A chemical genetic approach reveals that p38alpha MAPK activation by diphosphorylation aggravates myocardial infarction and is prevented by the direct binding of SB203580

Abstract

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 p38alpha alleles were targeted to cause substitution of the gatekeeper residue and resistance to inhibition. In homozygous drug-resistant compared with wild-type 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 p38alpha kinase activity is necessary to elicit protection. These results indicate that, during myocardial ischemia, p38alpha (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.

FIGURE 1.

p38 MAPK activation during myocardial ischemia and sensitivity to myocardial infarction in hearts expressing the WT or DR form of p38α. A, DR and WT hearts were subjected to 10 min of ischemia in the presence and absence of SB203580 (SB). The values below each band represent quantification of band density from three separate experiments expressed as mean ± S.E. B, shown is the normalized infarction volume of DR and WT hearts in the presence of intraischemic SB203580 or vehicle. p38 in the DR/DR hearts was resistant to inhibition by SB203580 based on intact TAB1 and HSP27 phosphorylation, and this was accompanied by a failure of SB203580 to reduce infarct size. ns, not significant; *, p < 0.05 compared with the control; #, p < 0.05 compared with ischemia without SB203580.

FIGURE 2.

Effect of BIRB796 on p38 diphosphorylation and sensitivity to myocardial infarction. A and B, the format was as described in the legend to Fig. 1, but DR and WT hearts were exposed to BIRB796 or vehicle for 30 min prior to ischemia. *, p < 0.05 compared with the control and ischemia + BIRB796. C, to ensure that the targeting of p38α in the DR hearts did not increased the expression of the SB203580-resistant p38γ isoform, heart homogenates were probed with the pan-isoform (T-p38)-selective and p38γ-selective primary antibodies. 16 ng of glutathione S-transferase-tagged recombinant p38α and p38γ was used to index antibody selectivity. The T-p38 antibody preferentially indicated p38α, but both isoforms seemed equally expressed in WT and DR hearts.

FIGURE 3.

Effect of SB203580 on hearts from mice heterozygous for the p38α DR allele. A, hearts from mice harboring one (DR/WT) or no (WT/WT) p38α DR allele were subjected to 10 min of ischemia in the presence and absence of SB203580 (SB). The values below each band represent quantification of band density from three separate experiments expressed as mean ± S.E. B, shown is the normalized infarction volume of DR/WT and WT/WT hearts in the presence of intraischemic SB203580 or vehicle. *, p < 0.05 compared with the control. ns, not significant. C, shown are the results of an in vitro kinase assay with p38 precipitated from crude heart homogenate using ATF2 as substrate. The DR/WT hearts behaved like DR/DR hearts in that SB203580 did not prevent p38 activation or the phosphorylation of the downstream endogenous substrates HSP27 and TAB1 or the exogenous substrate ATF2 in an in vitro kinase assay.

FIGURE 4.

Di- or monophosphorylation of p38 during myocardial ischemia. A, HEK293 cells were transfected as indicated. The three-letter codes denote the amino acid sequence in the activation motifs of ectopically expressed p38α with a tag that slowed migration (arrowheads). The anti-diphospho-p38 polyclonal antibody (P-p38) recognized phosphorylation of threonine alone (TGF = TGY), but the anti-diphospho-p38 monoclonal antibody was more selective for the true diphospho form (TGY ≫ TGF). The anti-phospho-Tyr¹⁸² antibody (pY182), unlike P-p38, was insensitive to threonine phosphorylation (TGY = AGY ≫ TGF). B, 10 min of ischemia resulted in phosphorylation of Tyr¹⁸², which was sensitive to SB203580 (SB). C, shown is the confirmation of TGY phosphorylation by the similarity in the patterns of immunoreactivity between anti-diphospho-p38 monoclonal and polyclonal antibodies.