Establishment of cardiac myocyte cell line

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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.