Regulation of antisense RNA expression during cardiac MHC gene switching in response to pressure overload

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Abstract
Hypertension has been shown to cause cardiac hypertrophy and a shift in myosin heavy chain (MHC) gene expression from the faster α- to slower β-MHC isoform. The expression of the β- and α-MHC pre-mRNAs, mRNAs, as well as the newly discovered antisense β-RNA were analyzed in three regions of the normal control (NC) and 12-day pressure-overloaded (AbCon) hearts: the left ventricle apex, left ventricle base, and the septum. The RNA analyses in the AbCon heart targeted both the 5′ and the 3′ ends of each RNA molecule. β-MHC mRNA expression significantly increased relative to control in all three regions, regardless of the target site (5′ or 3′ end). In contrast, β-MHC pre-mRNA expression in the AbCon heart depended on the site of the measurement (5′ vs. 3′ end). For example, whereas the pre-mRNA did not change when targeted at the 3′ end (last intron), it increased significantly in the AbCon heart when measurement targeted the 5′ end (2nd intron) of the 25-kb molecule. Analyses of the antisense β-RNA revealed that its expression in the AbCon heart was significantly decreased relative to control regardless of its measurement site. A negative correlation was observed between the β-mRNA expression and the antisense β-RNA (P < 0.05), suggesting an inhibitory role of antisense RNA on the sense β-MHC gene expression. In contrast, a positive correlation was observed between the antisense β-RNA and the α-MHC pre-mRNA (P < 0.05). This latter observation along with the α-MHC gene position relative to that of the β-antisense suggest that the α-MHC sense and β-antisense transcription are coregulated likely via common intergenic regulatory sequences. Our results suggest that the increased β-MHC expression in the AbCon heart not only is the result of increased β-MHC transcription but also involves an antisense β-RNA regulation scheme. Although the exact mechanism concerning antisense regulation is not clear, it could involve modulation of both transcriptional activity of the β-MHC gene and posttranscriptional processing.