Establishment of cardiac myocyte cell line

我们利用胚胎干细胞建立了心脏分化系统。ES细胞悬浮培养后，从第8天开始发育为类胚体(EB)，并开始自发搏动。Northern印迹分析表明，EB在悬浮培养5d开始表达心脏特异性同源蛋白CSX和锌指蛋白GATA4，8d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。我们利用该系统研究了丝裂原活化蛋白激酶(MAPK)在心脏分化中的作用。显性负性MAPK强烈抑制CSX的转录，而结构性激活MAPK则相反地激活CSX的转录。接下来，我们分离了永久性地转入MAPK负性调节因子MAPK磷酸酶(MKP)的ES细胞系(简称ES-MKP)。这些结果表明，MAPK在心肌细胞的发育中是必需的。

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.