To study this, mice hearts were perfused in Langendorff mode for 40 min of baseline and subjected to 20 or 30 min of global no-flow ischemia followed by 40 min of reperfusion. C57BL6 mice perfused with 11,12-EET (1 渭M) had improved postischemic recovery, whereas co-perfusion with PI3K伪 inhibitor, PI-103 (0.1 渭M), abolished the EET-mediated effect. In contrast, blocking of PI3K尾 or PI3K纬 isoforms failed to inhibit EET-mediated cardioprotection. In addition to the improved post-ischemic recovery, increased levels of p-Akt, decreased calcineurin activity and decreased translocation of proapoptotic protein BAD to mitochondria were noted in EET-treated hearts. Perfusion of 11,12-EET to Kir6.2 deficient mice (pmKATP) failed to improve postischemic recovery, decrease calcineurin activity and translocation of proapoptotic protein BAD, however increased levels of p-Akt were still observed. Patch-clamp experiments demonstrated that 11,12-EET could not activate pmKATP currents in myocytes pre-treated with PI-103. Mechanistic studies in H9c2 cells demonstrate that 11,12-EET limits anoxia-reoxygenation triggered Ca2 + accumulation and maintains mitochondrial 螖唯m compared to controls. Both PI-103 and glibenclamide (10 渭M, pmKATP inhibitor) abolished EET cytoprotection.
Together our data suggest that EET-mediated cardioprotection involves activation of PI3K伪, upstream of pmKATP, which prevents Ca2 + overload and maintains mitochondrial function.