Global gene expression analysis of quercetin bioactivity in cultured rat cardiomyocytes

Despite extensive studies, the fundamental mechanisms responsible for the development and progression of heart failure have not yet been fully elucidated. Recent experimental and clinical studies have suggested that reactive oxygen species (ROS) play a major pathological role. Quercetin, a major flavonoid present in the human diet, has been studied widely and its biological properties are consistent with its protective role in the cardiovascular system. However, it remains unknown whether the cardioprotective effects of quercetin may also occur through the modulation of genes involved in cell survival. This study was therefore designed to examine the gene expression profiling of cultured rat primary cardiomyocytes supplemented with quercetin using DNA-microarrays and relating these data to functional effects. Keywords: nutritional intervention, dose response Primary heart cell cultures were obtained by isolation of cardiomyocytes from the ventricles of 2–4 day old Wistar rats and grown until complete confluence. In some dishes, 30 μM quercetin was added to the culture medium for 6, 12 or 24 h. Total RNA was extracted using Total RNA Isolation Mini Kit (Agilent Technologies, Palo Alto, CA), following the manufacturer’s protocol. The yield and purity of the RNA were measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Rockland, Del, USA). The 28s and 18s ribosomal bands were checked using Agilent 2100 bioanalyzer with an RNA 6000 Nano LabChip Kit. For each sample, mRNA was amplified starting from 1.5 μg of totRNA by Amino Allyl MessageAmp I aRNA Kit (Ambion Inc.) to obtain amino allyl antisense RNA (aaRNA). Labeling of aaRNA was performed using NHS ester Cy3 or Cy5 dies (Amersham Biosciences, Piscataway, NJ - USA). One hybridization to Agilent 22K-gene arrays (Rat oligo array G4130A) was performed to compare quercetin supplemented cells versus unsupplemented ones. Each analysis was replicated swapping the labeling with the two cyanine dyes.