[Effect of iron death inhibitor on hypoxia/reoxygenation injury of cardiomyocytes and its mechanism].

OBJECTIVE: To investigate the effects of ferrostatin-1 (Fer-1) on cardiomyocyte hypoxia/reoxygenation injury and its mechanisms. METHODS: The original generation of myocardial cells were extracted from 1～3 d newborn SD rats, which were randomly divided into normal control group (control), hypoxia reoxygenation (H/R) group and hypoxia reoxygenation + iron death inhibitors group (H/R + Fer-1). After 52 h of culture, cells in H/R group were added with 4 mmol/L Na2S2O4 solution. After 1 h of hypoxia, cells were reoxygenated with DMEM medium containing 10% calf serum for 3 h.The H/R+ Fer-1 group was pretreated with Fer-1 (2 µmol/L) for 24 h and then subjected to hypoxia and reoxygenation. The release rate of lactate dehydrogenase (LDH) was measured by UV spectrophotometry, the cell survival rate was measured by CCK-8 method, SOD was measured by xanthine oxidase method, MDA was measured by chemical coloration, and the changes of mitochondrial membrane potential and reactive oxygen species (ROS) were observed by immunofluorescence. Western blot was used to detect the expressions of ACSL4 and GPX4. RESULTS: Compared with the control group, the cell activity, SOD release and MMP level were decreased (P＜0.05), the levels of LDH, MDA and ROS were increased (P＜0.05), the protein expression of ACSL4 was increased (P＜0.05), and the protein expression of GPX4 was decreased (P＜0.05) in H/R group. Compared with the H/R group, the cell activity, SOD release and MMP level were increased (P＜0.05), the level of LDH, MDA and ROS were decreased (P＜0.05), the protein expression of ACSL4 was decreased (P＜0.05), and the protein expression of GPX4 was increased (P＜0.05) in H/R+Fer-1 group. CONCLUSION: Fer-1 can inhibit the production of intracellular reactive oxygen species by regulating ACSL4 and GPX4, thereby alleviating the hypoxia and reoxygenation injury of primary cardiomyocytes caused by iron death.

miR­320­3p is involved in morphine pre­conditioning to protect rat cardiomyocytes from ischemia/reperfusion injury through targeting Akt3.

Morphine pre­conditioning (MPC) can significantly reduce myocardial ischemic injury and inhibit cardiomyocyte apoptosis, but the underlying mechanism still remains unclear. The aim of the present study was to investigate the protective mechanism of MPC in myocardial hypoxia/reoxygenation (H/R) injury at the microRNA (miR) level. H9c2 cells were used as a model of H/R and subjected to morphine pre­treatment. The protective effects of MPC on H/R injury in cardiomyocytes were evaluated using MTT and colorimetric assay, as well as flow cytometry. In addition, reverse transcription­quantitative PCR, western blotting and dual­luciferase reporter assay experiments were performed to determine the relationship between MPC, miR­320­3p and Akt3, and their effects on H/R injury. The present study demonstrated that MPC enhanced cell activity, decreased LDH content, and reduced apoptosis in rat cardiomyocytes, suggesting that MPC could protect these cells from H/R injury. Moreover, MPC partially reversed the increase in miR­320­3p expression and the decrease in Akt3 levels caused by H/R injury. Inhibition of miR­320­3p expression also attenuated the effects of H/R on cardiomyocyte activity, LDH content and apoptosis. Furthermore, Akt3 was predicted to be a target gene of miR­320­3p, and overexpression of miR­320­3p inhibited the expression of Akt3, blocking the protective effects of MPC on the cells. The current findings revealed that MPC could protect cardiomyocytes from H/R damage through targeting miR­320­3p to regulate the PI3K/Akt3 signaling pathway.

Aldehyde dehydrogenase 2 preserves mitochondrial morphology and attenuates hypoxia/reoxygenation-induced cardiomyocyte injury.

BACKGROUND: Disturbance of mitochondrial fission and fusion (termed mitochondrial dynamics) is one of the leading causes of ischemia/reperfusion (I/R)-induced myocardial injury. Previous studies showed that mitochondrial aldehyde dehydrogenase 2 (ALDH2) conferred cardioprotective effect against myocardial I/R injury and suppressed I/R-induced excessive mitophagy in cardiomyocytes. However, whether ALDH2 participates in the regulation of mitochondrial dynamics during myocardial I/R injury remains unknown. METHODS: In the present study, we investigated the effect of ALDH2 on mitochondrial dynamics and the underlying mechanisms using the H9c2 cells exposed to hypoxia/reoxygenation (H/R) as an in vitro model of myocardial I/R injury. RESULTS: Cardiomyocyte apoptosis was significantly increased after oxygen-glucose deprivation and reoxygenation (OGD/R), and ALDH2 activation largely decreased the cardiomyocyte apoptosis. Additionally, we found that both ALDH2 activation and overexpression significantly inhibited the increased mitochondrial fission after OGD/R. Furthermore, we found that ALDH2 dominantly suppressed dynamin-related protein 1 (Drp1) phosphorylation (Ser616) and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation (Thr172) but not interfered with the expression levels of mitochondrial shaping proteins. CONCLUSIONS: We demonstrate the protective effect of ALDH2 against cardiomyocyte H/R injury with a novel mechanism on mitochondrial fission/fusion.

Erythropoietin helix B surface peptide modulates miR-21/Atg12 axis to alleviates cardiomyocyte hypoxia-reoxygenation injury.

BACKGROUND: The erythropoietin helix B surface peptide (HBSP) has been shown to have neuroprotective and repair-damaging myocardium effects similar to erythropoietin (EPO). However, the protective mechanism of HBSP on cardiomyocyte hypoxia-reoxygenation (H/R) injury is not clear. METHODS: H9C2 cells were pretreated with HBSP and subjected to hypoxia/reoxygenation (H/R), changes in cell function, autophagy and apoptosis were assessed, respectively. Cells were transfected with miR-21 mimic and miR-NC, and the relative expression of miR-21 and Atg12 were detected by qRT-PCR. The target role of miR-21 and Atg12 was evaluated by dual-luciferase reporter. After transfected with si-Atg12 and si-NC, western blot was used to assess autophagy and apoptosis proteins, flow cytometry assay was used to detect apoptosis rate. RESULTS: We found the expression of miR-21 was significantly down-regulated, accompanied by remarkably activated of autophagy and apoptosis in H9C2 cells during H/R injury. Pleasantly, HBSP pretreatment has a similar effect as transfection of miR-21 mimic, which is to evidently inhibit autophagy and apoptosis by up-regulating miR-21 expression. Moreover, Bioinformatics analysis and luciferase reporter assay revealed that Atg12 was directly bond to miR-21. To further understand whether Atg12 is involved in the process of miR-21 regulating autophagy, si-Atg12 and si-NC were transfected into H9C2 cell, the results showed that knockdown of Atg12 enhances the inhibition autophagy and apoptosis effect of HBSP. CONCLUSION: These results demonstrate that HBSP inhibits myocardial H/R injury induced by autophagy over-activation and apoptosis via miR-21/Atg12 axis.

Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) alleviates myocardial hypoxia-reoxygenation injury by inhibiting oxidative stress and ameliorating mitochondrial dysfunction.

Oxidative stress and mitochondrial dysfunction are considered to be activators of apoptosis and serve a pivotal role in the pathogenesis of myocardial ischemia-reperfusion (MI/R) injury. Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) is a multifunctional protein that processes the cellular response to DNA damage and oxidative stress. Little is known about the role of APE1 in the pathogenesis of MI/R injury. The aim of the present study was to investigate the effects of APE1 on hypoxia-reoxygenation (H/R)-induced H9c2 cardiomyocyte injury and the underlying mechanism responsible. It was demonstrated that H/R decreased cell viability and increased lactic dehydrogenase (LDH) release, as well as reducing APE1 expression in H9c2 cells. However, APE1 overexpression induced by transfection with APE1-expressing lentivirus significantly increased H9c2 cell viability, decreased LDH release, decreased apoptosis and reduced caspase-3 activity in H/R-treated H9c2 cells. APE1 overexpression ameliorated the H/R-induced increases in reactive oxygen species and NAPDH oxidase expression, as well as the decreases in superoxide dismutase activity and glutathione expression. Furthermore, APE1 overexpression increased mitochondrial membrane potential and ATP production, stabilized electron transport chain activity (as illustrated by increased NADH-ubiquinone oxidoreductase, succinate dehydrogenase, coenzyme Q-cytochrome c oxidoreductase and cytochrome c oxidase activities) and decreased the ratio of B-cell lymphoma 2-associated X protein/B-cell lymphoma 2 in H/R, improving mitochondrial dysfunction. In conclusion, the results of the present study suggest that APE1 alleviates H/R-induced injury in H9c2 cells by attenuating oxidative stress and ameliorating mitochondrial dysfunction. APE1 may therefore be used as an effective treatment for MI/R injury.