The activation of p38 alpha, and not p38 beta, mitogen-activated protein kinase is required for ischemic preconditioning

Abstract

Numerous studies show that pharmacological inhibition of p38 mitogen-activated protein kinases (p38s) before lethal ischemia prevents conditioning. However, these inhibitors have off-target effects and do not discriminate between the alpha and beta isoforms; the activation of which is thought to have diverse and perhaps opposing actions with p38 alpha aggravating, and p38 beta reducing, myocardial injury. We adopted a chemical genetic approach using mice in which either the p38 alpha (DR alpha) or p38 beta (DR beta) alleles were targeted to substitute the "gatekeeper" threonine residue for methionine, thereby preventing the binding of a pharmacological inhibitor, SB203580. Isolated, perfused wild-type (WT), DR alpha and DR beta mouse hearts underwent ischemic preconditioning with 4 cycles of 4 min ischemia/6 min reperfusion, with or without SB203580 (10 microM), followed by 30 min of global ischemia and 120 min of reperfusion. In WT and DR beta hearts, SB203580 completely abolished the reduction in myocardial infarction seen with preconditioning and also the phosphorylation of downstream substrates of p38. These effects of SB203580 were not seen in DR alpha hearts. Furthermore ischemic preconditioning occurred unaltered in p38 beta null hearts. Contrary to expectation the activation of p38 alpha, and not p38 beta, is necessary for ischemic preconditioning. Since p38 alpha is also the isoform that leads to lethal myocardial injury, it is unlikely that targeted therapeutic strategies to achieve isoform-selective inhibition will only prevent the harmful consequences of activation.

Fig. 1

Experimental protocol and infarct sizes. Panel (A) All hearts were subjected to 50 min of stabilization and 30 min of global ischemia. Preconditioning was by 4 cycles of 4 min global ischemia with 6 min of reperfusion with blinding to corresponding vehicle (DMSO 0.1%). SB203580 (10 μmol/L) started 6 min before, and during, preconditioning but was washed out during the last reperfusion. After 120 min of reperfusion the infarct size was determined by TTC staining. Hearts were harvested for immunoblotting on the last period of preconditioning ischemia. Panels (B) and (C) Infarct size is expressed as percent of the left ventricular volume with statistical comparisons as shown. The number of observations on which the data are based appears as the numeral inset in the bar labels. The “+” and “−” signs below each bar denote which p38 isoform(s) remains active under the set conditions. Panels (D), (E) and (F) Activation of p38 and phosphorylation of downstream substrates in the presence and absence of pharmacological inhibition with SB203580 in B6 (panel (D)), DR α (panel (E)) and DRβ (panel (F)) hearts. In the immunoblots below Tp38 is total p38, pp38 dual phospho-p38 (on Thr180 and Tyr182), pHSP27 phosphorylated 27 kDa heat shock protein (on Ser82) and pTAB1 phosphorylated TAB1 (on Ser423). Panels (D) and (F) demonstrate that SB present during preconditioning cycles of ischemia prevents the downstream phosphorylation of HSP27, a substrate of mitogen-activated protein kinase activated protein kinase-2 that is in turn activated by p38 and TAB1 a direct substrate of p38. The phosphorylation of these downstream substrates is still present in DRα hearts exposed to SB confirming the lack of p38 inhibition despite the presence of SB203580. Protein in each lane is from a different heart. The figures below each band represent quantification of band density from 3 separate experiments expressed as mean + SEM. ^⁎P < 0.05 Cont vs. PC; ^$P < 0.05 PC vs. PC + SB; ^#P < 0.05 Cont vs. PC + SB.