title: Reduced atrial angiotensin receptor type 1 mRNA content in end-stage human heart failure: assessment by a novel quantitative PCR-ELISA technique.
abstract: The number of atrial angiotensin II binding sites is reduced in end-stage human heart failure. The goals of our study were the development of a quantitative polymerase chain reaction for angiotensin II receptor type 1 mRNA to determine the angiotensin receptor type 1 (AT1) mRNA content in the atria of patients with end-stage heart failure. We established a quantitative PCR based on coamplification of AT1 wild-type and an internal standard in the same PCR, followed by liquid-phase hybridization of PCR products in microtiter plates and quantitation by ELISA. Glyceraldehyde phosphate dehydrogenase mRNA in the same samples was used to relate the AT1 mRNA content to a stably expressed reference gene. Atrial samples from 11 patients with end-stage heart failure obtained at cardiac transplantation were compared with atrial samples from 11 patients with normal cardiac function undergoing routine cardiac surgery. A PCR/ELISA system with a variance of about 6% after reverse transcription and a linear measuring range was established. In the samples from 11 patients with end-stage heart failure a 58% decrease in AT1 mRNA content was found in comparison with 11 controls (heart failure: 185,680 +/- 196,912 AT1 mRNA copies/microgram RNA, controls: 440,555 +/- 268,456, P < 0.02). When AT1 mRNA content was related to glyceraldehyde phosphate dehydrogenase mRNA, a 65% decrease was detected (AT1/glyceraldehyde phosphate dehydrogenase: heart failure: 4.84 +/- 5.18; controls: 13.74 +/- 7.77; P < 0.005). Standardization of PCR resulting in a low coefficient of variance, high reproducibility, and large sample capacity is possible using optimal internal standardization and the liquid-phase hybridization/ELISA system for detection. The optimized PCR procedure indicated downregulation of atrial AT1 in end-stage human heart failure, suggesting a reduced capacity of the atria to respond to angiotensin II stimulation in end-stage heart failure.
authors.affiliation: Innere Medizin/Kardiologie, Virchow Klinikum, Humboldt Universität, Berlin, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Bauer
authors.forename: V
authors.initials: V
authors.lastname: Regitz-Zagrosek
authors.forename: J
authors.initials: J
authors.lastname: Hofmeister
authors.forename: J
authors.initials: J
authors.lastname: Lokies
authors.forename: A
authors.initials: A
authors.lastname: Rolfs
authors.forename: A G
authors.initials: AG
authors.lastname: Hildebrandt
authors.forename: R
authors.initials: R
authors.lastname: Hetzer
authors.forename: E
authors.initials: E
authors.lastname: Fleck
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011945:Receptors, Angiotensin / Q000235:genetics* / Q000378:metabolism
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D017931:DNA Primers; D012333:RNA, Messenger; D011945:Receptors, Angiotensin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases