ARTICLE |
Correspondence to: Y. James Kang, Dept. of Medicine, U. of Louisville School of Medicine, 511 South Floyd St., MDR 530, Louisville, KY 40202. E-mail: yjkang01@athena.louisville.edu
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Summary |
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Previous studies have described a cardiac-specific, catalase-overexpressing transgenic mouse model that was used to study myocardial oxidative injury. This study was undertaken to demonstrate cellular and subcellular localization of catalase in the hearts of transgenic mice. By the light microscopic immunoperoxidase method, we found that the overexpressed catalase was exclusively localized in cardiomyocytes. The ratios of immunoreactive cardiomyocytes in the heart were quite different among three transgenic lines examined but agreed with the elevated levels of catalase activity. In the cardiac blood vessels, positive cells were found in the walls of pulmonary veins and the vena cava, which consist of cardiomyocytes, but not in the pulmonary arteries, aorta, or cardiac valves. The electron microscopic immunogold method revealed that the elevated catalase was in sarcoplasm, nucleus, and peroxisomes, but not in mitochondria. In contrast to these distributions, catalase in the non-transgenic cardiomyocytes was in peroxisomes only. In addition, the number and size of peroxisomes in the transgenic cardiomyocytes were markedly increased, but no other ultrastructural changes were observed in comparison with those of non-transgenic mice. These results demonstrated that the elevated catalase in transgenic mouse heart is localized in cardiomyocytes and is distributed to peroxisomal and extraperoxisomal, but not mitochondrial, compartments. (J Histochem Cytochem 48:585594, 2000)
Key Words: catalase, immunocytochemistry, myocardium, transgenic mice
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Introduction |
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Catalase (oxidoreductase, EC1.11.1.6) is a major enzyme involved in the decomposition of hydrogen peroxide (
Several studies have been undertaken to examine subcellular localization of catalase in myocardial tissues. It has been known that catalase is localized in peroxisomes of cardiomyocytes (
Regardless of the subcellular localization, catalase activity in the heart is very low in comparison with other tissues. For example, the catalase activity per gram of heart tissue is about 2% that of liver in rats and mice (
Production of reactive oxygen species by doxorubicin occurs mainly in mitochondria and cytosol. It is therefore important to know the cellular and subcellular localization of the elevated catalase in the hearts of transgenic mice in order to gain a comprehensive understanding of the role of catalase in cardiac protection against oxidative injury.
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Materials and Methods |
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All chemicals and reagents were obtained from Sigma Chemical (St Louis, MO) unless otherwise stated.
Animals
Cardiac-specific catalase-overexpressing transgenic mice and non-transgenic controls, aged 8 weeks and weighing about 25 g, were used for this study. The production of the transgenic mice and their characterization were described in detail previously (
Tissue Preparation
The hearts of anesthetized mice were perfused in situ as described previously (
Light Microscopic Immunocytochemistry
The heart tissue was cut into about 3-mm-thick slices, which were fixed with 10% formalin in PBS 0.01 M, pH 7.4, for 20 hr. The tissue slices were dehydrated in graded ethanol and embedded in Paraplast. Tissue sections of 5 µm were cut and mounted on silanized slides. Sections were treated with 3% H2O2 in PBS for 10 min to quench endogenous peroxidase activity. Sections were then incubated in 5% goat serum in PBS for 20 min to reduce nonspecific binding. After tapping the excess goat serum solution, sections were incubated overnight at 4C with a rabbit anti-catalase antibody (Calbiochem; La Jolla, CA) diluted 1:1000, then incubated for 20 min in biotinylated goat anti-rabbit IgG (Zymed Laboratories; San Francisco, CA), followed by incubation with HRPstreptavidin (Zymed) for 20 min. The antibody binding sites were visualized by incubating the tissue sections with DABH2O 2 solution provided by a DAB kit (Zymed). Finally, sections were counterstained in 0.5% methyl green, dehydrated, and mounted.
To test the specificity of the anti-catalase antibody, an immunoblot analysis was performed. Briefly, heart tissues were homogenized and aliquots containing 10 µg protein were fractionated by SDS-PAGE. After transfer to nitrocellulose membrane, the membranes were incubated with the rabbit anti-catalase diluted 1:2000, then with the HRP conjugate of goat anti-rabbit IgG. An ECL detection system was used to visualize the membranes. A single binding band was identified, which corresponds to the molecular weight of catalase.
Electron Microscopic Immunocytochemistry
Left ventricles were cut into pieces of about 1 mm3 and fixed in 2% freshly depolymerized paraformaldehyde with 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, at 4C for 2 hr. After rinsing in sodium cacodylate buffer, the samples were partially dehydrated with 50% and 70% ethanol and embedded in LR White. Ultrathin sections were cut on an LKB ultratome and collected on gold grids. The ultrathin sections were incubated with the rabbit anti-catalase antibody (1:250 dilution) overnight at 4C. After rinsing in 0.01 M PBS (pH 8.2), the ultrathin sections were incubated in protein Agold (10-nm gold particles; British BioCell, Cardiff, UK) diluted in 0.01 M PBS (pH 8.2) for 2 hr. The ultrathin sections were then rinsed in distilled water and counterstained with uranyl acetate and lead citrate. The labeled ultrathin sections were observed with a Philips transmission electron microscope.
Quantitative Analysis
The number of cardiomyocytes that were immunoreactive to the conjugated anti-catalase antibody on the tissue slides was counted and expressed as percentage of total cardiomyocytes. The number of peroxisomes in cardiomyocytes on the immunogold-labeled ultrathin sections was counted and expressed as number per 100 µm2 cytoplasmic area. The diameter of peroxisomes was measured and the difference between the transgenic and non-transgenic cardiomyocytes was analyzed by Student's t-test. At least 50 fields from each slide were counted at x600 and x10,000 magnification for the light and electron microscopic examination, respectively.
Immunocytochemical Controls
The controls were conducted by (a) preadsorption of the rabbit anti-catalase antibody solution with an excess amount of catalase for 1 hr before incubation and (b) substitution of the rabbit anti-catalase with nonimmune rabbit serum.
Identification of Cardiomyocytes in Cardiac Blood Vessels
A monoclonal antibody against -sarcomeric actin (Clone 5C5, isotype mouse IgM; Sigma) was used to identify cardiomyocytes in the walls of cardiac blood vessels because the
-sarcomeric actin is a specific protein that is present only in cardiac myocytes. Tissue sections were incubated with the antibody overnight at 4C, then with biotinylated rabbit anti-mouse IgM (Zymed), followed by alkaline phosphatasestreptavidin (Zymed). A red chromogen of New Fuchsin was used to visualize the binding sites.
Catalase Activity
Heart tissues were homogenized in 1% Triton X-100 in the assay buffer described below using a variable-speed tissue tearer (Biospec Products; Racine, WI) at about 20,000 rpm for 30 sec on ice. The homogenates were centrifuged at 6000 x g at 4C for 20 min. The supernatant was diluted with 1.5 volumes of the assay buffer (50 mM KH 2PO4/50 mM Na2HPO4, pH 7.0). The enzyme activity was determined by the method described by
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Results |
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Catalase activities in the hearts of the three transgenic lines are shown in Table 1. The results obtained from light microscopic immunocytochemistry to identify cell populations that contain high levels of catalase activities in the heart are presented in Fig 1. In contrast to the negative immunocytochemical reactions of the tissue slides obtained from the non-transgenic mouse heart (Fig 1A and Fig 1B), highly immunoreactive cells were found in the myocardium of all three transgenic lines (Fig 1C1H). The immunoreactivity was exclusively localized in the cardiomyocytes. The subcellular staining sites were mainly on the cytoplasm. However, nuclear staining was also found in all three transgenic lines. The ratios of the positive cells in the hearts were quite different among the three transgenic lines but agreed with the elevated activities of catalase in these transgenic lines. Further detailed examination revealed that in hearts containing catalase activity about 15-fold higher than normal, immunoreactive cells were present in both atrium and ventricle but occupied only about 25% of the total cell populations (Fig 1C and Fig 1D). In hearts containing 60-fold more catalase activity, a large number of positive cells were observed in both atrium and ventricle (53% of the cell populations were positive; Fig 1E and Fig 1F ). In hearts with 600-fold more catalase activity, almost all cardiomyocytes were positive (Fig 1G and Fig 1H). In the cardiac blood vessels, the antibody-stained cells were found in the walls of pulmonary veins and the vena cava (Fig 2A and Fig 2B), which consist of cardiomyocytes, as identified by the staining of the antibody against -sarcomeric actin ( Fig 2E), a specific actin protein in cardiomyocytes. Positive cells were not found in pulmonary arteries, aorta, or cardiac valves (Fig 2C and Fig 2D), which are not composed of cardiomyocytes (Fig 2F).
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To confirm the specificity of catalase expression in the hearts of transgenic mice, catalase immunoreactivities in the kidneys and livers of transgenic mice were compared with those in non-transgenic mice. As shown in Fig 3, similar staining patterns were observed in both kidney and liver between the transgenic (myocardial catalase activity 600-fold higher than normal) and non-transgenic mice. In the kidney, the staining of the proximal tubules in the superficial cortex was weak, and the distal tubules and all the glomeruli were negative ( Fig 3A and Fig 3C ). Strong staining was found on the cytoplasm of proximal tubules in the juxtamedullary cortex (Fig 3B and Fig 3D). The tubules of Henle's loops and collecting ducts in the medulla were negative. Liver tissue showed moderate staining, specifically in the hepatocytes (Fig 3E and Fig 3F).
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Electron microscopic immunocytochemistry was then applied to identify the subcellular localization of catalase in the transgenic heart. A few peroxisomes could be observed in the cardiomyocytes of non-transgenic mice (Fig 4A), and the gold particles that identify catalase were found only in peroxisomes (Fig 4B and Fig 4C). In the transgenic mice, no obvious ultrastructural changes were found, except that the number of peroxisomes markedly increased from 3.2 ± 1.8/100 µm2 in non-transgenic myocardium to 9.0 ± 3.4/100 µm2 in the transgenic myocardium that contained catalase activities about 60-fold higher than normal (Fig 5A and Fig 5D). The size of the peroxisomes was also significantly enlarged to 0.43 ± 0.12 µm (p<0.01) in the transgenic myocardium in comparison to 0.29 ± 0.09 µm in the non-transgenic control. Peroxisomes and myofibers showed strong labeling, whereas the mitochondria were totally negative (Fig 5B). The gold labeling in the nucleus was moderate (Fig 5C ). Strong labeling was also observed on the sarcoplasm between the bundles of myofibers and in the margin area where the sarcoplasm was plentiful (Fig 5E).
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Discussion |
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Light microscopic immunocytochemical examination of the cellular localization of catalase in the hearts of transgenic mice revealed that all of the immunoreactive cells were localized in the tissues that consisted of cardiac myocytes, including atrium, ventricle, pulmonary vein, and vena cava. The tissues that are not composed of cardiac myocytes, such as pulmonary artery, aorta, and all the valves of the heart, were not reactive to the anti-catalase antibody. This result demonstrates that the elevated catalase driven by the transgene, which contains a rat catalase cDNA that is linked behind the mouse -cardiac myosin heavy chain promoter (
Immunogold labeling and electron microscopic examination revealed that the elevated catalase activities were localized in peroxisomal and extraperoxisomal compartments. Extraperoxisomal catalase was found in both the sarcoplasm and the nucleus, but not in mitochondria. An important observation is that, in the immunoreactive cardiomyocytes of the transgenic myocardium, the number and size of peroxisomes dramatically increased. Among the positive cells, however, there were marked differences in the ratio of peroxisomal to extraperoxisomal catalase activity. In addition, some cells showed an increase in both the number and the size of peroxisomes that contain high levels of catalase activity, whereas others showed more increased numbers than enlarged size of peroxisomes, or vice versa.
Catalase has been considered to be a peroxisomal marker enzyme and has been used as an indicator of peroxisomal proliferation (
The heart tissue was much less immunoreactive to the anti-catalase antibody than were the kidney and liver in the non-transgenic mice. Furthermore, the immunogold labeling showed that catalase was present only in the matrix of peroxisomes, and the number of peroxisomes in the cardiomyocytes is very small in the non-transgenic mice. Therefore, the weak staining of the non-transgenic myocardium observed by immunocytochemical light microscopy agreed with the small number of peroxisomes in the cardiomyocytes observed by electron microscopy and with the low catalase activities determined by the enzymatic assay.
Mitochondrial localization of catalase in rat cardiomyocytes has been reported only in one study (
Although the transgenic catalase was not localized in mitochondria, which are critical organelles for production of reactive oxygen species from doxorubicin, the extramitochondrial localization of this enzyme in the transgenic mouse heart appears to be highly effective in protecting against the cardiotoxicity of doxorubicin. This is evidenced by the results obtained from the present study and those shown in our previous studies (
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Acknowledgments |
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Supported in part by National Institutes of Health grant CA68125 , by a US Department of Agriculture grant #9604531, and by the Jewish Hospital Foundation, Louisville, Kentucky. YJK is a university scholar at the University of Louisville.
We thank Donald Mosley and Cathie Caple for technical assistance.
Received for publication September 17, 1999; accepted January 25, 2000.
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