Oxidative Stress In H9c2 Cells Research Articles

Exogenous hydrogen sulfide protects H9c2 cardiac cells against high glucose-induced injury by inhibiting the activities of the p38 MAPK and ERK1/2 pathways

Hyperglycemia is a risk factor for the development of diabetic cardiovascular complications, which are associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we demonstrate the inhibitory effects of exogenous hydrogen sulfide (H₂S) on the activation of the MAPK pathway. The aim of the present study was to determine whether exogenous H₂S prevents high glucose (HG)-induced injury by inhibiting the activation of the p38 MAPK and extracellular signal-regulated kinase (ERK)1/2 (members of MAPK) pathways in cardiomyoblasts (H9c2 cells). The findings of the present study demonstrated that the treatment of H9c2 cells with HG (35 mM glucose) for 24 h not only significantly induced injury, including cytotoxicity, apoptosis, overproduction of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (MMP), but also upregulated the expression levels of phosphorylated (p)-p38 MAPK and p-ERK1/2. The increased expression levels of p-p38 MAPK and p-ERK1/2 were markedly reduced by pre-treatment of the H9c2 cells with 400 µM sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to 35 mM glucose. Importantly, pre-treatment of the cells with 400 µM NaHS or 3 µM SB203580 (a selective inhibitor of p38 MAPK) or 15 µM U0126 (a selective inhibitor of ERK1/2) attenuated the HG-induced cardiomyocyte injury, leading to an increase in cell viability and a decrease in the number of apoptotic cells, preventing ROS generation, as well as the loss of MMP. In addition, pre-treatment of the cells with 1,000 µM N‑acetyl‑L‑cysteine (a ROS scavenger) prior to exposure to HG ameliorated the HG-induced cytotoxicity. Taken together, the data from the present study demonstrate for the first time, to our knowledge, that exogenous H2S exerts a protective effect against HG‑induced injury by inhibiting the activation of the p38 MAPK and ERK1/2 pathways and preventing oxidative stress in H9c2 cells.

Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy

Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). Cardiac H9c2 cells were exposed to H2 O2 (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H2 DCFDA). A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H2 O2 compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H2 DCFDA. Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to.

Hydrogen sulfide attenuates doxorubicin-induced cardiotoxicity by inhibition of the p38 MAPK pathway in H9c2 cells

We previously demonstrated the protective effect of hydrogen sulfide (H2S) against doxorubicin (DOX)-induced cardiotoxicity through inhibition of endoplasmic reticulum stress. The aim of the present study was to explore the role of p38 mitogen-activated protein kinase (MAPK) in DOX-induced cardiotoxicity and ascertain whether exogenous H2S protects DOX-induced injury by inhibiting p38MAPK in cardiomyoblasts (H9c2). We observed that exposure of H9c2 cells to 5µM DOX not only markedly induced injuries, including cytotoxicity, apoptosis, overproduction of reactive oxygen species (ROS) and dissipation of mitochondrial membrane potential (MMP), but also enhanced the expression level of phosphorylated(p)-p38MAPK. The DOX-induced increase in expression of p-p38 MAPK was significantly attenuated by pretreatment of H9c2 cells with either 400µM sodium hydrogen sulfide (NaHS) (a donor of H2S) or 1,000µM N-acetyl-L-cysteine (NAC, an ROS scavenger) prior to exposure to DOX. Pretreatment with either 400µM NaHS or 3µM SB203580, a selective inhibitor of p38MAPK, ameliorated DOX-induced cardiomyocyte injuries, as evidenced by an increase in cell viability, and decreases in the number of apoptotic cells, ROS generation as well as dissipation of MMP. In conclusion, the findings of the present study demonstrated that the activation of p38MAPK contributes to DOX-induced injuries, including cytotoxicity, apoptosis, mitochondrial damage and oxidative stress in H9c2 cells. We also provide novel evidence that exogenous H2S protects H9c2 cells against DOX-induced cardiotoxicity by inhibition of the p38 MAPK pathway.

Mitochondrial Ogg1 prevents loss of mitDNA content, decreases mitochondria fission and apoptosis under conditions of oxidative stress in H9C2 cells

Oxidative stress induces damage and depletion of mitochondrial (mit) DNA and suppresses ATP production leading to defects in cell function and initiating cell death. 8‐oxoguanosine DNA glycosylase‐1 (OGG1) acts in the mitochondria to repair DNA damage. In this study H9C2 cells were infected with either adv‐ OGG1 or empty vector and, challenged with menadione, to induce mitochondrial DNA damage. The cells were used to evaluate the effects of OGG1 on mitochondrial DNA content and damage and on apoptotic events induced by oxidative stress. Levels of 8‐oxodG and AP‐sites, metrics of mitochondrial DNA damage, were decreased by 30% and 35%, respectively, in adv‐Ogg1 infected cells. Cells over‐expressing mitOgg1 showed increased membrane potential (p<0.05) and decreased mitochondrial fragmentation (p<0.005). mitOgg1 over‐expression preserved mitDNA content and lowered levels of fission and apoptotic factors such as DRP‐1, FIS1, cytoplasmic cytochrome c, caspase‐3 and caspase‐9. These observations suggest that mitOgg1 may be an alternative to protect cardiac cells against oxidative stress damage.

2,3,7,8-Tetrachlorodibenzo- p-dioxin and β-naphthoflavone induce cellular hypertrophy in H9c2 cells by an aryl hydrocarbon receptor-dependant mechanism

Cigarette smoke is a major risk factor for cardiovascular diseases. It contains thousands of compounds that activate the aryl hydrocarbon receptor (AhR). In addition, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), the most potent AhR ligand, has been shown to cause cardiotoxic effects in several in vivo models. Although induction of CYP1 family is the most important effect of AhR activation, the role of CYP1 induction in mediating the cardiotoxic effect of TCDD is usually overlooked. Therefore, we investigated whether AhR activation causes a hypertrophic effect in H9c2 cells and we related this effect to changes in CYP gene expression. In the current study, the cardiac derived H9c2 cells were treated with two AhR ligands, TCDD and β-naphthoflavone (BNF), for 24 and 48 h. The expression of the hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and several CYP genes were measured by real-time PCR. Treatment of H9c2 cells with TCDD or BNF for 24 h caused a significant induction of CYP1A1, CYP1B1, and CYP4A1; however, there was no change in the expression of other genes. On the other hand, treatment of the cells with TCDD or BNF for 48 h caused a significant induction of the hypertrophic markers, ANP and BNP, and several CYP genes such as CYP1A1, CYP1B1, CYP2E1, CYP2J3, and CYP4F4 parallel to a significant increase in the cell surface area. Neither TCDD nor BNF increased the oxidative stress in H9c2 cells at all concentrations tested. Interestingly, resveratrol, an AhR antagonist, protected the cells from TCDD-induced hypertrophy. In conclusion, AhR ligands caused a hypertrophic effect in H9c2 cells which was associated with induction of several CYP genes which can be prevented by resveratrol.

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H 2O 2 stimulation

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H 2O 2 (100 μM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H 2O 2 stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 μM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H 2O 2-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H 2O 2 stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H 2O 2. Under these conditions, the H 2O 2-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H 2O 2, and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.