INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IMPAIRMENT OF MYOCARDIAL FUNCTION and tissue injury after ischemic events is a well-known pathophysiological phenomenon (14). Enhanced oxidative stress by free radicals generated during the reperfusion phase appears to be a major contributor to this effect (20). One approach to ameliorate oxidative cellular damage is the delivery of exogenous antioxidants during and after myocardial ischemia-reperfusion. This strategy may not be sufficient to protect intracellular targets against reactive oxidant species because tissue distribution of exogenous antioxidants is mainly restricted to the interstitial space (3). The increase of cell defenses by inducing endogenous pathways that are selectively engaged in restoring cellular homeostasis represents a valid and complementary alternative. Heat shock proteins (HSPs), or stress proteins, exemplify this concept because of their function as molecular chaperones and their high inducibility in stress conditions (21). HSPs favor the correct folding of newly synthesized proteins, and they are actively involved in repairing denatured proteins or promoting their degradation (12). Studies on cardiac muscle have revealed a direct correlation between elevated expression of the 70-kDa heat shock protein (HSP 70) and improved contractile function after ischemia (19). Recent work (18) has confirmed the ability of HSP 70 to decrease infarct size in hearts of transgenic HSP 70 mouse models.

In contrast with the classic HSPs family that facilitate the rearrangement of cellular components, some inducible stress proteins function as antioxidant enzymes. One of these, heme oxygenase-1 [HO-1 or heat shock protein 32 (HSP 32)], has attracted particular interest because of its unique ability to generate products that might have important biological activities (16). Heme oxygenase is the rate-limiting enzyme in heme degradation to iron, carbon monoxide (CO), and bilirubin (34). HO-1 is the stress-inducible isoform and is very sensitive to upregulation by a variety of different stimuli, such as the substrate heme, endotoxin, various hemoglobins, heavy metal ions, NO donors, and peroxynitrite (2, 7, 8, 10, 16, 22, 29, 30); heme oxygenase-2 (HO-2) is the constitutive isozyme expressed under physiological conditions (16). The possible beneficial effects of HO-1 induction in stress situations are mainly ascribed to the vasoactive gas CO and the antioxidant bilirubin. In analogy to NO, constitutively generated CO by HO-2 has been suggested to act as a neurotransmitter and to participate in the control of vasorelaxation (15, 35, 36). Motterlini et al. (24) have recently shown that increased CO production by HO-1 in vascular tissue contributes to the suppression of acute hypertensive responses under stress conditions in vivo. Similarly, Sammut et al. (31) have reported that HO-1-derived CO significantly suppresses phenylephrine-mediated contraction in isolated aortic rings. The other catabolite of heme degradation, bilirubin, is usually regarded as a potentially cytotoxic waste product when accumulated at abnormally high concentrations in biological tissues. However, Stocker and colleagues (28, 33) elegantly demonstrated that low concentrations of bilirubin are as effective as vitamin E in inhibiting lipid peroxidation in vitro, suggesting that the bile pigment is a physiological, chain-breaking antioxidant. Foresti et al. (10) recently reported that increased bilirubin levels after induction of HO-1 are associated with reduced apoptosis mediated by peroxynitrite in vascular endothelial cells. Despite these promising features, little has been done to elucidate the biological importance of bilirubin in the cardiovascular system. In particular, the inherent cytoprotective characteristics of endogenous bilirubin derived from HO-1 during stress situations have yet to be explored. Here, we investigate the induction of the HO-1-bilirubin pathway in cardiac tissue and its potential involvement in mitigating ischemia-reperfusion injury.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolated heart preparation. Langendorff heart preparations were performed using male Lewis rats (300-350 g) as previously described (26). Hearts were excised, the aorta was cannulated, and retrograde perfusion was established at a constant flow of 15 ml/min using Krebs-Henseleit buffer (in mM: 119 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.66 MgSO₄, 24.9 NaHCO₃, 1.18 KH₂PO₄, 5.55 glucose, 2.00 sodium pyruvate, and 0.5 EGTA) bubbled with 95% O₂-5% CO₂ at 37°C (pH 7.4). Coronary perfusion pressure (CPP) was measured by a pressure transducer (Grass Instruments, Warwick, RI) connected to the aortic cannula. A latex balloon filled with saline was inserted into the left ventricle through the atrium and connected by a catheter to a second pressure transducer. The balloon was inflated to provide an initial end-diastolic pressure (EDP) of 10 mmHg. Both transducers were connected to a computer, and data were acquired with BioPac instrumentation and analyzed with the accompanying AcqKnowledge software (BIOPAC System). Left ventricular developed pressure (LVDP), heart rate (HR), CPP, and EDP were continuously recorded throughout the period of perfusion. Cardiac performance was calculated at each time point from the following formula: (HR × LVDP)/1,000.

Reagents. Hemin (ferriprotoporphyrin IX chloride) and tin protoporphyrin IX dichloride were purchased from Porphyrin Products (Logan, UT). Stock solutions of both porphyrins were prepared by dissolving the compounds in 0.1 M NaOH and then adjusting the pH to 7.4 by the addition of 0.01 M phosphate buffer. Bilirubin was obtained from Fluka Chemicals (Gillingham, Dorset, UK) and solubilized in NaOH before the pH was adjusted to 7.4 with Krebs-Henseleit buffer. 2,3,5-Triphenyl-tetrazolium chloride (tetrazolium red) solution (3% wt/vol) was prepared in Krebs-Henseleit buffer at the end of each experimental protocol just before infusion into the isolated heart.

Experimental protocol. Twenty-four hours before heart isolation and perfusion, animals received an injection of either hemin (75 µmol/kg ip) or vehicle. One hour before excision of the heart was performed, animals received a second injection of either tin protoporphyrin IX (20 or 40 µmol/kg) or vehicle. Isolated hearts were allowed to equilibrate at constant flow for 20 min and were then made globally ischemic by interrupting the buffer perfusion. Hearts were kept at 37°C in the water-jacketed chamber for 30 min and then reperfused. After 60 min of reperfusion, hearts were removed from the aortic cannula and either dissected and fixed for electron microscopy analysis or stained for tissue viability. In an additional set of experiments, hearts from untreated animals were removed and perfused at constant flow for 10 min with oxygenated Krebs buffer containing bilirubin (0.05 or 0.1 µM) and subsequently subjected to ischemia (30 min) and reperfusion (60 min). Considering that hearts were perfused at a flow rate of 15 ml/min for 10 min, the total amount of bilirubin delivered to the organ was 7.5 or 15 nmol when the Krebs buffer contained 0.05 or 0.1 µM bilirubin, respectively. All the hemodynamic parameters were continuously monitored throughout the experimental protocol as reported above. Because both tin protoporphyrin IX and bilirubin are sensitive to light, all the experiments requiring these compounds were performed in the dark.

Heme oxygenase activity assay and Western blot analysis. Heme oxygenase activity and HO-1 protein expression were measured in the myocardial microsomal fraction. Microsomes were prepared as previously described (25). Hearts were perfused in situ with cold saline, removed, and immediately homogenized in 5 vol of 0.05 M Tris · HCl/0.25 M sucrose (pH 7.4). The homogenate was centrifuged at 27,000 g for 10 min, the supernatant was removed and centrifuged again at 105,000 g for 90 min, and the microsomal pellet was resuspended in 0.1 M phosphate buffer/2 mM MgCl₂ (pH 7.4). Heme oxygenase activity was determined in the microsomal fraction (1 mg of protein) by a spectrophotometric assay that measures the formation of bilirubin as a difference in absorbance between 464 and 530 nm (extinction coefficient for bilirubin 40 mM¹ · cm¹) (23). Expression of HO-1 protein was analyzed by Western blot as previously described (8). Briefly, microsomes (60 µg of protein) from each sample were added to a gel loading buffer (0.5 M Tris · HCl, pH 6.8, 10% glycerol, 2% SDS, 2% -mercaptoethanol, and 0.03% bromophenol blue) and boiled for 5 min. Proteins were separated by SDS-PAGE using a 12% acrilamide resolving gel and transferred overnight onto a nitrocellulose membrane. Nonspecific binding of antibodies was blocked with 3% dried milk in 0.01 M phosphate buffer (pH 7.4) for 2 h at room temperature. Membranes were then incubated for 2 h with a polyclonal rabbit anti-HO-1 antibody (StressGen, Victoria, Canada) using a 1:1,000 dilution in phosphate buffer containing 0.1% Tween 20. After being washed, blots were visualized using an amplified alkaline phosphatase kit from Sigma, and the relative density was analyzed using a GS-700 Densitometer (Bio-Rad). HSP 70 protein expression was determined in the myocardial cytosol by Western blotting using specific antibodies (Labvision).

HPLC method. Bilirubin was extracted from myocardial tissue and quantified by HPLC. Because bilirubin is highly sensitive to light, all the extraction procedures were performed in tubes covered with aluminium foil. Briefly, hearts were homogenized in 5 vol of 0.25 M sucrose and 0.05 M Tris · HCl (pH 7.4), mixed with 2 vol of chloroform, and shaken vigorously for 5 min. The chloroform phase containing the extracted bilirubin was separated from the aqueous phase by centrifugation at 1,000 g for 20 min. This extraction step was repeated twice, and the extracts were pooled. The organic phase was transferred to a dry tube and evaporated under a stream of nitrogen gas, and the residue was resuspended in 1 ml of DMSO and stored at 4°C until analysis. All HPLC equipment and software were purchased from Gyncotek (Macclesfield, Cheshire, UK). Chromatographic separations of bilirubin were performed using a Techsphere 5-µm C18 column (150 × 4.6 mm). The eluent used consisted of 40% ammonium acetate (100 mM, pH 5.0)-60% methanol running a linear gradient over 25 min to methanol (100%) at a flow rate of 1 ml/min. Peak identity for bilirubin was confirmed by measuring the retention time, spiking the sample with commercially available standards, and determining absorbance spectra using an ultraviolet photodiode array detector (model VVD-340, Gyncotek) set at 450 nm. Authentic standards of bilirubin were prepared in DMSO.

Electron microscopy and tissue viability. Heart muscle biopsies were fixed in 3% glutaraldehyde (Agar Scientific, Essex, UK) in 0.1 M phosphate buffer for 2 h. After two washes, a second fixation with 1% osmium tetroxide in 0.1 M phosphate buffer was carried out for 1 h at room temperature. The specimens were then dehydrated through an increasing acetone series, infiltrated with acetone-araldite CY-212 resin (1:1) overnight in the specimen rotator, and embedded in araldite CY-212 resin, and blocks were polymerized at 60°C for 18 h. Ultra-thin sections (70-100 nm) were cut, collected on copper grids, and stained with 2% uranyl acetate (10 min) followed by Reynolds lead citrate (10 min). Mitochondrial integrity was analyzed in the stained sections using a JEOL-1200CX electron microscope. The analysis was performed in a blind manner on two sections of the left ventricle per group (~400 mitochondria/section), and a representative picture of mitochondrial integrity for each group is reported. A representative number of hearts from each experimental group (n = 3-4 hearts) were also stained for tissue viability. Hearts were perfused through a side arm of the aortic cannula for 20 min with tetrazolium red (3%) in Krebs-Henseleit buffer at 37°C. After they were stained, hearts were removed and stored in 2% Formalin in the dark before analysis. Hearts were carefully cut into 2-mm-thick sections and scanned into a computer using an AGFA Arcus II scanner, and the total ischemic size was determined by volumetric analysis software (Scion Image, Scion).

Statistical analysis. Statistical analysis was performed by the Student's two-tailed t-test, and an ANOVA was performed when more than two treatments were compared. P values < 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hemin pretreatment induces HO-1 in cardiac tissue. We first examined whether treatment with hemin (75 µmol/kg ip), a substrate and a potent inducer of the heme oxygenase pathway, stimulates the expression of HO-1 in rat hearts. Figure 1, A and B, shows the time course of HO-1 protein expression measured by Western blot. Hemin markedly increased HO-1 expression (fourfold, P < 0.05) in the microsomal fraction of hearts taken from animals 1 day after injection. The HO-1 protein levels gradually returned to control values within 4-5 days after treatment. Induction of HO-1 observed at 1 day after hemin administration was associated with increased heme oxygenase activity (P < 0.05 vs. control); this effect was suppressed by treatment with tin protoporphyrin IX (40 µmol/kg ip), a competitive inhibitor of heme oxygenase enzymes (Fig. 1A, inset). These data indicate that maximal upregulation of the HO-1 pathway occurs in the myocardium at 1 day after treatment of animals with hemin. Therefore, this time point was chosen to examine the possible involvement of the HO-1 pathway in protection against myocardial ischemia-reperfusion injury.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Hemin pretreatment stimulates heme oxygenase-1 (HO-1) induction in cardiac tissue. A: time course of myocardial HO-1 expression at various time points after treatment of animals with an injection of hemin (75 µmol/kg ip). HO-1 protein was measured in microsomal fraction of hearts by Western blot using a polyclonal antibody. Data points are means ± SE; n = 3 hearts). * P < 0.05 vs. time 0. Inset: heme oxygenase (HO) activity (expressed as pmol of bilirubin · mg protein1 · h1) was determined in cardiac tissue 1 day after treatment of animals with vehicle (control), hemin, or hemin plus tin protoporphyrin IX (PP, 40 µmol/kg), an inhibitor of heme oxygenase activity. Data are means ± SE, n = 6 hearts per group. * P < 0.05 vs. control;  P < 0.05 vs. hemin. B: representative Western blots showing HO-1 and HSP 70 protein expression in heart samples at different time points after treatment of animals with hemin.

Hemin pretreatment improves myocardial function after ischemia-reperfusion. To determine the effect of HO-1 induction on the functional recovery of hearts subjected to ischemia-reperfusion, cardiac performance (LVDP × HR) and other hemodynamic parameters were continuously monitored in the Langendorff preparation of hearts from vehicle-treated (control group) and hemin-treated animals. Figure 2 shows the time course of all the parameters measured during reperfusion in the groups examined. For simplicity and clarity, we will refer to the hemodynamic parameters evaluated at 60 min of reperfusion. As shown in Fig. 3A, cardiac performance decreased to 65% of baseline after ischemia-reperfusion in hearts from the control group, whereas hearts removed after hemin treatment showed an improved recovery (81% of baseline, P < 0.05). CPP and EDP were also better maintained in hearts expressing high levels of HO-1 compared with the control group. The increase in CPP over the baseline value (CPP), which is indicative of coronary vessel contractility, was more pronounced in hearts from vehicle-treated animals (41 mmHg) compared with those treated with hemin (14 mmHg) (P < 0.05). A similar pattern was seen for EDP changes (EDP). Interestingly, the use of tin protoporphyrin IX (40 µmol/kg), a potent inhibitor of heme oxygenase activity, completely abolished the postischemic recovery of cardiac function of hemin-treated hearts (Fig. 3A), resulting in reduction of cardiac performance (70% of baseline) and increase in both EDP (31 mmHg) and CPP (33 mmHg). The doses of tin protoporphyrin IX used in our study (20 and 40 µmol/kg) did not have any apparent effect on the functional recovery of postischemic hearts removed from vehicle-treated animals (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Time course of myocardial function during reperfusion after 30 min of ischemia in isolated hearts. Cardiac performance [left ventricular developed pressure (LVDP) × heart rate (HR)] (A), increase in coronary perfusion pressure over baseline (CPP) (B), and change in end-diastolic pressure (EDP) (C) were determined in hearts from animals pretreated with vehicle (), hemin (), or hemin plus PP (). Animals received hemin (75 µmol/kg) or vehicle 1 day before removal of hearts. PP (40 µmol/kg) was given to animals 1 h before heart isolation. Hearts were perfused according to Langendorff technique as described in MATERIALS AND METHODS. Results are means ± SE; n = 6-8. * P < 0.05 vs. vehicle;  P < 0.05 vs. hemin.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Hemin pretreatment improves myocardial function after ischemia-reperfusion (I/R). A: myocardial function [cardiac performance (LVDP × HR), CPP, and EDP] was determined at 60 min of reperfusion after 30 min of ischemia in hearts isolated from animals pretreated with vehicle, hemin (75 µmol/kg), or hemin plus PP. B: effect of PP (20 and 40 µmol/kg) on myocardial function determined at 60 min of reperfusion after 30 min ischemia. Results are means ± SE; n = 6-8 hearts. * P < 0.05 vs. vehicle;  P < 0.05 vs. hemin.

Hemin pretreatment results in increased bilirubin production in the myocardium. Heme oxygenase-mediated heme degradation results in the production of CO and biliverdin. Biliverdin is promptly converted to bilirubin by the cytosolic enzyme biliverdin reductase, an enzyme present in most cell types. We detected a considerable biliverdin reductase activity in both control and hemin-treated hearts (data not shown). The bilirubin IX isomer was measured in cardiac tissue by HPLC as described in MATERIALS AND METHODS. A representative chromatogram and absorbance spectrum of bilirubin extracted from cardiac tissue of heme-treated animals are shown in Fig. 4A. An increase of 23% in bilirubin was measured in hearts from animals treated with hemin compared with vehicle-treated rats (P < 0.05, Fig. 4B). Bilirubin was also measured in the heart perfusate, which was circulated continuously through the isolated organ for 2 h. The rate of bilirubin released into the perfusion buffer from hemin-treated hearts was also markedly increased by 55% (P < 0.05) compared with hearts removed from vehicle-treated animals (Fig. 4C).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Hemin pretreatment results in increased bilirubin production in myocardium. A: representative elution curve from HPLC analysis of myocardial bilirubin extracted from a hemin-treated rat. Chromatograms show peak and absorption spectra at 450 nm typical of bilirubin. B: rate of bilirubin released into the circulating buffer of isolated-perfused hearts. Hearts were removed from animals 1 day after pretreatment with vehicle (control) or hemin (75 µmol/kg) and perfused according to Langendorff technique as described in MATERIALS AND METHODS. After 20 min equilibration, 100 ml of buffer were recirculated through system for 2 h to allow bilirubin released from heart to accumulate in perfusion buffer. Bilirubin was extracted with chloroform at end of perfusion and measured spectrophotometrically as a difference in absorbance between 460 and 530 nm. C: tissue bilirubin content in hearts removed from animals 1 day after treatment with vehicle or hemin (75 µmol/kg). Tissue bilirubin was extracted with chloroform and measured by HPLC as described in MATERIALS AND METHODS. Results are expressed as means ± SE; n = 5-7 hearts. * P < 0.05; ** P < 0.01 vs. vehicle.

Exogenously applied bilirubin increases functional recovery of the myocardium after ischemia-reperfusion. The effect of exogenously applied bilirubin on our ex vivo system was investigated after having demonstrated that upregulation of the heme oxygenase enzymatic pathway in cardiac tissue results in increased bilirubin production and enhanced myocardial function after ischemia-reperfusion. Hearts removed from untreated animals and perfused for 10 min with buffer containing low levels of bilirubin (0.1 µM) before ischemia showed an 87% recovery in cardiac performance at 60 min of reperfusion as opposed to 65% recovery in untreated hearts (P < 0.05, Fig. 5). The increased myocardial performance in hearts pretreated with bilirubin was associated with a significant reduction in CPP (25 vs. 41 mmHg in untreated hearts, P < 0.05) and EDP (19 vs. 44 mmHg in untreated hearts, P < 0.05) at 60 min of reperfusion. These data demonstrate that, at concentrations as low as 0.1 µM, exogenously applied bilirubin partially prevents reperfusion-mediated impairment of myocardial contractility.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Exogenously applied bilirubin increases functional recovery of hearts after I/R. Hearts were removed from untreated animals and perfused according to the Langendorff technique as described in MATERIALS AND METHODS. After 10 min of equilibration, hearts were perfused with Krebs-Henseleit buffer containing 0.05 or 0.1 µM bilirubin for 10 min and then subjected to 30 min of ischemia plus 60 min of reperfusion. Cardiac performance (LVDP × HR) (A), CPP (B), and EDP (C) were measured at the end of reperfusion period and compared with untreated hearts (0 µM). Results are means ± SE; n = 6-7 hearts. * P < 0.05 vs. untreated control.

Protective effect of hemin pretreatment and bilirubin against myocardial ischemia-reperfusion injury. To assess the damage caused by ischemia-reperfusion in cardiac tissue, hearts were stained with tetrazolium red or fixed for electron microscopy analysis at the end of the reperfusion period. The tetrazolium salt stains viable myocardium brick red, whereas the damaged tissue remains unstained and appears white. Figure 6A shows the ischemic volume (infarct size) measured at 60 min of reperfusion in the experimental groups examined as well as a representative heart section from each group. Hearts from vehicle-treated animals that were subjected to ischemia-reperfusion showed a significantly larger infarct size compared with nonischemic, control hearts (11.7 vs. 0.4%, P < 0.05). The infarct size was reduced to 6.3% in hearts from animals-treated with hemin. The protective effect of hemin was abolished by treatment with tin protoporphyrin IX (infarct size = 10.5%), which has been previously shown to inhibit heme oxygenase activity. Perfusion of the hearts with bilirubin (100 nmol) significantly (P < 0.05) reduced the infarct size to 3.9%. Figure 6B shows four electron micrographs of mitochondria from the left ventricular cardiac muscle. Compared with nonischemic tissue (control), mitochondria from hearts subjected to ischemia-reperfusion appeared swollen and the cristae structure was disrupted. However, both heme treatment and exogenous bilirubin perfusion preserved mitochondrial integrity after ischemia-reperfusion.

View larger version (58K): [in this window] [in a new window]   Fig. 6.   Protective effect of hemin pretreatment and exogenously applied bilirubin against myocardial I/R injury. A: hearts isolated from animals pretreated with vehicle, hemin (75 µmol/kg), or hemin plus PP (40 µmol/kg) were perfused according to Langendorff technique and subjected to I/R as reported in Fig. 2. Bilirubin (0.1 µM) was infused in isolated hearts for 10 min before I/R. Ischemic area was determined after I/R using triphenyltetrazolium chloride staining. Control group (Con) is represented by hearts perfused for 20 min at constant flow without I/R. Area of unstained white tissue is indicative of cellular damage and ischemic size was calculated as a percentage of total tissue volume. Results are means ± SE; n = 3-4 hearts. * P < 0.05 vs. control;  P < 0.05 vs. vehicle. B: effect of I/R on integrity of cardiac mitochondria. Representative electron micrographs of hearts from each of treatments described in A are shown. Compared with control hearts, cardiac tissue subjected to I/R resulted in severe injury with swollen mitochondria and disrupted cristae. Pretreatment of animals with hemin or perfusion of isolated hearts with bilirubin showed intact mitochondria with well-preserved double membrane and parallel cristae (bar, 235 nm).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In isolated perfused hearts, we found that reperfusion after 30 min of ischemia caused a significant decrease in myocardial contractility and impairment of hemodynamic function, as well as tissue damage and mitochondrial disruption. Myocardial ischemia-reperfusion is a well-documented pathological condition characterized by depletion of high-energy phosphates, loss of cellular integrity, and, ultimately, irreversible injury and cell death (14). It is also well established that oxidative stress resulting from increased production of reactive oxygen species plays a pivotal role in the progression of cell damage in postischemic tissue (20). Paradoxically, increased resistance to reperfusion injury has been shown to occur when brief ischemic episodes precede a prolonged ischemic insult, a phenomenon termed "preconditioning" (27). Two temporally distinct phases distinguish the cardioprotective effect of ischemic preconditioning: an early phase, which develops immediately after preconditioning and wanes within few hours, and a late phase associated with increased expression of inducible stress proteins or HSPs (17). In several experimental models of myocardial ischemia, cardioprotection directly correlates with the overexpression of either HSP 70, HSP 90, or HSP 60, the most widely studied molecular chaperones in the HSPs family (3). Because diverse stimuli can enhance HSPs levels, the possibility to pharmacologically or genetically manipulate the expression of these proteins to limit ischemic injury is attainable (21).

Our study shows that maximal upregulation of the stress protein HO-1 (or HSP 32), the inducible isoform of heme oxygenase, occurs in cardiac tissue 24 h after treatment of animals with hemin, both a substrate and inducer of HO-1. Interestingly, the recovery of postischemic myocardial function was markedly increased in hearts isolated after treatment of animals with hemin compared with hearts from vehicle-treated rats. Moreover, hearts displaying high HO-1 levels showed a considerably reduced infarct size and preserved mitochondrial integrity after ischemia-reperfusion. These data are indicative of a major role for HO-1 in cardioprotection. Although such a role for HO-1 has not been described in the context of ischemia-reperfusion yet, recent studies highlighted the importance of this stress protein in cardiac xenograft survival and prevention of chronic rejection in heart allografts (11, 32). Our results also exclude the possibility that HSP 70 may play a role in protection against reperfusion injury in our system, because the levels of this HSP remained unchanged after hemin treatment. Pretreatment of animals with hemin could, however, have affected the levels of other HSPs, and at present we cannot rule out the involvement of other chaperones in the observed cardioprotection. Nonetheless, a direct contribution of the heme oxygenase pathway in minimizing reperfusion damage was ascertained in our study by using an inhibitor of its activity, tin protoporphyrin IX. Predictably, blockade of heme oxygenase activity completely abolished the improved postischemic myocardial performance observed after hemin treatment. Likewise, cardiac tissue injury was exacerbated by treatment of animals with tin protoporphyrin IX.

How can heme oxygenase activation provide protection against the cytotoxicity caused by ischemia-reperfusion? Because heme oxygenase is the rate-limiting step in heme degradation, increased HO-1 protein is expected to result in augmented production of its catabolites, bilirubin and CO. Bilirubin is regarded as a waste product but has been known to possess antioxidant properties for a long time (4). This peculiarity of bilirubin has been examined by Stocker and colleagues (33), who showed that physiological levels of the bile pigment suppress the oxidation of lipid membranes more than -tocopherol, which hitherto had been regarded as the best antioxidant against lipid peroxidation. Bilirubin is normally present in plasma as either free bilirubin or bound to serum albumin to form a bilirubin-albumin complex. Interestingly, free bilirubin protects low-density lipoproteins from oxidation more efficiently than albumin-bound bilirubin (28). Motterlini and colleagues (10, 23) also reported that both exogenous and endogenously generated bilirubin can effectively prevent endothelial cell death mediated by hydrogen peroxide and peroxynitrite, respectively. Remarkably, while this paper was in preparation, Dore et al. (6) demonstrated that accumulation of bilirubin due to enhancement of HO-2 catalytic activity by phosphorylation is also protective against hydrogen peroxide-induced cytotoxicity in neuronal cultures. The results of the present study provide strong evidence for a direct involvement of HO-1-derived bilirubin in reducing reperfusion injury. Indeed, consistent with the significant recovery of postischemic myocardial function and preserved tissue viability, we found that hearts displaying high levels of HO-1 and increased heme oxygenase activity exhibited elevated intracellular bilirubin content. In addition, the rate of bilirubin released into the circulating buffer was higher in hearts showing increased HO-1 expression. The notion that bilirubin is most likely a candidate for cardioprotection was further corroborated by our findings showing that bilirubin, exogenously delivered to isolated hearts before ischemia at concentrations as low as 100 nM, significantly restored myocardial function and minimized both infarct size and mitochondrial damage on reperfusion.

Our findings on increased intracellular bilirubin as a consequence of HO-1 induction imply that production of CO, the other catabolite of heme degradation, is also enhanced. CO, as a vasoactive molecule, could be partially responsible for the beneficial effects mediated by activation of the heme oxygenase pathway. Notably, we found that the rise in CPP, a parameter indicative of coronary vessel contractility, was less pronounced in postischemic cardiac tissue expressing high levels of HO-1. These results suggest that increased endogenous CO may play a role in the maintenance of vascular tone during the reperfusion of ischemic hearts. The recent reports of Motterlini and colleagues (24, 31) sustain this hypothesis by demonstrating that HO-1-derived CO is a major regulator of pressor responses in vivo and in vitro.

Our studies delineate a plausible mechanism by which HO-1 protein confers protection against reperfusion injury in the myocardium. The induction of this stress-sensitive enzyme, which is triggered by a range of oxidant-related stimuli, appears to act as a ubiquitous defensive system in a variety of cell types (1). In addition, a fundamental role for heme oxygenases in the maintenance and restoration of cellular homeostasis has been recently postulated (9). The results presented here substantiate this concept by direct assessment of the cardioprotective action of HO-1-derived bilirubin. The possibility that the protection provided by bilirubin in the ischemic myocardium could be physiologically significant is indicated by reports showing an inverse correlation between plasma bilirubin levels and the risk of coronary artery disease (5, 13). Therefore, manipulation of the HO-1 pathway to raise endogenous bilirubin levels may represent a feasible strategy to counteract oxidative stress and, ultimately, may have relevant clinical implications in the prevention of cardiovascular disease.