Abstract
A clonal derivative named P19CL6 has been isolated from pluripotent P19 mouse embryonic carcinoma cells, and this subline efficiently differentiates into beating cardiomyocytes when treated with 1% dimethyl sulfoxide (DMSO). It offers a valuable model to study cardiomyocytes differentiation in vitro. In this study, comparative proteomic analysis was used to characterize the protein profiles associated with the DMSO‐induced cardiomyocytes differentiation of P19CL6 cells. We demonstrated that P19CL6 cells indeed differentiated into cardiomyocytes after DMSO inducement as they expressed sarcomeric myosin heavy chain (MHC) as well as three cardiac‐specific transcription factors (Csx/Nkx‐2.5, GATA‐4, and MEF2C). Image analysis of silver‐stained two‐dimensional gels was used to find protein spots that exhibited an at least 1.5‐fold change in abundance after successful differentiation. Seventeen protein spots were selected for further analysis by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐TOF‐MS) and/or nano‐electrospray ionization MS/MS (ESI‐MS/MS), and 16 protein spots were identified. The identified proteins are involved in different cellular functions such as metabolism, signal transduction, and cellular organization. To confirm the expression changes of the identified proteins during differentiation, the mRNA levels of six identified proteins (including seven protein spots) were assessed by the real‐time polymerase chain reaction and three showed a correlation between mRNA level and protein abundance. As an initial step toward identifying proteins involved in maintaining the differentiated state of cardiomyocytes derived from P19CL6 cells, our data provide some helpful information that may lead to a better understanding of the molecular mechanisms by which P19CL6 cells differentiate into cardiomyocytes after treatment with DMSO. J. Cell. Biochem. 102: 149–160, 2007. © 2007 Wiley‐Liss, Inc.