J013 Inhibition of mTOR survival pathway by an embryonic developmental Wnt pathway suppresses preconditioning induced cardioprotection

Abstract

Ischemic preconditioning (IPC) protects against heart prolonged lethal ischemia by activating a cardioprotective signalling cascade involving Akt and GSK3β; however molecular pathways are still incompletely known. In an in vivo model of IPC, we previously demonstrated that the Wnt pathway can inhibit cardioprotection via a direct target, GSK3β. Here we go further in the preconditioning pathway comprehension. On a retrograde isolated heart perfusion model, two models of preconditioning were set up: IPC via four sequences of 5 min 0 flow ischemia followed by 5 min reperfusion ; a Pharmacological Preconditioning (PCP), by 25 min perfusion of diazoxide, a direct activator of mitoKATP. Preconditioning signalling was studied by Western blot analysis, in hearts harvested just after preconditioning, and cardioprotection was analyzed after 40 min 0 flow ischemia followed by 120 min reperfusion, by TTC staining. We verified that PCP, like IPC, induce an inhibition/phosphorylation of GSK3β (ser9) by activation of Akt (ser479). These results were correlated with a significant reduction of infarct size after ischemiareperfusion (10,8 % and 18 % (n=6, p<0.05) of risk area, after PCP and IPC respectively, vs 37,5 % (n=7, p<0.05) in non preconditioned control hearts). We showed then that both IPC and PCP were able to induce the mTOR survival pathway by increasing P-mTOR (ser2448) and its subcellular targets P70S6K (thr389) and 4E-BP1 (ser65) via Akt activation. Wortmanin, an AKT inhibitor, blocked preconditioning induced mTOR activation and infarct size reduction (39,9 % of risk area, n=6, p<0.05 vs control, NS). These effects of preconditioning were also inhibited either by 5-hydroxydecanoate, an antagonist of mitoKATP (44,4 % of risk area, n=6, p<0.05 vs control, NS) or by rapamycin, a specific inhibitor of mTOR (40 % of risk area, n=7, p<0.05 vs control, NS). Hearts of transgenic mouse overexpressing sFRp1 (α-MHC/sFRP1), a Wnt/Frz antagonist, cardioprotections induced by IPC and PCP were inhibited: sFRP1 impaired GSK3β inhibition and mTOR activation (P70S6K and 4E-BP1 phosphorylation), independently of Akt. We evidenced for the first time that cardioprotection involves a crosstalk between an embryonic developmental Wnt pathway and a survival pathway, mTOR/P70S6K.