Transcription profiling of cultured rat cardiomyocytes grown with eicosapentaenoic acid or docosahexaenoic acid supplementation in the culture media
ABSTRACT: Despite the recognized protective effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in cardiovascular diseases, and the demonstration of the control of gene expression by polyunsaturated fatty acids (PUFAs), the effects of these n-3 fatty acids on the whole genoma has never been investigated in cardiac cells. Using rat arrays, the effects of Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) supplementation on the global gene expression profile were evaluated in cultured neonatal rat cardiomyocytes. Experiment Overall Design: Primary cardiomyocyte cultures were obtained from the ventricles of newborn Wistar rats and grown in HAM F10 plus 10% fetal calf serum and 10% horse serum medium (controls), or in the same medium supplemented with 60 M Eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Media were changed every 48 hrs; cells were grown until complete confluence, then they were scraped off in ice cold PBS, and RNA isolation, labeling of complementary RNA (cRNA), hybridization to Agilent 22K-gene arrays (Rat oligo array G4130A) and assessment of expression ratios were performed. About one million cells treated with RNAlater were homogenized and total RNA was extracted by column technology (Rneasy Protect mini kit) and analyzed on both a spectrophotometer and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Only samples with 28S/18S ratio of >2.0 and no evidence of ribosomal peak degradation were included. The cRNA was generated by in vitro transcription with the use of T7 RNA polymerase (Low RNA input fluorescent linear amplification kit) and labeled with Cy3-CTP or Cy5-CTP. Direct comparisons were performed between n-3 PUFAs supplemented cells versus unsupplemented ones (controls); each analysis was replicated swapping the labeling with the two cyanine dyes.