Calpain-Mediated Impairment of Na + /K + –ATPase Activity During Early Reperfusion Contributes to Cell Death After Myocardial Ischemia

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Abstract
Na+ overload and secondary Ca2+ influx via Na+/Ca2+ exchanger are key mechanisms in cardiomyocyte contracture and necrosis during reperfusion. Impaired Na+/K+–ATPase activity contributes to Na+ overload, but the mechanism has not been established. Because Na+/K+–ATPase is connected to the cytoskeleton protein fodrin through ankyrin, which are substrates of calpains, we tested the hypothesis that calpain mediates Na+/K+–ATPase impairment in reperfused cardiomyocytes. In isolated rat hearts reperfused for 5 minutes after 60 minutes of ischemia, Na+/K+–ATPase activity was reduced by 80%, in parallel with loss of α-fodrin and ankyrin-B and detachment of α1 and α2 subunits of Na+/K+–ATPase from the membrane–cytoskeleton complex. Calpain inhibition with MDL-7943 during reperfusion prevented the loss of these proteins, increased Na+/K+–ATPase activity, attenuated lactate dehydrogenase release, and improved contractile recovery, and these beneficial effects of MDL-7943 were reverted by ouabain. The impairment of Na+/K+–ATPase was not a mere consequence of cell death because it was not altered in hearts in which contracture and cell death had been prevented by contractile blockade with 2,3-butanedione monoxime. In these hearts, concomitant calpain inhibition preserved Na+/K+–ATPase content and function and attenuated cell death occurring on withdrawal of 2,3-butanedione monoxime. In vitro assay showed no detectable degradation of Na+/K+–ATPase subunits after 10 minutes of incubation with activated calpain. Thus, we conclude that calpain activation contributes to the impairment of Na+/K+–ATPase during early reperfusion and that this effect is mainly mediated by degradation of the anchorage of Na+/K+–ATPase to the membrane cytoskeleton.