REDUCED ATRIAL ANGIOTENSIN RECEPTOR-TYPE-1 MESSENGER-RNA CONTENT IN END-STAGE HUMAN HEART-FAILURE - ASSESSMENT BY A NOVEL QUANTITATIVE PCR-ELISA TECHNIQUE

The number of atrial angiotensin IT binding sites is reduced in end-st age human heart failure. The goals of our study were the development o f a quantitative polymerase chain reaction for angiotensin II receptor type 1 mRNA to determine the angiotensin receptor type1 (AT1) mRNA co ntent in the atria of patients with end-stage heart failure. We establ ished a quantitative PCR based on coamplification of AT1 wild-type and an internal standard in the same PCR, followed by liquid-phase hybrid ization 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 ge ne. Atrial samples from 11 patients with end-stage stage heart failure obtained at cardiac transplantation were compared with atrial samples from 11 patients with normal cardiac function undergoing routine card iac surgery. A PCR/ELISA system with a variance of about 6% after reve rse 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 II controls (heart fa ilure: 185680+/-196912 AT1 mRNA copies/mu g RNA, controls: 440555+/-26 8456, P<0.02). When AT1 mRNA content was related to glyceraldehyde pho sphate dehydrogenase mRNA, a 65% decrease was detected (AT1/glyceralde hyde phosphate dehydrogenase: heart failure: 4.84+/-5.18; controls: 13 .74+/-7.77; P<0.005). Standardization of PCR resulting in a low coeffi cient of variance, high reproducibility, and large sample capacity is possible using optimal internal standardization and the liquid-phase h ybridization/ELISA system for detection. The optimized PCR procedure i ndicated 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.