Mechanisms by Which Arachidonic acid Metabolite, Epoxyeicosatrienoic acid Elicit Cardioprotection Against Ischemic Reperfusion Injury

Abstract

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that have cardioprotective properties but the exact mechanism(s) remains unknown. Evidence suggests phosphoinositide 3-kinase (PI3K) and plasma membraneATP-sensitive potassium channels (pmKATP) are important. However the role of the above two protective pathways and the corresponding intracellular cardioprotective mechanism involved is unknown. To investigate this, in the current study hearts from CYP2J2 transgenic mice or WT treated with either vehicle, EET, soluble epoxide hydrolase inhibitor or an EET analog (UA-8) were perfused in Langendorff mode for 40min of baseline and subjected to 20 or 30 min of global no-flow ischemia followed by 40 min of reperfusion. Hearts with elevated EETs had improved ventricular functional recovery and reduced injury compared to WT hearts following ischemia/ reperfusion. Blocking of pmKATP channel activity abolished these EET mediated protective effects. Similarly inhibition of PI3K activity, either with the pan specific PI3K inhibitor wortmannin or class-I, PI3Kα specific inhibitor, PI-103, abolished the EET-mediated protective effect, but other PI3K isoform specific inhibitors failed to block the functional recovery. In addition to the improved post-ischemic functional recovery, increased expression of p-Akt an PI3K downstream target, decreased calcineurin activity, Ca2+ activated enzyme, and decreased translocation of proapoptotic protein BAD to mitochondria were noted in EET elevated hearts. All these protective actions of EETs were abolished when pmKATP channel activity was inhibited however, increased expression of p-Akt was still observed in these hearts, suggesting PI3K pathway is still active. Further in patch clamp experiments pre-treatment of myocytes with the PI3Kα inhibitor PI-103 significantly reduced the EET activation of pmKATP channels. Mechanistic studies using H9c2 cells demonstrate that EETs limit anoxia-reoxygenation triggered Ca2+ accumulation, decrease casapase-3 activity and maintain mitochondrial ΔΨm and decrease cell death compared to control. Both blocking of pmKATP channel and PI3Kα abolished EET mediated cytoprotection. Together our data suggest that EET-mediated cardioprotection involves activation of PI3Kα, upstream of pmKATP, which prevents Ca2+ overload and maintains mitochondrial function.