Introduction

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Reperfusion of myocardium subjected to a transient period of ischemia rapidly induces severe ventricular arrhythmias (Manning and Hearse, 1984). The main pathogenetic factors of such reperfusion-induced sudden arrhythmogenesis include a burst of oxygen free radical discharge (Woodward and Zakaria, 1985), a Ca²⁺ overload in the intracellular space (Manning and Hearse, 1984; Kihara and Morgan, 1991), and the rapid washout of extracellular protons (Avkiran and Ibuki, 1992). Moreover, myocardial ischemia is associated with the release of massive amounts of noradrenaline within the ischemic myocardium (Schömig et al., 1984, 1987; Obata et al., 1994). This results in excess sympathetic stimulation that not only contributes to the development of ventricular arrhythmias (Akiyama et al., 1991) but also accelerates the development of irreversible cellular damage (Rona, 1985). Following a sufficiently long period of ischemia, reperfusion can enhance the damage to the myocardium with an increase in swelling of the fibers, possible disruption of the sarcolemma, activation of degradative enzymes (such as proteases, endonucleases, and phospholipases), cell death, and enlargement of the necrotic area (Van der Vusse et al., 1994). A large body of evidence indicates that an inflammatory response also plays an important role in the pathophysiology of myocardial ischemia-reperfusion injury (Engler et al., 1986; Lucchesi, 1990).

In both animals and humans, several melanocortin peptides [ACTH-(1-24), -melanocyte-stimulating hormone, and other fragments or fragment analogs of the ACTH molecule] have a prompt and sustained resuscitating effect in conditions of severe tissue hypoxia, either due to hypoperfusion (hemorrhage-induced hypovolemic shock, hypovolemic shock produced through the graded occlusion of the inferior vena cava, cardiogenic shock, splanchnic artery occlusion shock) (Bertolini et al., 1986, 1987; Noera et al., 1989, 1991; Pinelli et al., 1989; Bertolini, 1995; Ludbrook and Ventura, 1995; Squadrito et al., 1999) or to prolonged respiratory arrest (Guarini et al., 1997a). We have previously shown that the hemorrhagic shock reversal produced by the melanocortin peptide ACTH-(1-24), as well as its resuscitating effect in a condition of prolonged respiratory arrest, are associated with a substantial reduction in circulating free radicals. Also nitric oxide level is decreased, by a direct inhibition of inducible nitric-oxide synthase induction at the level of mRNA transcription. Free radicals, including nitric oxide, are massively overproduced in such conditions, suggesting that melanocortins prevent their generation during the phase of recovery, characterized by blood mobilization and subsequent tissue reperfusion (Guarini et al., 1996, 1997b, 1998; Altavilla et al., 2000). Finally, accumulating data indicate that melanocortins have a peculiar anti-inflammatory activity (for reviews, see Lipton and Catania, 1997; Wikberg, 1999). On the whole, the above-mentioned knowledge prompted us to study the possible effect of melanocortins on the consequences of short-term coronary ischemia followed by reperfusion, and on the damage induced by a permanent coronary occlusion.

	Materials and Methods

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Animals and Surgery. Adult Wistar rats of either sex, weighing 280 to 320 g, were used. They were kept in air conditioned colony rooms (temperature 21 ± 1°C; humidity 60%) on a natural light/dark cycle, with food in pellets and tap water available ad libitum. Housing conditions and experimental procedures were in strict accordance with the European Community regulations on the use and care of animals for scientific purposes. The animals were acclimatized to our housing conditions for at least 1 week before use. Rats were anesthetized with urethane (1.25 g/kg i.p.) and fixed in the supine position on a heated operating platform, so to maintain rectal temperature at 37.5°C. Under clean dissection, indwelling catheters were inserted into a common carotid artery and into a femoral vein. The arterial catheter was connected to a Statham P23 Db transducer for the recording of arterial pressure on a polygraph (Mortara-Rangoni, Bologna, Italy); the venous catheter was used for drug administration. Rats were given i.v. heparin (600 IU/kg). After cannulation of the trachea, the animals were ventilated with room air by means of a respirator for small rodents with a stroke volume of approximately 20 ml/kg and a rate of 70 strokes/min. These ventilation parameters maintained arterial pO₂, pCO₂, and pH within the normal range. The lead II ECG was recorded by means of needle electrodes placed s.c. on the limbs. The chest was then opened by a left thoracotomy, the pericardium incised, and the heart gently exteriorized by pressure on the abdomen (Selye et al., 1960). A loose loop [5/0 braided silk suture attached to a 10-mm micropoint reverse cutting needle (Ethicon K-890)] was placed around the left anterior descending coronary artery, close to its origin. A polyethylene tube was threaded over the suture and the heart was replaced in the chest cavity with the ligature ends exteriorized, and any animal in which this procedure itself produced dysrhythmias or a sustained fall in mean arterial pressure to less than 60 mm Hg was discarded from the study at this point. After an equilibration period of 15 min the ligature was tied. In a group of animals, after a 5-min period of coronary occlusion, reperfusion was obtained by cutting the suture according to the device of Manning et al. (1989), and the animals were then monitored for a further 5 min, recording lethality and occurrence of dysrhythmias. In another group of animals, the left coronary artery was permanently ligated, and after the heart had been replaced in the chest cavity, the surgical incision of the chest wall was sutured. Sham-operated animals underwent all the previously described procedures, apart from the fact that the suture passing around the left coronary artery was not tied.

Drugs and Treatments. ACTH-(1-24), NDP-MSH, lidocaine hydrochloride, and heparin sodium were purchased from Sigma (St. Louis, MO); Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) from Aldrich (Milwaukee, WI), and urethane from Fluka AG (Buchs, Switzerland). All drugs were freshly dissolved in saline immediately before use. The i.v., s.c., and i.p. injections were in a volume of 1 ml/kg.

Measurement of Arrhythmias. The ECG was continuously monitored and recorded up to the 5th min after reperfusion. Chart speed was set at 50 mm/s a few seconds before reperfusion so as to obtain a permanent high-speed recording of the changes in the ECG during early reperfusion. The ECG was retrospectively analyzed, in a blinded manner, for the incidence of VT and VF. All analyses were carried out in accordance with the Lambeth Conventions (Walker et al., 1988). VT was defined as four or more consecutive premature beats of ventricular origin, and VF was defined as a signal in which individual QRS deflections could no longer be distinguished from one another and for which the rate could not be determined.

Histology. The rats that were subjected to permanent occlusion of the left coronary artery, and still surviving 72 h after artery ligature, had their hearts removed under urethane anesthesia, washed in isotonic saline, and fixed in 10% neutral buffered formalin for 24 to 48 h. The hearts were then cut from apex to base in slices of 2.5 mm in thickness, according to the step section technique. Histological and histochemical routine procedures were applied to process the heart slices. They were embedded in paraffin blocks, sectioned (5-6 µm in thickness), and collected on glass slides coated with aminoalkylsilane (Dako, Glostrup, Denmark).

Blood Sampling, Extraction of Radical Species, and ESR Spectra Determination. A technique modified from Tortolani et al. (1993) was used to avoid the injection of the spin-trapping agent in vivo. Each animal had 3 to 4 ml of whole blood rapidly withdrawn via the arterial catheter into a syringe containing 2 ml of a 0.1 M solution of -phenyl-N-tert-butylnitrone (PBN; Sigma) in isotonic saline. Each animal served for a single sample. The samples were immediately centrifuged (1680g for 10 min) and the plasma/PBN supernatant was added to 12 ml of 2:1 (v/v) chloroform/methanol for radical extraction. The chloroform layer was separated, dried under nitrogen flow, the resulting pellet was resuspended in 250 µl of chloroform, and the ESR spectrum was taken. ESR spectra were recorded at room temperature using a Bruker 300 ESR spectrometer (Bruker Spectrospin, Karlsruhe, Germany). Typical instrumental settings were as follows: microwave power, 20 mW; modulation amplitude, 0.1 mT; field width, 10 mT; and microwave frequency, 9.14 GHz. The ESR peak height of the central absorption was measured and expressed in arbitrary units (a.u.), as a direct function of adduct concentration; for statistical analysis, the values (a.u.) were normalized to a fixed sample volume of 1 ml of whole blood.

Statistics. Gaussian-distributed variables were expressed as mean ± S.E. and analyzed for statistical significance by Student's t test for unpaired data, or by means of ANOVA followed by Student-Newman-Keuls test. Binomially distributed variables, such as the incidence of VT, VF, or lethality were compared using Fisher's exact probability test. A value of P < 0.05 was considered significant.

Animals Ethics. Experimental procedures were carried out in accordance with the guidelines of the European Community, local laws, and policies (D.L.vo 116/92).

	Results

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Effect of Melanocortins on Ischemia/Reperfusion-Induced Myocardial Injury. As shown in Table 1, in control (saline-treated) rats, coronary reperfusion following 5-min occlusion caused the abrupt (within a few seconds) occurrence of severe ventricular arrhythmias (paroxysmal VT in all rats; VF in 13 of 15 rats), with death of 13 of 15 rats within the first 5 min following reperfusion. The i.v. injection of ACTH-(1-24) 2.5 min before reperfusion (i.e., 2.5 min after coronary occlusion) dose dependently reduced the incidence of arrhythmias and the lethality: survival (within the first 5 min following reperfusion) was 100% starting from the dose of 0.32 mg/kg, and no episode of VF occurred with the dose of 0.48 mg/kg. Lidocaine, i.v. injected at the dose of 5 mg/kg, and used as reference antiarrhythmic compound, was almost as effective as the highest dose of ACTH-(1-24) only when administered 10 min before coronary ligature (VT in four of eight, VF in zero of eight, and survival in 100% of rats; data not shown), and less effective when administered 2.5 min before reperfusion (Table 1). On the other hand, the antioxidant vitamin E analog Trolox, administered for comparison at a dose (0.041 mg/kg i.v.) equimolar (162 nmol/kg) to 0.48 mg/kg ACTH-(1-24), was ineffective (Table 1).

Histological Evaluation of Melanocortin Effects on Myocardial Tissue after a Permanent Coronary Artery Occlusion. The amount of the healthy myocardial tissue (Fu et al., 1993), which was measured 72 h after coronary ligature on immunohistologically stained serial sections by means of an image analyzing computer, was much reduced in saline-treated rats (24.81 ± 1.92; n = 8), as shown in Fig. 1. At the histological examination (Fig. 2A) the ischemic tissue had a transmural appearance extending from endocardium to epicardium. Subendocardial tissue often appeared involved, as a circumferential necrosis. Ischemic areas showed an increase of acid glycosaminoglycan and a decrease of glycoprotein content. A remarkable inflammatory cell accumulation (polymorphonuclear leukocytes and monocytes) was found inside the ischemic tissue. The expression of the cytoplasm apoptotic body (Olivetti et al., 1997) was very high in the injured tissue of saline-treated rats (Fig. 2C).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Amount of volume of healthy myocardial tissue, as estimated 72 h after permanent coronary occlusion (CO) calculated as a percentage of the myocardial volume of the lateral wall of the left ventricle (LWLV), in sham-operated (n = 6), saline-treated (n = 8), and NDP-MSH-treated (0.27 mg/kg s.c. every 12 h; n = 8) rats (mean values ± S.E). *P < 0.001 versus saline-treated rats (Student-Newman-Keuls test).

View larger version (177K): [in this window] [in a new window]   Fig. 2.   Microphotographs of the lateral wall of the left ventricle after 72 h of coronary ligature. Saline-treated rat (A) shows a low amount of healthy myocardium (dark gray; brown in the stained section) and a corresponding high amount of ischemic tissue (). NDP-MSH-treated rat (B) shows few ischemic tissue () in a subepicardium position. Note in C (saline-treated rat) the higher amount of black granules (brown in the stained section), corresponding to the positive cytoplasm expression of apoptosis, and the nuclei (gray; blue in the stained section) of cells without apoptotic bodies. No apoptosis is present in NDP-MSH-treated rat (D); only nuclei of cells without apoptotic bodies (gray; blue in the stained section) are present in D. A and B, anti-actin immunoreaction. C and D, anti-bcl2 (apoptosis) immunoreaction. Harris hematoxylin counterstaining. Field width, 3 mm (A), 3.75 mm (B), 3.86 mm (C and D).

Effect of Melanocortins on Free Radical Levels in the Blood of Rats Subjected to Heart Ischemia/Reperfusion. As shown in Fig. 3, the heart ischemia/reperfusion injury caused a large increase in the blood levels of free radicals (measured using an ex vivo spin-trapping technique) (basal, preischemia level: 62 ± 7 × 10² a.u./ml whole blood; 2 min after reperfusion: 635 ± 28 × 10² a.u./ml whole blood). The 2-min time period was chosen because, starting from 2.5 to 3 min after reperfusion, control rats begun to die. Treatment with ACTH-(1-24) almost completely prevented the ischemia/reperfusion-induced increase in free radical blood levels (98 ± 9 × 10² a.u./ml whole blood). Figure 4 depicts representative ESR spectra. The spectrum obtained during reperfusion in saline-treated rats is characterized by a well defined three line-signal due to the trapping of circulating free radical species; the signal is not well defined in ACTH-(1-24)-treated rats. The spectral feature hints to trapping of multiple radical species (lipids and proteins) arising from the radical burst.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Influence of ACTH-(1-24) (ACTH, 0.48 mg/kg i.v.) on PBN-adduct signal (ESR) intensity in rat blood, 2 min after myocardial reperfusion. Saline, ; ACTH, . Mean values ± S.E. for six animals per group. Treatments with ACTH-(1-24) or saline (1 ml/kg) were performed 2.5 min after left anterior descending coronary ligature (that is, 2.5 min before reperfusion). *P < 0.001 versus saline-treated rats (Student's t test).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Representative ESR spectra of PBN-adduct obtained with 3-ml samples of rat blood. A, basal condition. B, 2 min after myocardial reperfusion in saline-treated rat. C, 2 min after myocardial reperfusion in ACTH-(1-24)-treated rat. Treatments as in Fig. 3. Hyperfine splittings: aN = 14.84 and aH = 3.32.

	Discussion

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Our present results demonstrate that melanocortin peptides are able to exert an effective protection against the outcomes either of a short-term myocardial ischemia followed by reperfusion or of the permanent occlusion of a coronary artery. In the rat model of myocardial ischemia/reperfusion used in our present experiment, which causes a very high incidence of severe ventricular arrhythmias and lethality, ACTH-(1-24) was able to significantly reduce, or to completely prevent, according to the dose, the development of serious arrhythmias, with a consequently high or even complete survival rate. In the rat model of myocardial infarction, produced by the permanent ligature of the left anterior descending coronary artery, the long-acting NDP-MSH {s.c. injected twice daily starting 5 min after coronary artery ligature up to sacrifice (3 days after ligature), at the dose of 0.27 mg/kg [equimolar to 0.48 mg/kg of ACTH-(1-24)]} was able to cause a significant reduction of both infarct size and leukocyte infiltration. The healthy myocardial tissue in the lateral wall of the left ventricle was significantly lower in saline-treated rats (24.81 ± 1.92%, n = 8) than in NDP-MSH-treated rats (69.03 ± 1.39%, n = 8) (P < 0.001). As a consequence, the injured tissue was almost twice as much in saline-treated rats. Moreover, the induced expression of cell apoptotic bodies was very high in saline-treated rats, while it was not detectable in ACTH-(1-24)-treated ones.

Besides being highly effective, melanocortins also were considerably potent in these experimental conditions. Indeed, some signs of the ischemia/reperfusion injury were significantly reduced already by an i.v. dose of 0.16 mg/kg ACTH-(1-24) (significant reduction of lethality), and a virtually complete protection was obtained with an i.v. dose of 0.48 mg/kg (maximum effective dose in our experimental conditions). On the contrary, an equimolar dose of the vitamin E analog Trolox, an extremely efficient antioxidant, had no protective effect. This dose of ACTH-(1-24) also completely prevented the ischemia/reperfusion-induced sharp increase in free radical blood levels.

The pathophysiology of myocardial ischemia/reperfusion injury is complex and not yet fully understood. However, the role of some factors is fairly well established. So, the overproduction of free oxygen radicals must be considered of primary importance in the reperfusion injury (Manning and Hearse, 1984; McCord, 1985; Woodward and Zakaria, 1985). The main sources of these metabolites are 1) the oxidation of hypoxanthine to xanthine and on to uric acid by the oxidase form of xanthine oxidoreductase, and 2) leukocytes accumulating in ischemic and reperfused tissue. Indeed, a classical inflammatory response takes place when heart is subjected to ischemia followed by reperfusion (Lucchesi, 1990).

The possible mechanisms of the protective effect of melanocortins in myocardial ischemia and ischemia/reperfusion most likely involve the capacity of these peptides to inhibit the overproduction of oxygen free radicals (data that have been confirmed also in the present study) in conditions of tissue hypoxia (Guarini et al., 1996, 1998), and their quite peculiar anti-inflammatory activity (for reviews, see Lipton and Catania, 1997; Wikberg, 1999).

Already in previous experiments (Guarini et al., 1996) we produced evidence suggesting that ACTH-(1-24) prevents the burst of free radical generation during tissue reperfusion. This has been confirmed also in the present experimental conditions: the blood levels of free radicals were 635 ± 28 × 10² a.u./ml 2 min after reperfusion in saline-treated rats, and 98 ± 9 × 10² a.u./ml in ACTH-(1-24)-treated rats (basal, preischemia level: 62 ± 7 × 10² a.u./ml). The mechanism by which melanocortins prevent free radical formation cannot be ascribed to a direct radical scavenging activity (Guarini et al., 1996) both because of their chemical structure (a peptide is in se a poor radical scavenger) and because of the extremely low concentrations used. In fact, the vitamin E analog Trolox, an extremely efficient antioxidant, when used at melanocortin equimolar concentration, has no protective activity. Thus, the mechanism(s) responsible of such an effective antioxidant activity of melanocortins remains elusive; a central nervous system involvement could be postulated, on the basis of previous data (Guarini et al., 1999) and of recent findings (Borovikova et al., 2000).

On the other hand, melanocortin peptides have been shown to inhibit the inflammation in many experimental conditions, such as arthritis, brain inflammation/ischemia, and kidney ischemia (for reviews, see Lipton and Catania, 1997; Wikberg, 1999). The anti-inflammatory effects of melanocortin peptides are often associated with a reduced production of proinflammatory cytokines, such as interleukin-1, interleukin-1, interleukin-6, and tumor necrosis factor- (Luger et al., 1997; Lipton et al., 1998), and with an increased production of the anti-inflammatory interleukin-10 and of the angiogenic factor interleukin-8 (Luger et al., 1997). It is possible that other mechanisms of action may be involved in this cardioprotective activity of melanocortins, as is the case for other cardiovascular effects of these peptides, particularly in shock conditions (Bertolini, 1995; Guarini et al., 1999).

In conclusion, our present data demonstrate for the first time an unforeseen property of melanocortin peptides, i.e., their ability to significantly attenuate the consequences either of myocardial ischemia/reperfusion or of permanent coronary occlusion. It must of course be stressed that these results have been obtained in animal models. However, in our opinion they are rather exciting, both because they may disclose a completely new therapeutic approach to these so frequent and serious pathological conditions, and because melanocortins are practically devoid of acute toxicity (particularly cardiotoxicity, contrary to all available antiarrhythmic drugs).