Endothelin-1 responsiveness of a 1.4 kb phospholamban promoter fragment inrat cardiomyocytes transfected by the gene gun

The transcriptional regulation of an isolated rat phospholamban (PL) promot er fragment in rat cardiomyocytes was analyzed by applying a new method to reach substantially higher transfection efficiencies: gene gun biolistics. The gene gun transfection method was optimized for application to primary c ultures of rat neonatal cardiomyocytes. Cells, cultured at different densit ies (0.75-1.50 x 10(5) cells/cm(2)) in serum-free medium, were transfected with DNA coated gold particles. A transfection efficiency of up to 10% coul d be achieved (compared to <1% with other methods) by the gene gun as check ed using a RSV-beta-Gal construct. Cardiomyocytes were stimulated by endoth elin-1 (ET-1) (10(-8) M) to induce hypertrophy, thereby yielding the charac teristic changes in gene expression (upregulation of Atrial Natriuretic Fac tor (ANF) and downregulation of FL). The basal activity of an ANF promoter fragment (increasing from the lowest to highest density 2.6-fold) and its E T-1 inducibility (only significant upregulation of 2.6-fold, at lowest dens ity) appeared to be dependent on the plating density of the cardiomyocytes. A PL promoter fragment was isolated, sequenced and 1.4 kb was subcloned in a luciferase reporter vector. The basal activity of the PL promoter fragme nt was not dependent on the plating density. ET-1 did not downregulate the PL promoter, rather a significant upregulation (1.4-fold) was found at the highest plating density. In conclusion, plating density of the cardiomyocyt es can influence promoter activity as shown with an ANF promoter fragment. A newly isolated and sequenced rat PL promoter fragment did not direct gene expression as expected on basis of downregulation of the PL gene by ET-1 o bserved in this model. (C) 2000 Academic Press.