Shyu K G, Chen J J, Shih N L, Wang D L, Chang H, Lien W P, Liew C C
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
Biochem Biophys Res Commun. 1995 May 16;210(2):567-73. doi: 10.1006/bbrc.1995.1697.
We examined the effect of cyclical mechanical stretch on the regulation of cardiac myosin heavy chain genes using an isolated neonatal rat cardiocyte culture system. Cultured cardiocytes grown on a flexible membrane were deformed by vacuum to 20% of maximum elongation, at 60 cycles/min in a serum-free medium. Cyclical stretch did not cause myocyte damage as assessed by supernatant LDH measurement and trypan blue exclusion test. The levels of myosin heavy chain (MyHC) mRNA increased as early as 1 h after stretch, reaching 12-fold over the control in 24 h, as shown by Northern blot analysis. However, the proximal 5'-flanking regions of the alpha- and beta-MyHC gene which were linked to chloramphenicol acetyltransferase (CAT) reporter gene did not exhibit enhanced CAT activity following cyclical stretch. Deletion of the chimeric constructs to shorten the 5'-flanking regions of the MyHC genes generated by polymerase chain reaction amplification did not enhance the CAT activity under cyclical stretch. This finding suggests that the stretch-response element of the alpha- and beta-MyHC gene promoter is probably not present in the proximal region of either the alpha- or beta-MyHC genes.
我们使用分离的新生大鼠心肌细胞培养系统，研究了周期性机械拉伸对心肌肌球蛋白重链基因调控的影响。在无血清培养基中，以60次/分钟的频率通过真空将生长在柔性膜上的培养心肌细胞拉伸至最大伸长率的20%。通过上清液乳酸脱氢酶测量和台盼蓝排斥试验评估，周期性拉伸未导致心肌细胞损伤。如Northern印迹分析所示，肌球蛋白重链（MyHC）mRNA水平早在拉伸后1小时就开始升高，在24小时内达到对照的12倍。然而，与氯霉素乙酰转移酶（CAT）报告基因相连的α-和β-MyHC基因的近端5'-侧翼区域在周期性拉伸后并未表现出增强的CAT活性。通过聚合酶链反应扩增产生的缩短MyHC基因5'-侧翼区域的嵌合构建体缺失，在周期性拉伸下并未增强CAT活性。这一发现表明，α-和β-MyHC基因启动子的拉伸反应元件可能不存在于α-或β-MyHC基因的近端区域。
