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.

Ankyrin-B Protein in Heart Failure

“…10). Previous work supports that both ankyrin-B and Na/K-ATPase levels are protected from ischemia reperfusion by MDL28170 (38), further supporting our findings. Collectively, our findings demonstrate a role for ankyrin-B as a component of the protective response to cardiac injury.…”

“…Injury-Based on the findings above as well as past work from Inserte et al (38), we hypothesized that ankyrin-B plays a cardioprotective role in the response of the heart to stress/injury (increased Ca 2ϩ , ROS, calpain) and structural, electrical, transcriptional, and metabolic remodeling. As a first step to test this hypothesis, we evaluated the hearts from mice lacking 50% of their ankyrin-B expression (ankyrin-B ϩ/Ϫ mice) for response to myocardial ischemia, a common cause of myocardial infarction and death in humans (39).…”

“…Subsequently, animal and cell models lacking ankyrins show loss of spectrin isoform targeting, whereas models lacking specific spectrin gene products display reduced ankyrin expression (2, 24, 27, 32, 33, 50 -52). In fact, multiple studies now support differential regulation of ankyrin or spectrin proteolysis depending on the disease state and even the cell type (38,53). Clearly, additional research will be required to unravel the complex relationships of these functionally related proteins in health and disease.…”

“…Calpain Alters Cardiac Ankyrin-B Expression-Calpains are calcium-dependent cysteine proteases, downstream from both elevated Ca 2ϩ and ROS, implicated in both structural and electrical modeling in cardiovascular disease, and previously linked with ankyrin-and spectrin-based pathways in both heart and brain, albeit primarily studied downstream of membrane ion channels and transporters (37,38). Based on altered ankyrin-B expression in response to altered calcium and ROS, we directly FIGURE 2.…”

Myocardial and Renal Ischemia/Reperfusion Injury and Experimental Models: Review

“…46 Bu olaylar sonucunda hücreler reperfüzyon başlar başlamaz inflamatuar kaskadın etkinleşmesi ve sitokinler ile katekolaminler gibi biyoaktif molekül-lerin salıverilmesi ile hücresel ölüme ya da miyokardiyal kasılma işlev bozukluğuna daha duyarlı duruma gelir. 47 48 …”