Original Articles Korean Circulation J 2002;328:689-696 성장에따른백서심근세포의허혈손상에대한 Protein Kinase C 의보호효과차이 손지원 1 김영대 2 PKC Activation Protects the Cardiomyocytes from Ischemic Insult in Adult, but not in Neonatal Rat Heart Ji-Won Son, MD 1 and Young-Dae Kim, MD 2 1 Division of Cardiology, Gachon Medical School, Inchon, 2 Department of Internal Medicine, Dong-A University, Busan, Korea ABSTRACT Background and ObjectivesProtein kinase C PKC has been known to play a central role in mediating ischemic preconditioning. The isoform of the PKC changes during the development of the heart in rats. Therefore, the protective effects of PKC activation may vary between neonatal and adult hearts. Materials and Methods To test this hypothesis, primary cultures of neonatal and adult rat ventricular myocytes NRVM and ARVM, respectively were subjected to ischemic condition, which consisted of a deoxygenated air supply and glucose deprivation in the media. The survival was evaluated by counting trypan blue excluding cells. The effect of PKC activation was analyzed by the addition of a PKC agonist 12-o-tetradecanoylphorbol 13-acetate, TPA, or an antagonist staurosporin to cultured myocytes. ResultsUnder ischemic condition, ARVMs were more susceptible than NRVM. The survival of the ARVMs were 63.18.3, 42.86.1, 10.15.8 after 3, 6, 12 hours of ischemia, respectively, while those of the NRVMs were 68.96.4, 60.37.3, 34.37.5, and 8.26.6 after 6, 12, 24, 36 hours of ischemia, respectively p0.031. However, the activation of the PKC following the addition of 100 nm TPA to the media significantly enhanced the survival of the ARVM, from 38.58.3 to 62.17.3, after 6 hours of ischemia, which was similar to that of the controls 65.46.2. In contrast, the activation of the PKC by the addition of TPA did not change the survival of the NRVM, from 31.85.8 to 28.57.3, after less than 24 hours of ischemia. ConclusionThese findings demonstrate that the protective effect of PKC activation in adult hearts differs from that in neonatal hearts, indicating that PKC isoform variance between two tissues may affect the biologic consequence of its activation. Korean Circulation J 2002;328:689-696 KEY WORDSProtein kinase CIschemic preconditioningmyocardium. 서 론 - 689
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원래의 막대 형태(rod shape)를 유지하고 있으면 생존 세포로 간주하였다. 통계학적 분석 모든 실험은 3회 이상 반복 시행하였다. 대조군과 허 혈군 사이의 생존 비교는 unpaired t-test로 하였으며 p 값이 0.05 미만을 유의한 것으로 하였다. PKC 활성화 및 억제 19) PKC를 활성화하는 물질로서 phorbol ester 의 일종 결 인 12-o-tetradecanoylphorbol 13-acetate(TPA 과 Sigma)를 100 nm의 농도로서 배지에 첨가하였으며 PKC 의 억제제로는 staurosporin(sigma)을 100 nm 농도 로 첨가하였다. 허혈 상태에서의 성체쥐와 신생쥐 심근세포의 생존 차이 허혈 상태에서 trypan blue 염색으로 산출한 단위 면 적 당의 평균 생존율은 신생쥐에서는 6시간 경과 시 68.9 Fig. 1. Survival of myocyte under control (white bar) and ischemic condition (black bar) as determined by trypan blue exclusion. A neonatal rat ventricular myocyte, B adult rat ventricular myocyte. * p<0.5 between control and ischemia group. A B C D Fig. 2. Trypan blue staining of neonatal rat ventricular myocyte cyultured for 24 hours under various conditions. A control, B ischemia, C ischemia+ staurosporin 100 mm, D ischemia+ 12-o-tetradecanoylphorbol 13-acetate (TPA) 100 mm. 692 Korean Circulation J 2002; 32(8):689-696
±6.4%, 12시간 60.3±7.3%, 24시간에서 34.3±7.5%, 즉 신생쥐의 경우 허혈 24시간에서 성체쥐의 경우는 허 36시간 경과시 8.2±6.6%로 나타났으며(시간에 따른 혈 6시간 경과 시점에 실험을 하였다. Fig. 2는 신생쥐 유의성 p 0.004) 이에 비해 성체쥐에서는 허혈 3시간 에서의 trypan blue 염색으로 허혈 24시간에 다수의 세 경과 시에 63.1±8.3%, 6시간 경과 시 42.8±6.1%, 12 포 사망을 보이고 있으며 생존 세포는 staurosporin 투 시간 경과 시 10.1±5.8%로서(p 0.031) 분리된 성체 여에 의해서 약간의 감소를 보이나 100 nm TPA를 투 심장의 심근세포에서 허혈 손상에 의한 세포 사멸이 이 여한 군의 세포 생존 비율은 큰 변화를 보이지 않았다. 반 른 시간 내에 더 많이 발생하였다(Fig. 1). 면 Fig. 3에서 성체쥐의 심근세포는 허혈 6시간에서 생 신생쥐와 성체쥐에서 PKC 활성화에 따른 심근 보호 효과 urosporin에 의해서 생존 세포를 거의 찾아볼 수 없을 의 차이 정도로 세포 사망이 늘어났다. 그러나 PKC 활성제제인 존 세포의 감소를 보이고 있으며 PKC 억제제인 sta- 각각의 군에서 충분하게 세포의 사망이 나타나는 시간 100 nm의 TPA를 투여한 군에서는 거의 비허혈 대조 A B C D Fig. 3. Trypan blue staining of adult rat ventricular myocyte cyultured for 6 hours under various conditions. A control, B ischemia, C ischemia+ staurosporin 100 mm, D ischemia+ 12-o-tetradecanoylphorbol 13-acetate (TPA) 100 mm. Fig. 4. Survival of myocyte under control (white bar) and ischemic condition (black bar) under various conditions as determined by trypan blue exclusion. A neonatal rat ventricular myocyte (24 hr culture), B adult rat ventricular myocyte (6 hr culture). * p<0.5 between control and ischemia group. Concentration of staurosporin and 12-o-tetradecanoylphorbol 13-acetate (TPA) was 100 mm respectively. 693
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결론 : 중심단어 REFERENCES 1) Fibrinolytic Therapy Trialist FTT Collaborative Group. Indications for fibrinolytic therapy in suspected acute myocardial infarction collaborative overview of early mortality and major morbidity from all randomized trials of more than 1000 patients. Lancet 1994343311-22. 2) Weaver WD, Simes RJ, Betriu A, Grines CL, Zijlstra F, Garcia E, Grinfeld L, Gibbsons RJ, Ribeiro EE, De- Wood MA, Ribichini F. Comparison of primary coronary angioplasty and intravenous thrombolytic therapy for acute myocardial infarction a quantitative review. JAMA 19972782093-8. 3) Rogers WJ, Bowlby LJ, Chandra NC, French WJ, Gore JM, Lambrew CT, Rubison RM, Tiefenbrunn AJ, Weaver WD. Treatment of myocardial infarction in the United States 1990 to 1993. Circulation 1994902103-14. 4) Reimer KA, Murry CE, Yamagawa I, Hill ML, Jennings RB. Four brief periods of ischemia cause no cumulative ATP loss or necrosis. Am J Physiol 1986251H1306-15. 5) Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia a delay of lethal cell injury in ischemic myocardium. Circulation 1986741124-36. 6) Baxter GF. Ischaemic preconditioning of myocardium. Ann Med 199729345-52. 7) Nishizuka Y. Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C. Science 1992258607-14. 8) Steinberg SF, Goldberg M, Rybin VO. Protein kinase C isoforms diversity in the heart. J Mol Cell Cardiol 1995 27141-53. 9) Goldberg M, Zhang HL, Steinberg SF. Hypoxia alters the subcellular distribution of protein kinase C isoforms in neonatal rat ventricular myocytes. J Clin Invest 1997 9955-61. 10) Inagaki K, Kihara Y, Hayashida W, Izumi T, Iwanaga Y, Yoneda T, Takeuchi Y, Suyama K, Muso E, Sasayama S. Anti-ischemic effect of a novel cardioprotective agent, JTV519, is mediated through specific activation of disoform of protein kinase C in rat ventricular myocardium. Circulation 2000101797-804. 11) Gray MO, Karliner JS, Mochly-Rosen D. A selective e- protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem 199727230945-51. 12) Miyamae M, Rodriguez MM, Camacho SA, Diamond I, Mochly-Rosen D, Figueredo VM. Activation of epsilon protein kinase C correlates with a cardioprotective effect of regular ethanol consumption. Proc Natl Acad Sci USA 1998958262-7. 13) Clerk A, Bogoyevitch MA, Fuller SJ, Lazou A, Parker PJ, Sugden PH. Expression of protein kinase C isoforms during cardiac ventricular development. Am J Physiol 1995269H1087-97. 14) Park SK, Kim HS, Sohn CS, Dokko YC, Jeon YH. Expression of protein kinase C isoform mrnas in the developing rat heart. Korean Circ J 1998281341-9. 15) Berger HJ, Prasad SK, Davidoff AJ, Pimental D, Ellingsen O, Marsh JD, Smith TW, Kelly RA. Continued electric field stimulation preserves contractile function of adult ventricular myocytes in primary culture. Am J Physiol 1994266H341-9. 16) Volz A, Piper HM, Sigmund B, Schwartz P. Longevity of adult ventricular rat heart muscle cells in serum-free priamry culture. J Mol Cell Cardiol 199123161-73. 17) Springhorn JP, Claycomb WC. Preproenkephalin mrna expression in developing rat heart and in cultured ventricular cardiac muscle cells. Biochem J 198925873-8. 18) McGahon AJ, Martin SJ, Bissonnette RP, Mahboubi A, Shi Y, Mogil RJ, Nishioka WK, Green DR. The end of the cell line methods for the study of apoptosis in vitro. Methods Cell Biol 199546153-85. 19) Parker PJ. Protein kinase C and its relatives. In Woodget JR, editor. Protein kinases. Oxford IRL press1996. p.68-84. 20) Ruoslahti E. Stretching is good for a cell. Science 1997 2761345-6. 21) Koh JS, Lieberthal W, Heydrick S, Levine S. Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signalling pathway. J Clin Invest 1998 102716-27. 22) Rybin VO, Steinberg SF. Protein kinase C isoform expression and regulation in the developing rat heart. Circ Res 199474299-309. 23) Zhao J, Renner O, Wightman L, Sugden PH, Stewart L, Miller AD, Latchman DS, Marber MS. The expression of constituitively active isotype of protein kinase C to investigate preconditioning. J Biol Chem 199827323072-9. 24) Dorn GW 2nd, Souroujon MC, Liron T, Chen CH, Gray MO, Zhou HZ, Csukai M, Wu G, Lorenz JN, Mochly- Rosen D. Sustained in vivo cardiac protection by rationally designed peptide that causes epsilon protein kinase C translocation. Proc Natl Acad Sci USA 19999612798-803. 696 Korean Circulation J 2002;328:689-696