心肌细胞核钙调素I介导的bcl-2转录调节在大鼠心肌肥厚中的作用

Role of bcl-2 transcriptional regulation induced by calmodulin I in pressure overload rat hypertrophic hearts

为探讨心肌细胞核钙调素I(calmodulin I,CaM I)介导的bcl-2转录调节在大鼠心肌肥厚中的作用及其可能机制, 实验随机分为对照组和心肌肥厚组,采用腹主动脉缩窄法制备大鼠心肌肥厚模型。模型复制成功后4周,以改良差速离心和密度梯度离心提取并纯化细胞核;蛋白印迹法测定心肌细胞核cAMP反应元件结合蛋白(cAMP response-element binding protein,CREB)及磷酸化CREB(phosphorylated cAMP response-element binding protein,pCREB)表达;免疫组化法观察左室心肌组织CaM I蛋白表达及分布;延续转录分析法观察阻断CaM I后心肌细胞核bcl-2 mRNA的变化。结果表明,心肌肥厚组pCREB蛋白表达较对照组明显增加(P<0．05),CREB蛋白表达无明显变化(P>0．05);CaM I分布于细胞核及细胞浆,心肌肥厚组CaM I蛋白表达较对照组明显增加(P<0．05);使用CaM抑制剂后心肌细胞核bcl-2 mRNA表达明显上调(P<0．05)。结果提示,压力超负荷时心肌细胞核内CaM I激活,抗凋亡基因bcl-2表达下调,核转录因子CREB磷酸化增加,但CREB 在调节bcl-2基因转录过程中可能发挥次要作用。

This study was designed to evaluate the role of bcl-2 transcriptional regulation induced by calmodulin Ⅰ （CaM Ⅰ ） in pressure overload rat hypertrophic hearts. The model of hypertensive Sprague-Dawley rat was established by abdominal aortic constriction. The hearts were collected four weeks after abdominal aortic constriction. Velocity and isopyknic gradient centrifugation was employed to fractionate rat myocardial nuclei. Western blot analysis revealed a marked increase in phosphorylated cAMP response-element binding protein （pCREB） of cardiac hypertrophy group compared with that in control group （P〈0.05）, while the protein level of cAMP response-element binding protein （CREB） was constant （P〉0.05）. Immunohistochemistry results showed a significant increase of CaM Ⅰ protein in cardiac hypertrophy group relative to the control group （P〈0.05）. Nuclear run off transcription assay displayed a significant increase in bcl-2 mRNA treated with trifluoperazne compared with non-drug treatment （P〈0.05）.The results obtained suggest that the transcription of bcl-2 is possibly regulated by CaM I in hypertrophic rat hearts, and CREB phosphorylation seems to be a minor factor in bcl-2 transcriptional regulation.