Cultured Neonatal Rat Cardiomyocytes Continue Beating Through Upregulation of CTGF Gene Expression

Abstract

Background: Some cultured neonatal rat cardiomyocytes continue spontaneous beating even in serum-free medium. The present study explored the cause and genes responsible for this phenomenon. Methods: Ingenuity Pathway Analysis (IPA) software was used to analyze fold changes in gene expression in beating neonatal rat cardiomyocytes, as compared with non-beating cardiomyocytes, which were obtained from DNA microarray data of total RNA extracts of cardiomyocytes. To confirm the involvement of the 8 genes selected by IPA prediction, cellular protein abundances were determined by Western blot. The gene expression of connective tissue growth factor (CTGF) was substantially higher in beating cardiomyocytes than in non-beating cardiomyocytes; thus, CTGF protein content released from cardiomyocytes into the culture medium was examined. Results: IPA showed that the "Apelin Cardiac Fibroblast Signaling Pathway" was significantly inhibited and that microtubule dynamics and cytoskeleton organization were significantly activated. Each fluctuation in the cellular abundances of the 8 proteins in beating cardiomyocytes, as compared with non-beating cardiomyocytes, was primarily in the same direction as that of gene expression. However, the cellular CTGF protein abundance as well as CTGF content released into the medium did not substantially differ between beating and non-beating cardiomyocytes. Conclusions: The present results suggest that the large increase in CTGF gene expression in beating cardiomyocytes is not a cause but a result of beating, which may provide a putative pathway for controlling beating. Beating is sustained by developed cardiomyofibrils and directly upregulates CTGF gene expression, which is not followed by CTGF protein synthesis.