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Updated: Mar 18, 2026
Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury
Published on: March 7, 2022
Phosphorylated eEF2 is SUMOylated and induces cardiomyocyte apoptosis during myocardial ischemia reperfusion
Chao Zhang1, Xiaojuan Liu2, Chi Zhang1
1Department of Cardiology, Affiliated Hospital of Nantong University, Nantong, PR China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
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.
Insights
Eukaryotic elongation factor 2 (eEF2) has a dual role in heart recovery after myocardial ischemia reperfusion (MIR). Phosphorylated eEF2 entering the nucleus can promote cardiomyocyte apoptosis, hindering cardiac function recovery.
Area of Science:
- Cardiology
- Molecular Biology
- Cellular Biology
Background:
- Cardiomyocyte apoptosis impedes cardiac function recovery post-myocardial ischemia reperfusion (MIR).
- Protein synthesis via eukaryotic elongation factor 2 (eEF2) is crucial for MIR recovery, but its specific role remains unclear.
Purpose of the Study:
- To investigate the function of eEF2 in cardiomyocyte apoptosis during MIR.
- To elucidate the mechanisms by which eEF2 influences cardiac function recovery.
Main Methods:
- Induction of MIR in a rat model and hypoxia/reoxygenation (H/R) in H9c2 cells.
- Assessment of eEF2 and phosphorylated eEF2 localization, SUMOylation, and protein expression (Bax, Bcl-2) via western blot and immunofluorescence.
- Evaluation of cell viability and apoptosis using CCK8 assay and flow cytometry.
Main Results:
- Phosphorylated eEF2 translocated to the nucleus of cardiomyocytes during MIR and H/R.
- SUMOylation of eEF2 was observed in cardiomyocytes.
- Overexpression of eEF2 upregulated Bcl-2, suggesting a role in reducing apoptosis, while the N-terminal fragment promoted apoptosis.
Conclusions:
- eEF2 exhibits a bidirectional role in regulating cardiomyocyte apoptosis during MIR.
- Phosphorylated eEF2, upon SUMOylation and nuclear translocation, can promote cardiomyocyte apoptosis, impacting cardiac recovery.
