk De Rijdt (Pharmacia & Upjohn, Inc., Stockholm, Sweden) is acknowledged for generously providing the rhGH (Genotropin).

We thank Prof. Cy Bowers for providing the GHRP-2 and for the valuable discussions, Prof. Willem Flameng for manuscript review, Prof. Johannes Veldhuis and Dr. Parlow for providing the chemicals for rabbit GH measurements, Prof. Roger Bouillon, Ms. Viviane Celis and Susanna Bengtsson for laboratory support, and Prof. Patrick Wouters, Dr. Marco Dewolf, and Mrs. Veerle Leunens for help with the initiation of the model.


   Footnotes
 
1 Presented in part at the Annual Meeting of the Belgian Endocrine Society, Brussels, November 20, 1999 (Young Investigator Award Winning abstract). This work was in part supported by a FUTURA Research Award (Voorzorgskas voor Geneesheren) 1998–1999 (to F.W.), research grants from the University of Leuven (OT 99/32 to G.V.d.B.), the Belgian Fund for Scientific Research (FWO G. 0144.00 & FWO G.3C05.95N) (to G.V.d.B.), and the Belgian Foundation for Research in Congenital Heart Diseases (to G.V.d.B. and F.W.). Back

Received May 4, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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