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

Abstract

Na + overload and secondary Ca 2+ influx via Na + /Ca 2+ 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 α 1 and α 2 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.