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N-n-butyl haloperidol iodide protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy.

ABSTRACT: N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expression of autophagy marker protein LC3. Our results indicated that treatment with F2 inhibited autophagy in H9c2 cells exposed to H/R. 3-methyladenine, an inhibitor of autophagy, suppressed H/R-induced autophagy, and decreased apoptosis, whereas rapamycin, a classical autophagy sensitizer, increased autophagy and apoptosis. Mechanistically, macrophage migration inhibitory factor (MIF) was inhibited by F2 treatment after H/R. Accordingly, small interfering RNA (siRNA)-mediated MIF knockdown decreased H/R-induced autophagy. In summary, F2 protects cardiomyocytes during H/R injury through suppressing autophagy activation. Our results provide a new mechanistic insight into a functional role of F2 against H/R-induced cardiomyocyte injury and death.

SUBMITTER: Wang B

PROVIDER: S-EPMC4694790 | biostudies-other | 2015 Sep

REPOSITORIES: biostudies-other

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N-n-butyl haloperidol iodide protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy.

Wang Bin B Zhong Shuping S Zheng Fuchun F Zhang Yanmei Y Gao Fenfei F Chen Yicun Y Lu Binger B Xu Han H Shi Ganggang G

Oncotarget 20150901 28

N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. In this study, we hypothesized the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by inhibiting autophagy in H9c2 cells. The degree of autophagy by treatment with F2 exposed to H/R in H9c2 cell was characterized by monodansylcadaverine, transmission electron microscopy, and expre ...[more]

PMID: 26359352

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Project description:Early growth response-1 (Egr-1), a transcription factor which often underlies the molecular basis of myocardial ischemia/reperfusion (I/R) injury, and oxidative stress, is key to myocardial I/R injury. Silent information regulator of transcription 1(SIRT1) not only interacts with and is inhibited by Egr-1, but also downregulates reactive oxygen species (ROS) via the Forkhead box O1(FOXO1)/manganese superoxide dismutase (Mn-SOD) signaling pathway. N-n-butyl haloperidol iodide (F2), a new patented compound, protects the myocardium against myocardial I/R injury in various animal I/R models in vivo and various heart-derived cell hypoxia/reoxygenation (H/R) models in vitro. In addition, F2 can regulate the abnormal ROS/Egr-1 signaling pathway in cardiac microvascular endothelial cells (CMECs) and H9c2 cells after H/R. We studied whether there is an inverse Egr-1/ROS signaling pathway in H9c2 cells and whether the SIRT1/FOXO1/Mn-SOD signaling pathway mediates this. We verified a ROS/Egr-1 signaling loop in H9c2 cells during H/R and that F2 protects against myocardial H/R injury by affecting SIRT1-related signaling pathways. Knockdown of Egr-1, by siRNA interference, reduced ROS generation, and alleviated oxidative stress injury induced by H/R, as shown by upregulated mitochondrial membrane potential, increased glutathione peroxidase (GSH-px) and total SOD anti-oxidative enzyme activity, and downregulated MDA. Decreases in FOXO1 protein expression and Mn-SOD activity occurred after H/R, but could be blocked by Egr-1 siRNA. F2 treatment attenuated H/R-induced Egr-1 expression, ROS generation and other forms of oxidative stress injury such as MDA, and prevented H/R-induced decreases in FOXO1 and Mn-SOD activity. Nuclear co-localization between Egr-1 and SIRT1 was increased by H/R and decreased by either Egr-1 siRNA or F2. Therefore, our results suggest that Egr-1 inhibits the SIRT1/FOXO1/Mn-SOD antioxidant signaling pathway to increase ROS and perpetuate I/R injury. F2 inhibits induction of Egr-1 by H/R, thereby activating SIRT1/FOXO1/Mn-SOD antioxidant signaling and decreasing H/R-induced ROS, demonstrating an important mechanism by which F2 protects against myocardial H/R injury.

Project description:Myocardial ischemia/reperfusion (MIR) injury, which occurs following acute myocardial infarction, can cause secondary damage to the heart. Tripartite interaction motif (TRIM) proteins, a class of E3 ubiquitin ligases, have been recognized as critical regulators in MIR injury. Zenglv Fumai Granule (ZFG) is a clinical prescription for the treatment of sick sinus syndrome, a disease that is associated with MIR injury. The present study aimed to investigate the effect of ZFG on MIR injury and to determine whether ZFG exerts its effects via regulation of TRIM proteins. In order to establish an in vitro MIR model, human cardiomyocyte cell line AC16 was cultured under hypoxia for 5 h and then under normal conditions for 1 h. Following hypoxia/reoxygenation (H/R) treatment, these cells were cultured with different ZFG concentrations. ZFG notably inhibited H/R-induced cardiomyocyte apoptosis. The expression levels of four TRIM proteins, TRIM7, TRIM14, TRIM22 and TRIM28, were also detected. These four proteins were significantly upregulated in H/R-injured cardiomyocytes, whereas their expression was inhibited following ZFG treatment. Moreover, TRIM28 knockdown inhibited H/R-induced cardiomyocyte apoptosis, whereas TRIM28 overexpression promoted apoptosis and generation of reactive oxygen species (ROS) in cardiomyocytes. However, the effects of TRIM28 overexpression were limited by the action of ROS inhibitor N-acetyl-L-cysteine. In addition, the mRNA and protein levels of antioxidant enzyme glutathione peroxidase (GPX)1 were significantly downregulated in H/R-injured cardiomyocytes. TRIM28 knockdown restored GPX1 protein levels but had no effect on mRNA expression levels. Co-immunoprecipitation and ubiquitination assays demonstrated that TRIM28 negatively regulated GPX1 via ubiquitination. In sum, the present study revealed that ZFG attenuated H/R-induced cardiomyocyte apoptosis by regulating the TRIM28/GPX1/ROS pathway. ZFG and TRIM28 offer potential therapeutic options for the treatment of MIR injury.

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N-n-butyl haloperidol iodide protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy.

Publications

N-n-butyl haloperidol iodide protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy.

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