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Ann Thorac Surg 1999;68:1266-1271
© 1999 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Myocardial protection with endogenous overexpression of manganese superoxide dismutase

^a First Department of Surgery, Osaka University, Osaka, Japan
^b Institute for Cellular and Molecular Biology, Osaka University, Osaka, Japan

Address reprint requests to Dr Matsuda, First Department of Surgery, Osaka University, 2-2 Yamada-Oka, Suita, Osaka, 565 Japan;
e-mail: matsuda{at}surg1.med.osaka-u.ac.jp

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Superoxide dismutase (SOD) is a potent candidate for myocardial protection against ischemia-reperfusion injury; however, its clinical significance by means of exogenous administration remains controversial.

Methods. To determine a role of endogenously overexpressed manganese SOD (Mn-SOD) in myocardial tolerance, rat hearts were transfected with Mn-SOD gene (group M) or no gene (group C) through intracoronary infusion of hemagglutinating virus of Japan (HVJ) liposome. Each group was divided into two subgroups to be subjected to ischemia-reperfusion using Langendorff apparatus with (subgroups M+ and C+) or without (M- and C-) administration of recombinant SOD.

Results. Mn-SOD overexpression was confirmed in M with ELISA, activity measurement, and immunohistochemistry. The highest recoveries of maximum and minimum dp/dt and the least creatine phosphokinase (CPK) leakage were observed in M+. These recoveries were higher in M- than in C- and C+.

Conclusions. Thus, endogenous overexpression of Mn-SOD improved myocardial tolerance and its protective effect was enhanced by exogenous administration of SOD. These results suggest a possible strategy for myocardial protection with SOD: a combination of endogenous introduction through gene transfer with exogenous administration.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
There is no doubt that the development of myocardial protection methods in cardiac surgery has brought substantial improvements in cardiac surgery results; however, even the latest method shows limited efficacy, especially in critical cases with severe heart failure or hypertrophy. Accordingly, establishment of further advanced methods is desirable. Superoxide dismutase (SOD) is an effective protein for self-preservation because it scavenges free radicals in reperfusion injury [1, 2]_. Therefore, many studies have been done to investigate whether SOD could be a reagent for myocardial protection [1, 2]. However, the significance of exogenously administered SOD is still controversial, probably because it can not sufficiently permeate into cardiomyocytes, especially into mitochondria, where SOD can show its ability to scavenge superoxide [3, 4].

Mammals have three distinct SODs: copper and zinc-SOD (Cu, Zn-SOD), mitochondrial manganese SOD (Mn-SOD), and extracellular SOD [5, 6]. Of them, Mn-SOD reportedly exists abundantly in mitochondria to attenuate reperfusion injury by dismutating the superoxide occurring in mitochondria [1, 2]. Accordingly, Mn-SOD is expected to play a significant role in protecting against reperfusion injury of cardiomyocytes, which have an abundance of mitochondria. We therefore hypothesized that endogenously overexpressed Mn-SOD in the myocardium, rather than exogenously administered recombinant SOD, can be more effective for scavenging superoxide occurring in cardiomyocytes, thus resulting in attenuation of reperfusion injury.

We have developed a gene transfection method for rat hearts involving intracoronary infusion of HVJ (hemagglutinating virus of Japan)-liposome, which provides overexpression of a protein with little toxicity and high efficiency [7, 8]. Here, we investigated the above-mentioned hypothesis with this method, demonstrating a significant gene transfection of Mn-SOD. Additionally, we examined whether exogenous SOD could enhance the protective effect of endogenously introduced SOD.

	Material and methods

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Animal care
All studies were performed with the approval of the Committee for Animal Research Ethics, Osaka University Medical School. The investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Gene construction
Full-length human Mn-SOD cDNA [9] was provided by Drs Kunitaka Hirose and Kouji Matsushima (Laboratory of Molecular Immunoregulation, Biological Response Modifiers Program, Division of Cancer Treatment, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD). It was cloned at the EcoRI site of pcDNA3, which has a cytomegalovirus promoter (Invitrogen Corporation, San Diego, CA).

Preparation of HVJ-liposome
The preparation of HVJ-liposome has been previously described [7, 8]. Briefly, 10 mg of a lipid mixture (phosphatidylserine, phosphatidylcholine, and cholesterol) was deposited on the side of a flask by removing the tetrahydrofuran in a rotary evaporator. The dried lipid was then hydrated in 200 µL of balanced salt solution (137.0 mmol/L NaCl, 5.4 mmol/L KCl, 10.0 mmol/L Tris-HCl; pH 7.6) containing a DNA (200 µg)-HMG1 (high mobility group 1 nuclear protein, 64 µg) complex. A liposome-DNA-HMG1 complex suspension was prepared by vortexing, sonication, and shaking to produce the liposome. The liposome suspension was incubated with 30,000 hemagglutinating units of HVJ, which was inactivated by ultraviolet irradiation, first at 4°C and then at 37°C. Finally, 4 mL of the sucrose gradient layer containing HVJ-liposome was collected for use.

Gene transfection by intra-coronary infusion of HVJ-liposome
Gene transfection was performed on the hearts of Sprague-Dawley rats (250 g) as described before [7, 8]. Briefly, the hearts were arrested with cold crystalloid cardioplegia (4°C) and removed under anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneal) and anticoagulation with heparin (200 USP units, intravenous). The hearts from group M (n = 23) were infused with 1 mL of HVJ-liposome containing pcDNA3 with human Mn-SOD cDNA via the coronary artery, with the venae cavae, pulmonary arteries, and veins ligated. The control hearts (group C, n = 23) were infused with the same volume of HVJ-liposome containing pcDNA3 but without the Mn-SOD gene. After incubation on ice for 10 min, the hearts were then heterotopically transplanted into the abdomens of recipient rats (300 g) of the same strain. The cold ischemic time was 39 ± 9 minutes. These rats were sacrificed on the fourth day after gene transfection, thus allowing the introduced gene to express proteins stably and giving enough time for rat intrinsic SOD induced by the surgical stress to disappear [10].

Immunohistochemical analysis
Seven rats from each group (group M or C) were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal) and anticoagulated by intravenous injection of heparin. These hearts were removed and their left ventricles were divided into two parts quickly. One part was immediately frozen in an embedding medium, OCT compound (Miles Inc, Diagnostics Division, Elkhart, IN), with liquid nitrogen for immunohistochemical analysis. The embedded samples were cut into thin sections (5 µm) and immersed in phosphate buffered saline containing Triton X-100. After blocking with 5% fetal bovine serum, the sections were incubated first with a 1:100 dilution of monoclonal anti-human Mn-SOD antibody (The Binding Site, Birmingham, England), followed by incubation with a 1:180 dilution of FITC-conjugated goat anti-mouse IgG monoclonal antibody (Medical & Biological Laboratories Co, Ltd, Nagoya, Japan). The sections were observed with a fluorescence microscope (PM-30, Olympus, Tokyo, Japan).

Mn-SOD content and activity
The other part of the left ventricle was immediately frozen in liquid nitrogen, homogenized thoroughly, centrifuged, sonicated on ice, and then centrifuged at 8,000 rpm for 5 minutes. The supernatant of these samples was used to measure Mn-SOD content and activity after determination of protein concentration with the bicinchoninic acid method. Mn-SOD content was determined by ELISA with a Mn-SOD ELISA system (Nippon-Yushi Co, Ltd, Tokyo, Japan) [11]. Mn-SOD activity in the myocardium was determined by means of the NBT method [10, 11]. Briefly, the supernatant was added to the reaction mixture of NBT with xanthine-xanthine oxidase, and the SOD activity in the supernatant measured colorimetrically in the form of inhibitory activity toward blue formazan formation by SOD in the reaction mixture. To determine Mn-SOD activity, the assay was performed in the presence of potassium cyanide (1 nM) to inhibit Cu, Zn-SOD activity. The Mn-SOD content and activity were corrected for the protein concentration of the sample.

Myocardial tolerance to ischemia-reperfusion injury
The remaining 16 rats of group M or C were divided into two subgroups (M+, M-, C+, and C-; n = 8 in each subgroup). These hearts were used for the perfused heart experiment to evaluate myocardial tolerance to ischemia and following reperfusion injury (subgroups M+ and C+) or without (subgroups M- and C-) administration of recombinant SOD throughout reperfusion period.

The recipient rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal) and anticoagulated by intravenous injection of heparin. The transfected hearts were quickly excised from the abdomen of the recipient rats and perfused with modified Krebs-Henseleit buffer (120.0 mmol/L NaCl, 4.5 mmol/L KCl, 20.0 mmol/L NaHCO₃, 1.2 mmol/L KH₂PO₄, 1.2 mmol/L MgCl₂, 2.5 mmol/L CaCl₂, and 10.0 mmol/L glucose; gassed with 95% O₂ + 5% CO₂ to obtain pH 7.4 at 37°C) at a pressure equal to 1 mH₂O by means of a Langendorff apparatus. A thin-wall latex balloon was inserted into the left ventricle through the left atrium to monitor left ventricular pressure and control left ventricular volume. After 30 minutes of stabilization, heart rate (HR), left ventricular developed pressure (LVDP), maximum dp/dt (max dp/dt), minimum dp/dt (min dp/dt), and coronary flow (CF) were measured with LV diastolic pressure stabilized at 10 mm Hg. The hearts were then subjected to global ischemia at 37°C for 30 minutes, followed by 60 minutes of reperfusion. The balloon was deflated during ischemia. For the hearts from subgroups M+ and C+, recombinant human Cu, Zn-SOD (150 IU/min; Sigma, St. Louis, MO) was administered in an intracoronary fashion throughout the reperfusion. On the other hand, for the hearts from subgroups M- and C-, no recombinant SOD was administered during either ischemia or reperfusion. Coronary effluent was collected for the initial 10 minutes of reperfusion to measure creatine phosphokinase (CPK) leakage as a marker of cardiomyocyte damage and mitochondrial aspartate aminotransferase (m-AST) leakage as a marker of mitochondrial damage [12].

Statistical analysis
All values are expressed as means ± SEM. The difference in the data for content or activity of Mn-SOD between the two groups was determined with Student’s t-test. The differences in the data for cardiac parameters or enzyme leakage among the four subgroups were determined with Fisher’s Protected Least Significant Difference post-hoc test. All analyses were performed with the aid of the Statview v4.0 statistical package (Abacus Concepts Inc, Berkeley, CA). A probability value (p) of less than 0.05 was considered statistically significant.

	Results

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Expression of Mn-SOD
To determine whether endogenous overexpression of Mn-SOD introduced by gene transfection plays a role in enhancement of myocardial ischemic tolerance, rat hearts were transfected with HVJ-liposome containing human Mn-SOD gene (group M) or no gene (group C) through the coronary artery. Consequently, significantly higher levels both of Mn-SOD content and of Mn-SOD activity were detected in group M (Fig 1). The content of Mn-SOD as measured by enzyme-linked immunosorbent assay (ELISA) was larger in group M than in group C (53.7 ± 4.5 versus 20.9 ± 2.1 µg/mg protein, p < 0.0001, n = 7 in each group). The activity of Mn-SOD measured with the nitroblue tetrazolium (NBT) method was also higher in group M than in group C (1.13 ± 0.07 versus 0.43 ± 0.04 U/mg protein, p < 0.0001, n = 7 in each group).

View larger version (24K): [in this window] [in a new window]   Fig 1. Content and activity of Mn-SOD. Mn-SOD content and activity were measured with ELISA and nitroblue tetrazolium method respectively. Both of them were larger in group M than in group C. M, Mn-SOD gene transfected heart; C, control transfected heart. *p < 0.0001 vs group C, n = 7 in each group.

View larger version (115K): [in this window] [in a new window]   Fig 2. Immunohistochemical analysis of Mn-SOD. Distribution of Mn-SOD expression was investigated immunohistochemically by using anti-human Mn-SOD monoclonal antibody and FITC conjugated second antibody. It was observed that apparent and extensive overexpression of Mn-SOD in cardiomyocytes was distributed globally in the heart from group M as compared with that from group C. Left panel, Mn-SOD gene transfected; right panel, control transfected heart.

View larger version (45K): [in this window] [in a new window]   Fig 3. Myocardial tolerance to ischemia-reperfusion injury. The isolated hearts from the four subgroups were subjected to 30 minutes of normothermic ischemia followed by 60 minutes of reperfusion to evaluate myocardial tolerance among the four subgroups. Recoveries of LVDP and coronary flow were shown in (A) as well as those of maximum and minimum dp/dt in (B). CPK and m-AST leakage for the initial 10 minutes of reperfusion were compared among the four subgroups (C). (C-, control transfected heart without recombinant SOD administration; C+, control transfected heart with recombinant SOD administration during reperfusion; M-, Mn-SOD gene transfected heart without SOD administration; M+, Mn-SOD gene transfected heart with SOD administration during reperfusion.) *p < 0.05 vs subgroup C-; p < 0.05 vs subgroup C+; #p < 0.05 vs subgroup M-; n = 8 in each subgroup. All values are expressed as mean ± SEM.

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
We demonstrated that gene transfection with Mn-SOD could efficiently introduce a high level of expression of Mn-SOD into rat heart, resulting in reduction of myocardial ischemia-reperfusion injury. This indicates the significant role of Mn-SOD itself in protecting against ischemia-reperfusion injury in myocardium. Furthermore, it was demonstrated that, in crystalloid perfusion, gene transfection with Mn-SOD enhanced myocardial tolerance more effectively than did administration of recombinant SOD. This may suggest that endogenously overexpressed Mn-SOD in the myocardium can be more effective for scavenging superoxide occurring in cardiomyocytes than exogenously administered recombinant SOD.

It is still unknown where the superoxide radical is generated—intracellular or extracellular—and therefore where SOD would be most beneficial. Major primary sources of superoxide in myocardial reperfusion injury are reportedly migrated leukocytes into the ischemic area (NADPH oxidase), mitochondria (electron transfer system), endothelial cells (xanthine-xanthine oxidase), and others [13, 14]. It appears to be difficult for recombinant SOD, which is a large protein with a molecular weight of 32,000 (Cu, Zn-SOD), 80,000 (Mn-SOD), or 135,000 (EC-SOD) to permeate into cardiomyocytes, especially into mitochondria [3, 4]. Therefore, exogenously administered SOD can scavenge only free radicals derived from outside the cardiomyocytes (within the vasculature) but is not useful for dismutating superoxide occurring within cardiomyocytes. In the crystalloid perfusion used in this study, the role of superoxide occurring within cardiomyocytes is enhanced because of the absence of leukocytes. We demonstrated that, in this situation, gene transfection with Mn-SOD resulted in better recovery of cardiac function after ischemia, as well as less damage of the myocardium, than exogenously administration of recombinant SOD did. In addition, m-AST leakage after ischemia and reperfusion, which is a marker of mitochondrial damage [12], was attenuated in the gene transfected heart with Mn-SOD. Accordingly, we speculate that this protective effect of overexpressed Mn-SOD by gene transfection could be mainly a result of efficient scavenging of the superoxide occurring in cardiomyocytes, perhaps in mitochondria of cardiomyocytes. Furthermore, superoxide derived from endothelial cells, which is another major source of free radicals, may also be scavenged by overexpressed Mn-SOD in the endothelial cells by gene transfection, because Mn-SOD is considered to be overexpressed in coronary endothelial cells as well as in cardiomyoctes as a result of our gene transfection method [15]. The better recovery of CF in the Mn-SOD gene transfected hearts in the present study may be caused by this protective effect of overexpressed Mn-SOD in endothelial cells.

In addition, we demonstrated that administration of recombinant SOD enhanced myocardial tolerance in the control transfected heart. This protective effect of administered SOD is thought to be achieved mainly by scavenging superoxide from outside of cardiomyocytes, which may be generated by the high pO₂ required during crystalloid perfusion or derived from endothelial cells. This cardioprotective effect of SOD administration was also observed in the Mn-SOD gene-transfected heart. This suggests that endogenous overexpression of Mn-SOD by gene transfection could not completely dismutate all of the superoxide, because it might not be sufficient for scavenging the superoxide derived from outside of cardiomyocytes. Therefore, we consider that a combination of gene transfection with Mn-SOD (against superoxide occurring in cardiomyocytes) and administration of SOD (against superoxide occurring outside of cardiomyocytes) may lead to a potent strategy to protect the myocardium from reperfusion injury.

The level of H₂O₂ in the SOD-overexpressing situation may be important for myocardial protection, because H₂O₂, which is generated by SOD, is more harmful than O₂^-. However, this issue is still controversial. Schmidt and associates [16] have demonstrated that H₂O₂ level is higher in SOD overexpressing cells than in wild type cells. On the other hand, Teixeira and associates [17] have shown that excess of SOD decreases the O₂^- level without increasing the endogenous formation of H₂O₂. They commented that O₂^- dismutation mediated by excess SOD prevents the formation of higher H₂O₂ levels by other reactions, probably including reaction mediated by catalase. In our study, we demonstrated that overexpression of SOD could decrease the myocardial damage in ischemia-reperfusion. If high levels of H₂O₂ were generated by excess SOD and it was not efficiently scavenged by other reactions, it would be unlikely that myocardial tolerance could be improved by overexpressed SOD. We, therefore, suspect that this observation might support the latter opinion, even though we did not measure the level of H₂O₂ in the present study.

Several methods to enhance Mn-SOD activity endogenously have been reported. Yen and associates [18] developed transgenic mice overexpressing Mn-SOD to investigate the protective role of Mn-SOD against adriamycin-induced cardiac toxicity. Maulik and associates [19] demonstrated that injection of recombinant interleukin-1 reduces myocardial ischemia reperfusion injury through enhancing self-preservation proteins including Mn-SOD. Yamashita and associates [10] and Das and associates [20] showed that induction of Mn-SOD by ischemic preconditioning plays a pivotal role in the acquisition of tolerance to ischemia. Although these methods are expected to result in a new myocardial protection method, they still display some problems for clinical application, such as duration and timing of Mn-SOD expression or harmful side effects in other organs. Our gene transfection method can be expected to overexpress Mn-SOD for a long period (longer than 2–3 weeks) with little cell toxicity both in heart and in other organs. [7, 8]. In addition, our method for gene transfection includes the procedure of heart transplantation, which is very close and applicable to the clinical settings in heart transplantation. Therefore, gene transfection may be a potential strategy for myocardial protection with Mn-SOD against ischemia and reperfusion injury, although many investigations are needed before it can be applied in clinical settings.

In conclusion, we have shown that endogenous overexpression of Mn-SOD by gene transfection improved myocardial tolerance to ischemia-reperfusion injury and its efficacy was enhanced by exogenously administrated SOD. This suggests a possible application of a novel strategy for myocardial protection with SOD: endogenous overexpression as a result of gene transfection supported by exogenous administration.

	Acknowledgments

 
This work was supported by a Grant-in-Aid for Scientific Research in Japan. Dr Ken Suzuki is a Research Fellow of the Japan Society for the Promotion of Science. We would like to thank Dr Kunitaka Hirose and Dr Kouji Matsushima, Laboratory of Molecular Immunoregulation, Biological Response Modifiers Program, Division of Cancer Treatment, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD, for providing the full length of human Mn-SOD cDNA and Dr Nobushige Yamashita, First Department of Internal Medicine and Department of Pathophysiology, Osaka University, Osaka, Japan, for his assistance with the measurement of myocardial Mn-SOD content and activity.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Hajime Ichikawa Hikaru Matsuda Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suzuki, K. Articles by Matsuda, H. Search for Related Content PubMed PubMed Citation Articles by Suzuki, K. Articles by Matsuda, H.