Fig. 4
Empagliflozin suppresses mitochondrial fission by inhibiting Fis1 phosphorylation. 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. A–C MitoTracker™ was used to detect changes in mitochondrial dynamics. The number cardiomyocyte with fragmented mitochondria as well as the average length of mitochondria were recorded. At least 100 mitochondria from 10 HCAECs were used to evaluate the number of HCAECs with fragmented mitochondria. D–G Western blotting was used to determine p-Drp1, p-Fis1 and p-Mff protein levels in HCAECs following sI/R injury or empagliflozin treatment. H–J Western blotting was used to assess cytoplasmic and mitochondrial Drp1 protein levels. HCAECs were transfected with a Drp1 phosphorylation-mimetic mutant (Drp1S616D) or a Fis1 phosphorylation-mimetic mutant (Fis1T34D) in the presence of empagliflozin. K–L The binding between Drp1 and Fis1 was determined using a co-immunoprecipitation assay in the presence of empagliflozin. HCAECs were transfected with Drp1S616D or Fis1T34D. M–O Immunofluorescence staining was used to observe the mitochondrial morphology in HCAECs transfected with Drp1S616D or Fis1T34D. Data are shown as mean ± SEM, n = ten independent cell isolations per group. *p < 0.05