Isoform Specific Effect of Ischemia/Reperfusion on Cardiac Na,K-ATPase: Protection by Ouabain Preconditioning

by Stebal, Cory J.

Ouabain and other digitalis drugs are specific inhibitors of the purified Na+,K+-ATPase, the ubiquitous enzyme that transports Na+ and K+ across the plasma membrane by hydrolysis of ATP. Independent of changes in ion pumping activity, ouabain binding to the cardiac Na+, K+-ATPase also triggers the assembly and activation of a cardioprotective signaling cascade initiated by the formation of the Na+,K+-ATPase/c-Src binary receptor. Activation of this complex by low doses of ouabain before ischemia/reperfusion protects the heart against infarction, a phenomenon called ouabain preconditioning. The aim of this study was to investigate whether ouabain preconditioning affects the fate of the cardiac membrane Na+,K+-ATPase enzyme complex following ischemia/reperfusion (IR) injury. In our model of Langendorff-perfused rat heart preparation exposed to 30 min of zero flow ischemia, Na+,K+-ATPase activity was significantly decreased in the IR group after 30 min of reperfusion, but not after 5 min of reperfusion. This decrease was prevented by ouabain preconditioning. This was correlated with total Na+,K+-ATPase ?1 and ?2 isoforms protein contents, which were not affected after 0 or 5 min, but decreased after 30 min of reperfusion and protected by ouabain. Finally, immunodetection following differential cell fractionation showed that, after IR, Na+, K+-ATPase ?1 was enriched in fractions enriched with the Early Endosome Antigen 1 (EEA1) marker in the IR group, and this was prevented by ouabain pre-treatment. Taken together, these data suggest that, during the first 5 min of reperfusion that follow 30 min of ischemia, the Na+,K+- ATPase complex is redistributed into intracellular compartments. After 30 min, Na+,K+-ATPase is degraded. Such redistribution may explain previous reports of decreased sarcolemmal activity without changes in total protein contents and contribute to the increase in intracellular Na+ observed during ischemia/reperfusion injury. Ouabain preconditioning prevented the redistribution of ?1 at 5 min reperfusion and protected against ?1 and ?2 protein degradation and loss of enzyme activity.