Kinases and phosphatases in ischaemic preconditioning: a re-evaluation

Abstract

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.

Cardioprotection by mild hypothermia during ischemia involves preservation of ERK activity

“…We therefore tested a much less selective PPase inhibitor, cantharidin. Five μM cantharidin during ischemia in rat hearts depressed post-ischemic function and did not limit infarct size [8]. While 1μM in our preliminary experiments was also ineffective (data not shown), a 10-fold lower dose (100 nM) had an anti-infarct effect (Fig.…”

“…We biopsied several cantharidin-treated hearts, but did not see any sign of preservation of ERK or MEK phosphorylation (data not shown). Since inhibition of PP2A, a prominent phosphatase in the heart, has previously been implicated in cardioprotection [8, 42], we investigated whether mild hypothermia could inhibit a protein phosphatase in vitro . The effect of mild hypothermia on an in vitro assay of PP2A is demonstrated in Fig.…”

Phosphatase PTEN is critically involved in post-myocardial infarction remodeling through the Akt/interleukin-10 signaling pathway

“…We also demonstrated that Erk1/2 phosphorylation and c-Jun phosphorylation were associated with TNF-α expression in the infarcted heart. Since Erk1/2 and AP-1 have been shown to promote cell survival and IL-10 expression [6,12,38], further studies are required to determine their roles in regulating TNF-α expression and cell death.…”

Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion

“…Activation of RISK signaling during reperfusion unites IPC, IPost and other agents that facilitate cardioprotection when offered either prior to ischemia or at reperfusion [10], and prolonging RISK activation by phosphatase inhibition during reperfusion also offers cytoprotection [8]. Of note, studies have questioned the importance of RISK in cardioprotection by IPost [27, 30] and in gentle reperfusion [23].…”