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* Departments of Preclinical and Clinical Pharmacology, University of Florence, 50139, Florence, Italy, and
Internal Medicine, University of Florence, 50134, Florence, Italy
Abstract
Carbon monoxide (CO) is a signaling gas produced intracellularly by heme oxygenase (HO) enzymes using heme as a substrate. During heme breakdown, HO-1 and HO-2 release CO, biliverdin, and Fe2+. In this study, we investigated the effects of manipulation of the HO-1 system in an in vivo model of focal ischemia–reperfusion (FIR) in the rat heart. Male Wistar albino rats, under general anesthesia and artificial ventilation, underwent thoracotomy, the pericardium was opened, and a silk suture was placed around the left descending coronary artery; ischemia was induced by tightening the suture and was monitored for 30 min. Subsequently, the ligature was released to allow reperfusion lasting for 60 min. The first group of rats was sham operated and injected intraperitoneally (ip) with saline. The second group underwent FIR. The third group was treated ip 18 hr before FIR with hemin (4 mg/kg). The fourth group was pretreated ip 24 hr before FIR and 6 hr before hemin with zinc protoporphyrin IX (ZnPP-IX, 50 µg/kg). Specimens of the left ventricle were taken for determination of HO expression and activity, infarct size, malonyldialdehyde (MDA) production, and tissue calcium content. FIR led to a significant increase in the generation of MDA and notably raised tissue calcium levels. Induction of HO-1 by hemin significantly decreased infarct size, incidence of reperfusion arrhythmias, MDA generation, and calcium overload induced by FIR. These effects were prevented by the HO-1 inhibitor ZnPP-IX. The present experiments show that the concerted actions of CO, iron, and biliverdin/bilirubin modulate the FIR-induced myocardial injury.
Key Words: calcium transport • arrhythmia • cell proliferation • ischemia
Carbon monoxide (CO) is a signaling gas produced intracellularly by heme oxygenase (HO) enzymes using heme as a substrate. During heme breakdown, HO-1 and -2 release CO, biliverdin, and Fe2+ (1). We have recently shown that overexpression of HO-1 inhibits cardiac anaphylaxis in the guinea pig through the endogenous generation of CO (2). Moreover, focal ischemia–reperfusion (FIR) in the pig heart leads to a coordinated expression of mRNA encoding HO-1, proposing that the myocardial adaptive response to FIR involves an up-regulation of HO-1 in cardiac cells (3). The HO/CO pathway has a crucial role in the regulation of blood pressure under stress conditions in vivo (4). Upregulation of HO-1 activity in human endothelial cells elicited by gene transfer prevented pyrrolidine–dithyocarbamate mediated abnormalities in DNA distribution and increased the resistance to toxicity produced by exposure to hydrogen peroxide, showing that an increase of HO-1 activity in endothelial cells is a potential therapeutic means of attenuating the effects of free radical induced oxidative stress (5, 6). The aim of this study was to investigate the effects of manipulation of the HO-1 pathway in an in vivo model of FIR in the rat heart.
Materials and Methods
Thirty-two male Wistar albino rats were divided in four groups. The rats were anesthetized by intraperitoneal (ip) injection of pentothal sodium (Abbott, Latina, Italy; 50 mg/kg bw). A cannula was inserted into the trachea and the animals were ventilated with air using a Palmer pump (U. Basile, Comerio, Italy). Subcutaneous peripheral limb electrodes were inserted and an electrocardiogram (ECG) was continuously recorded for the entire duration of the experiment. All rats underwent thoracotomy: the pericardium was opened and a silk suture was placed around the left descending coronary artery together with a small silicone ring to permit the release of the ligature. Ischemia was induced by tightening the suture and was monitored for 30 min. Subsequently, the ligature was released to allow reperfusion lasting for 60 min. The first group of rats was sham operated and injected intraperitoneally (ip) with saline 18 hr before the intervention. The other three groups underwent FIR: the second group was treated ip with saline 18 hr before FIR, the third group was treated ip with hemin (4 mg/kg bw) 18 hr before FIR, and the fourth group was pretreated with zinc protoporphyrin IX (ZnPP-IX, 50 µg/kg bw) ip 6 hr before hemin and 24 hr before FIR. Specimens of the left ventricle were taken for determination of HO expression and activity, malonyldialdehyde production and tissue calcium content.
Western Blot Analysis of HO-1 and -2.
Cardiac samples were homogenized in 1 ml of lysis buffer of the following composition: 50 mmol/l HEPES, 5 mmol/l EDTA, 50 mmol/l NaCl, and 1% Triton X100, pH 7.5, containing complete protease inhibitor (Boehringer, Mannheim). Samples were kept on ice for 1 hr and then centrifuged at 4°C for 30 min at 12,000g. The precipitated insolubilized fraction was discarded and electrophorized on a 12% SDS-polyacrylamide gel using a Bio-Rad system and transferred into a nitrocellulose membrane. The membrane was then probed with polyclonal HO-1 and HO-2 antibodies and visualized as previously reported (2, 4).
Determination of HO Activity.
Cardiac samples were washed, homogenized, and incubated for 30 min at 37°C with 50 µl of rat liver biliverdin reductase necessary to convert biliverdin to bilirubin (7). The level of bilirubin was measured spectrophotometrically using a Sigma diagnostic procedure (Sigma, St. Louis, MO) as previously reported (2).
Determination of Infarct Size.
At the end of reperfusion, or at the moment of cessation of the cardiac activity in the rats that did not survive the predetermined reperfusion period, the chest was re-opened and the hearts quickly removed. The extension of the left ventricular myocardium undergoing damage caused by ischemia–reperfusion was determined by the nitroblue tetrazolium dye exclusion method. The sham-operated hearts from the rats in Group 1 were treated with the same method and were used as negative controls. On removal, the hearts were attached to a Langendorff’s apparatus through a cannula introduced into the aorta and perfused with 10 ml of 1% nitroblue tetrazolium dissolved in a modified Tyrode solution, pH 7.4, at a constant pressure of 40 cm of water at 37°C for 20 min.
Determination of Malonyldíaldehyde (MDA) Production.
MDA levels in cardiac tissues were determined by measurement of the chromogen generated from the reaction of MDA with 2-thiobarbituric acid as described previously (8). Protein concentration was determined according to Bradford assay (9). The values are expressed as nmoles of thiobarbituric acid reactive substances (MDA equivalents) per mg of protein, using a standard curve of 1,1,3,3-tetramethoxypropane.
Calcium Content.
Total calcium content was determined by atomic absorption spectrometry in left ventricular samples. After washing the heart three times for 5 min with a calcium-free buffered solution, 30 mg of tissue was dried and digested overnight at 80°C with 65% HNO3. After acidification with 1 ml of HCl at 32%, the samples were dried at 45°C under nitrogen. LaCl3 was added to provide a final concentration of 1% and CaCl2 was used as a standard (8). The values were expressed as ng of calcium per mg of tissue (dwt).
Substances.
HEPES and EDTA were purchased from Sigma (Milano, Italy); NaCl, KCl, NH4Cl, and KHCO3 were purchased from Merck (Darmstadt, Germany); hemin and zinc protoporphyrin IX were purchased from Aldrich Chemical Co. (Milwaukee, WI), and rat bi liverdin reductase was from StressGen Biotech. Corp. (Canada).
Statistical Analysis.
Data are expressed as mean ± SEM. For ECG analysis, significance of difference between the experimental groups was assayed by the 
2 test. Statistical analysis was performed by either one-way analysis of variance test followed by Student-Newman-Keuls multiple comparison test or by Student t test for unpaired values. Calculations were conducted using a GraphPad Prism 2.0 statistical program (GraphPad Software, San Diego, CA). P < 0.05 was considered significant.
Results
The HO-1 inducer hemin given intraperitoneally 18 hr before FIR significantly increased the HO-1 activity in the area exposed to ischemia and reperfusion (Fig. 1A
). Pretreatment of animals with ZnPP-IX 6 h before hemin abolished the increase in HO activity (Fig. 1A). The hemin-induced increase in HO activity in cardiac samples exposed to ischemia–reperfusion correlated with an increase in the expression of HO-1 protein as shown by Western blot analysis (Fig. 1B). Consistently, the increase in the HO-1 expression was reduced in cardiac samples from animals pretreated with ZnPP-IX before hemin (Fig. 1B). HO-2, which is the constitutive isoform of the protein, was not modified after hemin treatment (Fig. 1B). The results of the morphometry in the ischemic-reperfused hearts stained with nitroblue tetrazolium showed that hemin significantly reduced the extension of the necrotic area (Fig. 2); the protective effect afforded by hemin was completely prevented by pretreatment of the animals with ZnPP-IX (Fig. 2). Examination of ECG recordings from the rats subjected to FIR showed that the incidence of ventricular arrhythmias during reperfusion decreased in hemin-treated animals, with a correspondent increase in the survival time; both effects were abrogated in the group pretreated with ZnPP-IX (Table I). The myocardial damage induced by FIR is conceivably related to the oxidative stress, as shown by the increase in the production of MDA and by calcium overload (Figs. 3 and 4). In sham-operated rats, MDA production and calcium content were negligible. In the ischemic-reperfused hearts, MDA production and tissue calcium content were significantly increased (Figs. 3 and 4). The FIR-induced increase in membrane lipoperoxidation and calcium overload was abated by hemin treatment, and the protective effect of HO-1 induction on FIR-induced damage was prevented in animals pretreated with ZnPP-IX (Figs. 3 and 4), an HO-1 inhibitor.
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The present experiments show that pretreatment of animals with hemin, an HO-1 inducer, provides protection against ischemia–reperfusion damage. In fact, the concerted actions of CO, iron, and biliverdin-bilirubin produced by the activation of HO-1 modulate the free radical-induced IR injury, as shown by the decrease both of the infarct area and of the increase of the markers of oxidative stress (MDA and tissue calcium). The effects are completely antagonized by ZnPP-IX, an HO-1 inhibitor. The treatment of the animals with hemin before FIR also results in a substantial reduction of the occurrence of severe ventricular arrhythmias and a significant increase of the survival time. The myocardial salvage induced by the HO-1/CO system could be attributable to the effect of bilirubin and ferritin, which can function as potent anti-oxidant molecules in vitro and in vivo (7). It is also possible that a codependent activation of soluble guanylyl-cyclase could lead to an increase in cGMP levels and to a decrease in calcium overload (1). We have previously shown in cardiac anaphylaxis in isolated, Langendorff-perfused guinea pig heart, that overexpression of HO-1 inhibits the response to antigen through a cyclic GMP- and a calcium-dependent mechanism (2). However, an upregulation of an antiapoptotic mechanism induced by the increase in cGMP levels could not be excluded (10). Finally, it is possible that the protective effect of hemin pretreatment on ischemia–reperfusion damage may be caused by the release of nitric oxide (NO) (11). The protective effects of NO on cardiac IR have been repeatedly described (12), and similarities between CO and NO have been recently recognized (11, 13). In conclusion, the experiments lend further support to the effect of CO donors as modulators of the ischemia-reperfusion injury.
Footnotes
This work was supported by grants of MURST and University of Florence.
1 To whom requests for reprints should be addressed Università degli Studi di Firenze, Dipartimento di Farmacologia Preclinica e Clinica, "Mario Aiazzi Mancini", Viale G. Pieraccini, 6 50139 Firenze, Italy. E-mail: emanuela.masini{at}unifi.it 
References
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