Expression of α-subunits of G proteins in failing human heart: A reappraisal utilizing quantitative polymerase chain reaction

Abstract

A decrease in the density of the β 1-adrenergic receptor and an increase in the functional activity of the G inhibitory protein G i accompany human heart failure; [ 1, 8] however, the molecular and biochemical mechanisms responsible for these changes are unclear. We previously reported that the steady-state levels of the mRNAs encoding both αG i-3 and αG s were significantly increased in failing human heart [ 4]. However, these results are not consistent with recent studies demonstrating that immunodetectable levels of αG proteins are not different in failing human hearts when compared with non-failing controls [ 6]. In addition, analysis of the 5′ flanking regions of αG i[ 10] and αG s[ 13] suggests that these two genes are unlikely to be co-regulated as their regulatory domains are quite different. Therefore, we hypothesized that the disparity between the measurements of αG protein gene expression and assessment of the actual levels of αG proteins might be due to technical limitations of the Northern blot technique utilized in previous studies for assessment of the mRNA levels; (i) cytoskeletal β-actin mRNA was used as a standard for normalization; and (ii) only relative levels of αG mRNAs were measured. The recent application of the polymerase chain reaction to quantification of mRNA levels in small quantities of human heart provided the tool with which to test this hypothesis [ 7]. When expressed in molecules of mRNA per microgram of total RNA, there were no differences in the levels of αG i and αG s mRNAs in failing human heart when compared with non-failing controls. Although steady-state levels of β 2-adrenergic receptor and α-myosin heavy chain mRNAs were also unaltered in heart failure, there was a 60% reduction in cytoskeletal β-actin mRNA.