Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and changes in sarcolemmal Na(+)-K(+)-ATPase subunits and activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, as well as reperfusion after 45 min of ischemia. Decreases in Na(+)-K(+)-ATPase activity at 30-60 min of global ischemia were accompanied by augmented activities of both calpain and matrix metalloproteinases (MMPs) and depressed protein content of β(1)- and β(2)-subunits, without changes in α(1)- and α(2)-subunits of the enzyme. Compared with control values, the activities of both calpain and MMP-2 were increased, whereas the activity and protein content for all subunits of Na(+)-K(+)-ATPase were decreased upon reperfusion for 5-40 min, except that α(1)- and α(2)-subunit content was not depressed in 5 min I/R hearts. MDL28170, a calpain inhibitor, was more effective in attenuating the I/R-induced alterations in cardiac contracture, Na(+)-K(+)-ATPase activity, and α(2)-subunit than doxycycline, an MMP inhibitor. Incubation of control sarcolemma preparation with calpain, unlike MMP-2, depressed Na(+)-K(+)-ATPase activity and decreased α(1)-, α(2)-, and β(2)-subunits, without changes in the β(1)-subunit. These results support the view that activation of both calpain and MMP-2 are involved in depressing Na(+)-K(+)-ATPase activity and degradation of its subunits directly or indirectly in hearts subjected to I/R injury.