Reperfusion of the heart after brief ischemia may lead to potentially lethal arrhythmias (1). Active oxygen species, such as superoxide and hydroxy radicals, have been suggested to play a role in the genesis of ventricular arrhythmias. Active oxygen species are generated from a variety of sources (2-4) during the early moments of reperfusion (5,6). Various studies (7-11) have shown that a variety of inhibitors of free radical production (e.g., allopurinol), free radical scavengers [e.g., superoxide dismutase (SOD), catalase, mannitol and glutathione], and spin trap agents reduce or abolish the ventricular fibrillation (VF) associated with reperfusion after brief ischemia.
Adult T-cell leukemia (ATL) is an endemic T-cell leukemia/lymphoma originally described in Japan in 1974 (12). Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of ATL and was the first human retrovirus discovered (13,14). ATL-derived factor (ADF), originally identified as a cytokine-like factor produced by ATL cell lines (15-17), is a 12-Kd polypeptide consisting of 104 amino acids; it exhibits a variety of biological activities and its center of activity is a -Cys-Gly-Pro-Cys- amino acid sequence. Subsequent studies showed that ADF is a human homologue of thioredoxin (17-19). Thioredoxin is a small, ubiquitous protein with two redox-active half-cysteine residues in an exposed active center. The reversible reaction of the two-Cys residues from dithiol to disulfide causes thioredoxin to be intimately involved in oxidation-reduction in the body. Escherichia coli thioredoxin has been well characterized as a hydrogen donor for ribonucleotide reductase (20), and Mitsui and colleagues (21) reported that human thioredoxin (h-thioredoxin) eliminates hydrogen peroxide and acts as a radical scavenger.
h-Thioredoxin also protected against reperfusion injury in an ischemic lung model (22,23). In the present study, we investigated whether h-thioredoxin suppressed reperfusion-induced arrhythmias in an isolated rat heart model. We used three concentrations of h-thioredoxin and compared its inhibitory effects with the effects observed in a control, a SOD (8 × 104 IU/L) treated, and a catalase (1 × 106 IU/L)-treated group.
MATERIALS AND METHODS
Animals and perfusion technique
Male Wistar rats weighing 330-450 g (Japan SLC) were anesthetized with diethyl ether and received heparin (200 U) intravenously (i.v.). After 30 s, the hearts were excised and placed in cold perfusion medium. Each heart was cannulated through the aorta and perfused by the Langendorff method with a constant perfusion pressure equivalent to 70 cm water at 37 °C. The perfusion medium was Krebs-Henseleit buffer (in mM): Na+ 143, K+ 4.3, Ca2+ 1.4, Mg2+ 1.2, H2PO4- 1.2, HCO3- 25, and glucose 11 gassed with 95% O2/5% CO2. During preparation of the perfusion medium, precautions were taken to prevent the precipitation of calcium, and all solutions were filtered through a 0.2-μm pore filter to remove any particulate contaminants before use.
A 6-0 polypropylene ligature was placed around the left anterior descending artery near the pulmonary valves. Both ends of the ligature were passed through a small plastic tube. An epicardial ECG was performed with three surface electrodes attached to the surface of the heart (Physio-Control VSM1, Nihon Kohden, Tokyo, Japan).
After 15-min stabilization, the hearts were subjected to 10-min regional ischemia by snaring the ligature, followed by 30-min reperfusion. The recording of the ECG (RJG-4124; Nihon Kohden) was started 1 min before reperfusion. ECG were recorded for almost 5 min and at 10, 15, 20, and 30 min after reperfusion. If VF occurred, ECG was recorded. Coronary flow was measured in the preischemic period and 3, 5, 10, 20, and 30 min after reperfusion.
The rats were assigned to six groups: control (n = 12), SOD group (n = 14), and catalase group (n = 12), and three h-thioredoxin groups (n = 12 in each group). In the control group, the hearts were perfused with Krebs-Henseleit buffer alone throughout the experiment. In the SOD and catalase groups, the hearts were perfused first with Krebs-Henseleit solution alone and then followed by Krebs-Henseleit solution containing SOD (8 × 104 IU/L) or catalase (1 × 106 IU/L), respectively, from 5 min before reperfusion. The dosages of SOD (8 × 104 IU/L) and catalase (1 × 106 IU/L) were determined according to a previous report (9). In the three h-thioredoxin groups (TRX-I group, 0.001 mg/min of h-thioredoxin; TRX II group, 0.01 mg/min; TRX-III, 0.1 mg/min), h-thioredoxin infusion was started an aortic line at 0.5 ml/min by a syringe pump (201B, Atom Medical, Tokyo, Japan) from 5 min before reperfusion. To avoid the denaturation of h-thioredoxin by oxygen, we dissolved h-thioredoxin in Krebs-Henseleit buffer during the period of preischemic stabilization and infused this solution into the aortic line.
Creatine phosphokinase (CPK), lactate dehydrogenase (LDH), and lactate levels in the coronary effluents were measured 2 min before onset of regional ischemia. Total amounts were also measured during early (5-min) reperfusion and during 30-min reperfusion.
Recombinant SOD was provided by Nihon Kayaku (Tokyo, Japan). Catalase was purchased from Sigma Chemical (St. Louis, MO, U.S.A.). Recombinant h-thioredoxin, produced by the Basic Research Laboratory of Ajinomoto (Kawasaki, Japan), was provided by that company. The cDNA of h-thioredoxin was expressed in E. coli, and h-thioredoxin protein in the bacterial cells was purified by ion-exchange chromatography. The purified protein was completely reduced by treatment with dithiothreitol and was >99% pure. The content of bacterial endotoxin was determined to be <4 pg/mg protein.
All values are mean ± SD except for the incidence of VF. The error bars in the figures show the SD of the mean. Statistical analyses were performed on a Macintosh computer PowerBook Duo 210, using StatView 4.0 (serial number XS10133, Abacus Concepts, Berkeley, CA, U.S.A.). Differences in the incidence of VF between groups were compared by the chi-square test, with Fisher's exact p-values. The mean values were compared by one-way analysis of variance (ANOVA) or by Kruskal-Wallis nonparametric analysis. If data had a normal distribution, ANOVA was used. Any significant group effect detected by ANOVA was reanalyzed with Fisher's protected least significant difference (PLSD) test to characterize significant differences between groups. If data did not have a normal distribution, nonparametric analysis was used. Any significant group effect determined by Kruskal-Wallis analysis was reanalyzed by the Mann-Whitney test to determine differences between groups. p-Values <0.05 represented significant differences.
Concentration of h-thioredoxin
There were no significant differences between the groups in body weight or coronary flow (data not shown). The concentration of h-thioredoxin calculated from the coronary flow is shown in Fig. 1; values at 3-min reperfusion were 0.0136 ± 0.0028 μM in the TRX-I group, 0.123 ± 0.031 μM in the TRX-II group, and 1.33 ± 0.32 μM in the TRX-III group.
Findings during early (3-min) reperfusion
Incidence of VF. The incidence of VF was 75% (9 of 12) in the control group, 43% (6 of 14) in the SOD group, 33% (4 of 12) in the catalase group, 67% (8 of 12) in the TRX-I group, 8% (1 of 12) in the TRX-II group, and 33% (4 of 12) in the TRX-III group (Fig. 2). In the SOD and catalase groups, the incidence of VF was reduced, but there was no significant difference as compared with that in the control group (p = 0.13 and p = 0.056, respectively). The incidence of VF in the TRX-II group was significantly less than that in the control group (p < 0.01), but there was no significant difference between the TRX-II and the SOD groups (p = 0.08), nor was there a significant difference between the TRX-III and the control groups (p = 0.056).
Duration of sinus rhythm. The duration of sinus rhythm was compared by nonparametric analysis (Fig. 3). Values in the control, SOD, catalase, TRX-I, TRX-II, and TRX-III groups were 24 ± 39, 25 ± 23, 79 ± 74, 20 ± 30, 65 ± 49, and 55 ± 61 s, respectively. SOD had no effect on the duration of sinus rhythm. The duration in the catalase group was not significantly longer than the duration in either the control group or the SOD group. The duration in the TRX-II group was significantly longer than the duration in the control group (p < 0.05), but not significantly longer than that in the SOD group. There was no difference between the control and the TRX-III group.
Findings throughout 30-min reperfusion
Incidence of VF. The first VF episode occurred within 10 min of reperfusion in all hearts. The incidence was 83% (10 of 12) in the control group, 79% (11 of 14) in the SOD group, 42% (5 of 12) in the catalase group, 83% (10 of 12) in the TRX-I group, 25% (3 of 12) in the TRX-II group, and 50% (6 of 12) in the TRX-III group (Fig. 2). Catalase significantly reduced the incidence as compared with the control value (p < 0.05). The incidence of VF in the TRX-II group was significantly different from that in the control (p < 0.01) and the SOD groups (p < 0.05).
Time to onset of VF. Differences between the groups in the time to onset of VF were compared by nonparametric analysis (Fig. 4). Values in the control, SOD, catalase, TRX-I, TRX-II, and TRX-III groups were 66 ± 38, 162 ± 118, 97 ± 91, 93 ± 68, 213 ± 183, and 137 ± 141 s, respectively. There was a significant difference between the control and the SOD groups (p < 0.01). However, there was no significant difference between the control and TRX-II groups (p = 0.063) due to the small numbers of hearts with VF in the TRX-II group.
Gaussian-distributed arrhythmia score. To determine arrhythmia, we used a Gaussian-distributed arrhythmia score (24)(Table 1). The score was applied in a hierarchical manner; a single value was given to each heart, this being the highest possible score. We defined fatal VF as long-lasting VF (>5-min duration). The arrhythmia scores in the control, SOD, catalase, TRX-I, TRX-II, and TRX-III groups were 6.3 ± 2.8, 5.4 ± 2.8, 4.2 ± 3.7, 6.3 ± 2.8, 2.9 ± 2.5, and 4.5 ± 2.9, respectively (Fig. 5). The scores were compared by nonparametric analysis. Catalase reduced the arrhythmia score, but the difference between the control and the catalase groups was not significant. There were significant differences between the control and the TRX-II groups (p < 0.01) and between the SOD and the TRX-II groups (p < 0.05).
There were no significant differences among groups in CPK, LDH, and lactate in the coronary effluent before ischemia, during early (5-min) reperfusion, or during the entire reperfusion period (data not shown). Thioredoxin of ≈1 μM had no adverse effects on the hearts.
Oxygen free radicals are generated by a variety of sources, e.g., xanthine oxide (2), leukocytes (3), arachidonic acid (4), catecholamines (25), or mitochondria (26). They are considered a contributory factor in the genesis of reperfusion-induced arrhythmia (8). These potentially cytotoxic species, including superoxide anion, hydrogen peroxide, and hydroxy radical, are believed to be formed during the early moments of reperfusion (5,6). They may exacerbate ischemia-induced injury by promoting unfavorable oxidative changes in membrane lipids and ion pumps.
These changes, which may include modification of membrane sodium-calcium exchange (27), calcium transporting ability (28), and sodium-potassium exchange (29), would be expected to increase the susceptibility of the myocardium to electrophysiological perturbations, which in turn may promote the genesis of arrhythmias. Systems that generate free radicals can cause changes in transmembrane action potential characteristics and enhance susceptibility to arrhythmias (30).
Yodoi and colleagues (12) first reported ATL in 1974. HTLV-I, the etiologic agent of ATL, was the first human retrovirus discovered (13,14). Overexpression of the interleukin-2 receptor (IL-2R) α chain is considered to be one of the characteristics of T-cells transformed by HTLV-I (31). However, the leukemic T-cells from some ATL patients do not require exogenous IL-2 for their growth in vitro (32), and most ATL cells and cell lines do not proliferate in response to IL-2 (31,33). There are no abnormalities in the IL-2R α-chain gene in ATL cells, and this gene is continuously activated in HTLV-I-infected T-cells (33,34).
ADF identified as a cytokine-like factor in the culture supernatant of ATL cell lines (15,16), was shown to induce IL-2R α-chain expression in the human large granular lymphocyte cell line, YT (35) and to act as an autocrine substance in ATL cells (36,37). Tagaya and co-workers (38) determined the amino acid sequence of ADF. ADF is a polypeptide consisting of 104 amino acids, has a molecular weight of ≈12,000 daltons, and is similar to the bacterial coenzyme thioredoxin (20).
Thioredoxin is a small, ubiquitous protein with two redox-active half-cysteine residues in an exposed active center, having the consensus amino acid sequence: -Cys-Gly-Pro-Cys-. Thioredoxin is found in many different prokaryotes and eukaryotes and appears to be present in all living cells. E. coli thioredoxin has been well characterized as a hydrogen donor for ribonucleotide reductase (20). E. coli thioredoxin was also shown to have protein disulfide-isomerase-like activity; i.e., it has a reactivating effect on denatured enzymes through the formation of protein disulfide (39). Because ADF has been confirmed to be a human homologue of thioredoxin (18,19), we use the term human thioredoxin rather than ADF in the present report.
h-Thioredoxin expression can be induced by a variety of stresses, including x-ray and ultraviolet irradiation, hydrogen peroxide, and mitogens (40,41). h-Thioredoxin also regulates the cytotoxic activity of tumor necrosis factor (42). Mitsui and colleagues (21) reported that h-thioredoxin reduced hydrogen peroxide. h-Thioredoxin is believed to protect living cells from cytotoxicity caused by oxygen free radicals, and its beneficial effect on reperfusion injury in ischemic lung models has been reported (22,23).
Bernier and associates (9) reported that the dose-response characteristics of SOD described an asymmetric U-shaped curve and that the optimal dose of SOD required to reduce reperfusion-induced arrhythmias was 8 × 104 IU/L. Using this optimal dose of SOD (8 × 104 IU/L) throughout the experiment, they noted the incidence of VF to be significantly decreased, from the control value of 87% to 27% (p < 0.05). Late administration of SOD (8 × 104 IU/L), from 2 min before reperfusion, reduced the incidence of VF to 60%, i.e., this administration had relatively little effect. Early administration of catalase reduced the incidence of VF in a linear dose-dependent manner from its control value of 87 to 7% at 1 × 106 IU/L (p < 0.05). Late administration of this dose (1 × 106 IU/L) reduced the incidence of VF from its control value of 87 to 27% (p < 0.05) (9).
Koerner and Dage (43) reported that SOD shifted the occurrence of reperfusion-induced VF to longer durations of ischemia without affecting the peak incidence of VF. They noted that SOD significantly reduced the incidence of reperfusion-induced VF when regional ischemia lasted 8 min. However, when regional ischemia lasted ≥15 min, the incidence of VF in the SOD group was higher than that in the control group (43).
Shuter and colleagues (6) reported the effect of oxygen free radical scavengers in a study using electron spin resonance spectroscopy. After 15-min global ischemia, control hearts exhibited a maximal burst of N-tert-butyl-α-phenylnitrone (PBN) adducts during the first minute (when signal intensity increased to about three times that of the baseline value), subsequently decreasing to the baseline value. SOD-treated hearts exhibited a maximal burst of PBN adducts after 10-min reperfusion (when signal intensity increased to about twice that of the baseline value). Catalase-treated hearts showed no evidence of a burst of free radical production at any time during reperfusion. The study of Shuter and colleagues (6) suggested that superoxide was indirectly toxic and that hydrogen peroxide was an important reactive oxygen species that mediated reperfusion-induced myocardial injury.
The study of Shuter and colleagues (6) prompted us to adopt a much longer reperfusion period than that used by most investigators. We administered recombinant SOD (rSOD), catalase, or h-thioredoxin from 5 min before reperfusion (late administration). During the early (3-min) reperfusion, rSOD (8 × 104 IU/L) and catalase (1 × 106 IU/L) reduced the incidence of VF from the control value of 75 to 43 and to 33%, respectively, but the differences were not significant. These findings were similar to those reported for late administration by Bernier and associates (9). Throughout 30-min reperfusion, rSOD had little effect but catalase significantly reduced VF. The time to onset of VF in the SOD group was significantly longer than that in the control group. Our study suggested that rSOD (8 × 104 IU/L) did not prevent the reperfusion-induced arrhythmias but did delay their occurrence. h-Thioredoxin, at a concentration of ≈0.1 μM (the TRX-II group), showed significant protection against reperfusion-induced arrhythmias throughout the reperfusion. h-Thioredoxin, at a concentration of ≈1 μM (the TRX-III group), showed protection similar to that in the catalase group. The findings of Shuter and colleagues (6) support our findings in this study.
Because xanthine oxidase is located primarily in endothelial cells (44,45), generated oxygen free radicals can cause endothelial injury after reperfusion. Exogenous h-thioredoxin may eliminate hydrogen peroxide (21) through interaction with cell membrane components such as thioredoxin reductases (46). Alternatively, hydrogen peroxide released from the endothelial cells may be directly catabolized by a thioredoxin-dependent process. Nakamura and co-workers (47) reported that h-thioredoxin inhibited the endothelial cytotoxicity induced by activated neutrophils or hydrogen peroxide. Iwata and associates (48) reported that exogenous h-thioredoxin enhanced L-cystine transport in phytohemagglutinin (PHA) blasts. Because L-cystine transport is a limiting step in glutathione synthesis, thioredoxin increases intracellular GSH and may act indirectly in the thiol oxidation system. Kim and co-workers and Chae and associates purified a 25-Kd enzyme from yeast (49) and from rat brain (50); this enzyme prevented the damage induced by the thiol oxidation system. They named this protein “thiol-specific antioxidant” (49,50). However, their further study (51) showed that this enzyme uses thioredoxin as an immediate hydrogen donor for hydrogen peroxide and various alkyl hydroperoxides. Therefore, they renamed this enzyme “thioredoxin peroxidase.” Because thioredoxin peroxidase is ubiquitous and abundant in mammalian tissues, it could provide a major pathway for the elimination of hydrogen peroxide together with glutathione peroxidase (51). Study of the target molecules of thioredoxin is in progress to clarify the mechanism of action of h-thioredoxin in protection against reperfusion injury.
rSOD (8 × 104 IU/L) delayed the onset of VF but did not prevent arrhythmias. Catalase (1 × 106 IU/L) significantly reduced the incidence of VF. h-Thioredoxin protected against reperfusion-induced arrhythmias; a concentration of ≈0.1-μM was the most effective both in reducing the incidence of VF and in maintaining sinus rhythms.
Acknowledgment: We thank Dr. Yukio Chiba, Second Department of Surgery, Fukui Medical College, for instruction regarding the isolated rat heart models and Miss Etsuko Hatanaka, Department of Cardiovascular Surgery, Kyoto University, for help with the experiments. We also thank Mr. Akira Mitsui and Mr. Tadashi Hirakawa, Basic Research Laboratory of Ajinomoto Company (Kawasaki, Japan).
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