No abstract

The mitogen-activated protein kinases (MAPKs) play an important role in ischemia/reperfusion (I/R) injury. Previous evidence suggests that p38 MAPK inhibition before ischemia is cardioprotective. However, whether p38 MAPK inhibition during ischemia or reperfusion provides cardioprotection is not well known. We tested the hypothesis that p38 MAPK inhibition at different times during I/R protects the heart from arrhythmias, reduces the infarct size, and attenuates ventricular dysfunction. Adult Wistar rats were subject to a 30-minute left anterior descending coronary artery occlusion, followed by a 120-minute reperfusion. A p38 MAPK inhibitor, SB203580, was given intravenously before left anterior descending coronary artery occlusion, during ischemia, or at the onset of reperfusion. The results showed that SB203580 given either before or during ischemia, but not at the onset of reperfusion, decreased the ventricular tachycardia/ventricular fibrillation (VT/VF) incidence and heat shock protein 27 phosphorylation, and increased connexin 43 phosphorylation. The infarct size and cytochrome c level was decreased in all SB203580-treated rats, without the alteration of the total Bax/Bcl-2 expression. The ventricular function was improved only in SB203580-pretreated rats. These findings suggest that timing of p38 MAPK inhibition with respect to onset of ischemia is an important determinant of therapeutic efficacy.

The use of nonselective pharmacological inhibitors has resulted in controversy regarding the mechanism and consequences of p38 activation during myocardial infarction. Classic p38 inhibitors such as SB203580 rely on a critical "gatekeeper" threonine residue for binding. We addressed these controversies by using mice in which the p38␣ alleles were targeted to cause substitution of the gatekeeper residue and resistance to inhibition. In homozygous drug-resistant compared with wildtype hearts, SB203580 failed to inhibit the activating phosphorylation of p38 or to reduce the infarction caused by myocardial ischemia. However, BIRB796, a p38 inhibitor not reliant on the gatekeeper for binding, similarly reduced p38-activating phosphorylation and infarction in both wild-type and knock-in mice, thereby excluding a nonspecific inhibitor-dependent phenotype resulting from the targeting strategy. Furthermore, the activation during myocardial ischemia involved phosphorylation of both the threonine and tyrosine residues in the activation loop of p38 despite the phosphorylation of the threonine alone being sufficient to create the epitope for dual phosphospecific antibody binding. Finally, SB203580 failed to reduce infarction in heterozygous drug-resistant hearts, suggesting that near complete inhibition of p38␣ kinase activity is necessary to elicit protection. These results indicate that, during myocardial ischemia, p38␣ (i) is the dominant-active p38 isoform, (ii) contributes to infarction, (iii) is responsible for the cardioprotective effect of SB203580, and (iv) is activated by a mechanism consistent with autodiphosphorylation despite this necessitating the phosphorylation of a tyrosine residue by an archetypal serine/threonine kinase.