Yao A, Takahashi T, Aoyagi T, Kinugawa K, Kohmoto O, Sugiura S, Serizawa T
Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
J Clin Invest. 1995 Jul;96(1):69-77. doi: 10.1172/JCI118081.
To investigate how cardiac myocytes recover from a brief period of ischemia, we used a metabolic inhibition (MI) model, one of the in vitro ischemic models, of chick embryo ventricular myocytes, and examined the induction of immediate-early (IE) genes mRNAs and the activity of mitogen-activated protein (MAP) kinase. We performed Northern blot analysis to study the expression of c-jun, c-fos, and c-myc mRNAs during MI using 1 mM NaCN and 20 mM 2-deoxy-d-glucose, and also during the recovery from MI of 30 min. The c-fos mRNA was induced transiently at 30 and 60 min during the recovery. The expression of c-jun mRNA was significantly augmented at 30, 60, 90, and 120 min during the recovery (3.0-, 4.7-, 2.4-, and 1.9-fold induction, respectively) and so did the expression of c-myc mRNA (1.4-, 1.7-, 1.8-, and 2.0-fold induction, respectively). In contrast, the levels of these mRNAs remained unchanged during MI. The electrophoretic mobility shift assay revealed that AP-1 DNA binding activity markedly increased at 120 min during the recovery. When the cells were pretreated with protein kinase C (PKC) inhibitors, 100 microM H-7 or 1 microM staurosporine, the induction of c-jun mRNA at 60 min during the recovery was markedly suppressed (95 or 82% reduction, respectively). The c-jun induction was partially inhibited when the cells were treated with 2 mM EGTA during MI and the recovery (42% reduction). MAP kinase activity quantified with in-gel kinase assay was unchanged during MI, but significantly increased at 5, 10, and 15 min during the recovery (3.0-, 4.1-, and 3.4-fold increase, respectively). S6 kinase activity was also augmented significantly at 15 min during the recovery. Thus, these data suggest that IE genes as well as MAP kinase may play roles in the recovery process of cardiac myocytes from MI, and that the augmentation of c-jun expression needs the activation of PKC and to some extent, [Ca2+]i.
为了研究心肌细胞如何从短暂的缺血状态中恢复，我们使用了一种代谢抑制（MI）模型，即鸡胚心室肌细胞的体外缺血模型之一，并检测了即刻早期（IE）基因mRNA的诱导情况以及丝裂原活化蛋白（MAP）激酶的活性。我们进行了Northern印迹分析，以研究在使用1 mM NaCN和20 mM 2-脱氧-D-葡萄糖进行MI期间以及MI 30分钟恢复过程中c-jun、c-fos和c-myc mRNA的表达。在恢复过程中，c-fos mRNA在30分钟和60分钟时短暂诱导。在恢复过程中的30、60、90和120分钟时，c-jun mRNA的表达显著增加（分别为3.0倍、4.7倍、2.4倍和1.9倍诱导），c-myc mRNA的表达也是如此（分别为1.4倍、1.7倍、1.8倍和2.0倍诱导）。相比之下，这些mRNA的水平在MI期间保持不变。电泳迁移率变动分析显示，在恢复过程中的120分钟时，AP-1 DNA结合活性显著增加。当细胞用蛋白激酶C（PKC）抑制剂100 microM H-7或1 microM星形孢菌素预处理时，恢复过程中60分钟时c-jun mRNA的诱导被显著抑制（分别减少95%或82%）。当细胞在MI和恢复过程中用2 mM EGTA处理时，c-jun的诱导被部分抑制（减少42%）。用凝胶内激酶测定法定量的MAP激酶活性在MI期间不变，但在恢复过程中的5、10和15分钟时显著增加（分别增加3.0倍、4.1倍和3.4倍）。S6激酶活性在恢复过程中的15分钟时也显著增强。因此，这些数据表明，IE基因以及MAP激酶可能在心肌细胞从MI恢复的过程中发挥作用，并且c-jun表达的增加需要PKC的激活以及在一定程度上需要细胞内钙离子浓度[Ca2+]i。
