Calpain-mediated impairment of Na+/K+-ATPase activity during early reperfusion contributes to cell death after myocardial ischemia.
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 alpha-fodrin and ankyrin-B and detachment of alpha1 and alpha2 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.
Inserte
Javier
J
Servicio de Cardiologia, Hospital Universitari Vall d'Hebron, Barcelona, Spain.
Garcia-Dorado
David
D
Hernando
Victor
V
Soler-Soler
Jordi
J
D000818:Animals; D017487:Ankyrins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D016923:Cell Death; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D017202:Myocardial Ischemia / Q000150:complications*; D015428:Myocardial Reperfusion Injury / Q000209:etiology*; D009206:Myocardium / Q000378:metabolism / Q000473:pathology*; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000502:physiology*; D016277:Ventricular Function, Left; D013049:Spectrin
D017487:Ankyrins; D002118:Calcium; D002154:Calpain; D002352:Carrier Proteins; D007770:L-Lactate Dehydrogenase; D008840:Microfilament Proteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D013049:Spectrin