Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice (original) (raw)

Abstract

A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the beta 2-adrenergic receptor (beta 2AR) is known to occur in the human population. The functional consequences of this polymorphism to catecholamine signaling in relevant cells or to end-organ responsiveness, however, are not known. To explore potential differences between the two receptors, site-directed mutagenesis was carried out to mimic the polymorphism. Transgenic FVB/N mice were then created overexpressing wild-type (wt) beta 2AR or the mutant Ile-164 receptor in a targeted manner in the heart using a murine alpha myosin heavy chain promoter. The functional properties of the two receptors were then assessed at the level of in vitro cardiac myocyte signaling and in vivo cardiac responses in intact animals. The expression levels of these receptors in the two lines chosen for study were approximately 1200 fmol/mg protein in cardiac membranes, which represents a approximately 45-fold increase in expression over endogenous beta AR. Myocyte membrane adenylyl cyclase activity in the basal state was significantly lower in the Ile-164 mice (19.5 +/- 2.7 pmol/min/mg) compared with wt beta 2AR mice (35.0 +/- 4.1 pmol/min/mg), as was the maximal isoproterenol-stimulated activity (49.8 +/- 7.8 versus 77.1 +/ 7.3 pmol/min/mg). In intact animals, resting heart rate (441 +/- 21 versus 534 +/- 17 bpm) and dP/dtmax (10,923 +/- 730 versus 15,308 +/- 471 mmHg/sec) were less in the Ile-164 mice as compared with wt beta 2AR mice. Similarly, the physiologic responses to infused isoproterenol were notably less in the mutant expressing mice. Indeed, these values, as well as other contractile parameters, were indistinguishable between Ile-164 mice and nontransgenic littermates. Taken together, these results demonstrate that the Ile-164 polymorphism is substantially dysfunctional in a relevant target tissue, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo. Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to diseased states, such as heart failure, this impairment may have important pathophysiologic consequences.

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Selected References

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  1. Black J. W., Leff P., Shankley N. P., Wood J. An operational model of pharmacological agonism: the effect of E/[A] curve shape on agonist dissociation constant estimation. Br J Pharmacol. 1985 Feb;84(2):561–571. doi: 10.1111/j.1476-5381.1985.tb12941.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bond R. A., Leff P., Johnson T. D., Milano C. A., Rockman H. A., McMinn T. R., Apparsundaram S., Hyek M. F., Kenakin T. P., Allen L. F. Physiological effects of inverse agonists in transgenic mice with myocardial overexpression of the beta 2-adrenoceptor. Nature. 1995 Mar 16;374(6519):272–276. doi: 10.1038/374272a0. [DOI] [PubMed] [Google Scholar]
  3. Bristow M. R., Ginsburg R., Umans V., Fowler M., Minobe W., Rasmussen R., Zera P., Menlove R., Shah P., Jamieson S. Beta 1- and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1-receptor down-regulation in heart failure. Circ Res. 1986 Sep;59(3):297–309. doi: 10.1161/01.res.59.3.297. [DOI] [PubMed] [Google Scholar]
  4. Bristow M. R., Hershberger R. E., Port J. D., Minobe W., Rasmussen R. Beta 1- and beta 2-adrenergic receptor-mediated adenylate cyclase stimulation in nonfailing and failing human ventricular myocardium. Mol Pharmacol. 1989 Mar;35(3):295–303. [PubMed] [Google Scholar]
  5. Brodde O. E., Schüler S., Kretsch R., Brinkmann M., Borst H. G., Hetzer R., Reidemeister J. C., Warnecke H., Zerkowski H. R. Regional distribution of beta-adrenoceptors in the human heart: coexistence of functional beta 1- and beta 2-adrenoceptors in both atria and ventricles in severe congestive cardiomyopathy. J Cardiovasc Pharmacol. 1986 Nov-Dec;8(6):1235–1242. doi: 10.1097/00005344-198611000-00021. [DOI] [PubMed] [Google Scholar]
  6. Green S. A., Cole G., Jacinto M., Innis M., Liggett S. B. A polymorphism of the human beta 2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor. J Biol Chem. 1993 Nov 5;268(31):23116–23121. [PubMed] [Google Scholar]
  7. Green S. A., Holt B. D., Liggett S. B. Beta 1- and beta 2-adrenergic receptors display subtype-selective coupling to Gs. Mol Pharmacol. 1992 May;41(5):889–893. [PubMed] [Google Scholar]
  8. Green S. A., Liggett S. B. A proline-rich region of the third intracellular loop imparts phenotypic beta 1-versus beta 2-adrenergic receptor coupling and sequestration. J Biol Chem. 1994 Oct 21;269(42):26215–26219. [PubMed] [Google Scholar]
  9. Green S. A., Turki J., Innis M., Liggett S. B. Amino-terminal polymorphisms of the human beta 2-adrenergic receptor impart distinct agonist-promoted regulatory properties. Biochemistry. 1994 Aug 16;33(32):9414–9419. doi: 10.1021/bi00198a006. [DOI] [PubMed] [Google Scholar]
  10. Hall I. P., Wheatley A., Wilding P., Liggett S. B. Association of Glu 27 beta 2-adrenoceptor polymorphism with lower airway reactivity in asthmatic subjects. Lancet. 1995 May 13;345(8959):1213–1214. doi: 10.1016/s0140-6736(95)91994-5. [DOI] [PubMed] [Google Scholar]
  11. Kobilka B. K., Dixon R. A., Frielle T., Dohlman H. G., Bolanowski M. A., Sigal I. S., Yang-Feng T. L., Francke U., Caron M. G., Lefkowitz R. J. cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc Natl Acad Sci U S A. 1987 Jan;84(1):46–50. doi: 10.1073/pnas.84.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Koch W. J., Rockman H. A., Samama P., Hamilton R. A., Bond R. A., Milano C. A., Lefkowitz R. J. Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor. Science. 1995 Jun 2;268(5215):1350–1353. doi: 10.1126/science.7761854. [DOI] [PubMed] [Google Scholar]
  13. Liggett S. B., Raymond J. R. Pharmacology and molecular biology of adrenergic receptors. Baillieres Clin Endocrinol Metab. 1993 Apr;7(2):279–306. doi: 10.1016/s0950-351x(05)80178-8. [DOI] [PubMed] [Google Scholar]
  14. Milano C. A., Allen L. F., Rockman H. A., Dolber P. C., McMinn T. R., Chien K. R., Johnson T. D., Bond R. A., Lefkowitz R. J. Enhanced myocardial function in transgenic mice overexpressing the beta 2-adrenergic receptor. Science. 1994 Apr 22;264(5158):582–586. doi: 10.1126/science.8160017. [DOI] [PubMed] [Google Scholar]
  15. Reihsaus E., Innis M., MacIntyre N., Liggett S. B. Mutations in the gene encoding for the beta 2-adrenergic receptor in normal and asthmatic subjects. Am J Respir Cell Mol Biol. 1993 Mar;8(3):334–339. doi: 10.1165/ajrcmb/8.3.334. [DOI] [PubMed] [Google Scholar]
  16. Schwinn D. A., Leone B. J., Spahn D. R., Chesnut L. C., Page S. O., McRae R. L., Liggett S. B. Desensitization of myocardial beta-adrenergic receptors during cardiopulmonary bypass. Evidence for early uncoupling and late downregulation. Circulation. 1991 Dec;84(6):2559–2567. doi: 10.1161/01.cir.84.6.2559. [DOI] [PubMed] [Google Scholar]
  17. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  18. Strader C. D., Candelore M. R., Hill W. S., Sigal I. S., Dixon R. A. Identification of two serine residues involved in agonist activation of the beta-adrenergic receptor. J Biol Chem. 1989 Aug 15;264(23):13572–13578. [PubMed] [Google Scholar]
  19. Turki J., Pak J., Green S. A., Martin R. J., Liggett S. B. Genetic polymorphisms of the beta 2-adrenergic receptor in nocturnal and nonnocturnal asthma. Evidence that Gly16 correlates with the nocturnal phenotype. J Clin Invest. 1995 Apr;95(4):1635–1641. doi: 10.1172/JCI117838. [DOI] [PMC free article] [PubMed] [Google Scholar]