Korean Circulation Journal

The Korean Society of Cardiology (대한심장학회)
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Cardioprotective Effect of IGF-1 in Mouse with Adriamycin Induced Cardiomyopathy

생쥐의 Adriamycin 유발 심근증 모델에서 IGF-1의 심근 보호 효과

Park, Chul-Soo;Youn, Ho-Joong;Cho, Eun-Joo;Oh, Yong-Seok;Baek, Sang-Hong;Lee, Jung-Hwa;Kang, Jin-Hyoung;Chung, Wook-Sung;Chae, Jang-Seong;Kim, Jae-Hyung;Choi, Kyu-Bo;Hong, Soon-Jo
박철수;윤호중;조은주;오용석;백상홍;이정화;강진형;정욱성;채장성;김재형;최규보;홍순조

  • Published : 20021200
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Abstract

Background and Objectives : The purpose of this study was to investigate the apoptotic pathway, and the effect and mechanism of IGF-1 (insulin like growth factor 1) as a therapeutic agent, in Adriamycin (ADR) induced cardiomyopathy. Materials and Methods : We divided 17 mice into ADR (n＝6), ADR +IGF-1 (n＝6) and saline (n＝5) groups. The following were injected into the intraperitoneal cavity for the specified periods : 2.5 mg/kg of ADR every week for 6 weeks, 0.1 mg/kg of IGF-1 in the 5th and 6th weeks and 10 mL/kg of saline every week for 6 weeks. Transthoracic echocardiography, with a 15MHz linear array, was performed before and after injection of the drugs. A TUNEL assay and immunohistochemical staining for Bax, bcl-2, capase-8 and 9, and western blotting for Bax and bcl-2 were performed on the myocardium. Results : The fractional shortening decreased (38.5$\pm$3.2% before vs. 29.2$\pm$5.5% after, p<0.05) and the left ventricular end systolic dimension increased (2.5$\pm$0.3 mm before vs. 3.1$\pm$0.5 mm after, p<0.05) following the injection of the ADR, but there were no interval changes in the mice treated with saline or ADR+IGF-1. The TUNEL assay showed a higher apoptotic index in the mice treated with ADR than in those treated with saline or ADR+IGF-1 (46$\pm$ 8% in ADR, 0.1$\pm$0.03% in control, 22$\pm$5% in ADR+IGF-1, p<0.05). The immunohistochemical staining and western blot showed an increase in the expression of Bax, and a decrease in the expression of bcl-2 in the mice treated with ADR than in those treated with ADR+IGF-1 or saline. Conclusion : The apoptosis caused by the increased expression of Bax, and the decreased expression of bcl-2, was an important pathogenetic mechanism, and the IGF-1 prevents the progression of cardiomyopathy by attenuating the expressions of Bax and bcl-2 in ADR induced cardiomyopaththy.

배경 및 목적 : 최근들어 여러 가지 심근증 모델에서 세포자멸의 관련성이 제기되어 Adriamycin심근증과 세포자멸의 연관성 및 IGF-1의 심근 보호효과와 작용기전에 대해 연구하고자 하였다. 방 법 : 41마리의 생쥐중 살아남은 17마리를 대상으로 세군으로 나누어 생리 식염수군 5마리는 10 mL/kg의 생리 식염수를 주 1회 6주간 복강내 투여하였고, Adriamycin 단독투여군 6마리는 Adriamycin 2.5 mg/kg를 주 1회 6주간 복강내 투여하였으며 IGF-1과 Adriamycin 병합투여군 6마리는 Adriamycin 2.5 mg/kg를 주 1회 6주간, IGF-1 0.1 mg/kg를 5주와 6주째에 복강내 병합투여 하였다. 약물 투여 전후로 심초음파를 이용하여 심기능 및 형태를 관찰하였으며 심장을 적출하여 hematxyline-eosin염색, TUNEL assay를 통해 세포자멸을 확인하고 Bax, bcl-2,caspase-8, caspase-9에 대한 면역 조직학적 염색 및 Bax, bcl-2에 대해 western blotting하여 세포자멸 인자에 대한 연구를 시행하였다. 결 과 : 대상 생쥐 41마리중 17마리가 생존하였으며(생리 식염수군 5마리, Adriamycin 단독투여군 6마리, Adriamycin, IGF-1 병합투여군 6마리) 심초음파 결과 Adriamycin 단독투여군에서 약물투여 전에 비해 수축기 좌심실 내경이 유의하게 증가되었으며(2.5$\pm$0.3mm vs 3.1$\pm$0.5 mm, p<0.05) 분획 수축률은 유의하게 감소되었으나(38.5$\pm$3.2% vs 29.2$\pm$5.5%, p<0.05) Adriamycin과 IGF-1 병합투여군에서는 약물 투여전후에 심기능 및 심장의 형태학적 변화는 없었다. TUNEL assay에서 자멸사 지수는 생리 식염수군 0.1$\pm$ 0.03%, Adriamycin 단독투여군은 46$\pm$8%, IGF-1, Adriamycin 병합투여군은 22$\pm$5%로 IGF-1, Adriamycin 병합투여군에서 Adriamycin 단독투여군에 비해 현저히 감소된 소견을 보였다. 면역 조직학적 검사상 Adrimamycin 단독투여군에서 IGF-1과 Adriamycin 병합투여군에 비해 Bax의 발현은 현저히 증가되어 있었고 bcl-2의 발현은 감소되어 있었으며 western blotting에서도 Adrimamycin 단독투여군에서 IGF-1과 Adriamycin 병합투여군에 비해 Bax의 발현은 현저히 증가되어 있었고 bcl-2의 발현은 감소되어 있었다. 결 론 : Adriamycin에 의한 심근증은 주로 Bax발현의 증가와 bcl-2발현의 감소에 따른 세포자멸로 인해 유도되며 여기서 IGF-1은 Bax와 bcl-2의 발현에 관여하여 심근 보호 효과를 나타냄을 추정할 수 있었다.

Keywords

References

  1. Rosen GM, Halpern HJ. Spin trapping biologically generated free radicals: correlating formation with cellular injury. Methods Enzymol 1990;186:611-21. https://doi.org/10.1016/0076-6879(90)86156-P
  2. Singal PK, Iliskovic N, Li T, Kumar D. Adriamycin cardiomyopathy: phthophysiology and prevention. FASEB J 1997;11:931-6. https://doi.org/10.1096/fasebj.11.12.9337145
  3. Wang L, Ma W, Markovic R, Chen JW, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I. Circ Res 1998;83:516-22. https://doi.org/10.1161/01.RES.83.5.516
  4. Kumar D, Kirshenbaum LA, Li T, Danelisen I, Singal PK. Apoptosis in adriamycin cardiomyopathy and its modulation by probucol. Antioxid Redox Signal 2001;3:135-45. https://doi.org/10.1089/152308601750100641
  5. Arola OJ, Saraste A, Pulkki K, Kallajoki M, Parvinen A, Voipio-Pulkki LM. Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res 2000;60: 1789-92.
  6. Sawyer DB, Fukazawa R, Arstall MA, Kelly RA. Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane. Circ Res 1999;84:257-65. https://doi.org/10.1161/01.RES.84.3.257
  7. Lee WL, Chen JW, Ting CT, Ishiwata T, Lin SJ, Korc M, Wang PH. Insulin-like growth factor 1 improves cardiovascular function and suppresses apoptosis of cardiomyocytes in dilated cardiomyopathy. Endocrinology 1999; 140:4831-40. https://doi.org/10.1210/en.140.10.4831
  8. Yamamura T, Otani H, Nakao Y, Hattori R, Osako M, Imamura H. IGF-1 differentially regulates Bcl-xL and Bax and confers myocardial protection in the rat heart. Am J Physiol Heart Circ Physiol 2001;280:H1191-200. https://doi.org/10.1152/ajpheart.2001.280.3.H1191
  9. Singal PK, Deally CM, Weinberg LE. Subcellular effects of adriamycin in the heart: a consice review. J Mol Cell Cardiol 1987;19:817-28. https://doi.org/10.1016/S0022-2828(87)80392-9
  10. Sinha BK, Katki AG, Batist G, Cowan KH, Myers CE. Adriamycin-stimulated hydroxyl radical formation in human breast tumor cells. Biochem Pharmacol 1987;36: 793-6. https://doi.org/10.1016/0006-2952(87)90164-X
  11. Muller I, Jenner A, Bruchelt G, Niethammer D, Halliwell B. Effect of concentration on the cytotoxic mechanism of doxorubicin: apoptosis and oxidative DNA damage. Biochem Biophys Res Commun 1997;230:254-7. https://doi.org/10.1006/bbrc.1996.5898
  12. Yen HC, Oberley TD, Gairola CG, Szweda LI, St Clair DK. Manganese superoxide dismutase protects mitochondrial complex I against adriamycin induced cardiomyopathy in transgenic mice. Arch Biochem Biophys 1999; 362:59-66. https://doi.org/10.1006/abbi.1998.1011
  13. Sawyer DB, Fukazawa R, Arstall MA, Kelly RA. Daunorubicin-induced apoptosis in rat cardiac myocyte is inhibited by dexrazoxane. Circ Res 1999;84:257-65. https://doi.org/10.1161/01.RES.84.3.257
  14. Nakamura T, Ueda Y, Juan Y, Katsuda S, Takahashi H, Koh E. Fas-mediated apoptosis in adriamycin-induced cardiomyopathy in rats. Circulation 2000;102:572-8. https://doi.org/10.1161/01.CIR.102.5.572
  15. Ambler GR, Johnson BM, Maxwell L, Gavin JB, Gluckman PD. Improvement of doxorubicin induced cardiomyopathy in rats treated with insulin-like growth factor I. Cardiovasc Res 1993;27:1368-73. https://doi.org/10.1093/cvr/27.7.1368
  16. Pecherskaya A, Solem M. IGF-1 activates PKC alphadependent protein synthesis in adult rat cardiomyocytes. Mol Cell Biol Res Commun 2000;4:166-71. https://doi.org/10.1006/mcbr.2001.0274
  17. Wu W, Lee WL, Wu YY, Chen D, Liu TJ, Jang A, Sharma PM, Wang PH. Expression of constitutively active phosphati-dylinositol 3-kinase inhibits activation of caspase-3 and apoptosis of cardiac muscle cells. J Biol Chem 2000; 275:40113-9. https://doi.org/10.1074/jbc.M004108200
  18. Hong F, Kwon SJ, Jhun BS, Kim SS, Ha J, Kim SJ, Sohn NW, Kang C, Kang I. Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways. Life Sci 2001; 68:1095-105. https://doi.org/10.1016/S0024-3205(00)01012-2