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.
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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
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
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
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
Bolli R (2007) Preconditioning: a paradigm shift in the biology of myocardial ischemia. Am J Physiol Heart Circ Physiol 292:H19
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
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
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
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
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
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
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
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
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
Jain SK, Schuessler RB, Saffitz JE (2003) Mechanisms of delayed electrical uncoupling induced by ischemic preconditioning. Circ Res 92:1138
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
Lampe PD, Lau AF (2000) Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys 384:205
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
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
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
Mackay K, Mochly-Rosen D (2001) Localization, anchoring, and functions of protein kinase C isozymes in the heart. J Mol Cell Cardiol 33:1301
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
Miyawaki H, Ashraf M (1997) Ca2+ as a mediator of ischemic preconditioning. Circ Res 80:790
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
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
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
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
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
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
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
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
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
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
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
Schulz R, Heusch G (2004) Connexin 43 and ischemic preconditioning. Cardiovasc Res 62:335
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
Severs NJ, Bruce AF, Dupont E, Rothery S (2008) Remodelling of gap junctions and connexin expression in diseased myocardium. Cardiovasc Res 80:9
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
Solan JL, Lampe PD (2009) Connexin43 phosphorylation: structural changes and biological effects. Biochem J 419:261
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
Steinberg SF (1999) The molecular basis for distinct beta-adrenergic receptor subtype actions in cardiomyocytes. Circ Res 85:1101
Wade MH, Trosko JE, Schindler M (1986) A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science 232:525
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
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
Xiao RP, Cheng H, Zhou YY, Kuschel M, Lakatta EG (1999) Recent advances in cardiac beta(2)-adrenergic signal transduction. Circ Res 85:1092
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).
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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)
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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
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DOI: https://doi.org/10.1007/s00424-010-0854-9