Aldose reductase modulates cardiac glycogen synthase kinase-3β

28 29 Objective: Earlier studies have demonstrated that aldose reductase (AR) plays a key role in 30 mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R 31 injury by influencing phosphorylation of glycogen synthase kinase 3β (p-GSK3β). 32 Methods: We utilized three separate models to study the effects of stress injury on the heart. 33 Hearts isolated from wildtype (WT), human expressing AR transgenic (ARTg) and AR knockout 34 (ARKO) mice were perfused with/without GSK3β inhibitors (SB216763 and lithium chloride 35 (LiCl)) and subjected to I/R. Ad-human AR (Ad-hAR) expressing HL-1 cardiac cells were 36 exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2) conditions, and I/R in a murine 37 model of transient occlusion and reperfusion of the left anterior descending coronary artery 38 (LAD). 39 Results: Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) 40 were measured. LVDP was decreased in hearts from ARTg mice compared to WT and ARKO 41 after I/R, while LDH release and apoptotic markers were increased (p<0.05). p-GSK3β was 42 decreased in ARTg hearts compared to WT and ARKO (p<0.05). In ARKO, p-GSK3β and 43 apoptotic markers were decreased compared to WT (p<0.05). WT and ARTg hearts perfused 44 with GSK3β inhibitors improved p-GSK3β expression and LVDP, and exhibited decreased LDH 45 release, apoptosis and mitochondrial pore opening (p<0.05). Ad-human AR (Ad-hAR) 46 expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2) had 47 greater LDH release compared to control HL-1 cells (p<0.05). p-GSK3β was decreased and 48 correlated with increased apoptotic markers in ad-hAR HL-1 cells (p<0.05). Treatment with 49 PI3K/Akt inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and 50 ARKO hearts and in ad-hAR expressing HL-1 cells. Cells treated with PKCα/β inhibitor 51 displayed significant increases in p-Akt and p-GSK3β expression, and resulted in decreased 52 LDH release. 53 Conclusion: AR mediates changes in p-GSK3β, in part, via PKCα/β and Akt during I/R. 54 55