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Reduced atrial angiotensin receptor type 1 mRNA content in end-stage human heart failure: assessment by a novel quantitative PCR-ELISA technique

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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 typel (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 liquidphase 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 endstage 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: 185680±196912 AT1 mRNA copies/μg RNA, controls: 440555±268456, 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 varince, 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.

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Abbreviations

ACE :

Angiotensin-converting enzyme

AT1 :

Angiotensin receptor typel

GAPDH :

Glyceraldehyde phosphate dehydrogenase

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Bauer, P., Regitz-Zagrosek, V., Hofmeister, J. et al. Reduced atrial angiotensin receptor type 1 mRNA content in end-stage human heart failure: assessment by a novel quantitative PCR-ELISA technique. J Mol Med 74, 447–454 (1996). https://doi.org/10.1007/BF00217520

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Key words

  • Angiotensin receptors
  • mRNA
  • Quantitative PCR
  • Human
  • Atrium