title: Reversible connexin 43 dephosphorylation during hypoxia and reoxygenation is linked to cellular ATP levels.
abstract: Altered gap junction coupling of cardiac myocytes during ischemia may contribute to development of lethal arrhythmias. The phosphoprotein connexin 43 (Cx43) is the major constituent of gap junctions. Dephosphorylation of Cx43 and uncoupling of gap junctions occur during ischemia, but the significance of Cx43 phosphorylation in this setting is unknown. Here we show that Cx43 dephosphorylation in synchronously contracting myocytes during ischemia is reversible, independent of hypoxia, and closely associated with cellular ATP levels. Cx43 became profoundly dephosphorylated during hypoxia only when glucose supplies were limited and was completely rephosphorylated within 30 minutes of reoxygenation. Similarly, direct reduction of ATP by various combinations of metabolic inhibitors and by ouabain was closely paralleled by loss of phosphoCx43 and recovery of phosphoCx43 accompanied restoration of ATP. Dephosphorylation of Cx43 could not be attributed to hypoxia, acid pH or secreted metabolites, or to AMP-activated protein kinase; moreover, the process was selective for Cx43 because levels of phospho-extracellular signal regulated kinase (ERK)1/2 were increased throughout. Rephosphorylation of Cx43 was not dependent on new protein synthesis, or on activation of protein kinases A or G, ERK1/2, p38 mitogen-activated protein kinase, or Jun kinase; however, broad-spectrum protein kinase C inhibitors prevented Cx43 rephosphorylation while also sensitizing myocytes to reoxygenation-mediated cell death. We conclude that Cx43 is reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation by a novel mechanism that is sensitive to nonlethal fluctuations in cellular ATP. The role of this regulated phosphorylation in the adaptation to ischemia remains to be determined.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami, Fla, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Turner
authors.forename: Guy A
authors.initials: GA
authors.lastname: Haywood
authors.forename: Peter
authors.initials: P
authors.lastname: Andreka
authors.forename: Lijing
authors.initials: L
authors.lastname: You
authors.forename: Patricia E
authors.initials: PE
authors.lastname: Martin
authors.forename: W Howard
authors.initials: WH
authors.lastname: Evans
authors.forename: Keith A
authors.initials: KA
authors.lastname: Webster
authors.forename: Nanette H
authors.initials: NH
authors.lastname: Bishopric
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D053119:Benzophenanthridines; D020126:Brefeldin A / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D015687:Cell Hypoxia; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D018031:Connexin 43 / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D003847:Deoxyglucose / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000502:physiology; D008301:Maleimides / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011190:Potassium Cyanide / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012265:Ribonucleotides / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D016559:Tacrolimus / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000470:Alkaloids; D053119:Benzophenanthridines; D002227:Carbazoles; D018031:Connexin 43; D005419:Flavonoids; D007093:Imidazoles; D007211:Indoles; D008301:Maleimides; D010617:Phenanthridines; D011725:Pyridines; D011758:Pyrroles; D011993:Recombinant Fusion Proteins; D012265:Ribonucleotides; C073601:KT 5823; D019319:Okadaic Acid; D020126:Brefeldin A; D000620:Aminoimidazole Carboxamide; C057416:KT 5720; D010042:Ouabain; D000968:Antimycin A; D000255:Adenosine Triphosphate; D003513:Cycloheximide; D003847:Deoxyglucose; C016299:chelerythrine; D048031:JNK Mitogen-Activated Protein Kinases; C031143:AICA ribonucleotide; D019311:Staurosporine; C088060:bisindolylmaleimide; D011190:Potassium Cyanide; D013755:Tetradecanoylphorbol Acetate; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D016559:Tacrolimus