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. 1994 Jul;94(1):301–310. doi: 10.1172/JCI117322

Increased angiotensin-I converting enzyme gene expression in the failing human heart. Quantification by competitive RNA polymerase chain reaction.

R Studer 1, H Reinecke 1, B Müller 1, J Holtz 1, H Just 1, H Drexler 1
PMCID: PMC296310  PMID: 8040271

Abstract

Local activation of the components of the renin angiotensin system in the heart is regarded as an important modulator of cardiac phenotype and function; however, little is known about their presence, regulation, and potential activation in the human heart. To investigate the gene expression of major angiotensin-II-forming enzymes in left ventricles of normal (n = 9) and failing human hearts (n = 20), we established a competitive RNA-polymerase chain reaction (PCR) for mRNA quantification of angiotensin-I converting enzyme (ACE) and human heart chymase. For each gene, competitor RNA targets with small internal deletions were used as internal standards to quantify the original number of transcripts and to control reverse transcription and PCR. In PCR, each target and the corresponding competitor were amplified by competing for the same primer oligonucleotides. The variability of ACE RNA-PCR was 11% indicating a high reproducibility of this method. In addition, ACE mRNA levels obtained by competitive RNA-PCR correlated favorably with traditional slot blot hybridization (r = 0.69, n = 10; P < 0.05). Compared with nonfailing hearts, the number of ACE transcripts referred to 100 ng of total RNA was increased threefold in patients with chronic heart failure (4.2 +/- 2.5 vs. 12.8 +/- 6 x 10(5); P < 0.0005). In contrast, no significant difference was found in chymase gene expression between normal and failing hearts. Thus, the expression of the cardiac ACE but not of human heart chymase is upregulated in failing human heart indicating an activation of the cardiac renin-angiotensin system in patients with advanced heart failure.

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Selected References

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  1. Aceto J. F., Baker K. M. [Sar1]angiotensin II receptor-mediated stimulation of protein synthesis in chick heart cells. Am J Physiol. 1990 Mar;258(3 Pt 2):H806–H813. doi: 10.1152/ajpheart.1990.258.3.H806. [DOI] [PubMed] [Google Scholar]
  2. Alhenc-Gelas F., Richard J., Courbon D., Warnet J. M., Corvol P. Distribution of plasma angiotensin I-converting enzyme levels in healthy men: relationship to environmental and hormonal parameters. J Lab Clin Med. 1991 Jan;117(1):33–39. [PubMed] [Google Scholar]
  3. Baker K. M., Aceto J. F. Angiotensin II stimulation of protein synthesis and cell growth in chick heart cells. Am J Physiol. 1990 Aug;259(2 Pt 2):H610–H618. doi: 10.1152/ajpheart.1990.259.2.H610. [DOI] [PubMed] [Google Scholar]
  4. Baker K. M., Booz G. W., Dostal D. E. Cardiac actions of angiotensin II: Role of an intracardiac renin-angiotensin system. Annu Rev Physiol. 1992;54:227–241. doi: 10.1146/annurev.ph.54.030192.001303. [DOI] [PubMed] [Google Scholar]
  5. Baker K. M., Chernin M. I., Wixson S. K., Aceto J. F. Renin-angiotensin system involvement in pressure-overload cardiac hypertrophy in rats. Am J Physiol. 1990 Aug;259(2 Pt 2):H324–H332. doi: 10.1152/ajpheart.1990.259.2.H324. [DOI] [PubMed] [Google Scholar]
  6. Becker-André M., Hahlbrock K. Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res. 1989 Nov 25;17(22):9437–9446. doi: 10.1093/nar/17.22.9437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brillantes A. M., Allen P., Takahashi T., Izumo S., Marks A. R. Differences in cardiac calcium release channel (ryanodine receptor) expression in myocardium from patients with end-stage heart failure caused by ischemic versus dilated cardiomyopathy. Circ Res. 1992 Jul;71(1):18–26. doi: 10.1161/01.res.71.1.18. [DOI] [PubMed] [Google Scholar]
  8. Cambien F., Poirier O., Lecerf L., Evans A., Cambou J. P., Arveiler D., Luc G., Bard J. M., Bara L., Ricard S. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature. 1992 Oct 15;359(6396):641–644. doi: 10.1038/359641a0. [DOI] [PubMed] [Google Scholar]
  9. Campbell D. J., Habener J. F. Angiotensinogen gene is expressed and differentially regulated in multiple tissues of the rat. J Clin Invest. 1986 Jul;78(1):31–39. doi: 10.1172/JCI112566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chan Y. L., Gutell R., Noller H. F., Wool I. G. The nucleotide sequence of a rat 18 S ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18 S ribosomal ribonucleic acid. J Biol Chem. 1984 Jan 10;259(1):224–230. [PubMed] [Google Scholar]
  11. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  12. Danilov S. M., Faerman A. I., Printseva OYu, Martynov A. V., Sakharov IYu, Trakht I. N. Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies. Histochemistry. 1987;87(5):487–490. doi: 10.1007/BF00496822. [DOI] [PubMed] [Google Scholar]
  13. Dostal D. E., Rothblum K. N., Conrad K. M., Cooper G. R., Baker K. M. Detection of angiotensin I and II in cultured rat cardiac myocytes and fibroblasts. Am J Physiol. 1992 Oct;263(4 Pt 1):C851–C863. doi: 10.1152/ajpcell.1992.263.4.C851. [DOI] [PubMed] [Google Scholar]
  14. Dzau V. J., Ellison K. E., Brody T., Ingelfinger J., Pratt R. E. A comparative study of the distributions of renin and angiotensinogen messenger ribonucleic acids in rat and mouse tissues. Endocrinology. 1987 Jun;120(6):2334–2338. doi: 10.1210/endo-120-6-2334. [DOI] [PubMed] [Google Scholar]
  15. Eberli F. R., Apstein C. S., Ngoy S., Lorell B. H. Exacerbation of left ventricular ischemic diastolic dysfunction by pressure-overload hypertrophy. Modification by specific inhibition of cardiac angiotensin converting enzyme. Circ Res. 1992 May;70(5):931–943. doi: 10.1161/01.res.70.5.931. [DOI] [PubMed] [Google Scholar]
  16. Finckh M., Hellmann W., Ganten D., Furtwängler A., Allgeier J., Boltz M., Holtz J. Enhanced cardiac angiotensinogen gene expression and angiotensin converting enzyme activity in tachypacing-induced heart failure in rats. Basic Res Cardiol. 1991 Jul-Aug;86(4):303–316. doi: 10.1007/BF02191528. [DOI] [PubMed] [Google Scholar]
  17. Foult J. M., Tavolaro O., Antony I., Nitenberg A. Direct myocardial and coronary effects of enalaprilat in patients with dilated cardiomyopathy: assessment by a bilateral intracoronary infusion technique. Circulation. 1988 Feb;77(2):337–344. doi: 10.1161/01.cir.77.2.337. [DOI] [PubMed] [Google Scholar]
  18. Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hellmann W., Suzuki F., Ohkubo H., Nakanishi S., Ludwig G., Ganten D. Angiotensinogen gene expression in extrahepatic rat tissues: application of a solution hybridization assay. Naunyn Schmiedebergs Arch Pharmacol. 1988 Sep;338(3):327–331. doi: 10.1007/BF00173408. [DOI] [PubMed] [Google Scholar]
  20. Hirsch A. T., Talsness C. E., Schunkert H., Paul M., Dzau V. J. Tissue-specific activation of cardiac angiotensin converting enzyme in experimental heart failure. Circ Res. 1991 Aug;69(2):475–482. doi: 10.1161/01.res.69.2.475. [DOI] [PubMed] [Google Scholar]
  21. Holtz J., Studer R., Reinecke H., Just H., Drexler H. Modulation of myocardial sarcoplasmic reticulum Ca(++)-ATPase in cardiac hypertrophy by angiotensin converting enzyme? Basic Res Cardiol. 1992;87 (Suppl 2):191–204. doi: 10.1007/978-3-642-72477-0_17. [DOI] [PubMed] [Google Scholar]
  22. Hubert C., Houot A. M., Corvol P., Soubrier F. Structure of the angiotensin I-converting enzyme gene. Two alternate promoters correspond to evolutionary steps of a duplicated gene. J Biol Chem. 1991 Aug 15;266(23):15377–15383. [PubMed] [Google Scholar]
  23. Kinoshita A., Urata H., Bumpus F. M., Husain A. Multiple determinants for the high substrate specificity of an angiotensin II-forming chymase from the human heart. J Biol Chem. 1991 Oct 15;266(29):19192–19197. [PubMed] [Google Scholar]
  24. Kunapuli S. P., Kumar A. Molecular cloning of human angiotensinogen cDNA and evidence for the presence of its mRNA in rat heart. Circ Res. 1987 May;60(5):786–790. doi: 10.1161/01.res.60.5.786. [DOI] [PubMed] [Google Scholar]
  25. Lindpaintner K., Lu W., Neidermajer N., Schieffer B., Just H., Ganten D., Drexler H. Selective activation of cardiac angiotensinogen gene expression in post-infarction ventricular remodeling in the rat. J Mol Cell Cardiol. 1993 Feb;25(2):133–143. doi: 10.1006/jmcc.1993.1017. [DOI] [PubMed] [Google Scholar]
  26. Linz W., Schaper J., Wiemer G., Albus U., Schölkens B. A. Ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats. Br J Pharmacol. 1992 Dec;107(4):970–975. doi: 10.1111/j.1476-5381.1992.tb13393.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Linz W., Schölkens B. A., Ganten D. Converting enzyme inhibition specifically prevents the development and induces regression of cardiac hypertrophy in rats. Clin Exp Hypertens A. 1989;11(7):1325–1350. doi: 10.3109/10641968909038172. [DOI] [PubMed] [Google Scholar]
  28. Murphy T. J., Alexander R. W., Griendling K. K., Runge M. S., Bernstein K. E. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991 May 16;351(6323):233–236. doi: 10.1038/351233a0. [DOI] [PubMed] [Google Scholar]
  29. Oikawa S., Imai M., Ueno A., Tanaka S., Noguchi T., Nakazato H., Kangawa K., Fukuda A., Matsuo H. Cloning and sequence analysis of cDNA encoding a precursor for human atrial natriuretic polypeptide. Nature. 1984 Jun 21;309(5970):724–726. doi: 10.1038/309724a0. [DOI] [PubMed] [Google Scholar]
  30. Paul M., Wagner J., Dzau V. J. Gene expression of the renin-angiotensin system in human tissues. Quantitative analysis by the polymerase chain reaction. J Clin Invest. 1993 May;91(5):2058–2064. doi: 10.1172/JCI116428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pfeffer J. M., Pfeffer M. A., Braunwald E. Influence of chronic captopril therapy on the infarcted left ventricle of the rat. Circ Res. 1985 Jul;57(1):84–95. doi: 10.1161/01.res.57.1.84. [DOI] [PubMed] [Google Scholar]
  32. Raynolds M. V., Bristow M. R., Bush E. W., Abraham W. T., Lowes B. D., Zisman L. S., Taft C. S., Perryman M. B. Angiotensin-converting enzyme DD genotype in patients with ischaemic or idiopathic dilated cardiomyopathy. Lancet. 1993 Oct 30;342(8879):1073–1075. doi: 10.1016/0140-6736(93)92061-w. [DOI] [PubMed] [Google Scholar]
  33. Rigat B., Hubert C., Alhenc-Gelas F., Cambien F., Corvol P., Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990 Oct;86(4):1343–1346. doi: 10.1172/JCI114844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rogg H., Schmid A., de Gasparo M. Identification and characterization of angiotensin II receptor subtypes in rabbit ventricular myocardium. Biochem Biophys Res Commun. 1990 Nov 30;173(1):416–422. doi: 10.1016/s0006-291x(05)81074-5. [DOI] [PubMed] [Google Scholar]
  35. Rosen K. M., Lamperti E. D., Villa-Komaroff L. Optimizing the northern blot procedure. Biotechniques. 1990 Apr;8(4):398–403. [PubMed] [Google Scholar]
  36. Saito Y., Nakao K., Arai H., Nishimura K., Okumura K., Obata K., Takemura G., Fujiwara H., Sugawara A., Yamada T. Augmented expression of atrial natriuretic polypeptide gene in ventricle of human failing heart. J Clin Invest. 1989 Jan;83(1):298–305. doi: 10.1172/JCI113872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Samani N. J., Swales J. D., Brammar W. J. Expression of the renin gene in extra-renal tissues of the rat. Biochem J. 1988 Aug 1;253(3):907–910. doi: 10.1042/bj2530907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sawa H., Tokuchi F., Mochizuki N., Endo Y., Furuta Y., Shinohara T., Takada A., Kawaguchi H., Yasuda H., Nagashima K. Expression of the angiotensinogen gene and localization of its protein in the human heart. Circulation. 1992 Jul;86(1):138–146. doi: 10.1161/01.cir.86.1.138. [DOI] [PubMed] [Google Scholar]
  39. Schunkert H., Dzau V. J., Tang S. S., Hirsch A. T., Apstein C. S., Lorell B. H. Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy. Effects on coronary resistance, contractility, and relaxation. J Clin Invest. 1990 Dec;86(6):1913–1920. doi: 10.1172/JCI114924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schunkert H., Ingelfinger J. R., Hirsch A. T., Pinto Y., Remme W. J., Jacob H., Dzau V. J. Feedback regulation of angiotensin converting enzyme activity and mRNA levels by angiotensin II. Circ Res. 1993 Feb;72(2):312–318. doi: 10.1161/01.res.72.2.312. [DOI] [PubMed] [Google Scholar]
  41. Schäfer R., Zischler H., Birsner U., Becker A., Epplen J. T. Optimized oligonucleotide probes for DNA fingerprinting. Electrophoresis. 1988 Aug;9(8):369–374. doi: 10.1002/elps.1150090804. [DOI] [PubMed] [Google Scholar]
  42. Seidman C. E., Duby A. D., Choi E., Graham R. M., Haber E., Homcy C., Smith J. A., Seidman J. G. The structure of rat preproatrial natriuretic factor as defined by a complementary DNA clone. Science. 1984 Jul 20;225(4659):324–326. doi: 10.1126/science.6234658. [DOI] [PubMed] [Google Scholar]
  43. Siebert P. D., Larrick J. W. Competitive PCR. Nature. 1992 Oct 8;359(6395):557–558. doi: 10.1038/359557a0. [DOI] [PubMed] [Google Scholar]
  44. Soubrier F., Alhenc-Gelas F., Hubert C., Allegrini J., John M., Tregear G., Corvol P. Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9386–9390. doi: 10.1073/pnas.85.24.9386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Takahashi T., Allen P. D., Izumo S. Expression of A-, B-, and C-type natriuretic peptide genes in failing and developing human ventricles. Correlation with expression of the Ca(2+)-ATPase gene. Circ Res. 1992 Jul;71(1):9–17. doi: 10.1161/01.res.71.1.9. [DOI] [PubMed] [Google Scholar]
  46. Takemura G., Fujiwara H., Horike K., Mukoyama M., Saito Y., Nakao K., Matsuda M., Kawamura A., Ishida M., Kida M. Ventricular expression of atrial natriuretic polypeptide and its relations with hemodynamics and histology in dilated human hearts. Immunohistochemical study of the endomyocardial biopsy specimens. Circulation. 1989 Nov;80(5):1137–1147. doi: 10.1161/01.cir.80.5.1137. [DOI] [PubMed] [Google Scholar]
  47. Tiret L., Kee F., Poirier O., Nicaud V., Lecerf L., Evans A., Cambou J. P., Arveiler D., Luc G., Amouyel P. Deletion polymorphism in angiotensin-converting enzyme gene associated with parental history of myocardial infarction. Lancet. 1993 Apr 17;341(8851):991–992. doi: 10.1016/0140-6736(93)91075-w. [DOI] [PubMed] [Google Scholar]
  48. Urata H., Boehm K. D., Philip A., Kinoshita A., Gabrovsek J., Bumpus F. M., Husain A. Cellular localization and regional distribution of an angiotensin II-forming chymase in the heart. J Clin Invest. 1993 Apr;91(4):1269–1281. doi: 10.1172/JCI116325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Urata H., Healy B., Stewart R. W., Bumpus F. M., Husain A. Angiotensin II-forming pathways in normal and failing human hearts. Circ Res. 1990 Apr;66(4):883–890. doi: 10.1161/01.res.66.4.883. [DOI] [PubMed] [Google Scholar]
  50. Urata H., Kinoshita A., Misono K. S., Bumpus F. M., Husain A. Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart. J Biol Chem. 1990 Dec 25;265(36):22348–22357. [PubMed] [Google Scholar]
  51. Urata H., Kinoshita A., Perez D. M., Misono K. S., Bumpus F. M., Graham R. M., Husain A. Cloning of the gene and cDNA for human heart chymase. J Biol Chem. 1991 Sep 15;266(26):17173–17179. [PubMed] [Google Scholar]
  52. Wharton J., Anderson R. H., Springall D., Power R. F., Rose M., Smith A., Espejo R., Khaghani A., Wallwork J., Yacoub M. H. Localisation of atrial natriuretic peptide immunoreactivity in the ventricular myocardium and conduction system of the human fetal and adult heart. Br Heart J. 1988 Oct;60(4):267–274. doi: 10.1136/hrt.60.4.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yamada H., Fabris B., Allen A. M., Jackson B., Johnston C. I., Mendelsohn A. O. Localization of angiotensin converting enzyme in rat heart. Circ Res. 1991 Jan;68(1):141–149. doi: 10.1161/01.res.68.1.141. [DOI] [PubMed] [Google Scholar]

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