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(Circulation Research. 2005;97:465.)
© 2005 American Heart Association, Inc.

Cellular Biology

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

Javier Inserte, David Garcia-Dorado, Victor Hernando, Jordi Soler-Soler

From the Servicio de Cardiologia, Hospital Universitari Vall d’Hebron, Barcelona, Spain.

Correspondence to Dr David Garcia-Dorado, Servicio de Cardiologia, Hospital Universitari Vall d’Hebron, Passeig Vall d’Hebron, 119-129, 08035 Barcelona, Spain. E-mail dgdorado{at}vhebron.net

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 -fodrin and ankyrin-B and detachment of ₁ and 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.

Key Words: calpain • Na⁺/K⁺–ATPase • Na⁺/Ca²⁺ exchanger • reperfusion injury

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