SIRT1 reduced DDX5 neddylation to attenuate myocardial ischemia/reperfusion injury.

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&#x2009;<&#x2009;0.001) and reduced SIRT1 protein levels (p&#x2009;<&#x2009;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&#x2009;<&#x2009;0.001) and increased DDX5 neddylation in H9C2 cells, while which were reversed by MLN4924 or LV-SIRT1 (p&#x2009;<&#x2009;0.05). Also, SIRT1 increased DDX5 protein level by enhancing DDX5 stability via reducing its neddylation. Functionally, hypoxia decreased cell viability (p&#x2009;<&#x2009;0.001) and increased cell apoptosis (p&#x2009;<&#x2009;0.001) and ROS level (p&#x2009;<&#x2009;0.001) in H9C2 cells, whereas they were all reversed by LV-SIRT1 (p&#x2009;<&#x2009;0.05, p&#x2009;<&#x2009;0.001) or LV-DDX5 (p&#x2009;<&#x2009;0.05, p&#x2009;<&#x2009;0.001). The in vivo experiments revealed that LV-DDX5 reversed the increased infarction area (p&#x2009;<&#x2009;0.05), necrotic myocardial fibers and cardiomyocytes apoptosis (p&#x2009;<&#x2009;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.