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Updated: Feb 12, 2026
Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury
Published on: March 7, 2022
SIRT1 reduced DDX5 neddylation to attenuate myocardial ischemia/reperfusion injury
Yan Liang1, Gongcheng Huang1, Xiaohua Zhu1
1Department of Cardiovascular Surgery, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe East Road, Zhengzhou, Henan Province, China.
Background:
SIRT1 exhibited a protective role in myocardial ischemia/reperfusion injury (MI/RI), but the related mechanisms remained unclear. In this study, the regulation of SIRT1 on neddylation modification in MI/RI was explored.
Methods:
H9C2 cells underwent hypoxia and reoxygenation (H/R) to mimic MI/RI in vitro, and C57BL6 mice were employed to establish MI/RI model for the in vivo experiments. Mass spectrometry analysis was employed to screen the possible modified substrates of NEDD8; Western blot was performed to detect protein level; CCK8 was performed to assess cell viability; flow cytometry, TUNEL, and Cardiac Troponin T (cTNT) double staining were performed to assess cardiomyocytes apoptosis; TTC and HE staining were performed to assess infarction area and pathological changes of cardiac tissues in MI/RI mice, respectively.
Results:
MLN4924 (an inhibitor of NEDD8-activating enzyme (NAE)) significantly reversed the elevated NEDD8 conjugated protein (p < 0.001) and reduced SIRT1 protein levels (p < 0.001) induced by H/R in H9C2 cells. Dead-box helicase 5 (DDX5) was screened as the possible modified substrate of NEDD8 via mass spectrometry. H/R further reduced DDX5 protein level (p < 0.001) and increased DDX5 neddylation in H9C2 cells, while which were reversed by MLN4924 or LV-SIRT1 (p < 0.05). Also, SIRT1 increased DDX5 protein level by enhancing DDX5 stability via reducing its neddylation. Functionally, hypoxia decreased cell viability (p < 0.001) and increased cell apoptosis (p < 0.001) and ROS level (p < 0.001) in H9C2 cells, whereas they were all reversed by LV-SIRT1 (p < 0.05, p < 0.001) or LV-DDX5 (p < 0.05, p < 0.001). The in vivo experiments revealed that LV-DDX5 reversed the increased infarction area (p < 0.05), necrotic myocardial fibers and cardiomyocytes apoptosis (p < 0.001) in MI/RI mice.
Conclusion:
These results suggested that SIRT1 increased DDX5 protein level to reduce cardiomyocytes apoptosis and ROS level via the inhibition of DDX5 neddylation, thus alleviating MI/RI.
Insights
Sirtuin 1 (SIRT1) protects against myocardial ischemia/reperfusion injury (MI/RI) by increasing Dead-box helicase 5 (DDX5) protein levels. This action reduces cardiomyocyte apoptosis and oxidative stress by inhibiting DDX5 neddylation.
Area of Science:
- Cardiovascular Biology
- Molecular Mechanisms of Disease
- Cellular Stress Response
Background:
- Sirtuin 1 (SIRT1) plays a protective role in myocardial ischemia/reperfusion injury (MI/RI).
- The precise mechanisms underlying SIRT1's protective effects in MI/RI remain incompletely understood.
- This study investigates SIRT1's regulation of neddylation modification in the context of MI/RI.
Purpose of the Study:
- To elucidate the role of SIRT1 in regulating neddylation in myocardial ischemia/reperfusion injury.
- To identify the downstream targets of SIRT1-mediated neddylation.
- To determine the functional consequences of SIRT1 and its target in cellular and animal models of MI/RI.
Main Methods:
- In vitro hypoxia/reoxygenation (H/R) in H9C2 cells and in vivo MI/RI models in C57BL6 mice.
- Mass spectrometry to identify NEDD8-conjugated substrates.
- Western blot, CCK8 assay, flow cytometry, TUNEL, TTC, and HE staining to assess protein levels, cell viability, apoptosis, and tissue damage.
Main Results:
- Hypoxia/reoxygenation (H/R) increased NEDD8 conjugation and decreased SIRT1 and Dead-box helicase 5 (DDX5) protein levels in H9C2 cells.
- DDX5 was identified as a substrate of NEDD8, and its neddylation was increased by H/R.
- SIRT1 overexpression or MLN4924 treatment reversed H/R-induced changes, with SIRT1 enhancing DDX5 protein stability by reducing its neddylation.
- SIRT1 and DDX5 overexpression protected H9C2 cells from hypoxia-induced apoptosis and ROS production.
- In vivo, DDX5 overexpression reduced infarct size and cardiomyocyte apoptosis in MI/RI mice.
Conclusions:
- SIRT1 alleviates MI/RI by increasing DDX5 protein levels through the inhibition of DDX5 neddylation.
- This mechanism reduces cardiomyocyte apoptosis and reactive oxygen species (ROS) levels.
- Targeting the SIRT1-DDX5 axis may offer a therapeutic strategy for MI/RI.
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