Lecithinized copper, zinc-superoxide dismutase ameliorates prolonged hypoxia-induced injury of cardiomyocytes

Free Radical Biology & Medicine, Vol. 29, No. 1, pp. 34 – 41, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(00)00290-2

Original Contribution LECITHINIZED COPPER, ZINC-SUPEROXIDE DISMUTASE AMELIORATES PROLONGED HYPOXIA-INDUCED INJURY OF CARDIOMYOCYTES HIROYOSHI NAKAJIMA,* NOBUKAZU ISHIZAKA,* MISAKO HANGAISHI,* JUN-ICHI TAGUCHI,* JOHBU ITOH,† RIE IGARASHI,‡ YUTAKA MIZUSHIMA,‡ RYOZO NAGAI,* and MINORU OHNO* *Department of Cardiovascular Medicine, University of Tokyo, Graduate School of Medicine, Tokyo, Japan; †Laboratories for Structure and Function Research, School of Medicine, Tokai University Bohseidai Isehara, Kanagawa, Japan; ‡Institute of Medical Science, St. Marianna University, Kawasaki, Japan (Received 1 September 1999; Revised 7 March 2000; Accepted 20 April 2000)

Abstract—Recent studies have suggested that prolonged hypoxia results in increased production of reactive oxygen species in cardiomyocytes, which leads to apoptosis of these cells. We previously showed that lecithinized recombinant human copper, zinc-superoxide dismutase (rhSOD) showed increased bioavailability through greater membrane affinity and a longer half-life than unmodified SOD. The purpose of this study was to investigate whether lecithinized SOD plays a protective role against hypoxic injury in cardiomyocytes. Cultured rat cardiomyocytes incubated with lecithinized SOD (100 U/ml), unmodified SOD (100 U/ml), or vehicle alone were subjected to hypoxia for up to 72 h. Lecithinized SOD, but not unmodified SOD, was successfully delivered intracellularly, which was verified by Western blot and confocal laser-scanning microscopy. Treatment of cells with lecithinized SOD significantly suppressed hypoxia-induced cell damage. Since lecithinized SOD also suppressed hypoxia-induced DNA fragmentation, the improved cell survival provided by lecithinized SOD is thought to be mediated by its antiapoptotic effect. In summary, lecithinization resulted in a facilitated rhSOD delivery into cultured cardiomyocytes, which reduced mortality of cardiomyocytes exposed to prolonged hypoxia. © 2000 Elsevier Science Inc. Keywords—Apoptosis, Cardiomyocyte, Superoxide, Hypoxia, Lecithinized superoxide dismutase, Reactive oxygen species, Superoxide dismutase, Free radicals

INTRODUCTION

cytes [7,8]. Since reactive oxygen species (ROS) induce myocardial apoptosis [9], increased ROS may be responsible for hypoxia-induced apoptosis of cardiomyocytes. However, the effectiveness of antioxidant therapy in preserving cardiac function in the setting of myocardial ischemia has been controversial [7,10 –14]. Such effectiveness may mainly depend on how easily these drugs are intracellularly delivered. It is possible that the ineffectiveness of SOD treatment in preserving ventricular function can be attributed to limited intracellular drug delivery [13,14]. To facilitate the intracellular delivery of SOD, modification of this enzyme has been tested, such as by liposome entrapment [15]. Modification of SOD with phosphatidylcholine derivatives is another means of facilitating cellular delivery [16,17]. We previously showed that recombinant human Cu, Zn-SOD (rhSOD) covalently bound to 2-(4-hydroxycarbonylbutyroyl) lysophosphatidylcholine (lecithinized SOD) improved cell

Recent studies have demonstrated that the mechanisms of myocardial death following coronary ligation with or without reperfusion include apoptosis as well as necrosis [1]. Hypoxia-induced apoptosis of cardiomyocytes has also been shown in primary cultures of neonatal cardiomyocytes [2,3]. The expressions of bcl-2, an inhibitor of apoptosis, and bax, an inducer of apoptosis, are regulated in the ischemic heart [4 – 6]. However, the proximity of apoptosis following myocardial hypoxia or ischemia is yet to be completely elucidated. Recently, it was shown that hypoxia increases mitochondrial superoxide generation with a subsequent increase of H2O2 in cardiomyoAddress correspondence to: Dr. Minoru Ohno, University of Tokyo, Graduate School of Medicine, Department of Cardiovascular Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; Tel: ⫹81 (3) 38155411, ext. 33016; Fax: ⫹81 (3) 5800-8806; E-Mail: [email protected]. 34

Lecithinized SOD and myocardial hypoxia

membrane affinity with a longer half-life compared to its unmodified counterpart [16,17]. Thus, the purpose of this study was to investigate whether lecithinization improved intracellular rhSOD delivery into cultured cardiomyocytes, and whether lecithinized SOD could ameliorated hypoxia-induced myocardial injury. MATERIALS AND METHODS

Synthesis of lecithinized SOD Lecithinized SOD was synthesized according to a method reported previously [16,17]. Purification was performed using ion-exchange column chromatography with Q-Sepharose FF (Pharmacia LKB Biotechnology Inc.; Piscataway, NJ, USA), and isoelectrofocusing was used to identify the target substance.

35

containing protease inhibitors (10 ␮g/ml aprotinin, 0.1 mmol/l PMSF, and 10 ␮g/ml leupeptin). Total protein content was determined by Bradford assay against a BSA standard [20]. Equal amounts of protein were loaded onto 15% SDS polyacrylamide gels and were subsequently blotted onto polyvinylidine difluoride membranes (Immobilon-P; Millipore Co.; Bedford, MA, USA). The membrane was hybridized with anti-SOD monoclonal antibody (Ube Kosan; Yamaguchi, Japan) (1:500 dilution), followed by horseradish peroxidase– conjugated secondary antibody (Jackson ImmunoResearch; West Grove, PA, USA) (1:2000 dilution). The ECL Western blotting system (Amersham Life Sciences; Braunschweig, Germany) was used for detection. The bands were visualized and quantified using luminoanalyzer, LAS-1000 (Fuji Photo Film; Tokyo, Japan). Fractionation of cell components

Preparation of neonatal rat cardiomyocyte-rich culture Neonatal ventricular myocytes were isolated from 1-d-old Wistar rats as described previously [18,19]. Briefly, removed ventricles were minced and placed in Hanks’ balanced salt solution with 80 U/ml collagenase (collagenase type 2; Worthington Biochemical Co.; Lakewood, NJ, USA). The cells were resuspended in Medium 199 supplemented with 6% fetal bovine serum, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, and 54% balanced salt solution (116 mmol/l NaCl, 1.0 mmol/l NaH2PO4, 0.8 mmol/l MgSO4, 26.2 mmol/l NaHCO3, 0.9 mmol/l CaCl2, and 5 mmol/l glucose). Following preplate dissociation, the resultant suspension was plated at a density of 1.5 ⫻ 105 cells/cm2 in a culture dish. Four days later, the culture media were exchanged with media containing 20% Medium 199, 20% Ham’s F-12 medium, 54% balanced salt solution, 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin, 0.6 mmol/l KCl, 270 mmol/l L-arginine, and 5 mg/ml insulin-transferrinselenium. The validity of the above-described procedure was confirmed by the observation of spontaneous beating in more than 90% of the cell population. Exposure of cultured cardiomyocytes to hypoxia Hypoxia of the cultured cardiomyocytes was achieved through the use of an anaerobic system (BBL GasPack Pouch System; Becton Dickinson and Co.; Cockeysville, MD, USA), that enabled O2 depletion at a concentration of ⬍2% within 2 h. Protein purification and Western blot analysis The protein was isolated with lysis buffer (50 mmol/l HEPES, 5 mmol/l EDTA, 50 mmol/l NaCl; pH 7.5)

Cells were scraped off the plates and pelleted by centrifugation at 800 ⫻ g for 10 min. Then, cells were resuspended in 1.2 ml of cold solution (0.25 mol/l sucrose, 10 mmol/l Tris-HCl; pH 7.4). Cells were homogenized with 10 strokes and centrifuged at 900 ⫻ g for 10 min. The pellet containing the nuclear fraction and the unbroken cells was removed. The supernatant was centrifuged at 5000 ⫻ g for 10 min. The mitochondrial pellet was resolved in 50 ␮l of cold solution, and the supernatant was centrifuged at 100,000 ⫻ g for 60 min. The resultant supernatant was used as cytosolic fraction. Measurement of SOD activity A nondenaturing gel assay for SOD activity was performed as described previously [21] with minor modifications. Samples (20 ␮g) were loaded onto a nondenaturing gel consisting of a 5% stacking gel (pH 6.8) and a 12% running gel (pH 8.8). The gels were first immersed in 2.4 mmol/l nitro blue tetrazolium (Wako Pure Chemical Industries; Osaka, Japan) for 20 min and then in 0.03 mmol/l riboflavin, 280 mmol/l TEMED in 50 mmol/l potassium phosphate buffer (pH 7.8) for 15 min in the dark. The gels were then washed in deionized water and illuminated under fluorescent light until clear zones of SOD activity were evident. Cell viability assay Cells exposed to hypoxia for up to 72 h were mixed with 50 ␮L trypan blue (Gibco; Grand Island, NY, USA). The unstained cells were considered to be viable, and the number of these cells was expressed as a percentage of the total cell number.

36

H. NAKAJIMA et al.

Measurement of hypoxia-mediated cell damage Lactate dehydrogenase (LDH) concentration in cultured medium was quantified as the biochemical indicator for cell damage using LDH assay kit (Wako Pure Chemical Industries, Osaka, Japan) [22]. Kinetic determination of LDH was based on the spectrophotometric method of Wroblewski and LaDue [23]. The activity of LDH was measured by the reductive ability of NADH, which was produced as lactate was oxidized to pyruvate. Immunocytochemical staining and confocal laser-scanning microscopy The cells were grown on a collagen cell disk (Iwaki Glass Co.; Tokyo, Japan). After fixation and permeabilization, cells were pretreated with 0.3% hydrogen peroxide in 70% methanol to exhaust endogenous peroxidase activity. The cells were preincubated with 10% horse serum, and then incubated with anti-SOD antibody (1:500 dilution) followed by incubation with biotinylated secondary antibody. After treatment of the cells with avidin-biotinylated HRP complex (Elite ABC kit; Vector Laboratories; Burlingame, CA, USA), the antigen was visualized with a 3,3-diamino-

benzidine tetrahydrochloride (Dako Japan; Kyoto, Japan) system. Counterstaining was performed with methyl green. The oxidized 3,3-diaminobenzidine was osmified in 0.05% osmic acid to enhance the detection signals for confocal laser-scanning microscopy (LSM410; Carl Zeiss; Jena, Germany) [24].

DNA ladder assay After trypsinization, the cells were incubated at 37°C for 24 h in a lysis buffer (10 mmol/l Tris (pH 8.0), 100 mmol/l NaCl, 25 mmol/l EDTA, 0.5% SDS, and 1.0 mg/ml Proteinase K), and subsequently treated with RNase. Equal amounts of genomic DNA samples were loaded on 1.4% agarose gels containing 0.01% SYBR Green (Molecular Probes; Eugene, OR, USA).

Statistics Data are expressed as mean ⫾ SEM. Differences were analyzed by one-way ANOVA followed by Fisher’s posthoc comparison. A value of p ⬍ .05 was considered statistically significant.

Fig. 1. Uptake of exogenous SOD by cardiomyocytes. Cells were cultured under normoxia or hypoxia with or without SOD (100 U/ml). Western blot analysis using anti-SOD antibody (Ube Kosan) and SOD activity gel assay are shown. The expression of SOD was extremely high in lecithinized SOD (Pc-SOD)–treated cells compared to control and unmodified SOD (Um-SOD)-treated cells independent of whether they were cultured under normoxia or hypoxia. The activity of SOD increased in lecithinized SOD-treated, but not in unmodified SOD-treated cells.

Lecithinized SOD and myocardial hypoxia

Fig. 2. Intracellular delivery of SOD into cardiomyocytes. (A) Immunocytochemistry using anti-SOD antibody followed by confocal laser-scanning microscopy. The following are shown: (a) cell cultured under normoxia, (b) unmodified SOD (Um-SOD)-treated cell cultured under normoxia, (c) lecithinized SOD (Pc-SOD)-treated cell cultured under normoxia, (d) cell cultured under hypoxia, (e) unmodified SOD-treated cell cultured under hypoxia, (f) lecithinized SOD-treated cell cultured under hypoxia. Immunocytochemistry was performed using anti-SOD antibody. The antigen was visualized with a 3,3-diaminobenzidine tetrahydrochloride system. Counterstaining was performed with methyl green (shown in green). Oxidized 3,3-diaminobenzidine was osmified in 0.05% osmic acid to enhance the detection signals for confocal laser-scanning microscopy. Overt intracellular uptake of SOD was seen in lecithinized SOD-treated cells cultured both under normoxia and under hypoxia (shown in red). (B) Intracellular localization of SOD in cardiomyocytes detected by Western blot. The expression of SOD was extremely high in cytosolic and mitochondrial fractions of lecithinized SOD (Pc-SOD)-treated cells whether they were cultured under normoxia or hypoxia. Cs ⫽ cytosolic fraction; Mt ⫽ mitochondrial fraction.

37

38

H. NAKAJIMA et al. RESULTS

Intracellular delivery of lecithinized and unmodified SODs Western blot demonstrated a strong band in the lane of lecithinized SOD-treated cells regardless of whether they were exposed to hypoxia, while no bands were seen in the lanes of control and unmodified SOD-treated cells (Fig. 1, upper panel). As expected, SOD activity in lecithinized SOD-treated cells was clearly increased compared to the untreated control and unmodified SODtreated cells (Fig. 1, lower panel). To further confirm the intracellular delivery of lecithinized SOD, immunocytochemistry and confocal laserscanning microscopy were performed. Abundant intracellular SOD was revealed in lecithinized SOD-treated cells cultured both under normoxic and hypoxic conditions, but not in unmodified SOD-treated cells (Fig. 2A). Western blotting suggested that lecithinized SOD was delivered to the cytosol and mitochondria (Fig. 2B). Effect of lecithinized SOD on hypoxia-induced cell damage The effects of lecithinized and unmodified SODs on hypoxia-induced cell death were examined. The cells were exposed to hypoxia for up to 72 h in the presence of lecithinized SOD (100 U/ml), unmodified SOD (100 U/ml), free lecithin derivative (2-(4-hydroxycarbonylbutyroyl) lysophosphatidylcholine; 1.6 ␮g/ml), or vehicle alone. The total amount of lecithin derivative contained in 100 U/ml of lecithinized SOD was equal to that of 1.6 ␮g/ml of free lecithin derivative. Hypoxia induced myocardial cell death in a time-dependent manner, while no significant cell death was noted in the serum-depleted group cultured under normoxia. Lecithinized SOD, but neither unmodified SOD nor free lecithin derivative, significantly suppressed cell death after 24 h of hypoxia (Fig. 3A). Hypoxia caused release of LDH into culture media in a time-dependent manner. Lecithinized SOD reduced LDH release compared to unmodified SOD or free lecithin derivative (Fig. 3B). Effect of lecithinized SOD on apoptotic cell death due to prolonged hypoxia We then examined whether hypoxia-induced apoptosis was suppressed by treatment with lecithinized SOD. Cardiomyocytes exposed to hypoxia for 48 h showed increased nuclear fragmentation. Treatment of cells with lecithinized SOD significantly suppressed nuclear fragmentation, but unmodified SOD did not produce a similar effect (Figs. 4A and B). DNA laddering was less

Fig. 3. Cell damage of rat neonatal cardiomyocytes subjected to prolonged hypoxia. (A) Survival curves for cells subjected to hypoxia. Cells were subjected to hypoxia for the indicated period in the presence of lecithinized SOD (Pc-SOD, 100 U/ml), unmodified SOD (Um-SOD, 100 U/ml), free lecithin derivative (2-(4-hydroxycarbonylbutyroyl) lysophosphatidylcholine, Free Pc, 1.6 ␮g/ml), or vehicle alone. Cell survival was assayed by trypan blue exclusion. Cells cultured under normoxia showed no significant cell death, while hypoxia induced myocardial cell death in a time-dependent manner. Lecithinized SOD suppressed cell death after 24 h. (B) LDH release into culture media. Hypoxia caused release of LDH into culture media in a time-dependent manner. Lecithinized SOD reduced LDH release compared to unmodified SOD or free lecithin derivative. †,#,*p ⬍ .05, Pc-SOD-treated cells vs. the cells subjected to hypoxia, free Pc-treated cells, and Um-SOD-treated cells, respectively. ††,##,**p ⬍ .01, Pc-SOD-treated cells vs. the cells subjected to hypoxia, free Pc-treated cells, and Um-SOD-treated cells, respectively.

obvious in lecithinized SOD-treated cells than in unmodified SOD-treated cells as well as in control cells (Fig. 4C). Effects of lecithinized SOD on bcl-2 and bax protein expression Next, we examined the expression of apoptotic regulatory proteins in cells subjected to hypoxia. Western blot demonstrated the existence of SOD in the lecithinized SOD-treated cells after 24 h (Fig. 5A) and that bcl-2 was downregulated in the cardiomyocytes sub-

Lecithinized SOD and myocardial hypoxia

39

Fig. 4. Effect of lecithinized SOD on hypoxia-induced nuclear fragmentation. (A) Cells were exposed to hypoxia for 48 h in the presence of lecithinized SOD (Pc-SOD, 100 U/ml), unmodified SOD (Um-SOD, 100 U/ml), or vehicle alone. The nuclei were stained with Hoechst 33342. Arrows indicate typical nuclear fragmentation. (B) The percentage of cells with nuclear fragmentation was significantly lower in cells treated with lecithinized SOD compared with the control. †,*p ⬍ .01, Pc-SOD-treated cells vs. the cells subjected to hypoxia and Um-SOD–treated cells, respectively. (C) DNA ladder formation. Formation of DNA ladder was examined using 1.4% agarose gels. DNA laddering was less obvious in lecithinized SOD-treated cells than in unmodified SOD-treated cells.

jected to hypoxia after 48 h (Fig. 5B). In the presence of lecithinized SOD, the cells retained protein expression of bcl-2 at 48 h, whereas no such effect was noted in the presence of unmodified SOD. However, cells expressed downregulated levels of bcl-2 protein at 72 h even if treated with lecithinized SOD. The expression of bax was not changed in either group at any time points (data not shown). DISCUSSION

In the present study, we demonstrated that modification of rhSOD with a phosphatidylcholine derivative (lecithinized SOD) facilitated intracellular drug delivery into cultured cardiomyocytes. We also showed that hypoxia-induced cellular injury was ameliorated by lecithinized SOD, a drug for which an anti-apoptotic effect was suggested. Although an increased formation of oxygen radicals has been suggested as one cause of cardiac injury in-

duced by hypoxia or ischemia [7,8,25,26], the effectiveness of SOD in preserving cardiomyocytes in these settings has not been established [22,26,27]. It is possible that the ineffectiveness of SOD treatment in inhibiting ischemia-induced cardiac injury may be, at least partially, due to the high molecular weight of this molecule, which makes intracellular penetration unlikely [28]. In fact, in ischemia/reperfusion cardiac injury models, transgene of rhSOD limited infarct area compared to untreated control [29], although unmodified SOD did not have a similar cardioprotective effect [14,30 –32]. In the present study, therefore, in order to investigate whether SOD can limit hypoxia-induced myocardial injury, we used rhSOD covalently bound to 2-(4-hydroxycarbonylbutyroyl) lysophosphatidylcholine (lecithinized SOD). By this modification, the improved affinity to cell membrane of rhSOD has shown to be provided [16,17]. The first major finding of the present study was that lecithinization of rhSOD markedly improved intramyocardial rhSOD transport in comparison with unmodified

40

H. NAKAJIMA et al.

Fig. 5. Time course expression of SOD and bcl-2 protein in cardiomyocytes. (A) Expression of SOD. Abundant SOD uptake was maintained in lecithinized SOD (Pc-SOD)-treated cells for up to 72 h. Trace uptake of SOD was detected in unmodified-SOD (Um-SOD)-treated cells at 72 h. (B) Expression of bcl-2. After exposure to hypoxia, bcl-2 expression was decreased after 48 h of hypoxia. In the presence of lecithinized SOD (Pc-SOD, 100 U/ml), bcl-2 expression was retained at 48 h, whereas no such retention was noted with unmodified SOD (Um-SOD, 100 U/ml).

SOD. In addition, we confirmed that SOD activity was well preserved after the intracellular transport of lecithinized SOD (Fig. 1). In previous papers, we demonstrated improved intracellular transport of lecithinized SOD in vascular endothelial cells in vitro [17] and in various tissues in vivo [16]. This study further demonstrated that lecithinization of rhSOD improved drug transport into cultured cardiomyocytes. The electron transport chain in mitochondria is an important intracellular site for the generation of the superoxide anion. Our data suggested that lecithinized SOD also localized at the mitochondria. We next demonstrated that successful intracellular delivery of rhSOD resulted in a significant suppression of the mortality of cardiomyocytes exposed to prolonged hypoxia. Recently, it was reported that the treatment of chick cardiomyocytes with antioxidative drugs attenuated a hypoxia-induced increase in reactive oxygen spe-

cies and a decrease in contractility of these cells [7]. Together with these findings, it is suggested that exposure to hypoxia results in the increased formation of oxygen radicals in cardiomyocytes, and that antioxidative treatment exerts a protective effect in preserving viability and function of these cells exposed to hypoxia. We also investigated the expression of pro- (bax) and antiapoptotic (bcl-2) regulatory proteins in cells exposed to hypoxia. We found that expression of bax was unchanged in cardiomyocytes after exposure to hypoxia, which is consistent with a previous report [33]. We also found that expression of bcl-2 protein was downregulated after the exposure of cardiomyocytes to hypoxia. Treatment of cells with lecithinized SOD retained bcl-2 expression after 48 h of hypoxia, although unmodified SOD did not inhibit hypoxia-induced bcl-2 downregulation. Therefore, lecithinized SOD may have improved viability of cardiomyocytes subjected to prolonged hypoxia via retaining antiapoptotic protein expression, thereby suppressing apoptosis. The finding that lecithinized SOD, but not unmodified SOD, decreased hypoxia-induced DNA laddering or nuclear fragmentation (Fig. 4) supports this notion. In summary, lecithinization of rhSOD facilitated intracellular drug delivery into cultured cardiomyocytes. Treatment with lecithinized SOD, but not with unmodified SOD, improved the viability of cardiomyocytes exposed to prolonged hypoxia through inhibiting hypoxiainduced apoptosis of cardiomyocytes. Acknowledgements — We would like to thank Dr. Eisei Noiri and Dr. Tatsuya Shimizu for their constructive comments.

REFERENCES [1] Li, Q.; Li, B.; Wang, X.; Leri, A.; Jana, K. P.; Liu, Y.; Kajstura, J.; Baserga, R.; Anversa, P. Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy. J. Clin. Invest. 100:1991–1999; 1997. [2] Long, X.; Boluyt, M. O.; Hipolito, M. L.; Lundberg, M. S.; Zheng, J. S.; O’Neill, L.; Cirielli, C.; Lakatta, E. G.; Crow, M. T. P53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes. J. Clin. Invest. 99:2635–2643; 1997. [3] Tanaka, M.; Ito, H.; Adachi, S.; Akimoto, H.; Nishikawa, T.; Kasajima, T.; Marumo, F.; Hiroe, M. Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ. Res. 75:426 – 433; 1994. [4] Misao, J.; Hayakawa, Y.; Ohno, M.; Kato, S.; Fujiwara, T.; Fujiwara, H. Expression of bcl-2 protein, an inhibitor of apoptosis, and bax, an accelerator of apoptosis, in ventricular myocytes of human hearts with myocardial infarction. Circulation 94:1506 –1512; 1996. [5] Maulik, N.; Goswami, S.; Galang, N.; Das, D. K. Differential regulation of Bcl-2, AP-1, and NF-kappaB on cardiomyocyte apoptosis during myocardial ischemic stress adaptation. FEBS Lett. 443:331–336; 1999. [6] Kajstura, J.; Cheng, W.; Reiss, K.; Clark, W. A.; Sonnenblick, E. H.; Krajewski, S.; Reed, J. C.; Olivetti, G.; Anversa, P. Apoptotic and necrotic myocyte cell deaths are independent contrib-

Lecithinized SOD and myocardial hypoxia

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

uting variables of infarct size in rats. Lab. Invest. 74:86 –107; 1996. Duranteau, J.; Chandel, N. S.; Kulisz, A.; Shao, Z.; Schumacker, P. T. Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes. J. Biol. Chem. 273:11619 –11624; 1998. Vanden Hoek, T. L.; Becker, L. B.; Shao, Z.; Li, C.; Schumacker, P. T. Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J. Biol. Chem. 273:18092–18098; 1998. Li, P. F.; Dietz, R.; von Harsdorf, R. Superoxide induces apoptosis in cardiomyocytes, but proliferation and expression of transforming growth factor beta1 in cardiac fibroblasts. FEBS Lett. 448:206 –210; 1999. Watanabe, B. I.; Limm, W.; Suehiro, A.; Suehiro, G.; Premaratne, S.; McNamara, J. J. Failure of deferoxamine to reduce myocardial infarct size in a primate model of ischemia-reperfusion injury. J. Surg. Res. 55:537–542; 1993. Kobayashi, A.; Watanabe, H.; Ozawa, K.; Hayashi, H.; Yamazaki, N. Oxygen-derived free radicals related injury in the heart during ischemia and reperfusion. Japan Circ. J. 53:1122– 1131; 1989. Tanaka, M.; Stoler, R. C.; FitzHarris, G. P.; Jennings, R. B.; Reimer, K. A. Evidence against the “early protection-delayed death” hypothesis of superoxide dismutase therapy in experimental myocardial infarction. Polyethylene glycol-superoxide dismutase plus catalase does not limit myocardial infarct size in dogs. Circ. Res. 67:636 – 644; 1990. Li, Q.; Bolli, R.; Qiu, Y.; Tang, X. L.; Murphree, S. S.; French, B. A. Gene therapy with extracellular superoxide dismutase attenuates myocardial stunning in conscious rabbits. Circulation 98:1438 –1448; 1998. Flaherty, J. T.; Pitt, B.; Gruber, J. W.; Heuser, R. R.; Rothbaum, D. A.; Burwell, L. R.; George, B. S.; Kereiakes, D. J.; Deitchman, D.; Gustafson, N. Recombinant human superoxide dismutase (h-SOD) fails to improve recovery of ventricular function in patients undergoing coronary angioplasty for acute myocardial infarction. Circulation 89:1982–1991; 1994. Briscoe, P.; Caniggia, I.; Graves, A.; Benson, B.; Huang, L.; Tanswell, A. K.; Freeman, B. A. Delivery of superoxide dismutase to pulmonary epithelium via pH-sensitive liposomes. Am. J. Physiol. 268:L374 –380; 1995. Igarashi, R.; Hoshino, J.; Takenaga, M.; Kawai, S.; Morizawa, Y.; Yasuda, A.; Otani, M.; Mizushima, Y. Lecithinization of superoxide dismutase potentiates its protective effect against Forssman antiserum–induced elevation in guinea pig airway resistance. J. Pharmacol. Exp. Ther. 262:1214 –1219; 1992. Igarashi, R.; Hoshino, J.; Ochiai, A.; Morizawa, Y.; Mizushima, Y. Lecithinized superoxide dismutase enhances its pharmacologic potency by increasing its cell membrane affinity. J. Pharmacol. Exp. Ther. 271:1672–1677; 1994. Kinugawa, K.; Kohmoto, O.; Takahashi, T.; Serizawa, T. Effects of a nonpeptide angiotensin II receptor antagonist (CV-11974) on [Ca2⫹]i and cell motion in cultured ventricular myocytes. Eur. J. Pharmacol. 235:313–316; 1993. Kinugawa, K.; Shimizu, T.; Yao, A.; Kohmoto, O.; Serizawa, T.; Takahashi, T. Transcriptional regulation of inducible nitric oxide synthase in cultured neonatal rat cardiac myocytes. Circ. Res. 81:911–921; 1997. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248 –254; 1976.

41

[21] Beauchamp, C.; Fridovich, I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44:276 –287; 1971. [22] Qian, Z. M.; Xu, M. F.; Tang, P. L. Superoxide dismutase does protect the cultured rat cardiac myocytes against hypoxia/reoxygenation injury. Free Radic. Res. 27:13–21; 1997. [23] Wroblewski, F.; La Due, J. Lactic dehydrogenase activity in blood. Proc. Soc. Exp. Biol. Med. 90:210 –213; 1955. [24] Itoh, J.; Sanno, N.; Matsuno, A.; Itoh, Y.; Watanabe, K.; Osamura, R. Y. Application of confocal laser scanning microscopy (CLSM) to visualize prolactin (PRL) and PRL mRNA in the normal and estrogen-treated rat pituitary glands using nonfluorescent probes. Microsc. Res. Tech. 39:157–167; 1997. [25] Vanden Hoek, T. L.; Li, C.; Shao, Z.; Schumacker, P. T.; Becker, L. B. Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion. J. Mol. Cell. Cardiol. 29:2571–2583; 1997. [26] Przyklenk, K.; Kloner, R. A. Effect of oxygen-derived free radical scavengers on infarct size following six hours of permanent coronary artery occlusion: salvage or delay of myocyte necrosis? Basic Res. Cardiol. 82:146 –158; 1987. [27] Ziegelstein, R. C.; Zweier, J. L.; Mellits, E. D.; Younes, A.; Lakatta, E. G.; Stern, M. D.; Silverman, H. S. Dimethylthiourea, an oxygen radical scavenger, protects isolated cardiac myocytes from hypoxic injury by inhibition of Na(⫹)-Ca2⫹ exchange and not by its antioxidant effects. Circ. Res. 70:804 – 811; 1992. [28] Silverman, H. S.; Siddoway, L. A.; Yavin, Z.; Gonenne, A.; Weisfeldt, M. L.; Becker, L. C. Pharmacokinetics of recombinant human superoxide dismutase. J. Cardiovasc. Pharmacol. 16:107– 111; 1990. [29] Wang, P.; Chen, H.; Qin, H.; Sankarapandi, S.; Becher, M. W.; Wong, P. C.; Zweier, J. L. Overexpression of human copper, zinc-superoxide dismutase (SOD1) prevents postischemic injury. Proc. Natl. Acad. Sci. USA 95:4556 – 4560; 1998. [30] Hess, M. L.; Kukreja, R. C. Free radicals, calcium homeostasis, heat shock proteins, and myocardial stunning. Ann. Thorac. Surg. 60:760 –766; 1995. [31] Uraizee, A.; Reimer, K. A.; Murry, C. E.; Jennings, R. B. Failure of superoxide dismutase to limit size of myocardial infarction after 40 minutes of ischemia and 4 days of reperfusion in dogs. Circulation 75:1237–1248; 1987. [32] Gallagher, K. P.; Buda, A. J.; Pace, D.; Gerren, R. A.; Shlafer, M. Failure of superoxide dismutase and catalase to alter size of infarction in conscious dogs after 3 hours of occlusion followed by reperfusion. Circulation 73:1065–1076; 1986. [33] Webster, K. A.; Discher, D. J.; Kaiser, S.; Hernandez, O.; Sato, B.; Bishopric, N. H. Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53. J. Clin. Invest. 104:239 –252; 1999.

ABBREVIATIONS

BSA— bovine serum albumin FITC—fluorescein isothiocyanate PMSF—phenylmethylsulfonyl fluoride ROS—reactive oxygen species SOD—superoxide dismutase TEMED—N,N,N⬘,N⬘-tetramethylethylenediamine