Division of Medical Physiology, Department of Biomedical Sciences, Faculty of Health Sciences, University of Stellenbosch, Tygerberg, Republic of South Africa.
Basic Res Cardiol. 2010 Jul;105(4):495-511. doi: 10.1007/s00395-010-0086-3. Epub 2010 Feb 2.
Activation of several protein kinases occurs during myocardial ischaemia and during subsequent reperfusion. In contrast to the intensive investigation into the significance of kinase activation in cardioprotection, relatively little is known about the role of the phosphatases in this regard. The aim of this study was to re-evaluate the putative roles of PP1 and PP2A in ischaemia/reperfusion and in triggering ischaemic preconditioning. Isolated perfused working rat hearts were subjected to sustained global (15 or 20 min) or regional ischaemia (35 min), followed by reperfusion. Hearts were preconditioned using global ischaemia (1 x 5 or 3 x 5 min, alternated with 5 min reperfusion). To inhibit both PP1 and PP2A cantharidin (5 muM) was used. To inhibit PP2A only, okadaic acid (7.5 nM) was used. The drugs were administered during the preconditioning protocol, before onset of sustained ischaemia (pretreatment) or during reperfusion. Endpoints were mechanical recovery during reperfusion, infarct size and activation of PKB/Akt, p38 MAPK and ERK p42/p44, as determined by Western blot. Pretreatment of hearts with okadaic acid or cantharidin caused a significant reduction in mechanical recovery after 15 or 20 min global ischaemia. Administration of the drugs during an ischaemic preconditioning protocol abolished functional recovery during reperfusion and significantly increased infarct size. Administration of the drugs during reperfusion had no deleterious effects and increased functional recovery in 3 x PC hearts. To find an explanation for the differential effects of the inhibitors depending on the time of administration, hearts were freeze-clamped at different time points during the perfusion protocol. Administration of cantharidin before 5 min ischaemia activated all kinases. Subsequent reperfusion for 5 min without the drug maintained activation of the kinases until the onset of sustained ischaemia. Cantharidin given during preconditioning was associated with activation of p38MAPK and PKB/Akt during reperfusion after sustained ischaemia. However, administration of the drug during reperfusion only after sustained ischaemia caused activation of both PKB/Akt and ERK p42/p44. Phosphatase inhibition immediately prior to the onset of sustained ischaemia or during preconditioning abolishes protection during reperfusion, while inhibition of these enzymes during reperfusion either had no effect or enhanced the cardioprotective effects of preconditioning. It is proposed that inhibition of phosphatases during reperfusion may prolong the period of RISK activation and hence protect the heart.
在心肌缺血和随后的再灌注期间，几种蛋白激酶的激活发生。与激酶激活在心脏保护中的重要性的深入研究相比，关于磷酸酶在这方面的作用知之甚少。本研究的目的是重新评估 PP1 和 PP2A 在缺血/再灌注和触发缺血预处理中的假定作用。分离的灌注工作大鼠心脏经历持续的全局（15 或 20 分钟）或局部缺血（35 分钟），随后进行再灌注。使用全局缺血（1x5 或 3x5 分钟，交替 5 分钟再灌注）对心脏进行预处理。使用斑蝥素（5μM）抑制两者 PP1 和 PP2A。仅抑制 PP2A 时，使用冈田酸（7.5 nM）。药物在预处理方案期间、持续缺血发作之前（预处理）或再灌注期间给予。再灌注期间的终点是机械恢复、梗死面积和通过 Western blot 确定的 PKB/Akt、p38MAPK 和 ERK p42/p44 的激活。用斑蝥素或斑蝥素预处理心脏导致 15 或 20 分钟全局缺血后机械恢复显著减少。在缺血预处理方案期间给予药物可消除再灌注期间的功能恢复，并显著增加梗死面积。药物在再灌注期间的给药没有有害影响，并增加了 3xPC 心脏的功能恢复。为了解释抑制剂根据给药时间的不同而产生的差异作用，在灌注方案的不同时间点对心脏进行冷冻夹闭。在 5 分钟缺血之前给予斑蝥素激活所有激酶。随后在没有药物的情况下再灌注 5 分钟，直到持续缺血发作，激酶的激活得以维持。在持续缺血后再灌注期间给予缺血预处理时给予斑蝥素与 p38MAPK 和 PKB/Akt 的激活相关。然而，在持续缺血后再灌注期间仅给予药物会导致 PKB/Akt 和 ERK p42/p44 的激活。在持续缺血发作之前或在预处理期间立即给予磷酸酶抑制剂可消除再灌注期间的保护作用，而在再灌注期间抑制这些酶要么没有影响，要么增强了预处理的心脏保护作用。据提议，在再灌注期间抑制磷酸酶可能会延长 RISK 激活的时间，从而保护心脏。
