Phosphorylated eEF2 is SUMOylated and induces cardiomyocyte apoptosis during myocardial ischemia reperfusion

Abstract Background Cardiomyocyte apoptosis after myocardial ischemia reperfusion (MIR) blocks the recovery of cardiac function during revascularization treatment. Protein synthesis mediated by eukaryotic elongation factor 2 (eEF2) is vital for the recovery of MIR. eEF2 promotes peptide elongation without phosphorylation of itself. However, the exact function of eEF2 during MIR is unknown. Methods We used suture tie-down of left coronary artery (LCA) to induce MIR in vivo, which was confirmed by electrocardiography and Evan's blue/triphenyltetrazolium chloride double staining. Hypoxia/reoxygenation (H/R) treatment was utilized to stimulate H9c2 cells, which was detected by CCK8 assay to evaluate cell viability. eEF2, phosphorylated eEF2, SUMO, Bax, and Bcl-2 protein expressions and location of eEF2 and phosphorylated eEF2 were determined by western blot, immunocytochemistry and immunofluorescent staining. H9c2 cell apoptosis was assessed by flow cytometry. The effects of eEF2 full-length plasmid and its fragments on H9c2 cells were also detected. Results In vivo, phosphorylated eEF2 to eEF2 ratio decreased gently in rat MIR model. Immunocytochemistry showed that phosphorylated eEF2 translocated to the nucleus of cardiomyocytes during myocardial reperfusion. Furthermore, double immunofluorescent staining in H9c2 cells after H/R treatment also showed phosphorylated eEF2 translocated to the nucleus. Meanwhile, SUMOylation of eEF2 was detected. The overexpression of eEF2 upregulated Bcl-2 expression after H/R treatment, suggesting that eEF2 might reduce cardiomyocyte apoptosis during MIR. In addition, the N-terminal fragment of eEF2 transfection could promote apoptosis. Conclusions eEF2 plays a bidirectional role in regulating cardiomyocyte apoptosis during MIR, in which eEF2 can be SUMOylated and translocate into nucleus of cardiomyocytes to promote cardiomyocyte apoptosis when eEF2 is phosphorylated.