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J Thorac Cardiovasc Surg 2002;124:775-784
© 2002 The American Association for Thoracic Surgery
Cardiopulmonary Support and Physiology (CSP) |
From the Department of Pharmacologya and Second Department of Surgery,b School of Medicine, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan.
Received for publication April 16, 2001. Revisions requested July 25, 2001; revisions received Feb 12, 2002. Accepted for publication Feb 21, 2002. Address for reprints: Satoshi Yamashiro, MD, Department of Pharmacology, School of Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan.
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Abstract |
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Introduction |
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1 Cardiovascular diseases, such as hypercholesterolemia, diabetes, and hypertension, are characterized by endothelial dysfunction and reduced endothelium-mediated vasodilatation. 2 The underlying defect might involve both decreased formation and increased breakdown of NO by means of reaction with superoxide anion.
The availability of tetrahydrobiopterin (BH4) is essential for the catalytic activity of NO synthase (NOS). 3 A close link between cellular availability of BH4 and NO synthesis has recently been demonstrated in a number of different cell types. 4,5 Biochemical evidence revealed that activation of purified constitutive NOS in the presence of suboptimal levels of BH4 results in uncoupling of oxygen reduction and arginine oxidation. 6 In agreement with these results, Cosentino and Katusic 7 recently proposed that, in isolated canine coronary arteries depleted of BH4, endothelial NOS might serve as a source of oxygen free radicals (OFRs). Furthermore, in human aortic endothelial cells exposed to prolonged stretching, inhibition of BH4 synthesis has been shown to increase markedly the production of superoxide anion. 8 In addition, Pritchard and colleagues 9 found that increased generation of superoxide anion in cultured endothelial cells exposed to low-density lipoprotein can be inhibited by NG-nitro-L-arginine methyl ester, a selective inhibitor of NOS. These results indicate that NOS itself can be a potential source for endothelial production of OFRs and that decreased availability of BH4 might cause a shift in balance between production of protective NO and toxic OFRs. 6 Such an imbalance, in turn, could result in endothelial dysfunction and oxidative vascular injury, as described in a number of vascular diseases. 1,2
Accordingly, these findings lead to the hypothesis that such dysfunctional NOS caused by insufficiency of BH4 participates in oxidative injury, especially under pathologic conditions, including ischemia and reperfusion. In fact, administration of exogenous BH4 has been shown to reduce postischemic endothelial dysfunction, 10 posttransplantation lung edema and OFR injury in grafts, 11 and ischemic renal injury. 12 Moreover, a recent experimental study suggested that intracellular BH4 levels are reduced after ischemia and reperfusion. 10
Thus, in the present study the major purpose was to clarify whether BH4 would exert a beneficial effect in a model of myocardial ischemia-reperfusion injury. The effects of administration of BH4 on myocardial ischemia-reperfusion injury were examined in isolated rat hearts by using the Langendorff method and compared with those of the representative antioxidants superoxide dismutase plus catalase (SOD/catalase) 13 and L-ascorbic acid (Vit C) 14 because BH4 itself has been reported to have radical scavenging activity, as assessed by means of spin trapping electron spin resonance (ESR) spectrometry in vitro. 15
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Materials and methods |
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In vitro free radical scavenging action of BH4
The free radical quenching capacity of BH4 in vitro was tested by using the method of breaching the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) with a free radical monitor (JES-FR 30; JEOL, Tokyo, Japan). The reaction mixture contains 100 µL of BH4 or water and 100 µL of 0.2 mmol/L DPPH. ESR spectra were measured by calculating the relative peak height of DPPH compared with that of the internal standard manganese oxide. Vit C was used as a standard agent for comparison of radical scavenging activity. BH4 and Vit C decreased the ESR spectra of DPPH in a concentration-dependent manner. The concentrations of BH4 and Vit C required to quench DPPH radicals by 50% were 46.0 and 32.0 µmol/L, respectively.
Experimental preparation
Male Sprague-Dawley rats (SLC, Shizuoka, Japan) weighing 260 to 310 g were anesthetized with diethylether and given 1000 IU/kg body weight sodium heparin intravenously. After thoracotomy, the heart was rapidly excised. Then the ascending aorta was cannulated, and retrograde perfusion of the heart was initiated on a Langendorff apparatus at a constant pressure of 100 cm H2O. The isolated heart was perfused with 37°C Krebs-Henseleit solution (KHS) of the following composition: NaCl, 120 mmol/L; KCl, 4.8 mmol/L; CaCl2, 1.25 mmol/L; MgSO4, 1.2 mmol/L; KH2PO4, 1.2 mmol/L; NaHCO3, 25.0 mmol/L; and glucose, 11.0 mmol/L. The perfusate was oxygenated with 95% O2/5% CO2 (PO2 >600 mm Hg).
A thin-wall latex balloon was inserted into the left ventricle through the left atrium to monitor left ventricular pressure (LVP). The balloon was filled with bubble-free saline. The ventricle was loaded with 5 to 10 mm Hg of the initial left ventricular end-diastolic pressure (LVEDP), and this balloon volume was maintained throughout the experiments. LVP was measured with a pressure transducer (TP-400T; Nihon Kohden, Tokyo, Japan), and the first derivative (dp/dt) of LVP was derived from differentiating the signal of LVP with an electronic differentiator (ED-601G, Nihon Kohden). Left ventricular developed pressure (LVDP) was estimated from left ventricular systolic pressure and LVEDP. The mean coronary flow (CF) was measured with an electromagnetic flow probe (FF-030T, Nihon Kohden) attached to the aortic cannula, which was connected to an electromagnetic flowmeter (MFV-3200, Nihon Kohden). Heart rate was counted by using a cardiotachometer (AT-600G, Nihon Kohden) triggered by the pressure pulse. After 10 minutes of equilibration, the hearts were atrially paced throughout the experiments with an electronic stimulator (SEN-3301, Nihon Kohden). Pacing rate was set at 110% of the own beat during the stabilizing period of Langendorff perfusion. All hemodynamic parameters were continuously recorded on an 8-channel thermal-pen recorder (WT-685G, Nihon Kohden).
Experimental protocol
All hearts were perfused for 10 minutes to stabilize hemodynamics before the experiment was started. Five minutes after the beginning of atrial pacing, baseline values of cardiovascular parameters were measured.
The hearts were divided into nonischemic and ischemic groups and were infused with vehicle (nonischemic KHS group and ischemic KHS group, n = 8 each) or BH4 (nonischemic BH4 group, ischemic BH4 group, and ischemic low BH4 group, n = 8 each). BH4 (0.6 or 1.25 mg/mL) and vehicle (KHS) were infused through a 3-way cock placed just proximal to the aortic cannula by using an infusion pump (Model 11 or 22; Harvard Apparatus Co, Holliston, Mass) at an infusion rate of 0.4 mL/min for 5 minutes just before ischemia and then again during the first 5 minutes of the reperfusion period. The dosages of BH4 in coronary perfusate (corresponding to nearly 50 and 100 µmol/L, respectively) used in this study were determined considering the previous in vitro findings that concentration-dependent NO production, which was estimated as L-citrulline formation, by human endothelial NOS was observed at doses of BH4 ranging from 10 to 100 µmol/L. 16
The hearts of rats from the ischemic groups were subjected to global ischemia (cessation of flow) at 37°C for 30 minutes, according to the method of Takeo and colleagues 17; the ischemic hearts were submerged in a chamber filled with the KHS, which was equilibrated with a gas mixture of 95% N2/5% CO2 (partial pressure of O2 <10 mm Hg) to support global ischemia and maintained at 37°C to prevent hypothermia-induced cardioprotection. During ischemia, peak LVEDP and the time to onset of ischemic contracture for LVEDP to increase more than 5 mm Hg were measured. After completion of ischemia, the KHS in the chamber was drained, and the hearts were reperfused with an aerobic KHS for 30 minutes. Sampling of perfusate was performed before ischemia (baseline) and at 10, 20, and 30 minutes after the initiation of reperfusion, and samples were stored at -20°C for measurement of lactate dehydrogenase (LDH) activity and nitrite plus nitrate (NOx) level.
The hearts of rats from the nonischemic groups were paced and perfused without ischemia. BH4 (1.25 mg/mL, nonischemic BH4 group) or vehicle (nonischemic KHS group) infusion and sampling of perfusate were performed in the same time course as for the ischemic groups. A low dose (0.6 mg/mL) of BH4 in the nonischemic group was omitted because there were no significant changes in functional and metabolic parameters with high-dose (1.25 mg/mL) BH4 in the preliminary experiment.
At the end of the experiments, sections of the left ventricular (LV) free wall were quickly excised and frozen with liquid nitrogen. These frozen myocardial sections were applied for determination of lipid peroxides and energy metabolites in myocardial tissues. Additionally, the effects of SOD (2000 U/mL) plus catalase (2000 U/mL; SOD/catalase group, n = 8) and Vit C (Vit C group, n = 6) at a dose (0.49 mg/mL) having an equipotent radical scavenging activity in vitro with BH4 (1.25 mg/mL) on myocardial ischemia-reperfusion injury were evaluated in the same time course as mentioned above.
In addition, effects of BH4 (2.5 mg/mL for 5 minutes) administered only just before ischemia (pre-BH4 group, n = 8) were compared with those of BH4 administered only during the first 5 minutes of the reperfusion period (post-BH4 group, n = 8) to estimate whether the time for BH4 treatment would influence the effects of BH4 on myocardial ischemia-reperfusion injury.
Determination of myocardial energy metabolites
The frozen myocardial tissue for measurement of energy metabolites was lyophilized for 6 hours. The dried tissue was homogenized with 0.6 mol/L perchloric acid. The mixture was centrifuged at 12,000 rpm for 15 minutes at 2°C, and the supernatant was used for assay. Adenosine triphosphate (ATP) was determined by using the firefly luminescence method with an ATP monitoring agent (LL-100-2; Toyo Ink, Tokyo, Japan) and a lumiphotometer (Minilumat LB9506; Berthold GmbH & Co KG, Calmabacher, Germany). Creatine phosphate (CrP) and inorganic phosphate (Pi) levels were determined by using the method of Fiske and Subbarow, as modified by Furchgott and DeGubareff, 18 with a spectrophotometer (UV-150-02; Shimadzu, Kyoto, Japan).
Determination of myocardial tissue lipid peroxidation
The extent of lipid peroxidation in the frozen myocardial tissue was measured by using the thiobarbituric acid (TBA) method, 19 with some modifications. The amount of TBA-reactive substances was estimated as malondialdehyde (MDA) equivalents per gram of wet myocardial weight. The developed color was read with a spectrophotometer (UV-2200A, Shimadzu) at 532 nm. Commercially available 1,1,3,3-tetraethoxypropane was used as a standard.
Determination of LDH activity in the effluent
LDH activity in the coronary effluent was estimated by using the method of Wróblewski and La Due 20 with an LDH monitoring kit (Wako Pure Chemical, Osaka, Japan) and a spectrophotometer (UV-2200A) at 340 nm.
Determination of NOx level in the effluent
NOx levels before ischemia and 10 minutes after the initiation of reperfusion in the effluent were analyzed by using the Griess method with an automated NO detector high-performance liquid chromatography system (ENO-20; EICOM, Kyoto, Japan). In the nonischemic groups the NOx level in the effluent was analyzed in the same time course as for the ischemic groups. The absorbance of the color of the product dye at 540 nm was measured. Appropriate concentrations of NaNO2 and NaNO3 were used for constructing a standard curve. The time-voltage change was traced by using a data processor (EPC-300, EICOM). By comparing a peak area shown on a chromatogram with a standard area, the concentration of NOx in the effluent was determined. The detection limit in this assay is 10 pmol/mL, according to the manufacturer.
Drugs
The drugs used in this study were (6R)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride (BH4, Wako), bovine erythrocyte SOD (5100 U/mg; Sigma Chemical Co, St Louis, Mo), bovine liver catalase (3260 U/mg, Sigma), Vit C (Wako), DPPH (Wako), TBA (Sigma), 1,1,3,3-tetraethoxypropane (Sigma), NaNO2 (Wako), and NaNO3 (Wako).
BH4, Vit C, SOD, and catalase were dissolved in KHS just before use. Vit C, at a concentration used in this study, caused no change in pH of coronary effluent.
Data analysis
The data were analyzed by using 1-way analysis of variance, and paired and unpaired observations were analyzed with paired and unpaired t tests, respectively. All values are presented as means ± SE.
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Results |
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). Significantly better recoveries of LVDP and maximum and minimum LV dp/dt during reperfusion were observed in ischemic BH4 (0.6 and 1.25 mg/mL) groups than those in the ischemic KHS group in a dose-related manner, whereas CF was comparable among these 3 groups (Figure 1). LVEDP of vehicle (KHS)-treated hearts increased during ischemia and increased again immediately after the onset of reperfusion (Figure 2, A). The increases in LVEDP during reperfusion in the ischemic BH4 and ischemic low-BH4 groups were significantly smaller than those in the ischemic KHS group (Figure 2, A). In addition, in the ischemic BH4 and ischemic low-BH4 groups, significantly delayed onset of ischemic contracture and lowered maximal values of LVEDP during ischemia were seen in comparison with the corresponding values in the ischemic KHS group (Figure 2, B and C). Likewise, SOD/catalase significantly improved contractile dysfunction and attenuated increases in LVEDP both during ischemia and reperfusion (Figures 1 and 2). On the other hand, in Vit C-treated ischemic hearts, changes in LVDP and LV dp/dt during reperfusion were indistinguishable from those in vehicle (KHS)-treated ischemic hearts (Figure 1), although increases in LVEDP during ischemia were significantly attenuated (Figure 2).
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). On the other hand, in ischemic BH4 and SOD/catalase groups, but not in the Vit C group, the MDA contents were significantly lower than that in the ischemic KHS group and showed nearly the same contents as those in nonischemic groups.
In all groups the LDH activities in the coronary effluent before ischemia were minimal, and those in nonischemic groups were unchanged throughout experiments (Figure 3). Release of LDH into the effluent in the ischemic KHS group was significantly increased after ischemia, but the LDH releases in the ischemic BH4 and ischemic low-BH4 groups and the SOD/catalase group were significantly lower than that in the ischemic KHS group throughout reperfusion (Figure 3). Moreover, LDH release in the Vit C (0.49 mg/mL) group was similar to that in the ischemic KHS group, and a higher dosage (4.9 mg/mL) of Vit C also did not alter postischemic functional and metabolic abnormalities (n = 4, data not shown).
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21 Furthermore, it is well known that both the mechanical function of the heart and myocardial energy metabolism are markedly impaired after ischemia. Consequently, the present results clearly indicate that BH4 exerts cardioprotective actions against postischemic injury in rat perfused hearts. In nonischemic hearts, on the other hand, BH4 did not alter any measured cardiovascular parameters and biochemical indexes. The lack of the coronary vasodilating action of BH4 at doses used in this study under the aerobic condition is consistent with previous findings, 22 although there is an in vivo study with healthy volunteers reporting vasodilator effects of intra-arterial BH4 infusion at much higher doses (16 or 32 mg/min) in the forearm circulation. 23 Thus, the possibility that the observed favorable effects of BH4 would be attributable to an improvement of coronary perfusion can be ruled out.
In the present study BH4, as well as SOD/catalase, suppressed increases in LVEDP both during ischemia and reperfusion. Additionally, the time to onset of ischemic contracture in our BH4 or SOD/catalase groups was significantly longer than that in the ischemic KHS group. Ischemic contracture has been suggested to be a sign of irreversible cell damage, and its appearance is thought to coincide with the time when glycolytic ATP production ceases. 24 Some studies with electron paramagnetic resonance spectroscopy support the theory that OFRs are generated during myocardial ischemia in addition to reperfusion. 25 These findings suggest that the beneficial effects of SOD/catalase are derived from scavenging of OFRs during not only reperfusion but also ischemia. In this study administration of BH4 only before ischemia (pre-BH4 group) improved postischemic contractile dysfunction in comparison with that seen in the post-BH4 group but to a lesser extent than that seen in the group in which BH4 was administered both before ischemia and just after reperfusion. Therefore, it is likely that the improvements of reperfusion injury by BH4 were derived from the effects of BH4 during ischemia in addition to reperfusion.
Kojima and colleagues 15 reported that BH4 has antioxidant activities and protects against paraquat-induced injury of rat hepatocytes, which is believed to be caused mainly by generation of OFRs. They considered that BH4 acts directly as a scavenger of superoxide anion generated by xanthine-xanthine oxidase or by rat macrophage-phorbol myristate acetate radical-generating systems. We also confirmed that BH4 itself possesses in vitro free radical scavenging activity with a similar potency to that of Vit C when compared on a molecular basis. Nevertheless, Vit C at 0.49 mg/mL, which was shown to have a radical scavenging activity equal to that of BH4 (1.25 mg/mL), or even at 4.9 mg/mL failed to improve ischemia and reperfusion-induced mechanical and metabolic abnormalities, which is different from the case of SOD/catalase or BH4. Because exogenous SOD, as well as catalase, is impermeable to the cell membrane because of its large molecular size, scavenging OFRs elicited by SOD/catalase leading to beneficial effects might occur solely in the intravascular space. Thus, the reason for the lack of cardioprotective effects of Vit C at doses used in this study might be explained by insufficiency of the antioxidant activity rather than by a poor membrane permeability. Considering these data, the free radical scavenging activity of BH4 itself might play a minor role, if any, in the protective effects against oxidative myocardial injury produced by 30 minutes of ischemia and 30 minutes of reperfusion at 37°C.
Vásquez-Vivar and coworkers 26 reported that BH4 has a protective effect against cerebral ischemia-reperfusion injury through inhibiting the generation of superoxide anion simultaneously with increasing levels of NO produced by NOS. Similarly, several investigators have demonstrated that treatment with BH4 reduces ischemia and reperfusion-induced tissue injury 10-12 and have suggested that decreased endothelium-derived NO activity might worsen the ischemic tissue damage, involving BH4 depletion. In the present study administration of BH4 attenuated decreases in NOx level in the coronary effluent during reperfusion, whereas SOD/catalase did not. These findings indicate that the effect of BH4 on perfusate NO level is due to improved NOS activity rather than decreased breakdown of NO by superoxide anion. Recently, Shen and associates 27 have suggested that endogenous NO increases myocardial metabolic efficiency by reducing myocardial O2 consumption. Thus, a part of the cardioprotective effects of BH4 might be explained by the increased generation of NO.
Although it remains unclear why no change in CF was observed with BH4 after ischemia in spite of increases in NOx level, it is unlikely that the beneficial effects of BH4 would attribute solely to increased generation of NO because SOD/catalase exhibited a cardioprotective effect similar to that of BH4 without an increase in NOx level. Therefore, these findings suggest a possibility that reduced generation of OFRs rather than increased generation of NO by NOS might be a key mechanism of the cardioprotective effect of BH4. In this regard recent investigations proposed that endothelial NOS-dependent superoxide formation plays an important role in pathologic conditions, such as diabetes, hypertension, and atherosclerosis, 28,29 which would be related to reduced levels of BH4. 7 In addition, enhancement of NO bioavailability by administration of BH4 has been reported in patients with hypercholesterolemia. 22 The current findings imply that supplementary administration of BH4, perhaps in combination with L-arginine, free radical scavengers, or both, might provide a novel strategy to prevent myocardial ischemia-reperfusion injury and associated complications.
The present data clearly demonstrate that BH4 has cardioprotective effects in a rat model of reperfusion injury and might provide important mechanistic information linking BH4 to improved cardiac function. However, the precise mechanism of action of BH4 involved in lessening myocardial ischemia-reperfusion injury remains to be clarified in this model at present. Further studies, including combined treatment with BH4 and SOD/catalase, might need to evaluate the concise role of BH4 in ischemia-reperfusion injury. Moreover, experiments with hemoglobin or carboxy-PTIO or other suitable NO-avid molecules that scavenge NO might help to evaluate the role of NO in cardioprotection.
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P < .05 and 