Fig. 5
Fis1 mutant transfection prevents empagliflozin from protecting HCAEC mitochondria. HCAECs were subjected to 45 min of hypoxia followed by two hours of reoxygenation to induce sI/R injury in vitro. The cells were incubated with empagliflozin (EMPA, 10 µM) for 12 h before sI/R injury. HCAECs were transfected with a Fis1 phosphorylation-mimetic mutant (Fis1T34D) to activate mitochondrial fission in empagliflozin-treated sI/R-injured HCAECs, or were transfected with a Fis1 phosphorylation-defective mutant (Fis1T34A) to inhibit mitochondrial fission in sI/R-treated HCAECs. A,B The MMP was measured using an MMP assay kit with the JC-10 fluorescence probe. C,D The mtROS levels in HCAECs were measured using a Mitochondrial ROS Detection Assay Kit. E ATP production was measured with an ELISA. F. The mPTP opening rate was determined with a tetramethylrhodamine ethyl ester fluorescence assay. G–I Western blotting was used to assess ET-1 and p-eNOS protein levels in HCAECs following sI/R injury or empagliflozin treatment. J,K FITC-dextran clearance and TER assays were performed to determine the alterations of endothelial barrier function and integrity. Data are shown as mean ± SEM, n = ten independent cell isolations per group. *p < 0.05