Kinetics of the translocation and phosphorylation of αB-crystallin in mouse heart mitochondria during ex vivo ischemia

Abstract

αB-crystallin (αBC) is a small heat shock protein expressed at high levels in the myocardium where it protects from ischemia-reperfusion damage. Ischemia-reperfusion activates p38 MAP kinase, leading to the phosphorylation of αBC on serine 59 (P-αBC-S59), enhancing its ability to protect myocardial cells from damage. In the heart, ischemia-reperfusion also causes the translocation of αBC from the cytosol to other cellular locations, one of which was recently shown to be mitochondria. However, it is not known whether αBC translocates to mitochondria during ischemia-reperfusion, nor is it known whether αBC phosphorylation takes place before or after translocation. In the present study, analyses of mitochondrial fractions isolated from mouse hearts subjected to various times of ex vivo ischemia-reperfusion showed that αBC translocation to mitochondria was maximal after 20 min of ischemia and then declined steadily during reperfusion. Phosphorylation of mitochondrial αBC was maximal after 30 min of ischemia, suggesting that at least in part it occurred after αBC association with mitochondria. Consistent with this was the finding that translocation of activated p38 to mitochondria was maximal after only 10 min of ischemia. The overexpression of αBC-AAE, which mimics αBC phosphorylated on serine 59, has been shown to stabilize mitochondrial membrane potential and to inhibit apoptosis. In the present study, infection of neonatal rat cardiac myocytes with adenovirus-encoded αBC-AAE decreased peroxide-induced mitochondrial cytochrome c release. These results suggest that during ischemia αBC translocates to mitochondria, where it is phosphorylated and contributes to modulating mitochondrial damage upon reperfusion.