Unknown,Transcriptomics,Genomics,Proteomics

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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 ïM 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.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Alessandra Bordoni

PROVIDER: E-GEOD-4327 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

N-3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure.

Bordoni Alessandra A Astolfi Annalisa A Morandi Luca L Pession Andrea A Danesi Francesca F Di Nunzio Mattia M Franzoni Monica M Biagi Pierluigi P Pession Annalisa A

FEBS letters 20070206 5

In cardiac cells the effects of n-3 PUFAs on the whole genome are still unknown despite their recognized cardioprotective effects and ability to modulate gene expression. We have evaluated the effects of n-3 PUFAs supplementation on the global gene expression profile in cultured neonatal rat cardiomyocytes, detecting many genes related to lipid transport and metabolism among the upregulated ones. Many of the downregulated genes appeared related to inflammation, cell growth, extracellular and car ...[more]

PMID: 17303130

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Project description:Background & Aims: Non-alcoholic fatty liver disease accompanies obesity and is independently associated with cardiovascular disease. Omega-3 fatty acids, such as docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid, reduce the risk of cardiovascular disease due to their anti-inflammatory and hypolipidemic effects. Recent data suggested that metabolic effects of EPA/DHA as marine phospholipids could be stronger than triglycerides (fish oil). We characterised the mechanisms underlying beneficial effects of EPA/DHA phospholipids alone or in combination with antidiabetic drugs on hepatosteatosis in dietary obese mice. Methods: Male C57BL/6N mice were fed for 7 weeks a corn oil-based high-fat diet (cHF) or subjected to cHF-based interventions: (i) cHF with DHA/EPA as phosphatidylcholine-rich concentrate replacing ~10 % of dietary lipids (PC); ii) cHF with a low-dose of thiazolidinedione rosiglitazone (10 mg/kg diet), which retains some of the beneficial effects but also potentiates hepatic lipid accumulation (R); and iii) PC+R. Metabolic profiling together with hepatic gene expression and lipidomic analyses were performed. Results: PC and PC+R prevented weight gain and glucose intolerance induced by cHF, while all interventions reduced abdominal fat and plasma triglycerides. In contrast to R, PC and PC+R lowered hepatic and plasma cholesterol and eliminated hepatosteatosis. Hepatic microarray analysis primarily revealed a complex downregulation of lipogenic and cholesterol biosynthesis pathways by PC. Lipidomic analysis identified arachidonic acid, DHA and EPA in hepatic phosphatidylcholine and phosphatidylethanolamine fractions as the most important variables. Importantly, down-regulation of genes within these pathways was enlarged by phospholipid versus triglyceride forms of EPA/DHA in an independent experiment. Conclusions: Obesity-associated hepatosteatosis and hypercholesterolemia were ameliorated by marine phospholipids in association with a complex inhibition of biosynthetic pathways in liver. Stronger effects of phospholipids versus triglyceride form of EPA/DHA suggest their preferential use in the prevention and possible treatment of obesity-associated metabolic hepatic dysfunctions.

Project description:Dietary polyunsaturated fatty acids (PUFA) act as potent natural hypolipidemics and are linked to many health benefits in humans and in animal models. Mice fed long-term a high fat diet, in which medium-chain alpha linoleic acid (ALA) was partially replaced by long-chain docosahexaenoic (DHA) and eicosapentaenoic (EPA) fatty acids, showed reduced accumulation of body fat and prevention of insulin resistance, besides increased mitochondrial beta-oxidation in white adipose tissue and decreased plasma lipids. ALA, EPA and DHA all belong to PUFA of n-3 series. The intestine is a gatekeeper organ for ingested lipids. To examine the potential contribution of the intestine in the beneficial effects of EPA and DHA, this study assessed gene expression changes using whole genome microarray analysis on small intestinal scrapings. The main biological process affected was lipid metabolism. Fatty acid uptake, peroxisomal and mitochondrial beta-oxidation, and omega-oxidation of fatty acids were all increased. Quantitative real time PCR and intestinal fatty acid oxidation measurements ([14C(U)]-palmitate) confirmed significant gene expression differences in a dose-dependent manner. Furthermore, no major changes in the expression of lipid metabolism genes were observed in colonic scrapings. In conclusion, we show that marine n-3 fatty acids regulate small intestinal gene expression patterns. Since this organ contributes significantly to whole organism energy use, this adaptation of the small intestine may contribute to the complex and observed beneficial physiological effects of these natural compounds under conditions that will normally lead to development of obesity and diabetes. Experiment Overall Design: Male 4-month-old C57BL/6J mice were maintained for 4 weeks on semisynthetic high-fat (20% wt/wt) diets differing in the composition of n-3 PUFA. Two isocaloric diets were used (n=12): control sHFf diet which contained flax-seed oil (rich in ALA) as the only lipid source, or the sHFf-F2 diet, which had the same composition except that 44 % of the lipids were replaced by a n-3 PUFA concentrate (EPA&DHA) containing 6 % EPA and 51 % DHA (EPAX 1050TG; EPAX AS, Lysaker, Norway). Quality control of RNA samples showed that four samples did not pass quality thresholds, and these samples were excluded from array analysis (two control small intestine samples and one EPA&DHA small intestine sample). The remaining RNA samples were pooled per tissue per diet group. Thus; the flax-seed (control) array was hybridized with RNA pooled from of 10 mice, the PUFA array was hybridized with RNA pooled from 11 mice.

Dataset Information

Transcription profiling of cultured rat cardiomyocytes grown with eicosapentaenoic acid or docosahexaenoic acid supplementation in the culture media

Publications

N-3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure.

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