SIRT1 reduced DDX5 neddylation to attenuate myocardial ischemia/reperfusion injury

Hum Cell. 2026 Feb 10;39(2):43. doi: 10.1007/s13577-026-01356-6.

Abstract

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.

Keywords: Cardiomyocytes apoptosis; DDX5; MI/RI; Neddylation; SIRT1.

MeSH terms

  • Animals
  • Apoptosis / genetics
  • Cell Line
  • Cyclopentanes / pharmacology
  • DEAD-box RNA Helicases* / genetics
  • DEAD-box RNA Helicases* / metabolism
  • Disease Models, Animal
  • Humans
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Myocardial Reperfusion Injury* / genetics
  • Myocardial Reperfusion Injury* / metabolism
  • Myocardial Reperfusion Injury* / pathology
  • Myocardial Reperfusion Injury* / prevention & control
  • Myocytes, Cardiac / metabolism
  • Myocytes, Cardiac / pathology
  • NEDD8 Protein* / metabolism
  • Pyrimidines / pharmacology
  • Rats
  • Sirtuin 1* / genetics
  • Sirtuin 1* / metabolism
  • Sirtuin 1* / physiology

Substances

  • Sirtuin 1
  • NEDD8 Protein
  • DEAD-box RNA Helicases
  • pevonedistat
  • Pyrimidines
  • Cyclopentanes