A new paradigm: cross talk of protein kinases during reperfusion saves life!

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Abstract
Timely reperfusion after myocardial ischemia is the prerequisite to reduce irreversible tissue injury ([16][1]). However, reperfusion itself after a prolonged ischemic period may contribute to tissue damage, including reversible contractile dysfunction of viable cardiomyocytes ([4][2], [14][3]) as well as cardiomyocyte necrosis and apoptosis ([8][4], [22][5]). To reduce the consequences of ischemia-reperfusion injury, many studies have been undertaken to define its underlying mechanisms. A better understanding of the signal transduction cascade involved in irreversible tissue injury came from the analysis of triggers and mediators involved in the endogenous cardioprotective phenomenon of ischemic preconditioning. Activation of certain sarcolemmal receptors during the preconditioning ischemic period triggers and subsequent activation of protein kinases during the sustained ischemia mediates the infarct size reduction by ischemic preconditioning. Depending on the animal species, some of these kinases act in parallel as demonstrated for protein kinase C and protein tyrosine kinases; in pigs, pharmacological inhibition of neither protein kinase C nor protein tyrosine kinase alone was sufficient to block the protection of ischemic preconditioning, but combined blockade completely abolished the infarct size reduction by ischemic preconditioning ([30][6]). Similar results were subsequently obtained in rat and dog hearts as well ([7][7], [18][8], [27][9]). Within a given family of protein kinases, certain isoforms appear to be important for mediating the cardioprotection by ischemic preconditioning, i.e., protein kinase C-α and/or -ε (for a review, see Ref. [25][10]), p38 MAPK-β ([26][11]), or p44 ERK MAPK ([6][12]), whereas other isoforms contribute to the ischemia-reperfusion-induced irreversible tissue injury, i.e., p38 ERK MAPK-α ([23][13]). Certain protein kinases that are activated during ischemia are activated to an even greater extent during the subsequent period of reperfusion. Such a pattern of activation is seen for p42/p44 ERK MAPK; ERK MAPKs are activated during ischemia ([2][14], [3][15], [6][12], [21][16]), but their activation is further increased early on during the subsequent reperfusion period ([2][14], [6][12], [21][16]), especially in preconditioned hearts ([6][12]). Also, many pharmacological interventions that were initiated at the time of reperfusion (for a review, see Ref. [13][17]) and reduced infarct size activated the ERK MAPK pathway. Activated ERK MAPK phosphorylates the proapoptotic factor BAD, thereby reducing its affinity and binding to the antiapoptotic factor BCLXL. In consequence, the ratio of unbound pro- (BAD) and antiapoptotic (BCLXL) factors is altered, resulting in a reduced incidence of apoptosis ([24][18]). Indeed, in isolated rat cardiomyocytes, blockade of ERK MAPK activation by PD-98059 increased the extent of apoptosis ([32][19]). In a recent study, Hausenloy et al. ([13][17]) also assessed the time course of protein kinase phosphorylation during global ischemia and subsequent reperfusion in nonpreconditioned and preconditioned isolated rat hearts. The authors reported a decrease of phosphatidylinositol 3-kinase (PI3K) and ERK MAPK phosphorylation during the sustained ischemic period but a marked increase in the phosphorylation of both kinases during the subsequent period of reperfusion in preconditioned hearts. This increase in kinase phosphorylation during reperfusion confirms previous data obtained for ERK MAPK ([6][12]) and PI3K [see a recently published study by the same group ([12][20])]. Such an increase in kinase phosphorylation was involved in the protection of ischemic preconditioning, because the blockade of either PI3K (using LY-29408) or ERK MAPK (using PD-98059) at the time of reperfusion abolished the infarct size reduction by ischemic preconditioning. These data are of great importance because they extend our view on ischemic preconditioning in that the mediation and execution of protection occurs to a large extent during the reperfusion period after the sustained ischemia. In a previously published study ([12][20]), the authors proposed a cross talk of PI3K and ERK MAPK, because blockade of PI3K phosphorylation increased ERK MAPK phosphorylation. Blockade of neither PI3K nor ERK MAPK phosphorylation alone was sufficient to block the phosphorylation of the downstream protein kinase target, i.e., phosphorylation of the p70S6K protein kinase, implying that both PI3K and ERK MAPK kinase acted in parallel on p70S6K protein kinase. This conclusion, however, contrasts to the data of the present study, in which blockade of either PI3K or ERK MAPK alone completely attenuated the reperfusion-induced phosphorylation of p70S6K protein kinase. However, given the fact that blockade of either p70S6K protein kinase (using rapamycin) ([12][20]) or PI3K (present study) or ERK MAPK (present study) at the time of reperfusion completely abolished the protection of ischemic preconditioning, the present findings on kinase phosphorylation appear more reasonable. As outlined above, the activation of ERK MAPK reduced cardiomyocyte apoptosis ([32][19]), a mechanism proposed by Hausenloy et al. ([13][17]) to be important for the protection observed in their experiments. However, the authors did not measure cardiomyocyte apoptosis (which might be difficult in the short time frame of reperfusion) but instead assessed a reduction in myocardial necrosis. Common cellular targets for both apoptosis and necrosis are the mitochondria. Tonic release of factors such as cytochrome c from mitochondria can induce cardiomyocyte apoptosis ([20][21]), whereas massive opening of the mitochondrial permeability transition pore (MPTP) leads to cardiomyocyte necrosis ([19][22]). Indeed, blockade of the MPTP at the time of reperfusion reduced irreversible tissue injury ([5][23], [9][24]–[11][25]), and ischemic preconditioning delayed opening of...