Expression of Several Cell Cycle Regulatory Genes in Cultured AT-1 Cardiomyocytes after Formation of Synchronously Beating Sheets

ABSTRACT

BACKGROUND AND OBJECTIVE: AT-1 cells have been derived from the left atrial tissue in which the ANF promoter targeted SV40 large T antigen expression. When cultured, clusters of spontaneously contracting cells were observed after 4-5 days and contiguous sheets of synchronously beating cardiomyocytes were formed after 10 days. In this study, expression of several cell cycle regulatory genes were monitored through Northern blot analyses in AT-1 cells during beating and after formation of beating sheets (BS). MATERIALS AND METHOD: AT-1 RNAs were obtained in 3 days after plating, during beating and after formation of BS, and used for Northern blot analyses. RESULTS: alpha-Cardiac myosin heavy chain expression was prominent in beating cells, as would be expected for this contractile protein isoform but ANF was decreased after beating. Gax was not expressed in cultured AT-1 cells but in AT-1 tumor and murine heart. p53 and p21 were decreased after beating which indicate transcription level of p53 and p21 correlated well in AT-1 cells. In contrast, pRB and p107 were increased after beating but p68 (2.4 kb) which arose by alternative splicing of p107 and lacks the pocket domain B was decreased in beating cells. pTCS2, murine tuberous sclerosis gene, represented similar levels during beating but a little was decreased after formation of BS. mRAD50, the murine homologue of yeast DNA recombinational repair gene RAD50, was increased in beating cells, a similar pattern to p107 and pRB. But the p50 arose by alternative splicing of mRAD50 and has 3' half of mRAD50 had unexpectedly appeared and maintained after beating. CONCLUSION: The expression of cell cycle regulatory genes after beating and formation of BS in AT-1 cells showed gene-specific pattern and the p50 which has homology to the mRAD50 may participate in differentiation of cardiomyocytes.