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(Circulation Research. 2003;93:e2.)
© 2003 American Heart Association, Inc.

UltraRapid Communications

Cardioprotective Actions by a Water-Soluble Carbon Monoxide–Releasing Molecule

James E. Clark, Patrick Naughton, Sandra Shurey, Colin J. Green, Tony R. Johnson, Brian E. Mann, Roberta Foresti, Roberto Motterlini

From the Vascular Biology Unit, Department of Surgical Research (J.E.C., P.N., S.S., C.J.G., R.F., R.M.), Northwick Park Institute for Medical Research, Harrow, Middlesex, UK; Department of Chemistry (T.R.J., B.E.M.), University of Sheffield, Sheffield, UK.

Correspondence to Dr Roberto Motterlini, Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex HA1 3UJ, UK. E-mail r.motterlini{at}imperial.ac.uk

	Abstract

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Carbon monoxide, which is generated in mammals during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator. Transition metal carbonyls have been recently shown to function as carbon monoxide–releasing molecules (CO-RMs) and to elicit distinct pharmacological activities in biological systems. In the present study, we report that a water-soluble form of CO-RM promotes cardioprotection in vitro and in vivo. Specifically, we found that tricarbonylchloro(glycinato)ruthenium(II) (CORM-3) is stable in water at acidic pH but in physiological buffers rapidly liberates CO in solution. Cardiac cells pretreated with CORM-3 (10 to 50 µmol/L) become more resistant to the damage caused by hypoxia-reoxygenation and oxidative stress. In addition, isolated hearts reperfused in the presence of CORM-3 (10 µmol/L) after an ischemic event displayed a significant recovery in myocardial performance and a marked and significant reduction in cardiac muscle damage and infarct size. The cardioprotective effects mediated by CORM-3 in cardiac cells and isolated hearts were totally abolished by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-dependent potassium channels. Predictably, cardioprotection is lost when CORM-3 is replaced by an inactive form (iCORM-3) that is incapable of liberating CO. Using a model of cardiac allograft rejection in mice, we also found that treatment of recipients with CORM-3 but not iCORM-3 considerably prolonged the survival rate of transplanted hearts. These data corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO and highlight the bioactivity and potential therapeutic features of CO-RMs in the mitigation of cardiac dysfunction. The full text of this article is available online at http://www.circresaha.org.

Key Words: transition metal carbonyls • carbon monoxide–releasing molecules • myocardial ischemia • heart transplantation • reperfusion injury

	Introduction

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Mammalian cells constantly generate carbon monoxide (CO) gas via the endogenous degradation of heme by a family of constitutive (HO-2) and inducible (HO-1) heme oxygenase enzymes.^1,2 Firstly described as a putative neural messenger,³ CO is now regarded as a versatile signaling molecule having essential regulatory roles in a variety of physiological and pathophysiological processes that take place within the cardiovascular, nervous, and immune systems. Indeed, CO produced in the vessel wall by heme oxygenase enzymes possesses vasorelaxing properties and has been shown to prevent vasoconstriction and both acute and chronic hypertension through stimulation of soluble guanylate cyclase.^4–10 Endogenous CO appears to modulate sinusoidal tone in the hepatic circulation,¹¹ control the proliferation of vascular smooth muscle cells¹² and suppress the rejection of transplanted hearts.¹³ The biological action of heme oxygenase–derived CO is substantiated by the pharmacological effects observed when this gas is applied exogenously to in vitro and in vivo systems. At concentrations ranging from 10 to 500 ppm, CO gas has been reported to mediate potent antiinflammatory effects,¹⁴ prevent endothelial cell apoptosis,¹⁵ inhibit human airway smooth muscle cell proliferation,¹⁶ and promote protection against hyperoxic as well as ischemic lung injury.^17,18 In view of the pivotal role exerted by the heme oxygenase pathway in the control of cellular homeostasis¹⁹ and the emerging pleiotropic properties attributed to CO,²⁰ it is conceivable that this diatomic gas could be used as a therapeutic tool for the treatment of vascular dysfunction and immuno-related disease states.

At present, three different approaches have been proposed for examining the therapeutic potential of CO: (1) direct administration of CO gas²⁰; (2) use of prodrugs (ie, methylene chloride) that are catabolized by hepatic enzymes to generate CO²¹; and (3) transport and delivery of CO by means of specific CO carriers.²² We have concentrated our efforts on the last strategic approach as we have recently reported that certain transition metal carbonyls possess the ability to liberate CO under appropriate conditions and function as CO-releasing molecules (CO-RMs) in biological systems. In particular, we have shown that CO-RMs induce vessel relaxation in isolated aortic tissue and prevent coronary vasoconstriction as well as acute hypertension in vivo through specific mechanisms that can be simulated by activation of the HO-1/CO pathway.²³ These unprecedented findings prompted us to intensify our studies on the chemical features of carbonyl complexes in an attempt to design novel compounds that have different rates of CO release and are more compatible with biological systems. This necessity is dictated by the fact that all carbonyl-based compounds described to date in the literature are soluble only in organic solvents and that physical (ie, irradiating light) or chemical (ie, steric ligands) stimuli are generally required to favor CO dissociation from these complexes.²² Interestingly, the versatile chemistry of transition metals allows them to be effectively modified by coordinating biological ligands to the metal center in order to render the molecule less toxic and more water soluble and to modulate the release of CO.

In the present study, we show that tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), a newly synthesized water-soluble form of metal carbonyl that liberates CO under physiological conditions, protects myocardial cells and tissues against ischemia-reperfusion injury as well as cardiac allograft rejection, and we report on the possible mechanisms involved in this protection. Our findings emphasize the notion that water-soluble metal carbonyls could be utilized as prototypic chemicals in the development of pharmacologically active compounds capable of delivering CO for therapeutic purposes.

	Materials and Methods

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Synthesis of Tricarbonylchloro(glycinato)ruthenium(II) (CORM-3)
Tricarbonylchloro(glycinato)ruthenium(II) ([Ru(CO)₃Cl(glycinate)] or CORM-3) was synthesized starting from a commercially available compound, tricarbonyldichlororuthenium(II) dimer ([Ru(CO)₃Cl₂]₂) (Sigma Aldrich). Briefly, [Ru(CO)₃Cl₂]₂ (0.129 g) and glycine (0.039 g) were placed under nitrogen in a round-bottomed flask. Methanol (75 mL) and sodium ethoxide (0.034 g) were added and the reaction was allowed to continue under stirring for 18 hours at room temperature. The solvent was then removed under pressure and the yellow residue redissolved in tetrahydrofuran (THF); this was filtered and excess 40 to 60 light petroleum added. The yellow solution was evaporated down to give a pale yellow solid (0.142 g, 96% yield). CORM-3 was stored in closed vials at -20°C and used freshly on the day of the experiments.

Reagents
Stock solutions of CORM-3 (0.11 mol/L) were prepared by solubilizing the compound in distilled water. Inactive CORM-3 (iCORM-3) was prepared by dissolving CORM-3 either in Krebs-Henseleit buffer or in 0.01 mol/L phosphate buffered saline (PBS) and allowing liberation of CO overnight (18 hours) at room temperature. Tricarbonyldichlororuthenium(II) dimer ([Ru(CO)₃Cl₂]₂), 5-hydroxydecanoic acid sodium salt (5-HD), 2,3,5-triphenyltetrazolium chloride (tetrazolium red), and all the other reagents were purchased from Sigma unless specified otherwise. Tetrazolium red solution (3% wt/vol) was prepared fresh in Krebs-Henseleit buffer before use.

Detection of CO Release
The release of CO from CORM-3 or iCORM-3 was assessed spectrophotometrically by measuring the conversion of deoxymyoglobin (deoxy-Mb) to carbonmonoxy myoglobin (MbCO) as previously described.²³

In Vitro Models of Hypoxia-Reoxygenation and Oxidative Stress
Rat H9c2 cardiac cells were purchased from the American Tissue Culture Collection (Manassas, Va) and cultured as previously described.²⁴ In experiments conducted under hypoxic conditions (95% N₂-5% CO₂, PO₂=2 mm Hg), cardiomyocytes at confluence were incubated with medium supplemented with 0.5% FBS and various concentrations of CORM-3 or iCORM-3 (10, 25 or 50 µmol/L) in an air tight chamber.²⁴ Additional experiments were performed by exposing cells to hypoxia in the presence of CORM-3 and 50 µmol/L 5-hydroxydecanoic acid (5-HD), a mitochondrial ATP-dependent potassium (mitoK_ATP) channel inhibitor. After 24-hour hypoxia, cardiomyocytes were returned to normoxic conditions (5% CO₂-95% air, PO₂=159 mm Hg), and cell viability assessed after 6-hour reoxygenation as previously described.²⁴ Cells were also exposed to hypoxia for 24 hours and reoxygenated for 6 hours in the presence of CORM-3. In a parallel model of oxidative stress, cardiac cells were treated with paraquat, a superoxide anion generator, in the absence or presence of CORM-3 and cell viability determined 24 hours after treatment.

Isolated Heart Preparation and Ischemia-Reperfusion Model
Isolated hearts from male Lewis rats (300 to 350 g) were perfused according to the Langendorff technique at constant flow as previously described by our group.²⁵ Coronary perfusion pressure (CPP), end-diastolic pressure, left ventricular developed pressure (LVDP), heart rate (HR), maximal contraction (+dP/dt), and relaxation (-dP/dt) rates were continuously recorded throughout the period of perfusion using PowerLab (AD Instruments). Isolated hearts were equilibrated for 20 minutes, made globally ischemic for 30 minutes, and then reperfused for 60 minutes. Krebs buffer was collected for 10 minutes from the pulmonary artery before the ischemic event and in the last 10 minutes of reperfusion for creatine kinase (CK) analysis. At the end of reperfusion, hearts were stained to assess tissue viability using tetrazolium red. In additional experiments, ischemic hearts were treated for the first 10 minutes of reperfusion with CORM-3 or iCORM-3 (10 µmol/L). To assess a possible role of mitoK_ATP channels in cardioprotection, control hearts or hearts receiving CORM-3 were pretreated for 10 minutes before ischemia with 5-HD (50 µmol/L), a specific blocker of mitoK_ATP.

Determination of Infarct Size and Cardiac Muscle Damage
Hearts were stained for tissue viability using tetrazolium red at the end of the reperfusion period as previously described.²⁵ Cardiac muscle damage was assessed by measuring the release of creatine kinase (CK) into the perfusate using a commercially available spectrophotometric assay kit (DG147-A) from Sigma Diagnostic.

Cardiac Transplant Rejection Model
Hearts from male BALB/c mice (25 to 30 g) were used as donor organs for transplantation into male CBA mice (25 to 30 g). Animals were anesthetized by an intraperitoneal injection of ketamine/xylazine during all procedures. The surgical technique involved the transplantation of the cardiac allograft into the recipient’s neck as previously described.¹³ Graft survival was assessed daily by palpation, and rejection was diagnosed by cessation of ventricular contractions. CORM-3 was freshly prepared in 0.1 mL saline and administered immediately into the peritoneum at a dose of 40 mg/kg. The donors received one dose of CORM-3 (or iCORM-3), respectively, at 1 day and 15 minutes before cardiac harvest. The recipients received one dose of CORM-3 (or iCORM-3) at 1 day before surgery, 30 minutes before cardiac reperfusion, and 1 hour after transplantation (Day 0). Thereafter, graft recipients received a daily dose of CORM-3 (or iCORM-3) from day 1 to day 8 after transplant. In the control group, recipients received an equivalent dose of saline (vehicle) 1 day before and each day (days 1 to 8) after cardiac transplantation.

Statistical Analysis
Statistical analysis was performed by the Student’s two-tailed t test, and an analysis of variance (ANOVA) was performed where more than two treatments were compared. Values of P<0.05 were considered statistically significant.

	Results

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Comparison Between CORM-3 and iCORM-3 in Their Ability to Release CO
CORM-3 (see chemical structure in Figure 1A) prepared in distilled water gives a pH of 3.0. When an aliquot of this solution was added to PBS (pH 7.4) containing Mb (66 µmol/L), a spectrum characteristic of MbCO was rapidly detected (Figure 1B). The amount of MbCO measured after this reaction revealed that 1 mole of CO is liberated per mole of CORM-3. Specifically, as shown in Figure 1C, addition of 40 µmol/L CORM-3 resulted in the formation of 36.4±0.9 µmol/L MbCO. When dissolved in water and left for 24 hours at room temperature, CORM-3 retained its full ability to liberate CO (data not shown). In contrast, CORM-3 prepared in Krebs buffer (pH 7.4) left overnight at room temperature became inactive and failed to convert deoxy-Mb to MbCO. As shown in Figures 1B and 1C, inactive CORM-3 (iCORM-3) added to the Mb solution did not liberate any detectable CO. Infrared spectroscopy suggests that iCORM-3 is a dicarbonyl species, consistent with the loss of one molecule of CO in its formation from CORM-3 (data not shown). These data reveal that CORM-3 prepared in water is relatively stable and that physiological buffers such as Krebs favor the release of CO from this metal carbonyl complex. The inactive form of CORM-3 (iCORM-3) also provided us with a negative control to assess the direct involvement of CO in the pharmacological activity mediated by CO-RMs.

View larger version (17K): [in this window] [in a new window]   Figure 1. Release of CO from CORM-3 in aqueous solutions. A, Chemical structure of [Ru(CO)3Cl(glycinate)] (CORM-3). B, Detection of MbCO and deoxy-Mb spectra after addition of 40 µmol/L CORM-3 or iCORM-3 to a PBS solution containing myoglobin (66 µmol/L). CORM-3 but not iCORM-3 promotes the formation of MbCO. C, Amount of MbCO formed 10 minutes after interaction of 40 µmol/L CORM-3 or iCORM-3 with myoglobin (66 µmol/L).

CORM-3 Protects Against Hypoxia-Reoxygenation and Oxidative Stress in Cardiac Cells
CORM-3 at a concentration ranging between 10 and 50 µmol/L did not cause any change in cell viability (data not shown). H9c2 cells exposed to hypoxia for 24 hours and reoxygenated for 6 hours exhibited approximately 70% loss in cell viability (Figure 2A). The presence of CORM-3 during the hypoxic period significantly attenuated cell injury at reoxygenation in a concentration-dependent manner. Interestingly, the inactive control, iCORM-3, did not protect cells against hypoxia-reoxygenation damage, implicating CO as a mediator of cytoprotection (Figure 2B). In addition, the cardioprotective effect mediated by CORM-3 was totally abolished by 50 µmol/L 5-HD, an inhibitor of mitoK_ATP channels (Figures 2C). Notably, CORM-3 also preserved cell viability even when present only during the reoxygenation phase after 24-hour hypoxia (Figure 3A). Similarly, CORM-3 markedly protected cardiac cells against injury caused by increasing concentrations of paraquat, a superoxide anion generator used to promote oxidative stress (Figure 3B).

View larger version (21K): [in this window] [in a new window]   Figure 2. Cytoprotective effects of CORM-3 against hypoxia-reoxygenation (H/R) in cardiomyocytes. Cardiomyocytes were exposed to hypoxia for 24 hours in the presence of increasing concentrations (10 to 50 µmol/L) of CORM-3 (A), iCORM-3 (B), or CORM-3 plus 50 µmol/L 5-HD (C), an inhibitor of mitoKATP potassium channels. Cells were then reoxygenated and cell viability determined after 6 hours. Control cells were incubated in complete medium under normoxic conditions for 30 hours. Data are expressed as the mean±SEM of 6 independent experiments. *P<0.01 vs control (CON); P<0.01 vs 0 µmol/L CORM-3.

View larger version (35K): [in this window] [in a new window]   Figure 3. Protective effects of CORM-3 against oxidative stress. A, Cell viability assessed in cardiomyocytes exposed to hypoxia for 24 hours and then reoxygenated for 6 hours in the presence of increasing concentrations (10 to 50 µmol/L) of CORM-3. B, Cells were exposed to increasing concentrations of paraquat (0.25 to 1 mmol/L) in the presence or absence of CORM-3 and cell viability determined after 24-hour incubation. Data are expressed as the mean±SEM of 6 independent experiments. *P<0.01 vs control (CON); P<0.01 vs 0 µmol/L CORM-3.

Cardioprotective Effects of CORM-3 Against Myocardial Ischemia-Reperfusion Injury and Possible Involvement of mitoK_ATP Channels
Hemodynamic, biochemical, and histological parameters were measured to assess the potential beneficial effects of CORM-3 on the functional recovery of hearts subjected to ischemia-reperfusion. As shown in Figure 4, control hearts displayed a 34% decrease in LVDP compared with baseline, whereas hearts reperfused in the presence of CORM-3 showed a 44% increase in this parameter (P<0.05; Figure 4A). This positive inotropic effect mediated by CORM-3 was also evident when analyzing the maximal rate of contraction (+dP/dt) and relaxation (-dP/dt) in postischemic hearts (see Figures 4B and 4C). CORM-3 was also capable of preventing the increases in EDP and CPP that are typical of postischemic myocardial dysfunction in this model. As shown in Figures 5A and 5B, control hearts showed an increase of 36.9±8.4 mm Hg in EDP and 31.6±8.8 mm Hg in CPP at the end of reperfusion, whereas CORM-3 significantly attenuated these effects (3±1.8 and 13±2.2 mm Hg for EDP and CPP, respectively; P<0.05). Creatine kinase (CK) activity, an index of cardiac tissue injury, was elevated in the buffer of reperfused control hearts but the activity was significantly attenuated in the presence of CORM-3 (P<0.05) (see Figure 6A). Similarly, the infarct size, measured at the end of the reperfusion, was significantly reduced in hearts treated with CORM-3 (2.3±0.6%) compared with control (9.5±2.1%; P<0.05) (Figures 6B and 6C). Predictably, iCORM-3, which is incapable of releasing CO, did not promote any protective effect on the hemodynamic, biochemical, and histological parameters measured (see Figures 4 through 6 ). In addition, the cardioprotective action of CORM-3 was significantly attenuated by the presence of 5-HD (see Figures 4 through 6). Collectively, these data demonstrate that CO is directly responsible for the myocardial recovery of ischemic hearts and suggest the involvement of mitoK_ATP channel activation in the cardioprotective actions mediated by CORM-3.

View larger version (34K): [in this window] [in a new window]   Figure 4. Preservation of myocardial contractility by CORM-3 after ischemia-reperfusion. Isolated rat hearts were subjected to 30 minutes of global ischemia and then reperfused for 60 minutes in the absence (CON) or presence of CORM-3 (10 µmol/L), iCORM-3 (10 µmol/L), 5-HD (50 µmol/L), or a combination of CORM-3 plus 5-HD. Left ventricular developed pressure (LVDP; A), maximal contraction (+dP/dt; B), and relaxation (-dP/dt; C) were continuously recorded during the reperfusion phase. Data are presented as the mean±SEM of 6 independent experiments. BAS indicates baseline value; *P<0.01 vs control (CON).

View larger version (33K): [in this window] [in a new window]   Figure 5. Preservation of coronary and end-diastolic pressures by CORM-3 after myocardial ischemia-reperfusion. Isolated rat hearts were treated as described in Figure 4 and coronary perfusion pressure (CPP; B) and end-diastolic pressure (EDP; A) were continuously recorded during the reperfusion phase. Data are presented as the mean±SEM of 6 independent experiments. BAS indicates baseline value; *P<0.01 vs control (CON).

View larger version (34K): [in this window] [in a new window]   Figure 6. Cardiac protection by CORM-3 against ischemia-reperfusion injury. Isolated rat hearts were treated as described in Figure 4. Cardiac muscle damage was assessed by measuring the release of creatine kinase (CK) into the perfusate after 60 minutes of reperfusion (A) and by quantifying the infarct size using tetrazolium red (B and C). Data are presented as the mean±SEM of 6 independent experiments. *P<0.01 vs control (CON).

CORM-3 Prevents Cardiac Allograft Rejection in Mice
To verify the pharmacological action of CORM-3 in vivo, we tested the effect of this transition metal carbonyl in a model of cardiac allograft rejection. The results of this study are shown in Figure 7. BALB/c hearts transplanted into CBA mice after treatment with saline (control group) underwent rejection very rapidly as all organs stopped beating within 9 days of transplantation. In contrast, the survival time of hearts transplanted into mice receiving CORM-3 was significantly prolonged (P<0.002) and all hearts were still beating 18 days after transplantation. At 25 days after heart transplantation, 60% of mice treated with CORM-3 did not show any sign of rejection (P<0.002), and at 30 days, 40% of the transplanted hearts were still viable. The inactive compound (iCORM-3) also showed some degree of protection against cardiac rejection but was significantly less effective than CORM-3 (P<0.01). These data demonstrate that CORM-3 prolongs the survival of murine cardiac grafts and attenuates organ rejection.

View larger version (15K): [in this window] [in a new window]   Figure 7. CORM-3 protects against cardiac allograft rejection. Hearts from BALB/c mice were transplanted into CBA mice after treatment with saline (control), CORM-3 (40 mg/kg), or iCORM-3 (40 mg/kg), and the survival rate assessed. Details on the administration of the drugs are reported in Materials and Methods. Treatment with CORM-3 is significantly different from control to P<0.001. Data are expressed as the mean±SEM of 6 independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transition metal carbonyls are a class of compounds that were discovered more than 100 years ago and were initially used as unique catalysts in the purification of metals such as nickel.²⁶ Until recently, the use of carbonyls and other metal-carbon complexes in medicine was unimaginable because most organometallic compounds are inherently sensitive to water and oxygen and, consequently, were thought to be incompatible with biological systems. However, in the last decade, remarkable studies have highlighted the potential of using newly synthesized metal-carbon complexes in various fields of research including cell biology, immunology, and pharmacology. Their application in cancer therapy, drug-receptor interaction, and malaria is so promising to the extent that "bio-organometallic chemistry" has now become an emerging discipline that may offer innovative solutions to biological problems.^27–30 Our group recently corroborated this view in finding that two metal carbonyls, dimanganese decacarbonyl and tricarbonyldichlororuthenium (II) dimer, modulate vessel relaxation and acute hypertension by liberating CO in vitro and in vivo.²³ These data not only provided the first direct evidence that transition metal carbonyls have the ability to function as CO-releasing molecules (CO-RMs) in biological systems, but also emphasized their use as important tools to better identify intracellular targets of CO and elucidate its mechanism(s) of action. Notably, due to the ubiquitous nature of endogenous CO and the increasing evidence supporting a crucial role for CO gas in physiology and disease states,²⁰ the possibility that CO-RMs could be utilized in the future for the therapeutic delivery of CO in disparate medical applications is a tantalizing hypothesis. However, despite the vast number of metal carbonyls described in the literature, only a few meet the criteria of being water soluble or containing biologically safe ligands.

In the present study, we provide the first example of a water-soluble metal carbonyl complex that liberates CO in in vitro, ex vivo, and in vivo biological models. Tricarbonylchloro(glycinato)ruthenium(II) (CORM-3) has been specifically designed to render the compound soluble in aqueous solutions by coordinating a biologically compatible ligand (glycine) onto the metal center. (Note: The water-soluble [Ru(CO)₃Cl(glycinate)] is the third in the list of transition metal carbonyls used by our group as CO-RMs after the initial identification of dimanganese decacarbonyl (CORM-1) and tricarbonyldichlororuthenium (II) dimer (CORM-2).²³) All the chemical features of CORM-3 are being characterized by our group and consistent results already indicate the following^31,32: (1) it is stable in water at 37°C and acidic pH for over 24 hours; (2) it liberates CO in physiological solutions and biological fluids such as PBS, Krebs buffer, cell culture media, and human blood plasma; and (3) in all tested solutions at pH 7.4, one mole equivalent of CO is released quite rapidly (t=4 to 18 minutes), and there are indications that the remaining CO may be released at a later time. Although the precise mechanism of CO liberation by CORM-3 is not yet fully understood, our results reveal that the chloride and glycinate ligands are labile and their substitution with higher affinity ligands present in the cellular environment (ie, glutathione) may favor or accelerate dissociation of CO from the metal center. Our data also indicate that modulation in the rate of CO release from this class of compounds is highly feasible and that the lability of the carbonyl groups could be strictly controlled by changing the type of ligand attached to the transition metal.

In the context of the bioactive properties of CORM-3, the present study directly implicates CO as an important mediator of cardioprotection. Using an in vitro model of hypoxia-reoxygenation in rat cardiomyocytes, we found that CORM-3 preserves cell viability against reoxygenation-mediated damage in a concentration-dependent manner. This cytoprotective effect was observed when CORM-3 was applied to the cells either during the hypoxic event or at the onset of reoxygenation. Interestingly, the fact that inactive compound (iCORM-3) did not show any protection against hypoxia-reoxygenation strongly emphasizes that CO liberated from the ruthenium carbonyl is required to exert the observed biological effect. We also found that the mitoK_ATP channel inhibitor, 5-HD, markedly reduced the protective effect mediated by CORM-3. Opening of the mitoK_ATP channels has been shown to decrease intracellular calcium overloading and protect the heart against ischemia-reperfusion injury, and has been directly implicated in the mechanism of preconditioning-associated cardioprotection.^33–35 Preservation of mitochondrial function during myocardial ischemia by the use of drugs such as nicorandil has also been attributed to activation of mitoK_ATP channels.^36,37 The protective effect promoted by the opening of mitoK_ATP channels appear to be linked to a delay in apoptosis as well as alteration in mitochondrial membrane potential.^35,36 Incidentally, there are precedents showing that CO gas increases the open probability of calcium-activated K⁺ channels in vascular smooth muscle cells and that histidine residues, which are constitutive components of the channel proteins, seem to be crucial for transducing the CO effect.^38,39 Vascular CO has also been shown to serve as an inhibitory modulator of vascular reactivity to vasoconstrictors via a mechanism that involves a tetraethylammonium-sensitive K⁺ channels.^40,41 Although a direct (or indirect) chemical interaction between CO and amino acid moieties remains to be demonstrated, those reports and the data presented in this study point to K⁺ channels as one of the possible targets of CORM-3–mediated biological action. Our results show that the loss in cell viability caused by paraquat, a superoxide anion generator, is also significantly attenuated by CORM-3, indicating that the cytoprotective action of CO may involve alteration of oxidative stress reactions. Notably, a recent report demonstrated that the opening of ATP-dependent potassium channels reduces reactive oxygen species generation in heart mitochondria.⁴² Collectively, these in vitro experiments reveal that CO liberated from CORM-3 protects cardiac cells against hypoxia-reoxygenation, an effect partly due to activation of mitoK_ATP channels.

The ex vivo and in vivo experiments conducted using models of ischemia-reperfusion injury in isolated rat hearts and cardiac allograft rejection in mice highlighted the potential pharmacological properties of metal carbonyl complexes as CO carriers. In the isolated heart preparation, the presence of CORM-3 but not iCORM-3 markedly improved myocardial functions after ischemia-reperfusion as indicated by a better maintenance of all the hemodynamic parameters measured. Moreover, both CK activity levels and infarct size measured at the end of reperfusion were significantly reduced in the presence of CORM-3. The cardioprotection mediated by CORM-3 was once again abolished by infusion of hearts with an inhibitor of mitoK_ATP channels. These data are complementary to our previous results demonstrating that induction of HO-1 in cardiac tissues is associated with increased production of endogenous bilirubin (and by inference CO) and amelioration of postischemic myocardial dysfunction.²⁵ Finally, our findings on the antirejection properties of CORM-3 support the possible utilization of water-soluble metal carbonyls in vivo. Consistent with recently published reports showing that CO gas reduces graft rejection in a model of mouse-to-rat cardiac transplant,¹³ we found that a daily dose of CORM-3 administered to the recipients for 8 days after transplant considerably prolonged the survival time of transplanted hearts. Although iCORM-3 provided significantly less protection against cardiac allograft rejection, the fact that this inactive form of CORM-3 still mediated some degree of protection could indicate that in vivo the metal carbonyl is further metabolized to release the residual CO groups. In addition, because preliminary data conducted in our laboratory showed that CORM-3 (but not iCORM-3) increases heme oxygenase activity in murine macrophages, we cannot exclude a priori that induction of HO-1 and other cytoprotective genes in vivo might partially contribute to the beneficial effects mediated by this metal CO-releasing agent. Additional studies on the chemistry of carbonyls in aqueous solutions and the pharmacokinetics of CO release from this new class of compounds will provide important information on the possible metabolic fate of CO groups in biological systems.

Thus, the present study identifies CORM-3 as the first prototype of metal carbonyl that can be utilized in aqueous solutions to study the bioactivity of CO. In support of the emerging physiological functions attributed to the HO-1/CO pathway in various disease states, our data on the cardioprotective actions of CORM-3 corroborate the notion that CO is an important and versatile cell mediator required as an adjuvant to alleviate interrelated pathological processes that may involve inflammatory reactions, oxidative stress, and apoptosis.^14,15,18,43 Although at very early stage, the studies on the chemical features of metal carbonyls and their potential therapeutic applications as CO-RMs will serve for a better understanding of the intracellular and molecular mechanisms controlled by CO.^31,32

	Acknowledgments

 
This work was supported in part by grants from the Medical Research Council (to B.E.M.), British Heart Foundation (FS/02/027 to R.M.), and the Dunhill Medical Trust (to R.M.).

	Footnotes

 
Original received May 5, 2003; revision received June 10, 2003; accepted June 20, 2003.

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