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PEP-1-CAT protects hypoxia/reoxygenation-induced cardiomyocyte apoptosis through multiple sigaling pathways.

ABSTRACT: Catalase (CAT) breaks down H2O2 into H2O and O2 to protects cells from oxidative damage. However, its translational potential is limited because exogenous CAT cannot enter living cells automatically. This study is aimed to investigate if PEP-1-CAT fusion protein can effectively protect cardiomyocytes from oxidative stress due to hypoxia/reoxygenation (H/R)-induced injury.H9c2 cardomyocytes were pretreated with catalase (CAT) or PEP-1-CAT fusion protein followed by culturing in a hypoxia and re-oxygenation condition. Cell apoptosis were measured by Annexin V and PI double staining and Flow cytometry. Intracellular superoxide anion level was determined, and mitochondrial membrane potential was measured. Expression of apoptosis-related proteins including Bcl-2, Bax, Caspase-3, PARP, p38 and phospho-p38 was analyzed by western blotting.PEP-1-CAT protected H9c2 from H/R-induced morphological alteration and reduced the release of lactate dehydrogenase (LDH) and malondialdehyde content. Superoxide anion production was also decreased. In addition, PEP-1-CAT inhibited H9c2 apoptosis and blocked the expression of apoptosis stimulator Bax while increased the expression of Bcl-2, leading to an increased mitochondrial membrane potential. Mechanistically, PEP-1-CAT inhibited p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways, resulting in blockade of Bcl2/Bax/mitochondrial apoptotic pathway.Our study has revealed a novel mechanism by which PEP-1-CAT protects cardiomyocyte from H/R-induced injury. PEP-1-CAT blocks Bcl2/Bax/mitochondrial apoptotic pathway by inhibiting p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways.

SUBMITTER: Zhang L

PROVIDER: S-EPMC3660214 | biostudies-other | 2013 May

REPOSITORIES: biostudies-other

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PEP-1-CAT protects hypoxia/reoxygenation-induced cardiomyocyte apoptosis through multiple sigaling pathways.

Zhang Lei L Wei Shuang S Tang Jun-Ming JM Guo Ling-Yun LY Zheng Fei F Yang Jian-Ye JY Kong Xia X Huang Yong-Zhang YZ Chen Shi-You SY Wang Jia-Ning JN

Journal of translational medicine 20130506

<h4>Background</h4>Catalase (CAT) breaks down H2O2 into H2O and O2 to protects cells from oxidative damage. However, its translational potential is limited because exogenous CAT cannot enter living cells automatically. This study is aimed to investigate if PEP-1-CAT fusion protein can effectively protect cardiomyocytes from oxidative stress due to hypoxia/reoxygenation (H/R)-induced injury.<h4>Methods</h4>H9c2 cardomyocytes were pretreated with catalase (CAT) or PEP-1-CAT fusion protein followed ...[more]

PMID: 23642335

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Project description:ObjectivesTo explore the effects of miR-16-5p and PTPN4 on the apoptosis and autophagy of AC16 cardiomyocytes after hypoxia/reoxygenation treatment.MethodsAC16 cells were divided into the control group (NC), hypoxia/reoxygenation group (H/R), knockdown miR-16-5p negative control group (NC inhibitor), knockdown miR-16-5p group (miR-16-5p inhibitor), overexpression miR-16-5p negative control group (NC mimics), overexpression miR-16-5p group (miR-16-5p mimics), silent PTPN4 negative control group (sh-NC), silent PTPN4 group (sh-PTPN4), and silent PTPN4 + knockdown miR-16-5p group (sh-PTPN4 + miR-16-5p inhibitor). Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting (WB) were used to measure the expression level of miR-16-3p, miR-16-5p, protein tyrosine phosphatase nonreceptor type 4 (PTPN4), and autophagy-related proteins (beclin-1, LC3 II/I, and P26) in AC16 cells. The apoptosis level of AC16 cells in each group was measured by flow cytometry and TUNEL. The dual-luciferase reporter gene experiment was also used to verify the targeting relationship between miR-16-5p and PTPN4.ResultsAfter H/R treatment, the levels of myocardial injury markers including LDH and CK-MB in AC16 cells were increased significantly (P < 0.05), and the levels of cell apoptosis and autophagy also increased significantly (P < 0.05). The level of miR-16-3p in AC16 cells did not change significantly after H/R treatment, whereas the level of miR-16-5p was increased significantly (P < 0.05). After miR-16-5p was knocked down, the levels of LDH and CK-MB in AC16 cells treated with H/R were significantly reduced (P < 0.05), and the rates of cell apoptosis and autophagy were also significantly reduced (P < 0.05). miR-16-5p negatively regulated the expression level of PTPN4 protein in AC16 cells (P < 0.05), and the dual-luciferase reporter gene experiment confirmed that PTPN4 was the downstream target of miR-16-5p. Silencing of PTPN4 significantly increased the damage of AC16 cells induced by H/R treatment (P < 0.05), but simultaneously inhibiting the expression of PTPN4 and miR-16-5p reversed the protective effect of miR-16-5p knockdown on AC16 cells (P < 0.05).ConclusionsThe expression of miR-16-5p is upregulated in AC16 cells after H/R treatment and the knockdown which can protect AC16 cells from H/R-induced cell damage that may be due to its regulation on the expression of PTPN4.

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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PEP-1-CAT protects hypoxia/reoxygenation-induced cardiomyocyte apoptosis through multiple sigaling pathways.

Publications

PEP-1-CAT protects hypoxia/reoxygenation-induced cardiomyocyte apoptosis through multiple sigaling pathways.

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