Aging has been shown to alter the spectrum of physiological and biochemical properties of the myocardium, including force production, excitation-contraction coupling, substrate use, and mitochondrial oxidative capacity (1). However, new insights into myocardial-reperfusion injury indicate that aged rats, besides a reduction of the myocardial antioxidant defense mechanisms (2), are affected by alteration of calcium handling in cardiac cells (3). In fact, abnormalities of regulation/modulation mechanisms normally involved in the restriction of calcium oscillation between sarcoplasmic reticulum and cytoplasm are associated with strong impairment of cardiac mechanics.
Recently it was shown (4) that growth hormone (GH) deficiency, induced experimentally in young male rats, is responsible for a marked aggravation of the ischemic damage in hearts subjected to global flow limitation and reperfusion. This aggravation, characterized by a significant increase of ventricular contracture during ischemia and an impaired contractility at reperfusion, is likely related to a defective function of GH/insulin-like growth factor-I (IGF-I) axis. In fact, it was completely counteracted by a GH-replacement therapy or by administration of hexarelin (5), a novel hexapeptide that is a strong GH secretagogue (GHS; (6,7).
In this vein, experimental and clinical evidence already established the pivotal role GH exerts in cardiac pathophysiology (8). Hypopituitary patients given hormone-replacement therapy, except for any GH substitution, had an increased mortality rate from cardiovascular diseases such as myocardial infarction and cardiac failure (9).
These findings and the awareness that GH secretion and its biological effects decline with aging in both experimental animals and humans (10,11) prompted us to investigate the protective action of hexarelin in comparison with that of GH against postischemic myocardial dysfunction in hearts from in vivo-treated senescent rats. Although strengthening of the somatotropic function would be instrumental in the antiischemic activity of hexarelin in aged rats, a direct action on the heart of the hexapeptide cannot be ruled out a priori. Favoring this view, previous findings of our laboratory demonstrated that hexarelin given to young male rats was very effective in the cardiac ischemia-reperfusion model, despite the lack of an overt stimulation of the GH/IGF-I axis (5).
Animals and treatments
Twenty-four-month-old male rats of the Sprague-Dawley strain in good health status, body weight 850 ± 70 g, were purchased (Harlan Nossan, Correzzana, MI, Italy) and housed under controlled conditions (22 ± 2°C; 65% humidity; artificial light from 06.00 to 20.00) with free access to food and water. They were randomly assigned to three experimental groups and treated subcutaneously with (a) 1 ml/kg saline (controls, n = 10); (b) biosynthetic human growth hormone (GH, n = 6); or (c) hexarelin (HEXA, n = 9).
Hexarelin (His-D-2-Me-Trp-Ala-Trp-D-Phe-Lys-NH2) and GH were given to rats at the dose of 80 μg/kg and 400 μg/kg b.i.d., respectively, for 21 days. The dose of hexarelin or GH was chosen on the basis of previous results showing their adequacy to restore somatotropic function in neuroendocrine (12,13) and cardiovascular studies (4). Animals were killed by cervical dislocation 14 h after the last injection. Pituitaries were removed, immediately frozen on dry ice, and stored at −20°C until used for determination of GH messenger RNA (mRNA) levels. Blood was collected into EDTA-containing tubes, and plasma was separated and stored at −20°C for IGF-I determination. The hearts were isolated and used for ischemia and reperfusion experiments.
Pituitary GH mRNA and plasma IGF-I levels
Pituitary GH mRNA levels were determined by Northern blot hybridization technique. Total RNA was isolated from each pituitary by the single-step acid guanidium-phenol-chloroform extraction (14). Total RNA samples (20 μg/sample) were electrophoresed on 1.2% formaldehyde-agarose gel and transferred to nylon membranes (Hybond N; Amersham, Little Chalfont, U.K.). The membranes were hybridized with a rat GH cDNA sequence (13,15) capable significantly to recognize the GH mRNA sequence. The probe was labeled by random primer with [α-32P]deoxycytidine triphosphate (dCTP) to a specific activity of 109 dpm/μg DNA. Hybridization conditions were as previously reported (13,15). Quantification of the hybridization signal was performed on a scanning densitometer (LKB XL Laser Densitometer; LKB, Uppsala, Sweden). Pituitary GH mRNA levels were expressed as a percentage of control values. GH mRNA levels were determined as they represent a reliable index of pituitary somatotropic function.
Plasma IGF-I levels were evaluated by a homologous radioimmunoassay in plasma after acid-ethanol extraction, according to the method described by Daughaday et al. (16). The reagents were provided by the National Hormone and Pituitary Program. The sensitivity of the assay was 100 pg/ml; intra- and interassay variations were <10%. The IGF-I plasma levels of six to 10 rats for each experimental group were determined and expressed in nanograms per milliliter.
Perfused rat heart preparations
Hearts from the three experimental groups of rats were perfused retrogradely at 37°C through the aorta by following a method described by Berti et al. (17). The perfusion medium contained (in mM): NaCl, 118; KCl, 2.8; KH2PO4, 1.2; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25; and glucose, 5.5. After a period of equilibration with 5% CO2 and 95% O2 gas mixture, the pH of the perfusate was 7.35, and the perfusion was maintained at 15 ml/min with a Minipuls3 roller pump (Gilson, Villiers le Bel, France). Left ventricular pressure (LVP) and coronary perfusion pressure (CPP) were recorded by using a HP-1280C pressure transducers (Hewlett-Packard, Waltham, MA, U.S.A.). LVP was obtained by inserting a small latex balloon filled with saline through the left atrium. Left ventricular end-diastolic pressure (LVEDP) was stabilized to 4-5 mm Hg, whereas CPP was maintained at 65-70 mm Hg. All these variables were displayed on a Hewlett-Packard dynograph (HP-7754A). The hearts were electrically paced at the frequency of 300 beats/min with rectangular impulses (1 ms duration, voltage 10% above threshold) by an S-88 Grass stimulator (Grass Instruments, Quincy, MA, U.S.A.). A moderate ischemia was induced by global reduction of the perfusion flow to 1 ml/min for a period of 20 min. A normal flow rate (15 ml/min) was then restored, and reperfusion continued for 30 min. Left ventricular developed pressure (LVDP, peak left ventricular systolic pressure minus LVEDP) was evaluated during reperfusion. At the beginning of each experiment, the activity of angiotensin II (from 0.25 to 4 μg as bolus in the perfusion system) on coronary vasculature was recorded.
6-Keto-PGF1α in heart perfusates
Prostaglandin (PGI2) generation by the cardiac tissues was measured in heart perfusates as 6-keto-PGF1α, according to the enzyme immunoassay method (detection limit, 3 pg/ml) of Pradelles et al. (18). The concentration of this stable metabolite was determined by collecting the perfusates for 5 min immediately before flow reduction and during the first 10 min of reperfusion. The rate of formation of 6-keto-PGF1α was expressed in nanograms per minute.
Creatine kinase in heart perfusates
The perfusate was collected every 150 s in an ice-cooled beaker before flow reduction and during reperfusion, and the activity of creatine kinase (CK) was evaluated according to the method of Bergmeyer et al. (19). The amount of the enzyme was determined on a Lambda16 spectrophotometer (Perkin Elmer Italia, Monza, MI, Italy) and expressed as mU/min/g wet tissue.
Differences of data among groups in individual experiments were analyzed for statistical significance by one-way analysis of variance (ANOVA) and Student's t test (two-tailed) for unpaired samples. A value of p < 0.05 was considered significant. The area under the curve (AUC) was assessed by using a computerized program, Microcal Origin.
The following drugs were used: hexarelin and biosynthetic human growth hormone (Pharmacia, Stockholm, Sweden); angiotensin II (Sigma Chemical Co., St. Louis, MO, U.S.A.); multiprime DNA labeling system (Rediprime; Amersham, Little Chalfont, U.K.); kit for 6-keto-PGF1α determination (Amersham Italia, Milan, Italy.); kit for CK determination (Boehringer-Mannheim Italia, MI, Italy).
Somatotropic functions of old male rats
Treatment of 24-month-old male rats with hexarelin or recombinant human growth hormone (rhGH) did not apparently affect the basal somatotropic function. In fact, pituitary GH mRNA and plasma IGF-I levels, reliable indices of somatotropic function, were in the range of values measured in saline-treated controls. During these treatments, rats did not lose weight nor showed any particular sign of toxicity, and systemic blood pressure and heart rate did not change. The heart weight/body weight ratio was not statistically different in the three experimental groups, indicating that neither hexarelin nor GH treatment had increased the cardiac ventricular mass (Table 1). Absence of either body-weight or heart-weight changes after GH treatment is not surprising because reportedly, the peripheral tissues of aged rats are less sensitive to GH than those of young rats, and a much longer exposure to increased GH doses is needed to elicit these changes in aged rats (20).
Ischemia and reperfusion in isolated rat hearts
The global reduction of flow for 20 min (from 15 ml/min to 1 ml/min) in isovolumic left heart preparations obtained from saline-treated rats induced a clear-cut decrease of left ventricular function associated with a substantial increase in coronary resistance. In fact, the recovery of postischemic LVDP was low and after 30 min of reperfusion, only the 37% of the preischemic strength of heart contractility was restored (from 90 ± 5.7 to 33.5 ± 3.8 mm Hg; p < 0.01); at this time, CPP was still 71% over the basal values (from 68.3 ± 5.2 to 116.8 ± 4.6 mm Hg; p < 0.01), and the event was not associated with stiffness of the hearts (Figs. 1 and 2). Furthermore, the partial functional recovery of the hearts during reperfusion was accompanied by a consistent release of CK into the perfusates. In fact, peak concentration of CK was increased 6.6-fold over preischemic values (from 45 ± 4 to 298 ± 25 mU/min/g wet tissue; p < 0.001) and, at the end of reperfusion, was still significantly increased (182%; p < 0.01; Fig. 3).
In contrast to rat heart preparations obtained from saline-treated control rats, there was a striking protective effect against the reperfusion damage in heart preparations from hexarelin-treated rats (Figs. 1 and 2). In fact, at the beginning of the reperfusion, a regular paced rhythm appeared, and the recovery of postischemic left ventricular function was in the range of 73% of the preischemic strength. After 30 min, LVDP values stabilized at 90% of those recorded during preischemia (from 93 ± 5.8 to 83.7 ± 5.9 mm Hg; p > 0.05; Fig. 2). In these preparations, CPP values increased only minimally in the first 5 min of reperfusion (from 67 ± 5.8 to 79.7 ± 6.9 mm Hg; p > 0.05), and basal values were attained at the end of this period. In keeping with these results, the kinetic profile of CK released in the effluent was significantly different from that observed in control preparations. At the peak of the concentration, CK was increased only twofold (from 45.8 ± 5.5 to 90 ± 7.2 mU/min/g wet tissue; p < 0.05) with a gradual return toward baseline at the end of reperfusion.
A lower protective activity against reperfusion damage was present in heart preparations obtained from GH-treated rats. In these series of experiments, the trend of postischemic left ventricular dysfunction was similar to that present in hearts from control rats, although, at the end of 30 min of reperfusion, the LVDP reached 55% of the preischemic values (from 91.5 ± 6.2 to 50 ± 3.5 mm Hg; p < 0.01; (Fig. 2). Furthermore, CPP was increased of 65% over the basal values at the beginning of reperfusion (from 68 ± 4.3 to 112.2 ± 5.2 mm Hg; p < 0.01) and was still markedly increased after 30 min (46% increase; p < 0.01; Fig. 2). These results were also reflected by a marked increase of CK in the effluent (from 43.8 ± 3.8 to 232 ± 16 mU/min/g wet tissue; p < 0.001), peaking between 8 and 15 min of reperfusion, and still evident at 30 min (109% increase; p < 0.01; Fig. 3).
6-Keto-PGF1α generation in perfused rat hearts and angiotensin II activity
The rate of release of 6-keto-PGF1α in the perfusates of hearts from the three experimental groups was not statistically different (2-2.5 ng/min). As expected, during the first 10 min of reperfusion, the generation of the prostacyclin metabolite increased approximately fivefold (8.5-10 ng/min) in hearts from controls, hexarelin or GH-treated rats (Fig. 4). This would indicate that the beneficial effect exerted by the two peptides in postischemic left ventricular dysfunction was not related to further stimulation of 6-keto-PGF1α formation by the heart tissues. Bolus injections of angiotensin II (0.25-4 μg) into heart preparations at the beginning of each experiment induced a dose-related increase in CPP. The dose-response curves of the vasopressor activity of angiotensin II were not statistically different in the three experimental groups of hearts, thus implying that either hexarelin or GH did not interfere with the endothelium-dependent relaxant function of coronary vasculature (data not shown).
Myocardial ischemia, defined as an imbalance between fractional uptake of oxygen and the rate of cellular oxidation, may have several potential outcomes, especially in senescent hearts that are more prone to this pathologic event. Under these circumstances, when ischemia is brief, a transient postischemic ventricular dysfunction may occur, and this condition reflects many disturbances of cardiomyocytes and insufficient cellular antioxidant activity (2,21). In our model of ischemia-reperfusion, hearts from old rats treated for the long term with hexarelin achieved a strong protection against myocardial stunning. Complete recovery of left ventricular function was present on reperfusion. Simultaneous blunting of CK leakage in the heart effluents bespoke the integrity of myocardial cell membranes and the preservation from the contractile impairment that follows oxygen readmission.
Under our experimental conditions, the beneficial effect disclosed by hexarelin in aged rats was not coupled to any apparent stimulation of the somatotropic function, because the levels of pituitary GH mRNA and plasma IGF-I were unchanged. This would indicate, albeit inferentially, that the hexapeptide had a direct myocardial action divorced from that of GH (see also later). Favoring this view, Grilli et al. (22) and Howard et al. (23) recently reported that mRNA coding for a receptor related to GHS is expressed in peripheral organs of male rats, heart included.
We still ignore what kind of intracellular signal transduction is triggered by GHS-receptor activation in peripheral organs, a point that deserves a thorough investigation. However, the striking hexarelin-induced inhibition of reperfusion damage in the isolated hearts would call for a restraint in the increase of cytosolic calcium that follows reperfusion (24,25). In this context, either the inhibitor of sarcoplasmic reticulum function, ryanodine, or the transsarcolemmal calcium channel blockers, diltiazem and verapamil, were shown capable of improving recovery of LVDP in rabbit hearts exposed to transient ischemia and hypoxia (26,27). However, because hexarelin, given directly to the heart through the perfusion system, does not depress myocardial contractility (authors' unpublished results), the mechanism/s responsible for its antiischemic action may be different from those of calcium entry blockers. In fact, these compounds are known to protect the isolated rabbit hearts against abnormalities produced by transient hypoxia and low-flow ischemia by a strong depression of myocardial contractility ultimately related to the inhibition of calcium entry into the myocardial cells (27,28). It is then possible that sustained administration of hexarelin in aged rats may have increased cardiomyocyte energy stores to an extent compatible with the maintenance of basic cell organization that allows a normal recovery of the contractile function at the termination of the ischemic insult. In this vein, it is noteworthy that the amount of CK released during reperfusion from the heart of hexarelin-treated rats was significantly less than that of control preparations. This may indicate, in the former setting, a better preservation of the integrity of myocardial cell membranes, which is indispensable for a favorable osmotic control (prevention of free radicals accumulation and continuance of calcium homeostasis) and that maintenance of energy-rich phosphates had occurred (28). Our study, however, still lacks data on both the concentration of energy-storing nucleotides and on the grade of density of glycogen granules in ischemic hearts of control and hexarelin-treated rats. These aspects, together with the evaluation of ultrastructural changes of myocardial cells associated with ischemia, deserve further investigations to provide a clearer picture of the mode of action of hexarelin in postischemic ventricular dysfunction.
Another interesting feature of our studies was the protective effect exerted by GH treatment in the heart preparations from senescent rats. The improvement of postischemic ventricular function was, however, modest and by no means comparable, under our experimental conditions, with that elicited by hexarelin, and it is likely attributable to a direct action of the hormone on the heart, where receptors for both GH (29) and IGF-I (30,31) have been identified. Reportedly, the GH-receptor gene is expressed to a greater extent in the myocardium than in any other tissue (29), and in hypophysectomized rats, GH administration induces IGF-I mRNA expression (32) and increases cardiac IGF content (33). IGF-I itself has a positive inotropic effect on the isolated perfused rat hearts (34), and it limits, after myocardial ischemia, the reperfusion damage by inhibiting apoptosis and leukocyte-induced cardiac necrosis (35).
In our study, we also investigated the ability of the cardiac tissues to generate 6-keto-PGF1α, the stable metabolite of prostacyclin, whose increase during the reperfusion period would contribute, with other biochemical events, to limit the reperfusion injury (17,36,37). Our data indicate that either chronic hexarelin or GH treatment failed to increase the production of 6-keto-PGF1α by the cardiac endothelium. These negative findings also are consistent with the inability of either treatment to alter the vasopressor activity of angiotensin II: the dose-response curves of this peptide on CPP during the preischemic period were, in fact, similar in the hearts of the three experimental groups. In whole, these data, in contrast with those obtained in hearts from GH-deficient young-adult rats, where the impairment of endothelial-dependent relaxant function was counteracted by the hormonal treatments (4,5), indicate that the latter in old rats do not improve the ability of the coronary vascular endothelium to modulate the effect of vasoconstrictors (38).
In conclusion, these findings clearly indicate that hexarelin, very likely through a mechanism divorced from its GH-releasing effect, strikingly reduces the reperfusion injury in isolated hearts from senescent rats. The protective effect of hexarelin, which under our experimental conditions overrides that exhibited by GH, opens new perspectives in the therapy of postischemic heart dysfunction in the elderly. This subject is of increasing interest because the aged population is continuously growing and is becoming one of the major targets of pharmacology; moreover, cardiac diseases are the first cause of mortality after age 65 years (39).
Acknowledgment: We are indebted to Dr. Muny Boghen and Dr. Magnus Nilsson of Pharmacia-Upjohn for the fruitful discussion and generous supply of hexarelin and rhGH.
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