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Patients suffer alleviated severity of ischemia-reperfusion with the use of rapamycin. Excessive activation of the mammalian target of rapamycin (mTOR) and decreased activation of STAT3 are implicated in infarction and repferfusion injury. Considering the potent cardioprotective effect of mTOR inhibitor, rapamycin, we hypothesized that reperfusion therapy with rapamycin would reduce infarct size in hearts through STAT3 signaling.
Eight week-old wild type mice (WT) inoculated intraperitoneally to low dose(50nM) and high dose(100nM) rapamycin were subjected to 30 min of ischemia followed by 24h reperfusion. Echocardiogram was performed for analysis of cardiac function. I/R injury was evaluated by IS(infarcted size)/AAR(Area at Risk) using TTC staining. The expressions of mTOR, Raptor, Rictor, p-AKT, p70s6k, STAT3 were analyzed by Western blot. WT and Cardiac conditional Raptor (component of mTORC1 complex) knockout (Raptor KO) mice were subjected to 30 min of ischemia followed by 24h reperfusion, respectively, To determine the cause and effect relationship of STAT3 in cardioprotection, a STAT3 inhibitor (5, 15-DPP) was given to both WT and Raptor (KO) mice at reperfusion. Cardiomyocytes isolated from Raptor (KO) mice were treated during reoxygenation following simulated ischemia. Necrosis and apoptosis of the cardiomyocytes were determined by Trypan blue staining and TUNEL assay.
IS/AAR was significantly reduced in low dose(LD) rapamycin(Rapa) treated group. LD Rapa+ I/R mice (45.3 ± 2.4%) compared to I/R mice (63.9 ± 0.9%) or HD (high dose) Rapa+ I/R mice (57.7 ± 1.1%). Left ventricular ejection fraction (LVEF) was prominently preserved in LD Rapa treated mice(65.6±1.54%) compared to I/R mice (53.44±2.25%) or HD (high dose) Rapa+ I/R mice (55.80±1.76%); LD Rapa treatment restored phosphorylation of STAT3 and enhanced AKT phosphorylation (target of mTORC2), but significantly reduced ribosomal protein S6 phosphorylation (target of mTORC1) in the I/R heart. Inhibition (5, 15-DPP) of STAT3 in both WT and Raptor (KO) mice at reperfusion, the cardioprotection was diminished during I/R in Raptor (KO) group. Necrosis and apoptosis analysis showed such protection of Raptor (KO) was also absent in cardiomyocytes treated with 5, 15-DPP.
Inhibition of mTORC1 reducing IS/AAR and attenuates cardiomyocyte death following reperfusion via STAT3 signaling pathway.