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Local ischemic postconditioning (IPost) and remote ischemic perconditioning (RIPer) are promising methods to decrease ischemia-reperfusion (I/R) injury. We tested whether the use of the two procedures in combination led to an improvement in cardioprotection through a higher activation of survival signaling pathways. Rats exposed to myocardial I/R were allocated to one of the following four groups: Control, no intervention at myocardial reperfusion; IPost, three cycles of 10-s coronary artery occlusion followed by 10-s reperfusion applied at the onset of myocardial reperfusion; RIPer, 10-min limb ischemia followed by 10-min reperfusion initiated 20 min after coronary artery occlusion; IPost+RIPer, IPost and RIPer in combination. Infarct size was significantly reduced in both IPost and RIPer (34.25 ± 3.36 and 24.69 ± 6.02%, respectively) groups compared to Control (54.93 ± 6.46%, both p < 0.05). IPost+RIPer (infarct size = 18.04 ± 4.86%) was significantly more cardioprotective than IPost alone (p < 0.05). RISK pathway (Akt, ERK1/2, and GSK-3β) activation was enhanced in IPost, RIPer, and IPost+RIPer groups compared to Control. IPost+RIPer did not enhance RISK pathway activation as compared to IPost alone, but instead increased phospho-STAT-3 levels, highlighting the crucial role of the SAFE pathway. In IPost+RIPer, a SAFE inhibitor (AG490) abolished cardioprotection and blocked both Akt and GSK-3β phosphorylations, whereas RISK inhibitors (wortmannin or U0126) abolished cardioprotection and blocked STAT-3 phosphorylation. In our experimental model, the combination of IPost and RIPer improved cardioprotection through the recruitment of the SAFE pathway. Our findings also indicate that cross talk exists between the RISK and SAFE pathways.

Recent studies reported cardioprotective effects of erythropoietin (EPO) against ischemia-reperfusion (I/R) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been reported to be impaired in diabetes and insulin resistance syndrome, we examined whether EPO-induced cardioprotection was maintained in rat models of type 1 diabetes and insulin resistance syndrome. Isolated hearts were obtained from three rat cohorts: healthy controls, streptozotocin (STZ)-induced diabetes, and high-fat diet (HFD)-induced insulin resistance syndrome. All hearts underwent 25 min ischemia and 30 min or 120 min reperfusion. They were assigned to receive either no intervention or a single dose of EPO at the onset of reperfusion. In hearts from healthy controls, EPO decreased infarct size (14.36 ± 0.60 and 36.22 ± 4.20% of left ventricle in EPO-treated and untreated hearts, respectively, p < 0.05) and increased phosphorylated forms of Akt, ERK1/2, and their downstream target GSK-3β. In hearts from STZ-induced diabetic rats, EPO did not decrease infarct size (32.05 ± 2.38 and 31.88 ± 1.87% in EPO-treated and untreated diabetic rat hearts, respectively, NS) nor did it increase phosphorylation of Akt, ERK1/2, and GSK-3β. In contrast, in hearts from HFD-induced insulin resistance rats, EPO decreased infarct size (18.66 ± 1.99 and 34.62 ± 3.41% in EPO-treated and untreated HFD rat hearts, respectively, p < 0.05) and increased phosphorylation of Akt, ERK1/2, and GSK-3β. Administration of GSK-3β inhibitor SB216763 was cardioprotective in healthy and diabetic hearts. STZ-induced diabetes abolished EPO-induced cardioprotection against I/R injury through a disruption of upstream signaling of GSK-3β. In conclusion, direct inhibition of GSK-3β may provide an alternative strategy to protect diabetic hearts against I/R injury.