Objectives This study is aimed to establish a simulated ischaemia/reperfusion (SI/R) model in cultured CMECs from adult rat, and investigate the role of mitochondrial fission in CMECs I/R injury and the underlying mechanisms.
CMECs were isolated from adult rat ventricles and exposed to simulated ischaemia/reperfusion (SI/R). CMECs were randomly divided into four groups: control group, SI/R group, SI/R + Mdivi-1 (a mitochondrial fission inhibitor) group and SI/R + NAC (an reactive oxygen species scavenger) group;
Cell viability was measured with MTT assay;
Migration ability was detected by the Transwell method;
Flow cytometry based on AnnexinV-FITC/PI double staining and TUNEL staining were used to detect apoptoticrates;
Mitochondrial morphology of CMECs was observed by fluorescence microscope following Mito-Tracker Green staining;
Protein levels of Drp1 and Fis1 were analysed by Western blot;
Intracellular ROS levels were tested by a reactive oxygen species assay kit;
Mitochondria ROS level was measured using mitochondria superoxide marker Mitosox;
Mitochondrial membrance potential was detected by a mitochondrial membrance potential assay kit (JC-1 staining).
Compared with control group, both cell viability and migration ability in the SI/R group were impaired, and apoptosis index was significantly increased. Mitochondrial fission was also enhanced following SI/R, as evidenced by increased mitochondrial fragments using fluorescence microscope. Cells in the SI/Rgroup exhibited significantly higher expression of Drp1 and Fisl protein than that in the control group.
Compared with SI/R group, both cell viability and migration ability in theSI/R + Mdivi-1 group were increased, with apoptosis index decreased by Mdivi-1 administered before reperfusion. Besides, mitochondrial fission was significantly confined. However, Drp1 and Fisl protein level showed no significant changes,and both cellular and mitochondrial ROS levels decreased remarkablely, along with increased mitochondrial membrance potential.
Antioxidant NAC reversed the SI/R-induced increase in both cellular and mitochondrial ROS levels, and further inhibited the SI/R-stimulated mitochondrial fission.
Conclusions Increased mitochondrial fission play an important role inCMECs ischaemia/ reperfusion injury. Underlying mechanisms involves its interaction with cellular and mitochondrial ROS generation and the maintenance of mitochondrial membrance potential. Thus inhibiting mitochondrial fission mayrepresent a new promising strategy in protecting CMECs from I/R injury, hence the salvage of I/R heart.