Analysis and functional characterization of alternatively spliced angiotensin II type 1 and 2 receptor transcripts in the human heart

Expression levels of angiotensin II type 1 and type 2 receptors (AT1, AT2) vary at different cardiac localizations and are regulated in cardiac diseas es. Differential splicing of the 5' untranslated exons of the primary AT1 m RNA transcripts may modulate translational efficiency and thus receptor exp ression. We therefore searched for ATI and AT1 mRNA splice patterns specifi c to chamber localization or to cardiac performance and analyzed their effe ct on protein expression in transfection experiments. The exon composition of the BT1 and AT2 mRNA transcripts in normal and diseased human hearts wer e analyzed using a reverse transcription polymerase chain reaction Followed by HPLC quantitation of the amplificates. We compared atrial (n=18) and ve ntricular (n=28) samples and endomyocardial biopsies (n=10) from patients w ith normal and severely impaired cardiac function and one donor heart, whic h was not used for transplantation. ATI transcripts with the exon compositi on 1/2/5 and 1/5 represented about 93-98% of all ATI mRNAs; transcript 1/2/ 3/5 represented 8% in the atria and 2% in ventricles. Since exon 2 reduces translational efficiency in vitro, the ratios of transcripts with and witho ut exon 2, (1/2/5+1/2/3/5) to (1/5), were compared. These were 1.24+/-0.07 in normal atria, 0.96+/-0.09 in atria 1 from failing hearts (P<0.05), 0.68 in the left ventricle of the donor heart, and 0.58+/-0.03 in failing left v entricles. Endomyocardial biopsy specimens showed significant differences b etween controls and heart failure (controls 0.63+/-0.04 vs. heart failure 0 .52+/-0.02, P<0.05). Of the two identified AT2 transcripts, mRNA 1/2/3 was the most abundant in the human heart (92%). Luciferase reporter gene assays were performed to test the effect of the various 5' untranslated regions ( 5' UTRs) on protein expression. Among the constructs which contained the AT I promoter/AT1 5' UTRs the plasmid Ex 1/2/5 exhibited 27% lower luciferase activity than Ex 1/5 (n=24%, P<0.001), and Ex 1/2/3/5 expressed only 35.9% of Ex 1/5 activity (P<0.001). Among the reporter gene plasmids with the AT2 promoter/AT2 5' UTRs the construct Ex 1/2/3 expressed a 31% lower lucifera se activity than Ex 1/3 (n=20, P<0.001). In conclusion, alternative splicin g may represent a mechanism of ATR regulation in vivo. In the human heart, ATI splice patterns differ distinctly between atria and ventricles and to a lesser degree between controls and failing hearts. This may lead to differ ences in ATI mRNA translation into protein in the various cardiac areas and under different pathophysiological conditions.