Calpain-mediated impairment of Na+/K+-ATPase activity during early reperfusion contributes to cell death after myocardial ischemia
Calpain-mediated impairment of Na+/K+-ATPase activity during early reperfusion contributes to cell death after myocardial ischemia
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DOI：
10.1161/01.res.0000181170.87738.f3
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发表时间：
2005-09-02
影响因子：
20.1
通讯作者：
Soler-Soler, J
中科院分区：
文献类型：
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作者：
Inserte, J;Garcia-Dorado, D;Soler-Soler, J
Na+超载和Na+/Ca 2+交换器介导的继发性Ca 2+内流是再灌注心肌细胞挛缩坏死的重要机制。Na+/K+-ATP酶活性受损是导致Na+超载的重要原因，但其机制尚未明确。由于Na+/K+-ATP酶通过钙蛋白酶的底物锚蛋白与细胞骨架蛋白fodrin连接，我们验证了钙蛋白酶介导再灌注心肌细胞Na+/K+-ATP酶损伤的假设。在离体大鼠心脏缺血60分钟后再灌注5分钟，Na+/K+-ATP酶活性降低80%，与α-胞衬蛋白和锚蛋白-B的丢失以及Na+/K+-ATP酶α（1）和α（2）亚基从膜-细胞骨架复合物上的分离平行。MDL-7943在再灌注期间抑制钙蛋白酶防止了这些蛋白的丢失，增加了Na+/K+-ATP酶活性，减弱了乳酸脱氢酶释放，并改善了收缩恢复，并且MDL- 7943的这些有益作用被哇巴因逆转。Na+/K+-ATP酶的损伤不仅仅是细胞死亡的结果，因为在心脏中，用2，3-丁二酮单肟收缩阻滞防止了挛缩和细胞死亡，但Na+/K+-ATP酶的损伤没有改变。在这些心脏中，伴随的钙蛋白酶抑制剂保护Na+/K+ -ATP酶的含量和功能，并减弱2，3-丁二酮单肟停药时发生的细胞死亡。体外实验表明，与活化的钙蛋白酶孵育10分钟后，Na+/K+-ATP酶亚基没有检测到降解。因此，我们得出结论，钙蛋白酶激活有助于损伤的Na+/K+ -ATP酶在再灌注早期，这种影响主要是通过降解锚定的Na+/ K+-ATP酶的膜细胞骨架介导的。
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 alpha(1) and alpha(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.