Establishment of cardiac myocyte cell line

我们利用胚胎干细胞建立了心脏分化系统。胚胎干细胞悬液培养8 d后发育为胚状体（EB）并开始自发跳动。Northern blot分析显示，EB细胞在开始悬浮培养后的第5天开始表达心脏特异性同型蛋白CSX和锌指蛋白GATA4，在第8天开始表达α肌球蛋白重链和肌球蛋白轻链等收缩蛋白基因。我们使用该系统检测了丝裂原活化蛋白激酶（MAPK）在心脏分化中的作用。显性负MAPK强烈抑制CSX的转录，并组成性激活MAPK，反过来激活CSX的转录。我们接下来分离ES细胞系（命名为ES-MKP），这些细胞系被MAPK磷酸酶（MKP）永久转染，MKP是MAPK的负调节因子。ES-MKP悬浮培养时，少量EB出现自发收缩，表达少量肌球蛋白重链蛋白。这些结果表明，MAPK是心肌细胞发育所必需的。

We have established cardiac differentiation system using embrryonic stem (ES) cells. When ES cells were cultured in suspension, they developed to embryoid bodies (EB) and started to beat spontaneously from 8 days. Northern blot analysis revealed that cardiac specific homeoprotein CSX and zinc finger protein GATA4 were expressed in EB from 5 days and contractile protein genes such as alpha myosin heavy chain and myosin light chain genes were expressed from 8 days after initiation of suspension culture. We examined the role of mitogen-activated protein kinase (MAPK) in cardiac differentiation using this system. Dominant negative MAPK strongly suppressed transcription of CSX and constitutively activated MAPK conversely activated transcription of CSX.We next isolated ES cell lines (designated as ES-MKP) which were permanently transfected by MAPK phosphatase (MKP), which is a negative regulator of MAPK.When ES-MKP was cultured in suspension, a few EB showed spontaneous contraction and expressed a little myosin heavy chain protein. These results suggest that MAPK is necessary for the development of cardiac myocytes.