title: Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning.
abstract: Na(+),K(+)-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na(+),K(+)-ATPase α(1)- and α(3)-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α(1)-cell surface abundance was significantly decreased. Na(+),K(+)-ATPase-activity in crude homogenates and (86)Rb(+) transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na(+),K(+)-ATPase activity and surface expression. This model also revealed that Na(+),K(+)-ATPase-mediated (86)Rb(+) uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na(+),K(+)-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na(+),K(+)-ATPase α(1) known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na(+),K(+)-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na(+),K(+)-ATPase may be independent of ion transport.
authors.affiliation: Department of Biochemistry, College of Medicine, University of Toledo, 3000 Arlington Ave., Toledo, OH 43614, USA.
authors.forename: Aude
authors.initials: A
authors.lastname: Belliard
authors.forename: Yoann
authors.initials: Y
authors.lastname: Sottejeau
authors.forename: Qiming
authors.initials: Q
authors.lastname: Duan
authors.forename: Jessa L
authors.initials: JL
authors.lastname: Karabin
authors.forename: Sandrine V
authors.initials: SV
authors.lastname: Pierre
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry; D017136:Ion Transport; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009154:Mutation; D015428:Myocardial Reperfusion Injury / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D010042:Ouabain / Q000494:pharmacology*; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D004791:Enzyme Inhibitors; D010042:Ouabain; D007770:L-Lactate Dehydrogenase; C497257:Prkce protein, rat; D051744:Protein Kinase C-epsilon; C499075:Atp1a1 protein, rat; C541459:Atp1a3 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase