Inconsistent relation of MAPK activation to infarct size reduction by ischemic preconditioning in pigs. Am J Physiol Heart Circ Physiol 279: H1111-H1119, 2000.-The importance of the activation of mitogen-activated protein kinases (MAPK) for the cardioprotection achieved by ischemic preconditioning (IP) is still controversial. We therefore measured infarct size and p38, extracellular signal-regulated kinase (ERK), and c-Jun NH 2terminal kinase (JNK) MAPK phosphorylation (by biopsies) in

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... -anesthetized pigs. After 90 min low-flow ischemia and 120 min reperfusion, infarct size averaged 18.3 Ϯ 12.4 (SD)% (group 1, n ϭ 14). At similar subendocardial blood flows, IP by 10 min ischemia and 15 min reperfusion (group 2, n ϭ 14) reduced infarct size to 6.2 Ϯ 5.1% (P Ͻ 0.05). An inconsistent increase in p38, ERK, and p54 JNK phosphorylation (by Western blot) was found during IP; p46 JNK phosphorylation increased with the subsequent reperfusion. At 8 min of the sustained ischemia, p38, ERK, and p54 JNK phosphorylation were increased with no difference between groups (medians: p38: 207% of baseline in group 1 vs. 153% in group 2; ERK: 142 vs. 144%; p54 JNK: 171 vs. 155%, respectively). MAPK phosphorylation and reduction of infarct size by IP were not correlated, thus not supporting the concept of a causal role of MAPK in mediating cardioprotection by IP. p38; extracellular signal-regulated kinase; c-Jun NH 2 -terminal kinase; Western blot; myocardial ischemia-reperfusion ONE OR SEVERAL SHORT EPISODES of myocardial ischemia and reperfusion delay the infarct development resulting from a subsequent, prolonged ischemic insult (17) . The infarct size-limiting effect of ischemic preconditioning (IP) is impressive, but its underlying mechanisms are still incompletely understood. Current concepts of IP propose that activation of G protein-coupled receptors results in the activation of intracellular protein kinases that ultimately activate the end effector, possibly the ATP-sensitive K ϩ channel (14) or the cytoskeleton (13). The proposed sequence of intracellular events is that activated phospholipase C or D degrades phosphatidylinositol 4,5-bisphosphate, thus producing diacylglycerol, which then activates specific protein kinase C (PKC) isoforms. There is also experimental evidence for the activation of a protein tyrosine kinase (PTK) pathway, either parallel to (12, 29) or downstream of PKC (2), which may vary among species. Although the exact pathway is not yet established, activation of PKC can result in activation of the mitogen-activated protein kinase (MAPK) cascade (11). Each subfamily of the MAPK family, p38 (16, 18, 31), extracellular signal-regulated kinase (ERK) (21, 28), or c-Jun NH 2 -terminal kinase (JNK) (3, 20) has been suggested to play a role in the cardioprotection achieved by IP. Evidence for the role of MAPK in IP is derived from investigations in which pharmacological inhibitors were used in isolated rabbit cardiomyocytes (1, 31), rat myoblasts (18), buffer-perfused rat hearts (16), and recently in two preliminary studies in pig (28) and dog hearts in situ (23). However, pharmacological inhibitors and activators may have nonspecific effects that could account for their effects on infarct size. A new approach to overcome the limitations of pharmacological inhibitors is the use of transfected cells with dominant negative mutants of the respective MAPK. However, this technique so far is restricted to cultured cardiomyocytes. An alternative approach to elucidate the role of MAPK in IP is to assess variations of MAPK activation in the time course of a preconditioning protocol. In a preliminary study in anesthetized pigs, activities of p38, ERK, and JNK were increased after short episodes of ischemia or ischemia-reperfusion (3). Several studies measured MAPK activation after a preconditioning procedure and infarct size reduction by IP. However, these studies did not assess MAPK activation and infarct size reduction by IP in the same hearts but used either different models (31) or different subgroups within a given experimental protocol (15, 16, 18) . Two recent studies by Ping et al. (20, 21) measured the activities of MAPK after IP in vivo using in-gel kinase assays: in conscious rabbits increased ERK (21) and JNK (20) activities were measured after an IP