164 Translational regulation of gene expression in cardiomyocytes following exposure to oxidative stress

Myocardial infarction leads to ischemia/reperfusion injury associated with an increase in oxidative stress. The cardiomyocyte response to oxidative stress is concentration-dependent: low concentrations may be protective, moderate levels promote apoptosis, and high levels lead to necrosis. These responses are associated with differential effects on cardiomyocyte mRNA expression. At the protein level, increasing levels of oxidative stress inhibit global protein synthesis, but some proteins continue to be synthesised. We investigated the effects of moderate and low levels of oxidative stress on translational regulation of mRNAs in cardiomyocytes. Total RNA and RNA associated with polysomes (i.e. mRNAs that are actively translated) were prepared from rat neonatal cardiomyocytes with or without treatment with H 2 O 2 (0.2 mM, 1 h). RNA expression profiling was performed using Affymetrix microarrays and analysed using GeneSpring GX 13. 35 RNAs were significantly changed (u003e1.5 fold; p 2 O 2 , with a significant difference in the degree of change in polysomal vs total RNA. Of these, 9 were increased to a greater extent in the polysomes (indicative of selective translation) including the small G protein Rasd1 and the transcription factor Nfil3. Other mRNAs were not significantly different between polysomal and total RNA (e.g. Atf3, Hmox1), whereas genes such as Mdm2 were upregulated to a greater extent in the total RNA pool. Microarray data were validated by quantitative PCR. Data for all genes studied were consistent with the microarrays. In cardiomyocytes exposed to moderate oxidative stress (0.2 mM H 2 O 2 ) or lower oxidative stress (0.1 mM H 2 O 2 ), Rasd1 and Nfil3 were upregulated to a greater extent in the polysomes and Hmox1 showed a similar degree of upregulation in polysomal and total RNA pools. However, although Mdm2 mRNA was increased to a greater extent in the total pool with 0.2 mM H 2 O 2 , it was increased to a similar extent in polysomal and total RNA pools with 0.1 mM H 2 O 2 . Furthermore, although Atf3 mRNA was similarly upregulated in total and polysomal RNA pools with 0.2 mM H 2 O 2 , there was greater upregulation in the polysomes with 0.1 mM H 2 O 2 . Rasd1 protein was studied in cardiomyocytes exposed H 2 O 2 . Consistent with the mRNA expression data, Rasd1 was upregulated over ˜2 h. Thus, oxidative stress has concentration-dependent effects on protein translation of specific mRNAs. Rasd1 is of particular interest because it is associated with circadian rhythm regulation that may influence the response to myocardial infarction. Furthermore, a genetic polymorphism in the Rasd1 gene is associated with coronary artery disease and ischaemic stroke.