Wnt-1 regulation of connexin43 in cardiac myocytes

Z Ai

A Fischer

D Spray

A Brown

G Fishman

^{Section of Myocardial Biology, Zena and Michael A. Wiener Cardiovascular Institute, Departments of Medicine, Physiology and Biophysics, and Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, USA}^{Departments of Neurosciences and Medicine, Albert Einstein College of Medicine, Bronx, New York 10583, USA}^{Department of Cell Biology, Weill Medical College of Cornell University and Strang Cancer Prevention Center, New York, New York 10021, USA}

Address correspondence to: Glenn I. Fishman, Section of Myocardial Biology, Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1269, New York, New York 10029, USA. Phone: (212) 241-2179; Fax: (212) 828-4178; E-mail: ude.mssm.cod@10gmhsif.

Received 1999 Jul 8; Accepted 1999 Dec 6.

Abstract

Gap junction channels composed of connexin43 (Cx43) are essential for normal heart formation and function. We studied the potential role of the Wnt family of secreted polypeptides as regulators of Cx43 expression and gap junction channel function in dissociated myocytes and intact hearts. Neonatal rat cardiomyocytes responded to Li^{, which mimics Wnt signaling, by accumulating the effector protein β-catenin and by inducing Cx43 mRNA and protein markedly. Induction of Cx43 expression was also observed in cardiomyocytes cocultured with Rat-2 fibroblasts or N2A neuroblastoma cells programmed to secrete bioactive Wnt-1. By transfecting a}Cx43 promoter-reporter gene construct into cardiomyocytes, we demonstrated that the inductive effect of Wnt signaling was transcriptionally mediated. Enhanced expression of Cx43 increased cardiomyocyte cell coupling, as determined by Lucifer Yellow dye transfer and by calcium wave propagation. Conversely, in a transgenic cardiomyopathic mouse model that exhibits ventricular arrhythmias and gap junctional remodeling, β-catenin and Cx43 expression were downregulated concordantly. In response to Wnt signaling, the accumulating Cx43 colocalized with β-catenin in the junctional membrane; moreover, forced expression of Cx43 in cardiomyocytes reduced the transactivation potential of β-catenin. These findings demonstrate that Wnt signaling is an important modulator of Cx43-dependent intercellular coupling in the heart, and they support the hypothesis that dysregulated signaling contributes to altered impulse propagation and arrhythmia in the myopathic heart.

Abstract

Acknowledgments

Supported in part by grants from the National Institutes of Health (NIH; RO1-HL38499 to G.I. Fishman, and RO1-CA47207 to A.M.C. Brown). The confocal laser scanning microscopy was performed at the Mount Sinai School of Medicine core facility supported by NIH grant 1 S10 RR09145-01 and NSF Major Research Instrumentation grant DBI-9724504. G.I. Fishman is a recipient of the Established Investigator Award of the American Heart Association. We thank J. Zhang for excellent technical assistance, E. Beyer for providing the rat Cx43 probe, E. Hertzberg for Cx43 antisera, R. Werner for the Cx43 reporter gene construct, J. Kitajewski for the pTOPFLASH, pFOPFLASH, and β-catenin expression plasmids, and S. Henderson for assistance with the confocal microscopy.

Acknowledgments

References

1. Loewenstein WRJunctional intercellular communication: the cell-to-cell membrane channel. Physiol Rev. 1981;61:829–913.[PubMed][Google Scholar]
2. Bennett MV, et al Gap junctions: new tools, new answers, new questions. Neuron. 1991;6:305–320.[PubMed][Google Scholar]
3. Kumar NM, Gilula NBThe gap junction communication channel. Cell. 1996;84:381–388.[PubMed][Google Scholar]
4. Spray, D.C., Suadicani, S., Srinivas, M., and Fishman, G.I. 1999. Gap junctions in the cardiovascular system. In Handbook of physiology: The heart. H. Fozzard, E. Page, and J. Solaro, editors. Oxford University Press. San Francisco, CA. In press.
5. Saffitz JE, et al The molecular basis of anisotropy: role of gap junctions. J Cardiovasc Electrophysiol. 1995;6:498–510.[PubMed][Google Scholar]
6. Coppen SR, Severs NJ, Gourdie RGConnexin45 (alpha 6) expression delineates an extended conduction system in the embryonic and mature rodent heart. Dev Genet. 1999;24:82–90.[PubMed][Google Scholar]
7. Smith JH, Green CR, Peters NS, Rothery S, Severs NJ. Altered patterns of gap junction distribution in ischemic heart disease. An immunohistochemical study of human myocardium using laser scanning confocal microscopy. Am J Pathol. 1991;139:801–821.
8. Peters NS, Green CR, Poole-Wilson PA, Severs NJReduced content of connexin43 gap junctions in ventricular myocardium from hypertrophied and ischemic human hearts. Circulation. 1993;88:864–875.[PubMed][Google Scholar]
9. Peters NS, Coromilas J, Severs NJ, Wit ALDisturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia. Circulation. 1997;95:988–996.[PubMed][Google Scholar]
10. Lee P, et al Conditional lineage ablation to model human diseases. Proc Natl Acad Sci USA. 1998;95:11371–11376.[Google Scholar]
11. Hagendorff A, Schumacher B, Kirchhoff S, Luderitz B, Willecke KConduction disturbances and increased atrial vulnerability in Connexin40-deficient mice analyzed by transesophageal stimulation. Circulation. 1999;99:1508–1515.[PubMed][Google Scholar]
12. Guerrero PA, et al Slow ventricular conduction in mice heterozygous for a connexin43 null mutation. J Clin Invest. 1997;99:1991–1998.[Google Scholar]
13. Reaume AG, et al Cardiac malformation in neonatal mice lacking connexin43. Science. 1995;267:1831–1834.[PubMed][Google Scholar]
14. Waldo KL, Lo CW, Kirby MLConnexin 43 expression reflects neural crest patterns during cardiovascular development. Dev Biol. 1999;208:307–323.[PubMed][Google Scholar]
15. Cadigan KM, Nusse RWnt signaling: a common theme in animal development. Genes Dev. 1997;11:3286–3305.[PubMed][Google Scholar]
16. Nusse R. WNT targets. Repression and activation. Trends Genet. 1999;15:1–3.[PubMed]
17. Olson DJ, Christian JL, Moon RTEffect of wnt-1 and related proteins on gap junctional communication in Xenopus embryos. Science. 1991;252:1173–1176.[PubMed][Google Scholar]
18. Olson DJ, Moon RTDistinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos. Dev Biol. 1992;151:204–212.[PubMed][Google Scholar]
19. Meyer RA, et al Developmental regulation and asymmetric expression of the gene encoding Cx43 gap junctions in the mouse limb bud. Dev Genet. 1997;21:290–300.[PubMed][Google Scholar]
20. van der Heyden MA, et al Identification of connexin43 as a functional target for Wnt signalling. J Cell Sci. 1998;111:1741–1749.[PubMed][Google Scholar]
21. Park M, Wu X, Golden K, Axelrod JD, Bodmer RThe wingless signaling pathway is directly involved in Drosophila heart development. Dev Biol. 1996;177:104–116.[PubMed][Google Scholar]
22. Park M, Venkatesh TV, Bodmer RDual role for the zeste-white3/shaggy-encoded kinase in mesoderm and heart development of Drosophila.Dev Genet. 1998;22:201–211.[PubMed][Google Scholar]
23. Clark CC, et al Molecular cloning of the human proto-oncogene Wnt-5A and mapping of the gene (WNT5A) to chromosome 3p14-p21. Genomics. 1993;18:249–260.[PubMed][Google Scholar]
24. Zakin LD, et al Structure and expression of Wnt13, a novel mouse Wnt2 related gene. Mech Dev. 1998;73:107–116.[PubMed][Google Scholar]
25. Duplaa C, Jaspard B, Moreau C, D’Amore PA. Identification and cloning of a secreted protein related to the cysteine-rich domain of frizzled. Evidence for a role in endothelial cell growth control. Circ Res. 1999;84:1433–1445.[PubMed]
26. Augustine K, Liu ET, Sadler TWAntisense attenuation of Wnt-1 and Wnt-3a expression in whole embryo culture reveals roles for these genes in craniofacial, spinal cord, and cardiac morphogenesis. Dev Genet. 1993;14:500–520.[PubMed][Google Scholar]
27. Wang YK, Sporle R, Paperna T, Schughart K, Francke UCharacterization and expression pattern of the frizzled gene Fzd9, the mouse homolog of FZD9 which is deleted in Williams-Beuren syndrome. Genomics. 1999;57:235–248.[PubMed][Google Scholar]
28. De Leon JR, Buttrick PM, Fishman GIFunctional analysis of the connexin43 gene promoter in vivo and in vitro. J Mol Cell Cardiol. 1994;26:379–389.[PubMed][Google Scholar]
29. Harding P, Carretero OA, LaPointe MCEffects of interleukin-1 beta and nitric oxide on cardiac myocytes. Hypertension. 1995;25:421–430.[PubMed][Google Scholar]
30. Yu W, Dahl G, Werner RThe connexin43 gene is responsive to oestrogen. Proc R Soc Lond B Biol Sci. 1994;255:125–132.[PubMed][Google Scholar]
31. Korinek V, et al Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC–/– colon carcinoma. Science. 1997;275:1784–1787.[PubMed][Google Scholar]
32. Young CS, Kitamura M, Hardy S, Kitajewski JWnt-1 induces growth, cytosolic beta-catenin, and Tcf/Lef transcriptional activation in Rat-1 fibroblasts. Mol Cell Biol. 1998;18:2474–2485.[Google Scholar]
33. McDonald TV, et al A minK-HERG complex regulates the cardiac potassium current I(Kr) Nature. 1997;388:289–292.[PubMed][Google Scholar]
34. Brown, A.M.C., and Dougherty, J.P. 1995. Retroviral vectors. In DNA cloning IV, mammalian systems: a practical approach. D.M. Glover and D.B. Hames, editors. Oxford University Press. Oxford, UK. 113–142.
35. Jue SF, Bradley RS, Rudnicki JA, Varmus HE, Brown AMThe mouse Wnt-1 gene can act via a paracrine mechanism in transformation of mammary epithelial cells. Mol Cell Biol. 1992;12:321–328.[Google Scholar]
36. Giarre M, Semenov MV, Brown AMWnt signaling stabilizes the dual-function protein beta-catenin in diverse cell types. Ann NY Acad Sci. 1998;857:43–55.[PubMed][Google Scholar]
37. Stambolic V, Ruel L, Woodgett JRLithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells [erratum 1997, 7:196] Curr Biol. 1996;6:1664–1668.[PubMed][Google Scholar]
38. Hedgepeth CM, et al Activation of the Wnt signaling pathway: a molecular mechanism for lithium action. Dev Biol. 1997;185:82–91.[PubMed][Google Scholar]
39. Darrow BJ, Fast VG, Kleber AG, Beyer EC, Saffitz JE. Functional and structural assessment of intercellular communication. Increased conduction velocity and enhanced connexin expression in dibutyryl cAMP-treated cultured cardiac myocytes. Circ Res. 1996;79:174–183.[PubMed]
40. Yokoya F, Imamoto N, Tachibana T, Yoneda YBeta-catenin can be transported into the nucleus in a Ran-unassisted manner. Mol Biol Cell. 1999;10:1119–1131.[Google Scholar]
41. McCrea PD, Turck CW, Gumbiner BA homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin. Science. 1991;254:1359–1361.[PubMed][Google Scholar]
42. Dodge SM, et al Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes. J Am Coll Cardiol. 1998;32:800–807.[PubMed][Google Scholar]
43. Bafico A, et al Interaction of frizzled related protein (FRP) with wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of wnt signaling. J Biol Chem. 1999;274:16180–16187.[PubMed][Google Scholar]
44. Chan SD, et al Two homologs of the Drosophila polarity gene frizzled (fz) are widely expressed in mammalian tissues. J Biol Chem. 1992;267:25202–25207.[PubMed][Google Scholar]
45. Hu E, et al Tissue restricted expression of two human Frzbs in preadipocytes and pancreas [erratum 1998, 248:941–943] Biochem Biophys Res Commun. 1998;247:287–293.[PubMed][Google Scholar]
46. Sagara N, et al Molecular cloning, differential expression, and chromosomal localization of human frizzled-1, frizzled-2, and frizzled-7. Biochem Biophys Res Commun. 1998;252:117–122.[PubMed][Google Scholar]
47. Shimizu H, et al Transformation by Wnt family proteins correlates with regulation of beta-catenin. Cell Growth Differ. 1997;8:1349–1358.[PubMed][Google Scholar]
48. Toyofuku T, et al Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes. J Biol Chem. 1998;273:12725–12731.[PubMed][Google Scholar]
49. Heasman J, et al Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell. 1994;79:791–803.[PubMed][Google Scholar]
50. Orsulic S, Huber O, Aberle H, Arnold S, Kemler RE-cadherin binding prevents beta-catenin nuclear localization and beta-catenin/LEF-1-mediated transactivation. J Cell Sci. 1999;112:1237–1245.[PubMed][Google Scholar]
51. Hotz-Wagenblatt A, Shalloway DGap junctional communication and neoplastic transformation. Crit Rev Oncog. 1993;4:541–558.[PubMed][Google Scholar]
52. Mehta PP, Hotz-Wagenblatt A, Rose B, Shalloway D, Loewenstein WRIncorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth. J Membr Biol. 1991;124:207–225.[PubMed][Google Scholar]
53. Blankesteijn WM, Essers-Janssen YP, Ulrich MM, Smits JFIncreased expression of a homologue of Drosophila tissue polarity gene “frizzled” in left ventricular hypertrophy in the rat, as identified by subtractive hybridization. J Mol Cell Cardiol. 1996;28:1187–1191.[PubMed][Google Scholar]
54. Blankesteijn WM, Essers-Janssen YP, Verluyten MJ, Daemen MJ, Smits JFA homologue of Drosophila tissue polarity gene frizzled is expressed in migrating myofibroblasts in the infarcted rat heart. Nat Med. 1997;3:541–544.[PubMed][Google Scholar]