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Mechanism of interactions between endoplasmic reticulum stress and autophagy in hypoxia/reoxygenation?induced injury of H9c2 cardiomyocytes.

ABSTRACT: Endoplasmic reticulum (ER) stress and autophagy are involved in myocardial ischemia?reperfusion (I/R) injury; however, their roles in this type of injury remain unclear. The present study investigated the roles of ER stress and autophagy, and their underlying mechanisms, in H9c2 cells during hypoxia/reoxygenation (H/R) injury. Cell viability was detected by CCK?8 assay. The autophagy flux was monitored with mCherry?GFP?LC3?adenovirus transfection. The expression levels of autophagy?related proteins and ER stress?related proteins were measured by western blotting. Apoptosis was detected by flow cytometry and western blotting. The results indicated that autophagy was induced, ER stress was activated and apoptosis was promoted in H9c2 cells during H/R injury. The inhibition of ER stress by 4?phenylbutyrate or C/EBP homologous protein (CHOP)?targeting small interfering RNA (siRNA) decreased autophagy and ameliorated cell apoptosis during H/R injury. Activation of autophagy by rapamycin attenuated ER stress and ameliorated cell apoptosis. Inhibition of autophagy by 3?methyladenine or Beclin1?targeting siRNA aggravated ER stress and exacerbated cell apoptosis, and activation of ER stress by thapsigargin decreased autophagy and induced cell apoptosis. Collectively, the findings of the present study demonstrated that H/R induced apoptosis and autophagy via ER stress in H9c2 cells, and that CHOP may serve an important role in ER stress?induced autophagy and apoptosis. Autophagy, as an adaptive response, was activated by ER stress and alleviated ER stress?induced cell apoptosis during H/R injury.

SUBMITTER: Guan G

PROVIDER: S-EPMC6580049 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Mechanism of interactions between endoplasmic reticulum stress and autophagy in hypoxia/reoxygenation‑induced injury of H9c2 cardiomyocytes.

Guan Gaopeng G Yang Lei L Huang Wenyin W Zhang Jun J Zhang Puhua P Yu Huan H Liu Shengyuan S Gu Xiang X

Molecular medicine reports 20190509 1

Endoplasmic reticulum (ER) stress and autophagy are involved in myocardial ischemia‑reperfusion (I/R) injury; however, their roles in this type of injury remain unclear. The present study investigated the roles of ER stress and autophagy, and their underlying mechanisms, in H9c2 cells during hypoxia/reoxygenation (H/R) injury. Cell viability was detected by CCK‑8 assay. The autophagy flux was monitored with mCherry‑GFP‑LC3‑adenovirus transfection. The expression levels of autophagy‑related prote ...[more]

PMID: 31115545

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Project description:BackgroundMitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial's structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism.Methods and resultsThe H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2, 6, 12, 18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy. RFP-GFP-LC3 double-labeled adenovirus was used for testing.) were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed.ConclusionsDuring myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect.

Project description:Background:Constitutive nuclear factor kappa B (NF?B) activation has been shown to exacerbate during myocardial ischemia/reperfusion (I/R) injury. We recently showed that miR-181c-5p exacerbated cardiomyocytes injury and apoptosis by directly targeting the 3'-untranslated region of protein tyrosine phosphatase nonreceptor type 4 (PTPN4). However, whether miR-181c-5p mediates cardiac I/R injury through NF?B-mediated inflammation is unknown. Thus, the present study aimed to investigate the role of miR-181c-5p during myocardial I/R injury and explore its mechanism in relation to inflammation in H9C2 cardiomyocytes. Methods and Results:In hypoxia/reoxygenation (H/R, 6?h hypoxia followed by 6?h reoxygenation)-stimulated H9C2 cardiomyocytes or postischemic myocardium of rat, the expression of miR-181c-5p was significantly upregulated, which was concomitant increased NF?B activity when compared to the nonhypoxic or nonischemic control groups. This is indicative that miR-181c-5p may be involved in NF?B-mediated inflammation during myocardial I/R injury. To investigate the potential role of miR-181c-5p in H/R-induced cell inflammation and injury, H9C2 cardiomyocytes were transfected with the miR-181c-5p agomir. Overexpression of miR-181c-5p significantly aggravated H/R-induced cell injury (increased lactate dehydrogenase (LDH) level) and exacerbated NF?B-mediated inflammation (greater phosphorylation and degradation of I?B?, phosphorylation of p65, and increased levels of proinflammatory cytokines tumor necrosis factor ? (TNF?), interleukin (IL)-6, and IL-1?). In contrast, inhibition of miR-181c-5p by its antagomir transfection in vitro had the opposite effect. Furthermore, overexpression of miR-181c-5p significantly enhanced lipopolysaccharide-induced NF?B signalling. Additionally, knockdown of PTPN4, the direct target of miR-181c-5p, significantly aggravated H/R-induced phosphorylation and degradation of I?B?, phosphorylation of p65, and the levels of proinflammatory cytokines. PTPN4 knockdown also cancelled miR-181c-5p antagomir mediated anti-inflammatory effects in H9C2 cardiomyocytes during H/R injury. Conclusions:It is concluded that miR-181c-5p may exacerbate myocardial I/R injury and NF?B-mediated inflammation via PTPN4, and that targeting miR-181c-5p/PTPN4/NF?B signalling may represent a novel strategy to combat myocardial I/R injury.

Dataset Information

Mechanism of interactions between endoplasmic reticulum stress and autophagy in hypoxia/reoxygenation?induced injury of H9c2 cardiomyocytes.

Publications

Mechanism of interactions between endoplasmic reticulum stress and autophagy in hypoxia/reoxygenation‑induced injury of H9c2 cardiomyocytes.

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