Ann Thorac Surg 1998;66:1647-1652
© 1998 The Society of Thoracic Surgeons
Lazaroid U74500A is superior to U74006F in preserving rabbit heart for 24 hours
Takahiro Nishida, MDa,
Shigeki Morita, MDa,
Munetaka Masuda, MDa,
Ryuji Tominaga, MDa,
Yoshito Kawachi, MDa,
Hisataka Yasui, MDa
a Department of Cardiovascular Surgery, Faculty of Medicine, Kyushu University, Fukuoka, Japan
Accepted for publication May 17, 1998.
Address reprint requests to Dr Morita, Department of Cardiovascular Surgery, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
e-mail: (morita{at}heart.med.kyushu-u.ac.jp)
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Abstract
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Background. Lazaroid, a series of 21-aminosteroids, has been shown to reduce free-radical–mediated injury after ischemia and reperfusion. Recent in vitro studies have demonstrated that, among the various compounds studied, the most efficient agent was U74500A. The question is whether these findings apply to the whole heart experiencing ischemia-reperfusion injury. In this study we compared the myocardial protective effects of U74006F, the only clinical candidate, and U74500A.
Methods. An isolated rabbit heart preparation perfused with the blood from a support rabbit was used. All hearts were divided into three groups according to the administration of U74500A (4 mg/kg, group A; n = 7), U74006F (4 mg/kg, group F; n = 7), or solvent (group S; n = 7) to the donor rabbit before preservation. After 24 hours of preservation with University of Wisconsin solution at 0°C, all hearts were perfused with cross-circulated blood for 60 minutes with the Langendorff mode followed by 40 minutes in the working mode.
Results. After 10 minutes of reperfusion the serum lipid peroxide levels were significantly (p < 0.05) lower in group A (0.62 ± 0.31 nmol/mL) than those in group S (2.1 ± 1.3 nmol/mL) and group F (1.0 ± 0.6 nmol/mL). The aortic flow rate at 10 mm Hg of left atrial pressure was significantly higher in group A (164 ± 37 mL/min) than that of other groups (71 ± 28 mL/min in group S and 97 ± 28 mL/min in group F). There were no significant differences in high-energy phosphate levels after reperfusion among the three groups.
Conclusion. These data imply that U74500A inhibits lipid peroxidation and prevents ischemia-reperfusion injury more efficiently than U74006F.
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Introduction
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Reactive oxygen species such as superoxide anions and hydroxyl radicals are thought to play an important role in ischemia-reperfusion injury [1]. Failure to neutralize reactive oxygen species at the reperfusion period results in the initiation of lipid peroxidation of the cell membrane [2]. The initiation of lipid peroxidation requires an electron-deficient species capable of abstracting an unstable hydrogen atom from a polyunsaturated fatty acid. Radicals initiating lipid peroxidation are derived from a combination of oxygen and the transition metal iron [3]. Thus, lipid peroxide inhibition by chelating iron is crucial to preventing ischemia-reperfusion injury.
Lazaroids are 21-aminosteroids without glucocorticoid and mineralcorticoid side effects and strongly inhibit lipid peroxidation by chelating iron. Despite the close similarities in structure among the compounds, different mechanisms of lipid peroxidation inhibition have been reported [4–6]. Although only U74006F was selected as a clinical candidate, Killinger and colleagues [5] demonstrated that U74500A was the most effective compound among the lazaroids. Previously we reported the superior cardioprotective effects of U74500A using an isolated rabbit heart preparation [7–9] and a canine orthotopic heart transplantation model [10]. Although some reports [11, 12] described the favorable effects of U74006F on ischemia-reperfusion injury of the heart, there have been conflicting reports on the cytoprotective effects of U74006F [13, 14]. Therefore, it is not clear which agent is more efficient for myocardial protection.
The purpose of this study was to compare the cardioprotective effects of U74500A and U74006F on prolonged hypothermic ischemia. An isolated rabbit heart perfused with cross-circulated blood was used for functional evaluation. We also measured the serum lipid peroxide (LPO) level to elucidate the effect of this drug.
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Material and methods
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A total of 51 adult Japanese white rabbits (21 for heart donors, 15 for support rabbits, and 15 for blood donors) weighing from 2.8 to 3.4 kg were used. All animals received humane care in compliance with the Principals of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23, Rev 1985). This experiment was also reviewed by the Committee of the Ethics on Animal Experiments in the Faculty of Medicine, Kyushu University, and was performed under the Guidelines for Animal Experiments in the Faculty of Medicine, Kyushu University and the law (no. 105) and notification (no. 6) of the Japanese Government. The experimental methods have been described previously [7, 8]. We describe them here briefly.
Donor heart management
The rabbits were anesthetized with 10 mg/kg of sodium thiamylal, which was administered to an ear vein, and were intubated with a tracheal tube connected to a mechanical ventilator with 100% oxygen. For further anesthesia, 1 mg/kg vecuronium bromide and 70 µg/kg fentanyl citrate were given intravenously. After a median sternotomy was performed, the thymus and pericardium were carefully removed and then the heart and aortic arch were exposed. After heparinization (1,500 U/kg) the innominate artery was cannulated to administer University of Wisconsin (UW) solution (ViaSpan; DuPont Pharmaceuticals, Wilmington, DE). Next, the inferior vena cava was transected to decompress the heart. Immediately after decompression, the aortic arch was cross-clamped and 20 mL/kg of cold (0°C) UW solution was infused from the innominate artery at a constant flow rate of 12 mL/min with a syringe infusion pump. The heart was topically cooled with ice slush. The hearts were excised quickly and immersed in cold UW solution (0°C) during preservation.
Support rabbit and cross-circulation system
The support rabbits were anesthetized and ventilated mechanically in the same way as donor rabbits. After heparinization (1,500 U/kg) the common carotid artery and the external jugular vein were exposed and cannulated. As shown in Figure 1, oxygenated blood from the common carotid artery of the support rabbit was introduced by a microtube pump to a cannula connected to the ascending aorta. The blood draining from the system was then returned to the jugular vein by another microtube pump. Anesthesia was maintained with a constant infusion of fentanyl citrate (200 µg/h) and vecuronium bromide (1.5 mg/h). In addition, 1,000 U/h sodium heparin was infused. An arterial blood gas analysis of the support rabbit was made using a pH–blood gas analyzer. The femoral artery pressure of the support rabbit was also continuously monitored.
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Fig 1. Cross-circulated rabbit heart preparation. (A) In the Langendorff mode, oxygenated blood from the common carotid artery of the support rabbit was introduced to an aortic inflow tract of this system. The coronary venous blood was collected from the pulmonary artery (PA) and then returned to the support rabbit. (B) In working mode, the left atrium was cannulated with a double lumen cannula. The blood collected in the atrial reservoir was ejected by the heart. (AoP = aortic pressure; LA = left atrium; LAP = left atrial pressure.)
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Measurement of left ventricular function in working mode
The donor heart was perfused through the aorta and Langendorff mode perfusion was initiated. The perfusion pressure was maintained at 60 mm Hg and the temperature of the circulating blood was maintained at 37°C. The superior and inferior vena cava and the pulmonary veins were closed. The pulmonary artery was cannulated to drain the coronary venous blood. The coronary blood flow rate was measured directly by time collection. The left atrium was cannulated with a double lumen cannula. One lumen of the double lumen cannula was connected to a pressure transducer to measure the left atrial pressure. The other lumen was connected to an atrial reservoir (Fig 1). The aortic flow rate was measured by an in-line electromagnetic flow probe (4 mm in diameter; Model FF-040-T; Nihon-Koden, Tokyo, Japan) connected to a flow meter (model MFV-1200; Nihon-Koden). The aortic pressure was measured from a sidearm in the aortic tract.
In the working mode, all hearts were paced atrially at 250 beats per minute and left atrial pressure and aortic afterload pressure were fixed at 10 and 60 mm Hg, respectively.
Measurement of myocardial oxygen consumption and purine catabolites
We measured oxygen consumption (MVO2) based on the following equation: MVO2 = CBF x (A - V)/100, where CBF is the coronary blood flow rate measured by time collection as described above, A is the oxygen content of the blood in the atrial reservoir, and V is oxygen content of the coronary effluent. The values of A and V are expressed as milliliters of O2 per minute. The oxygen content was calculated as (1.34 x hematocrit/3 x percent saturation of hemoglobin)/100 + (0.003 x PO2).
To measure purine catabolite levels at the end of reperfusion, we freeze-clamped the hearts with a Wollenberg clamp precooled in liquid nitrogen. The specimens were then immediately cooled in liquid nitrogen and stored until analysis. Neutralized perchlonic acid extracts were analyzed for adenine nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and inosine monophosphate) by means of high-performance liquid chromatography (Shimazu, Kyoto, Japan) on an anion exchange column (DEAE-2SW; Tosoh, Tokyo, Japan). The peak identification was based on the retention times, which were checked daily with a synthetic mixture of standards. The tissue levels were expressed as mmole per gram of dry weight.
Experimental protocol
The rabbits were divided into three groups according to the use of U74500A (21-[4-[5,6-bis-(diethylamino)-2-pyridinyl]-1-piperazinyl]-16a-methyl-pregna-1,4,9(11)-triene-3,20 dione, hydrochloride), U74006F (21-[4-(2,6-di-1-ryrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16a-metyl-pregna-1,4,9(11)-triene-3,20 dione, monomethane sulfonate), or solvent. In group A (n = 7), 4 mg/kg U74500A dissolved in a mixture of 6 mL of solvent (CS-4), 14 mL of water, and 0.6 g of albumin (bovine albumin; Sigma Chemicals, St. Louis, MO) was given through the ear vein for 20 minutes before preservation. In group F, 4 mg/kg U74006F was administrated in the same way. In the control group, only solvent was given (group S; n = 7). The solvent CS-4 consists of 0.02 mol/L citric acid monohydrate, 0.032 mol/L sodium citrate dehydrate, and 0.077 mol/L NaCl.
After the 24-hour preservation period, the hearts were perfused with blood from the support rabbit for 60 minutes in the Langendorff mode at 60 mm Hg of perfusion pressure. We measured the serum creatine kinase, its MB isozyme, troponin-T, and the serum lipid peroxide levels in the coronary effluents after 10 and 40 minutes of reperfusion. The level of creatine phosphokinase, its isozyme, and troponin-T and TnT were measured by the ultraviolet method, a chemiluminescent immunoassay, and an enzyme immunoassay, respectively (SRL, Tokyo, Japan). Serum LPO levels were measured by the modified Yagi method (DeterminerLPO; SRL) using a Methylene blue derivative [15].
After 60 minutes of Langendorff perfusion, the working mode was started. Forty minutes after the initiation of the working mode, we measured the aortic flow rate and calculated MVO2 at a rate of 250 beats per minute of atrial pacing. Finally, the hearts were freeze-clamped for analysis of high-energy phosphate levels.
Statistical analysis
Statistical analysis was performed using Stat View (Abacus Concepts, Berkeley, CA) on a Macintosh computer (Apple, Cupertino, CA). All values are expressed as the mean ± standard deviation. Differences in parameters among the three groups were examined by one-way analysis of variance. Bonferroni’s modification was used for multiple comparisons. Significance was designated as a p value of less than 0.05.
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Results
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The hemodynamic state of the donor rabbit was not affected by the administration of either lazaroids or solvent. Among the three groups no significant differences in heart rate and systolic blood pressure before (group S, 232 ± 32 beats/min and 128 ± 28 mm Hg; group A, 248 ± 36 beats/min and 120 ± 17 mm Hg; and group F, 253 ± 28 beats/min and 121 ± 23 mm Hg, respectively) and after the administration (group S, 221 ± 38 beats/min, and 120 ± 22 mm Hg; group A, 234 ± 41 beats/min and 123 ± 28 mm Hg; and group F, 255 ± 30 beats/min and 124 ± 26 mm Hg, respectively) were observed. During the experiment the hemodynamics of the support rabbits was stable and the systolic blood pressure was maintained over 100 mm Hg. An arterial blood gas analysis of the support rabbit was made at least three times during the experiment to confirm a stable acid–base balance. Minimum alternations in the arterial carbon dioxide tension, pH, and arterial bicarbonate level were observed during the experiment.
Biochemical measurements
As shown in Table 1, biochemical variables such as creatine kinase, its MB isozyme, troponin-T, and serum lipid peroxide levels of the coronary effluent after 10 minutes of reperfusion were significantly lower in group A than those in groups S and F. After 40 minutes of reperfusion, these variables were also significantly lower in group A, except for the LPO level.
Myocardial oxygen consumption and high-energy phosphate level
Oxygen consumption at a left atrial pressure of 10 mm Hg was significantly higher in group A (0.66 ± 0.09 mL O2/min) compared with those in group S (0.43 ± 0.07 mL O2/min) and group F (0.48 ± 0.13 mL O2/min) (Table 2). These data indicate more efficient oxygen use after long-time preservation in hearts pretreated by U74500A. There were no significant differences in high-energy phosphate levels at the end of ischemia and reperfusion among the three groups (Table 3).
Cardiac function at a left atrial pressure of 10 mm Hg
As shown in Figure 2, the aortic flow rate and total cardiac output, the sum of the aortic flow rate and coronary blood flow rate, were significantly higher in group A (164 ± 37 and 178 ± 39 mL/min) than those in group F (97 ± 28 and 109 ± 29 mL/min) and group S (71 ± 28 and 82 ± 29 mL/min).
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Fig 2. Cardiac function at a left atrial pressure (LAP) of 10 mm Hg after 24-hour preservation followed by reperfusion for 100 minutes. The aortic blood flow (A) and total cardiac output (B) were significantly higher than those in the other groups. Group S indicates hearts without pretreatment with 21-aminosteroids; group A indicates heart pretreated with U74500A; group F indicates heart pretreated with U74006F. (AoP = aortic pressure; PA = pulmonary artery.)
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For the purpose of comparing the function results, we studied ten freshly prepared hearts under an identical setup except for ischemia and pretreatment. The hearts excised from the donor rabbits were immediately mounted to the cross-circulated blood perfusion system. The functional measurement was performed during the working mode as described above. At a left atrial pressure of 10 mm Hg we obtained an aortic flow rate of 193 ± 20 mL/min. Comparing this non-ischemic control value with those of the experimental group, the aortic flow rates of groups A, F, and S were 90%, 55%, and 40% of nonischemic control, respectively.
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Comment
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In this study, we compared the cardioprotective effects of the lazaroids U74500A and U74006F when they were used as pretreatment preservation. We selected these two compounds because U74500A was found to be the most effective agent by an in vivo experiment [5], and U74006F was selected as a clinical candidate [16]. U74500A was more efficient than U74006F in inhibiting lipid peroxidation and preserving cardiac function after 24 hours of hypothermic ischemia. U74500A allowed 90% recovery of cardiac function in nonischemic control hearts.
Lipid peroxidation is a major factor causing cell membrane injury after ischemia reperfusion. The generation of oxygen-free radicals initiates lipid peroxidation of the cell membrane. Although the initial oxygen radicals produced on reperfusion are hydrogen peroxide and superoxide radicals, they have a relatively low cellular reactivity in the absence of a transition metal [17]. The subsequent production of hydroxyl radical generated by the iron-catalyzed Haber–Weiss or Fenton reaction [18, 19] is presumably the most detrimental factor to cells after ischemia and reperfusion. LPO is generated by the interaction of hydroxyl radicals and membrane phospholipids [17]. Once the LPO is generated, an enormous amount of it is produced by a lipid free-radical chain reaction. Thus, the inhibition of lipid peroxidation is crucial for preventing ischemia-reperfusion injury.
Lazaroids, a series of 21-aminosteroids, are potent inhibitors of iron-mediated lipid peroxidation [4–6, 20]. Several studies have shown the protective effects of lazaroids on ischemia-induced injury to various organs [21–25] and they are thought to inhibit lipid peroxidation by chelating iron and scavenging oxygen and lipid radicals. Few reports, however, have described the cardioprotective effects of U74006F and U74500A. Holzgrefe and associates [11] demonstrated that pretreatment of U74006F enhanced posterior wall thickening after 15 minutes of occlusion of the circumflex coronary artery in dog hearts. Carrea and colleagues [12] reported the cardioprotective effects of U74006F using an isolated rabbit heart with a normothermic global ischemia of 30 minutes and reperfusion of 30 minutes in a Langendorff preparation. However, the recovery rate of left ventrical–developed pressure was limited to only 50% of the control level even in U74006F-treated hearts. In our previous studies we showed the superb cardioprotective effects of U74500A in the isolated blood-perfused rabbit heart model [7–9]. The U74500A-pretreated hearts allowed over 90% recovery of cardiac function (left ventrical–developed pressure in the Langendorff model [9] and aortic flow rate in the working model [7, 8]). Although these reports indicate the efficiency of both U74500A and U74006F, no reports have compared the differences between the two compounds in a solid organ preservation model. In this study, U74500A was found to be an efficient inhibitor of lipid peroxidation and a beneficial agent for long-term heart preservation, whereas U74006F had little effect on ischemia-reperfusion injury.
Despite close similarities in their structures, the ability of U74500A and U74006F to inhibit lipid peroxidation was found to be different in an in vitro study. Braughler and associates [6] reported that U74500A was two to ten times more potent than U74006F when the efficacy to inhibit lipid peroxidation was compared in brain homogenate with Fe2+. Braughler and associates considered the pyridine structure with an N–C
C–N molecular fragment in U74500A to be related to the mechanism of the differences because this structure was generally associated with metal–ligand coordination. Killinger and colleagues [5] observed that U74500A was the most potent preservative agent among the various available lazaroids in a study using the cultured human umbilical endothelium. Ryan and associates [4] reported that U74500A inhibited lipid peroxidation by directly affecting iron redox chemistry, whereas U74006F-mediated inhibition required the competent microsomal system. The different potencies to inhibit lipid peroxidation between the two compounds may be based on these mechanisms.
Recent studies have indicated mechanisms of lazaroids other than iron-dependent lipid peroxidation inhibition. In our previous studies [9, 10] we demonstrated that administration of U74500A resulted in a higher myocardial adenosine triphosphate level at the end of ischemia. Ishizaki and associates [25] reported that U74500A more efficiently suppressed adenine nucleotide degradation during warm ischemia of liver than the other compounds, U74006F and U74389G. In the current study, there was no significant difference in the level of high-energy phosphates at the end of reperfusion. Although we could measure the adenosine triphosphate level only at the end of experiment, serial measurement of adenosine triphosphate using a larger heart during ischemia (preservation) and reperfusion might reveal some relationship between lazaroids and the metabolism of high-energy phosphates. U74500A might have some beneficial effect not only after reperfusion but also during ischemia. A report from our laboratory [9] showing the prevention of ischemic contracture suggested that this drug has some favorable effects during ischemia. Because this fact cannot be fully explained on the basis of the inhibition of lipid peroxidation, there appear to be some other mechanisms that have not yet been identified. Further study is therefore warranted to elucidate the underlying mechanism.
Recently, U74006F has been chosen as a clinical candidate and is currently undergoing trial for the treatment of neurologic disease [16]. Although a phase II trial of U74006F in aneurysmal subarachnoid hemorrhage revealed the favorable results of this agent, the results of our experiment clearly showed that U74500A was more effective for long-term heart preservation. We conclude that U74500A should be considered a clinical candidate to prevent ischemia-reperfusion injury of the heart. Although we have already shown the preferable effects of U74500A for organ preservation using a clinically relevant heart or lung transplantation model [10, 22] further study examining the toxicity and optimal dosage for clinical application are called for to confirm our findings before it is used for humans.
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Acknowledgments
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U74500A, U74006F, and its solvent CS-4 were kindly provided by The Upjohn Company (Kalamazoo, MI). We gratefully acknowledge the assistance of Miss Yumi Murakami in the biochemical analysis.
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References
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Abstract
Introduction
