Skip to main content

Advertisement

Log in

Epac stimulation induces rapid increases in connexin43 phosphorylation and function without preconditioning effect

  • Cardiovascular physiology
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

It has been recently shown that β-adrenergic receptors are able to activate phospholipase C via the cyclic adenosine monophosphate-binding protein Epac. This new interconnection may participate in isoproterenol (Iso)-induced preconditioning. We evaluated here whether Epac could induce PKCε activation and could play a role in ischemic preconditioning through the phosphorylation of connexin43 (Cx43) and changes in gap junctional intercellular communication (GJIC). In cultured rat neonatal cardiomyocytes, we showed that in response to Iso and 8-CPT, a specific Epac activator, PKCε content was increased in particulate fractions of cell lysates independently of protein kinase A (PKA). This was associated with an increased Cx43 phosphorylation. Both Iso and 8-CPT induced an increase in GJIC that was blocked by the PKC inhibitor bisindolylmaleimide. Interestingly, inhibition of PKA partly suppressed both Iso-induced increases in Cx43 phosphorylation and in GJIC. The same PKCε-dependent Cx43 phosphorylation by β-adrenergic stimulation via Epac was found in adult rat hearts. However, in contrast with Iso that induced a preconditioning effect, perfusion of isolated hearts with 8-CPT prior to ischemia failed to improve the post-ischemia functional recovery. In conclusion, Epac stimulation induces PKCε activation and Cx43 phosphorylation with an increase in GJIC, but Epac activation does not induce preconditioning to ischemia in contrast with β-adrenergic stimulation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Bao X, Reuss L, Altenberg GA (2004) Regulation of purified and reconstituted connexin 43 hemichannels by protein kinase C-mediated phosphorylation of serine 368. J Biol Chem 279:20058

    Article  CAS  PubMed  Google Scholar 

  2. Beardslee MA, Lerner DL, Tadros PN, Laing JG, Beyer EC, Yamada KA, Kleber AG, Schuessler RB, Saffitz JE (2000) Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia. Circ Res 87:656

    CAS  PubMed  Google Scholar 

  3. Boengler K, Dodoni G, Rodriguez-Sinovas A, Cabestrero A, Ruiz-Meana M, Gres P, Konietzka I, Lopez-Iglesias C, Garcia-Dorado D, Di Lisa F, Heusch G, Schulz R (2005) Connexin43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning. Cardiovasc Res 67(2):234–244

    Article  CAS  PubMed  Google Scholar 

  4. Boengler K, Konietzka I, Buechert A, Heinen Y, Garcia-Dorado D, Heusch G, Schulz (2007) Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43. Am J Physiol Heart Circ Physiol 292:H1764–H1769

    Article  CAS  PubMed  Google Scholar 

  5. Bolli R (2007) Preconditioning: a paradigm shift in the biology of myocardial ischemia. Am J Physiol Heart Circ Physiol 292:H19

    Article  CAS  PubMed  Google Scholar 

  6. Britz-Cunningham SH, Shah MM, Zuppan CW, Fletcher WH (1995) Mutations of the Connexin43 gap-junction gene in patients with heart malformations and defects of laterality. N Engl J Med 332:1323

    Article  CAS  PubMed  Google Scholar 

  7. Burghardt RC, Barhoumi R, Sewall TC, Bowen JA (1995) Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43. J Membr Biol 148:243

    CAS  PubMed  Google Scholar 

  8. Cazorla O, Lucas A, Poirier F, Lacampagne A, Lezoualc'h F (2009) The cAMP binding protein Epac regulates cardiac myofilament function. Proc Natl Acad Sci U S A 106:14144

    Article  CAS  PubMed  Google Scholar 

  9. de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL (1998) Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396:474

    Article  PubMed  Google Scholar 

  10. Doble BW, Dang X, Ping P, Fandrich RR, Nickel BE, Jin Y, Cattini PA, Kardami E (2004) Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell-cell contact forming cardiomyocytes. J Cell Sci 117:507

    Article  CAS  PubMed  Google Scholar 

  11. Doble BW, Ping P, Fandrich RR, Cattini PA, Kardami E (2001) Protein kinase C-epsilon mediates phorbol ester-induced phosphorylation of connexin-43. Cell Commun Adhes 8:253

    Article  CAS  PubMed  Google Scholar 

  12. Hucho TB, Dina OA, Levine JD (2005) Epac mediates a cAMP-to-PKC signaling in inflammatory pain: an isolectin B4(+) neuron-specific mechanism. J Neurosci 25:6119

    Article  CAS  PubMed  Google Scholar 

  13. Hund TJ, Lerner DL, Yamada KA, Schuessler RB, Saffitz JE (2007) Protein kinase Cepsilon mediates salutary effects on electrical coupling induced by ischemic preconditioning. Heart Rhythm 4:1183

    Article  PubMed  Google Scholar 

  14. Iwaki K, Sukhatme VP, Shubeita HE, Chien KR (1990) Alpha- and beta-adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells. fos/jun expression is associated with sarcomere assembly; Egr-1 induction is primarily an alpha 1-mediated response. J Biol Chem 265:13809

    CAS  PubMed  Google Scholar 

  15. Jain SK, Schuessler RB, Saffitz JE (2003) Mechanisms of delayed electrical uncoupling induced by ischemic preconditioning. Circ Res 92:1138

    Article  CAS  PubMed  Google Scholar 

  16. Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM (1998) A family of cAMP-binding proteins that directly activate Rap1. Science 282:2275

    Article  CAS  PubMed  Google Scholar 

  17. Lampe PD, Lau AF (2000) Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys 384:205

    Article  CAS  PubMed  Google Scholar 

  18. Lampe PD, TenBroek EM, Burt JM, Kurata WE, Johnson RG, Lau AF (2000) Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication. J Cell Biol 149:1503

    Article  CAS  PubMed  Google Scholar 

  19. Li X, Heinzel FR, Boengler K, Schulz R, Heusch G (2004) Role of connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions. J Mol Cell Cardiol 36:161

    Article  CAS  PubMed  Google Scholar 

  20. Lopez I, Mak EC, Ding J, Hamm HE, Lomasney JW (2001) A novel bifunctional phospholipase c that is regulated by Galpha 12 and stimulates the Ras/mitogen-activated protein kinase pathway. J Biol Chem 276:2758

    Article  CAS  PubMed  Google Scholar 

  21. Mackay K, Mochly-Rosen D (2001) Localization, anchoring, and functions of protein kinase C isozymes in the heart. J Mol Cell Cardiol 33:1301

    Article  CAS  PubMed  Google Scholar 

  22. Metrich M, Lucas A, Gastineau M, Samuel JL, Heymes C, Morel E, Lezoualc'h F (2008) Epac mediates beta-adrenergic receptor-induced cardiomyocyte hypertrophy. Circ Res 102:959

    Article  CAS  PubMed  Google Scholar 

  23. Miyawaki H, Ashraf M (1997) Ca2+ as a mediator of ischemic preconditioning. Circ Res 80:790

    CAS  PubMed  Google Scholar 

  24. Morel E, Marcantoni A, Gastineau M, Birkedal R, Rochais F, Garnier A, Lompre AM, Vandecasteele G, Lezoualc'h F (2005) cAMP-binding protein Epac induces cardiomyocyte hypertrophy. Circ Res 97:1296

    Article  CAS  PubMed  Google Scholar 

  25. Mylvaganam S, Zhang L, Wu C, Zhang ZJ, Samoilova M, Eubanks J, Carlen PL, Poulter MO (2010) Hippocampal seizures alter the expression of the pannexin and connexin transcriptome. J Neurochem 112:92–102

    Article  CAS  PubMed  Google Scholar 

  26. Naitoh K, Yano T, Miura T, Itoh T, Miki T, Tanno M, Sato T, Hotta H, Terashima Y, Shimamoto K (2009) Roles of Cx43-associated protein kinases in suppression of gap junction-mediated chemical coupling by ischemic preconditioning. Am J Physiol Heart Circ Physiol 296:H396

    Article  CAS  PubMed  Google Scholar 

  27. Nishida M, Sato Y, Uemura A, Narita Y, Tozaki-Saitoh H, Nakaya M, Ide T, Suzuki K, Inoue K, Nagao T, Kurose H (2008) P2Y(6) receptor-Galpha(12/13) signalling in cardiomyocytes triggers pressure overload-induced cardiac fibrosis. EMBO J 27:3104–3115

    Article  CAS  PubMed  Google Scholar 

  28. Oestreich EA, Malik S, Goonasekera SA, Blaxall BC, Kelley GG, Dirksen RT, Smrcka AV (2008) EPAC and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin-kinaseII. J Biol Chem 284:1514–1522

    Article  PubMed  Google Scholar 

  29. Oestreich EA, Wang H, Malik S, Kaproth-Joslin KA, Blaxall BC, Kelley GG, Dirksen RT, Smrcka AV (2007) Epac-mediated activation of phospholipase C(epsilon) plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2+ mobilization in cardiac myocytes. J Biol Chem 282:5488

    Article  CAS  PubMed  Google Scholar 

  30. Rouet-Benzineb P, Mohammadi K, Perennec J, Poyard M, Bouanani Nel H, Crozatier B (1996) Protein kinase C isoform expression in normal and failing rabbit hearts. Circ Res 79:153

    CAS  PubMed  Google Scholar 

  31. Ruiz-Meana M, Rodriguez-Sinovas A, Cabestrero A, Boengler K, Heusch G, Garcia-Dorado D (2008) Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia-reperfusion injury. Cardiovasc Res 77:325

    Article  CAS  PubMed  Google Scholar 

  32. Saez JC, Nairn AC, Czernik AJ, Fishman GI, Spray DC, Hertzberg EL (1997) Phosphorylation of connexin43 and the regulation of neonatal rat cardiac myocyte gap junctions. J Mol Cell Cardiol 29:2131

    Article  CAS  PubMed  Google Scholar 

  33. Saez JC, Spray DC, Nairn AC, Hertzberg E, Greengard P, Bennett MV (1986) cAMP increases junctional conductance and stimulates phosphorylation of the 27-kDa principal gap junction polypeptide. Proc Natl Acad Sci USA 83:2473

    Article  CAS  PubMed  Google Scholar 

  34. Schmidt M, Evellin S, Weernink PA, von Dorp F, Rehmann H, Lomasney JW, Jakobs KH (2001) A new phospholipase-C-calcium signalling pathway mediated by cyclic AMP and a Rap GTPase. Nat Cell Biol 3:1020

    Article  CAS  PubMed  Google Scholar 

  35. Schulz R, Heusch G (2004) Connexin 43 and ischemic preconditioning. Cardiovasc Res 62:335

    Article  CAS  PubMed  Google Scholar 

  36. Schwanke U, Konietzka I, Duschin A, Li X, Schulz R, Heusch G (2002) No ischemic preconditioning in heterozygous connexin43-deficient mice. Am J Physiol Heart Circ Physiol 283:H1740

    CAS  PubMed  Google Scholar 

  37. Severs NJ, Bruce AF, Dupont E, Rothery S (2008) Remodelling of gap junctions and connexin expression in diseased myocardium. Cardiovasc Res 80:9

    Article  CAS  PubMed  Google Scholar 

  38. Shi GX, Rehmann H, Andres DA (2006) A novel cyclic AMP-dependent Epac-Rit signaling pathway contributes to PACAP38-mediated neuronal differentiation. Mol Cell Biol 26:9136

    Article  CAS  PubMed  Google Scholar 

  39. Solan JL, Lampe PD (2009) Connexin43 phosphorylation: structural changes and biological effects. Biochem J 419:261

    Article  CAS  PubMed  Google Scholar 

  40. Somekawa S, Fukuhara S, Nakaoka Y, Fujita H, Saito Y, Mochizuki N (2005) Enhanced functional gap junction neoformation by protein kinase A-dependent and Epac-dependent signals downstream of cAMP in cardiac myocytes. Circ Res 97:655

    Article  CAS  PubMed  Google Scholar 

  41. Steinberg SF (1999) The molecular basis for distinct beta-adrenergic receptor subtype actions in cardiomyocytes. Circ Res 85:1101

    CAS  PubMed  Google Scholar 

  42. Wade MH, Trosko JE, Schindler M (1986) A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science 232:525

    Article  CAS  PubMed  Google Scholar 

  43. Wang H, Oestreich EA, Maekawa N, Bullard TA, Vikstrom KL, Dirksen RT, Kelley GG, Blaxall BC, Smrcka AV (2005) Phospholipase C epsilon modulates beta-adrenergic receptor-dependent cardiac contraction and inhibits cardiac hypertrophy. Circ Res 97:1305

    Article  CAS  PubMed  Google Scholar 

  44. Weng S, Lauven M, Schaefer T, Polontchouk L, Grover R, Dhein S (2002) Pharmacological modification of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation. FASEB J 16:1114

    CAS  PubMed  Google Scholar 

  45. Xiao RP, Cheng H, Zhou YY, Kuschel M, Lakatta EG (1999) Recent advances in cardiac beta(2)-adrenergic signal transduction. Circ Res 85:1092

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Valérie Nicolas and the Imaging Platform facility of IFR141, Valérie Domergue-Dupont and the animal core facility of IFR141, Dominique Fortin for her technical assistance, and Magali Breckler for her participation in this work.

Funding sources

This work was supported in part by grants from ANR (Physio06, Genopath09; F.L) and Fondation pour la Recherche Médicale (équipe FRM F.L; Fin de Thèse, M.M).

Disclosures

None.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Bertrand Crozatier.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1

Compared with adjacent cells, which recovered fluorescence after photobleaching (Ctl), no recovery was observed either in isolated cells or with adjacent cells treated with carbenoxolone (Cbx) or heptanol that close GJIC. (DOC 78 kb)

Control.avi (AVI 19500 kb)

Iso.avi (AVI 19500 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Duquesnes, N., Derangeon, M., Métrich, M. et al. Epac stimulation induces rapid increases in connexin43 phosphorylation and function without preconditioning effect. Pflugers Arch - Eur J Physiol 460, 731–741 (2010). https://doi.org/10.1007/s00424-010-0854-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-010-0854-9

Keywords

Navigation