Effect of Erythropoietin on bcl-2 Gene Expression in Rat Cardiac Myocytes After Traumatic Brain Injury M. Emir, K. Ozisik, K. Cagli, M. Misirlioglu, P. Ozisik, Z. Iscan, E. Yildirim, K. Kilinc, and E. Sener ABSTRACT The purpose of this study was to investigate whether erythropoietin (EPO) has an effect on the expression of bcl-2 in rat cardiac myocytes following experimental isolated traumatic brain injury (TBI). Forty-eight Wistar-Albino female rats were randomly allocated into eight groups. Groups AC and BC were controls; groups AS and BS were sham-operated animals. Groups A1 and B1 underwent head trauma without treatment. Groups A2 and B2, head traumas plus EPO intraperitoneally (1000 IU/kg); groups A3 and B3, the vehicle groups, head traumas and intraperitoneal albumin (0.4 ml/rat). The method of weight drop was used to produce impact trauma at 24 hours after injury. Samples obtained from the left ventricle were assayed for lipid peroxidation and bcl-2 gene expression using real-time quantitative polymerase chain reactions. Lipid peroxidation in the heart tissue was determined by the concentration of thiobarbituric acid reactive substances (TBARs). The results showed that administration of EPO significantly reduced the increase in lipid peroxidation by-products after moderate or severe trauma. The bcl-2 expression was significantly higher in EPO (A2 and B2) compared to trauma groups (A1 and B1) suggesting a protective effect. These findings suggest that EPO may play an important role in the expression of bcl-2 and decrease in TBARs—the end product of lipid peroxidation in myocytes—after moderate or severe TBI.
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ECENT STUDIES have identified multiple paracrine/ autocrine functions of erythropoietin (EPO) to coordinate local responses to injury by maintaining vascular autoregulation and attenuating both apoptotic and inflammatory causes of cell death.1 Apoptosis, which occurs by an active process via energy consumption, is involved in the etiology of myocytic lesions in cardiac diseases with or without an ischemic origin.2 A number of bcl-2 family proteins are expressed in cardiac myocytes, including bcl-2, bcl-xl, bad, bax, and bid. As in other cells bcl-2 family proteins are therefore key regulators of apoptosis in cardiac myocytes. The expression of various family members is likely to influence the rate of myocyte death.3–5 Since it is currently unknown whether EPO plays a role in myocardial tissue, we examined the effects of EPO on bcl-2 expression and lipid peroxidation in cardiac myocytes after moderate and severe TBI. MATERIALS AND METHODS The local Institutional Animal Care Committee approved the protocols used in this study. The rats were randomly allocated into eight groups: groups AC and BC were control groups (n ⫽ 6):
Tissue samples were obtained immediately after midline sternotomy; no head injury was performed; groups AS and BS were sham-operated groups (n ⫽ 6): scalp was closed after craniotomy; no trauma was induced; groups A1 and B1: trauma (n ⫽ 12): injury of 200 g-cm or 300 g-cm was produced; groups A2 and B2: EPO (n ⫽ 12): In addition to 200 g-cm or 300 g-cm injuries, r-Hu-EPO (1000 IU/kg; Eprex, Cilag AG, Zug, Switzerland) was administered intraperitoneally immediately after TBI; and groups A3 and B3: albumin (vehicle) (n ⫽ 12): injury with 200 g-cm or 300 g-cm accompanied by treatment with 0.4 mL albumin immediately administered intraperitoneally. Tissue samples were obtained at 24 hours after trauma in all groups.
From the Department of Cardiovascular Surgery (M.E., K.C., Z.I., E.S.), TYIH; the Department of Cardiovascular Surgery (K.O., E.Y.), Numune Education and Research Hospital, Metis Biotechnology Ltd (P.O.), Institute of Neurological Sciences and Psychiatry, and Department of Biochemistry (K.K.), Hacettepe University, Ankara, Turkey. Address reprint requests to Kanat Ozisik, Ankara Numune Education and Research Hospital, 4.cad 71, Sokak No. 8/3, Ankara 6550, Turkey.
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0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.11.101
Transplantation Proceedings, 36, 2935–2938 (2004)
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Surgical Procedure The surgical procedure was performed under general anesthesia induced by intramuscular 10 mg/kg xylasine (Bayer, Istanbul, Turkey) and 60 mg/kg ketamine hydrochloride (Parke Davis, Istanbul, Turkey). Forty-eight female Wistar-Albino rats, weighing 190 to 230 g, were placed in the prone position. Following a midline longitudinal incision, the scalp was dissected over the cranium and retracted laterally. Right frontoparietal craniectomies were performed lateral to the sagittal suture using a dental drill system. The dura was exposed and left intact. Rats were injured by dropping stainless steel rods (5 mm diameter, weighing 200 or 300 g) vertically through a calibrated tube from a height of 10 cm onto the exposed dura using the method of Allen.6 The scalp was closed with silk sutures. Body temperature continuously monitored during the procedure with a rectal thermometer was maintained at 37°C, using a heating pad and an overhead lamp. Rats were neither intubated nor ventilated between the brain injury or the heart sampling.
Sample Obtaining From the Heart Twenty-four hours after injury, rats reanesthetized with ketamine and xylasine, were placed supine on operating table. Samples for lipid peroxidation and bcl-2 gene expression were obtained from the heart. Thereafter the hosts were decapitated under general anesthesia. Heart samples were collected in randomly numbered containers for analysis by blinded observers.
Lipid Peroxidation Assay Lipid peroxidation in heart tissues was assessed using the thiobarbituric acid method of Mihara and Uchiyama.7 The samples thoroughly cleansed of blood were immediately frozen and stored at ⫺20°C for malondialdehyde assays. Half a milliliter of a tissue homogenate prepared in 10 volumes (w/v) of cold phosphate buffer (pH 7.4) was mixed with 3 mL of 1% H3PO4. After the addition of 1 mL of a 0.67% solution of thiobarbituric acid, the mixture was heated in boiling water for 45 minutes. After the color was extracted into n-butane, the absorption at 532 nm was measured versus tetramethoxypropane as the standard, yielding tissue lipid peroxide levels (nanomole per gram wet tissue).
EMIR, OZISIK, CAGLI ET AL ATGTCACGCACGATT and 5=TCACCCACACTGTGCCCAT, respectively. The selected TaqMan probe between the primers was fluorescence labeled at the 5= end with 6-carboxyfluorescein (FAM) as a reporter dye and at the 3= end with 6-carboxytetramethylrhodamine (TAMRA) as the quencer 5=-FAM-ATCCTGCGTCTGGACCTG GCT-TAMRA (Tibmolbiol, Germany).9,10 Every sample was run for -actin along with the same cycling protocol described above for bcl-2 gene expression.
Statistical Analysis All data were coded, recorded, and analyzed by using commercially available statistical software packages. All the continuous data are represented as mean values ⫾ SD. Parametric data were compared for differences between groups, by ANOVA with Bonferroni corrections for post hoc analyses.
RESULTS
Figures 1 and 2 show the levels of lipid peroxide measured in the cardiac myocyte samples. The mean level of lipid peroxide in the control and sham groups were 65.1 nmol/g tissue (⫾9.8 nmol/g SD) and 75.9 nmol/g tissue (⫾9.5 nmol/g SD), respectively. These results showed no statistically significant difference in lipid peroxidation levels between control and sham groups. Two hundred g-cm trauma or 300 g-cm traumas were found to produce significant elevations in lipid peroxidation. The mean level of lipid peroxide in the A1 and B1 groups were 105.6 nmol/g tissue (⫾2.0 nmol/g SD) and 108.2 nmol/g tissue (⫾6.2 nmol/g SD) respectively. Intraperitoneal EPO administration significantly decreased thiobarbituric acid reactive substances (TBARs) in groups A2 (P ⬍ .0001) and B2 (P ⬍ .0001) compared with trauma groups. The difference between trauma and vehicle-treated groups A3 (P ⫽ .048) and B3 (P ⫽ .405) were not statistically significant. Figures 3 and 4 show the bcl-2 expression ratios measured in the cardiac myocyte samples. Intraperitoneal ad-
Isolation of RNA and Synthesis of cDNA Samples immediately frozen in liquid nitrogen and then stored at ⫺80°C. Total RNA of each heart tissues isolated using a high pure RNA tissue kit (Roche Diagnostics, Germany) were assessed for RNA integrity electrophoretically verified with ethidium bromide staining and by an OD260/OD280 nm absorption ratio ⬎ 1.95. One microgram of total RNA was used for cDNA synthesis using first strand cDNA synthesis kit for RT-PCR (AMV) (Roche Diagnostics, Germany) according to the manufacturer’s protocol.
Quantitative Real-Time Polymarase Chain Reaction Analysis Real time quantitative polymerase chain reaction analysis for bcl-2 gene expression was performed as described previously,8 using a Light Cycler instrument (Roche Diagnostics). The cycling parameters were 2 minutes at 95°C for denaturation, 40 cycles of 15 seconds at 95°C, and 30 seconds at 60°C for amplification and quantification. -actin mRNA was quantified to adjust the amount of mRNA in each sample with the -actin probe and the primer set. The upstream and downstream primer sequences were 5=TCTTTA
Fig 1. Figure shows the effect of 200 g-cm TBI on heart TBARS levels. Treatment with EPO decreased TBARs levels indicating preservation of heart.
ERYTHROPOIETIN AND
BCL-2
GENE EXPRESSION
Fig 2. Figure shows the effect of 300 g-cm TBI on heart TBARS levels. Treatment with EPO decreased TBARs levels indicating preservation of heart.
ministration of EPO produced a significant increase in bcl-2 expression in groups A2 (P ⫽ .009) and B2 (P ⫽ .001) compared with the trauma groups, suggesting that EPO protects myocytes after moderate or severe TBI. Treatment with vehicle solution did not produce a significant increase in bcl-2 expression in cardiac myocytes in groups A3 (P ⫽ .1) and B3 (P ⫽ .1). DISCUSSION
Physical forces activate apoptosis and gene expression, but the mechanism is unknown. Apoptosis, is regulated by the expression of genes, such as fas, p53, c-myc, and bax.11,12 The bcl-2 protein, which is encoded by a gene involved in
Fig 3. Figure shows the effect of 200 g-cm TBI on heart bcl-2 expression ratios. Treatment with EPO increased bcl-2 expression ratios.
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Fig 4. Figure shows the effect of 300 g-cm TBI on heart bcl-2 expression ratios. Treatment with EPO increased bcl-2 expression ratios.
the 14:18 chromosomal translocation,13,14 is a cytosolic protein with a lipid anchoring domain that can target it to mitochondria or to the nucleus. Elevated levels of bcl-2 impede apoptosis, whereas high levels of bax stimulate it.15 Calvillo and his coworkers showed that EPO markedly prevents apoptosis of cultured adult rat cardiomyocytes subjected to hypoxia.1 Additional studies employing a rat model of coronary ischemia-reperfusion showed that the administration of EPO reduces cardiomyocyte loss. These observations not only suggest a potential therapeutic role for EPO in the treatment of myocardial infarction by preventing apoptosis and attenuating postinfarct deterioration in hemodynamic function, but also predict that EPO is likely a tissue protective cytokine.1 Overexpression of bcl-2 has been reported to induce a plethora of apoptosis-associated changes, such as alterations of cellular redox state (depletion of glutathione and generation of reactive oxygen species [ROS]) and plasma membrane changes.16 bcl-2 may act as an antioxidant, regulator of intracellular ion fluxes, protease inhibitor, and mitochondriotropic agent.17,18 In our study, the bcl-2 expression was significantly higher among EPO (A2 and B2) than trauma groups (A1 and B1), suggesting that EPO protected myocytes after moderate or severe injury. The production of ROS is known to increase after brain injury by ischemic or hemorrhagic stroke or trauma. ROSmediated lipid peroxidation is one of the major mechanisms of secondary damage.19 The generation of ROS and peroxidation of lipids are extremely fast reactions, which are generally measured by their end products, mostly TBARs, which are believed to be markers of cell damage indicating increased production of ROS and lipid peroxidation. Despite our follow-up period of only 24 hours, heart tissue lipid peroxidation levels were increased in both trauma groups (A1 and B1).
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We previously determined that injury with 140 g-cm was produced by the weight-drop technique resulted in gradual damage to the ultrastructure of cardiac myocytes.20 In this study, we did not observe any correlation between the degree of bcl-2 expression and the grade of injury. Finally, apoptosis is a complex process, involving a wide range of gene products at each stage. We used only two gene expression products, thus this study must be considered to be limited in scope. Further examinations of bad, bak, fas, and apoptosis itself are warranted. We conclude that EPO may play an important role in the expression of bcl-2 and to decrease TBARs, the end product of lipid peroxidation, in myocytes after moderate or severe TBI. The protective effect of EPO may result in better recovery, therefore achieving higher heart donation rates. REFERENCES 1. Calvillo L, Latini R, Kajstura J, et al: Proc Natl Acad Sci USA 100:4802, 2003 2. Szabolcs M, Michler R, Yang X, et al: Circulation 94:1665, 1996 3. Cook SA, Sugden PH, Clerk A: Circ Res 85:940, 1999
EMIR, OZISIK, CAGLI ET AL 4. von Harsdorf R, Li PF, Dietz R: Circulation 99:2934, 1999 5. Chen M, He H, Zhan S, et al: J Biol Bhem 276:30724, 2001 6. Allen AR: JAMA 57:878, 1911 7. Mihara S, Uchiyama M: Anal Biochem 86:271, 1978 8. Yamada Y, Watanabe Y, Zhang J, et al: Neuroscience 114:165, 2002 9. Peirson SN, Butler JN, Foster RG: Nucleic Acids Res 31:e73, 2003 10. Kalinina O, Lebedeva I, Brown J, et al: Nucleic Acids Res 25:1999, 1997 11. Oh SI, Kim IW, Jung HC, et al: Transplantation 71:906 –9, 2001 12. Song H, Conte JV, Foster AH, et al: J Heart Lung Transplant 18:744, 1999 13. Cleary ML, Skar J: Proc Natl Acad Sci USA 82:7439, 1985 14. Tsujimoto Y, Gorham J, Cossman J, et al: Science 229:1390, 1985 15. Hockenbery D, Nunez G, Milliman C, et al: Nature 348:334, 1990 16. Minn AJ, Velez P, Schendel SL, et al: Nature 385:353, 1997 17. Tron VA, Krajewski S, Klein-Parker H, et al: Am J Pathol 146:643, 1995 18. Zamzami N, Susin SA, Kromer G: Immunol Today 18:44, 1997 19. Ercan M, Inci S, Kilinc K, et al: Neurosurg Rev 24:127, 2001 20. Ozisik K, Yildirim E, Kaplan S, et al: Am J Transplant 4:900, 2004