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: Aude Belliard, Yoann Sottejeau, Qiming Duan, Jessa L Karabin, Sandrine V Pierre
• Journal: American journal of physiology. Heart and circulatory physiology (Am J Physiol Heart Circ Physiol) Vol. 304 Issue 1 Pg. H94-103 (Jan 01 2013) ISSN: 1522-1539 [Electronic] United States
• PMID: 23086991 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Enzyme Inhibitors
• Ouabain
• L-Lactate Dehydrogenase
• Prkce protein, rat
• Protein Kinase C-epsilon
• Atp1a1 protein, rat
• Atp1a3 protein, rat
• Sodium-Potassium-Exchanging ATPase

Topics

• Animals
• Animals, Newborn
• Apoptosis (drug effects)
• Cell Membrane (drug effects, enzymology, pathology)
• Cell Survival (drug effects)
• Cells, Cultured
• Enzyme Inhibitors (pharmacology)
• Immunohistochemistry
• Ion Transport
• L-Lactate Dehydrogenase (metabolism)
• Mutation
• Myocardial Reperfusion Injury (enzymology, pathology, prevention & control)
• Myocytes, Cardiac (drug effects, enzymology, pathology)
• Ouabain (pharmacology)
• Protein Kinase C-epsilon (metabolism)
• Protein Transport
• Rats
• Sodium-Potassium-Exchanging ATPase (antagonists & inhibitors, genetics, metabolism)
• Time Factors
• Transfection