Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H₂O₂)-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H₂O₂-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H₂O₂-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H₂O₂-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.     

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in Human hepatoma cells

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H₂O₂-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H₂O₂-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H₂O₂-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H₂O₂-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H₂O₂-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance.     

TAK1 activates AMPK-dependent cell death pathway in hydrogen peroxide-treated cardiomyocytes, inhibited by heat shock protein-70

The aim of this current study is to investigate the potential role of AMP-activated protein kinase (AMPK) in hydrogen peroxide (H₂O₂)-induced cardiomyocyte death, and focused on the signaling mechanisms of AMPK activation by H₂O₂. We observed a significant AMPK activation in H₂O₂-treated cardiomyocytes (both primary cells and H9c2 line). Inhibition of AMPK by its inhibitor or RNAi-reduced H₂O₂-induced cardiomyocyte death. We here proposed that transforming growth factor-β-activating kinase 1 (TAK1) might be the upstream kinase for AMPK activation by H₂O₂. H₂O₂-induced TAK1 activation, which recruited and activated AMPK. TAK1 inhibitor significantly suppressed H₂O₂-induced AMPK activation and following cardiomyocyte death, while over-expression of TAK1-facilitated AMPK activation and aggregated cardiomyocyte death. Importantly, heat shock protein-70 (HSP-70)-reduced H₂O₂-induced reactive oxygen species (ROS) accumulation, the TAK1/AMPK activation and cardiomyocyte death. In conclusion, we here suggest that TAK1 activates AMPK-dependent cell death pathway in H₂O₂-treated cardiomyocytes, and HSP-70 inhibits the signaling pathway by reducing ROS content.     

SIRT3 protects cardiomyocytes from oxidative stress-mediated cell death by activating NF-κB

Oxidative stress-mediated cell death in cardiomyocytes reportedly plays an important role in many cardiac pathologies. Our previous report demonstrated that mitochondrial SIRT3 plays an essential role in mediating cell survival in cardiac myocytes, and that resveratrol protects cardiomyocytes from oxidative stress-induced apoptosis by activating SIRT3. However, the exact mechanism by which SIRT3 prevents oxidative stress remains unknown. Here, we show that exposure of H9c2 cells to 50 μM H₂O₂ for 6 h caused a significant increase in cell death and the down-regulation of SIRT3. Reactive oxygen species (ROS)-mediated NF-κB activation was involved in this SIRT3 down-regulation. The SIRT3 activator, resveratrol, which is considered an important antioxidant, protected against H₂O₂-induced cell death, whereas the SIRT inhibitor, nicotinamide, enhanced cell death. Moreover, resveratrol negatively regulated H₂O₂-induced NF-κB activation, whereas nicotinamide enhanced H₂O₂-induced NF-κB activation. We also found that SOD2, Bcl-2 and Bax, the downstream genes of NF-κB, were involved in this pathological process. These results suggest that SIRT3 protects cardiomyocytes exposed to oxidative stress from apoptosis via a mechanism that may involve the NF-κB pathway.     

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts

Background

Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca²⁺ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H₂O₂-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts.

Results

Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 μM H₂O₂ exposure for 30 min with and/or without 10 to 20 μM EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H₂O₂-induced injury in H9c2 cells. H₂O₂ exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca²⁺ overload, increases in glycolytic protein, α-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H₂O₂-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3β (GSK-3β)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3β inhibitor (SB 216763) significantly improved the H₂O₂-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3β (S9), and level of cyclin D1 in cells.

Conclusions

Collectively, these findings suggest that EGCG blunts the H₂O₂-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3β mediated cardiac cell injury.     

RXR agonists inhibit oxidative stress-induced apoptosis in H9c2 rat ventricular cells

Retinoid X receptor (RXR) plays a central role in the regulation of intracellular receptor signaling pathways. We examined its role in regulating oxidative stress-induced apoptosis in H9c2 rat ventricular cells. We showed for the first time that functional RXR protein was downregulated by hydrogen peroxide (H₂O₂) in H9c2 cardiomyocytes. Natural and synthetic agonists of RXR, 9-cis-RA, and LGD1069 respectively, prevented H₂O₂-triggered apoptosis, and this anti-apoptotic effect was inhibited by the RXR antagonist HX531. Further investigation into the protective mechanisms of RXR demonstrated that H₂O₂-induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and caspase-3 activation were all significantly attenuated by pretreatment with RXR agonists. Furthermore, this protection was associated with a reduction in intracellular reactive oxygen species and an upregulation in catalase activity. Thus, these data indicate that pharmacological activation of RXR exerts protective effects against H₂O₂-induced apoptosis in H9c2 rat ventricular cells through antioxidant and mitochondria-protective mechanisms.     

Hypothermia protects H9c2 cardiomyocytes from H2O2 induced apoptosis

The purpose of our study was to investigate underlying basic mechanisms of hypothermia-induced cardioprotection during oxidative stress in a cardiomyocyte cell culture model. For hypothermic treatment we cooled H9c2 cardiomyocytes to 20 °C, maintained 20 min at 20 °C during which short-term oxidative damage was inflicted with 2 mM H₂O_2, followed by rewarming to 37 °C. Later on, we analyzed lactate dehydrogenase (LDH), caspase-3 cleavage, reactive oxygen species (ROS), mitochondrial activity, intracellular ATP production, cytoprotective signal molecules as well as DNA damage. Hypothermia decreased H₂O₂ damage in cardiomyocytes as demonstrated in a lower LDH release, less caspase-3 cleavage and less M30 CytoDeath staining. After rewarming H₂O₂ damaged cells demonstrated a significantly higher reduction rate of intracellular ROS compared to normothermic H₂O₂ damaged cardiomyocytes_. This was in line with a significantly greater mitochondrial dehydrogenase activity and higher intracellular ATP content in cooled and rewarmed cells. Moreover, hypothermia preserved cell viability by up-regulation of the anti-apoptotic protein Bcl-2 and a reduction of p53 phosphorylation. DNA damage, proven by PARP-1 cleavage and H2AX phosphorylation, was significantly reduced by hypothermia. In conclusion, we could demonstrate that hypothermia protects cardiomyocytes during oxidative stress by preventing apoptosis via inhibiting mitochondrial dysfunction and DNA damage.     

Downregulation of IDH2 exacerbates H2O2-mediated cell death and hypertrophy

Objectives: Reactive oxygen species-mediated cell death contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. We recently showed that mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) functions as an antioxidant and anti-apoptotic protein by supplying NADPH to antioxidant systems.

Methods: In the present study, we demonstrated that H₂O₂-induced apoptosis and hypertrophy of H9c2 cardiomyoblasts was markedly exacerbated by small interfering RNA (siRNA) specific for IDH2.

Results: Attenuated IDH2 expression resulted in the modulation of cellular and mitochondrial redox status, mitochondrial function, and cellular oxidative damage. MitoTEMPO, a mitochondria-targeted antioxidant, efficiently suppressed increased caspase-3 activity, increased cell size, and depletion of cellular GSH levels in IDH2 siRNA-transfected cells that were treated with H₂O₂.

Discussion: These results indicated that the disruption of cellular redox balance might be responsible for the enhanced H₂O₂-induced apoptosis and hypertrophy of cultured cardiomyocytes by the attenuated IDH2 expression.     

Constitutive heat shock protein 70 interacts with α-enolase and protects cardiomyocytes against oxidative stress

Abstract

Constitutive heat shock protein 70 (Hsc70) is a molecular chaperone that has been shown to protect cardiomyocytes against oxidative stress. However, the molecular mechanism responsible for this protection remains uncertain. To understand the mechanism associated with the myocardial protective role of Hsc70, we have embarked upon a systematic search for Hsc70-interacting proteins. Using adenosine diphosphate (ADP) affinity chromatography and mass spectrometry, we have identified α-enolase, a rate-limiting enzyme in glycolysis, as a novel Hsc70-interacting protein in the myocardium of both sham and myocardial ischemia-reperfused Sprague–Dawley rat hearts. This interaction was confirmed by co-immunoprecipitation (IP) assays in the myocardial tissues and H9c2 cardiomyocytes and protein overlay assay (POA). It was further shown that Hsc70-overexpression alleviated the H₂O₂-induced decrease of α-enolase activity and cell damage, and Hsc70 deficiency aggravated the decrease of α-enolase activity and cell damage in H₂O₂ treated H9c2 cells. Our research suggests that the protective effect of Hsc70 on the cardiomyocytes against oxidative stress is partly associated with its interaction with α-enolase.     

Transient Receptor Potential Melastatin-4 Is Involved in Hypoxia-Reoxygenation Injury in the Cardiomyocytes

Ischemic heart disease still remains the most common cause of cardiac death. During ischemia-reperfusion (I/R), reactive oxygen species (ROS) are produced in excess in cardiac tissue, where they induce cell death. Our previous study showed that 9-phenanthrol (9-Phe), a specific inhibitor of the TRPM4 channel, preserves cardiac contractile function and protects the heart from I/R injury-related infarction in the excised rat heart. Accordingly, we hypothesized that TRPM4 channels are involved in the 9-Phe-mediated cardioprotection against ROS-induced injury. In rats, intravenous 9-Phe mitigated the development of myocardial infarction caused by the occlusion of the left anterior descending artery. Immunohistochemical analysis indicated that TRPM4 proteins are expressed in ventricular myocytes susceptible to I/R injury. Hydrogen peroxide (H₂O₂) is among the main ROS overproduced during I/R. In the cardiomyocyte cell line H9c2, pretreatment with 9-Phe prevented cell death induced by conditions mimicking I/R, namely 200 μM H₂O₂ and hypoxia-reoxygenation. Gene silencing of TRPM4 preserved the viability of H9c2 cardiomyocytes exposed to 200 μM H₂O₂. These results suggest that the cardioprotective effects of 9-Phe are mediated through the inhibition of the TRPM4 channels.     

Propofol Protects Against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Oxidative Injury in Differentiated PC12 Cells via Inhibition of Ca<Superscript>2+</Superscript>-Dependent NADPH Oxidase

Propofol (2,6-diisopropylphenol) is a widely used general anesthetic with anti-oxidant activities. This study aims to investigate protective capacity of propofol against hydrogen peroxide (H₂O₂)-induced oxidative injury in neural cells and whether the anti-oxidative effects of propofol occur through a mechanism involving the modulation of NADPH oxidase (NOX) in a manner of calcium-dependent. The rat differentiated PC12 cell was subjected to H₂O₂ exposure for 24 h to mimic a neuronal in vitro model of oxidative injury. Our data demonstrated that pretreatment of PC12 cells with propofol significantly reversed the H₂O₂-induced decrease in cell viability, prevented H₂O₂-induced morphological changes, and reduced the ratio of apoptotic cells. We further found that propofol attenuated the accumulation of malondialdehyde (biomarker of oxidative stress), counteracted the overexpression of NOX core subunit gp91^phox (NOX2) as well as the NOX activity following H₂O₂ exposure in PC12 cells. In addition, blocking of L-type Ca²⁺ channels with nimodipine reduced H₂O₂-induced overexpression of NOX2 and caspase-3 activation in PC12 cells. Moreover, NOX inhibitor apocynin alone or plus propofol neither induces a significant downregulation of NOX activity nor increases cell viability compared with propofol alone in the PC12 cells exposed to H₂O₂. These results demonstrate that the protective effects of propofol against oxidative injury in PC12 cells are mediated, at least in part, through inhibition of Ca²⁺-dependent NADPH oxidase.     

HMGB1 as an autophagy sensor in oxidative stress

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, actively released following cytokine stimulation as well as passively during cell injury and death. Autophagy is a tightly regulated cellular stress pathway involving the lysosomal degradation of cytoplasmic organelles or proteins. Organisms respond to oxidative injury by orchestrating stress responses such as autophagy to prevent further damage. Recently, we reported that HMGB1 is an autophagy sensor in the presence of oxidative stress. Hydrogen peroxide (H₂O₂) and loss of superoxide dismutase 1 (SOD1)-mediated oxidative stress promotes cytosolic HMGB1 expression and extracellular release. Inhibition of HMGB1 release or loss of HMGB1 decreases the number of autolysosomes and autophagic flux in human and mouse cell lines under conditions of oxidative stress. These findings provide insight into how HMGB1, a damage associated molecular pattern (DAMP), triggers autophagy as defense mechanism under conditions of cellular stress.     

Increase of autophagy and attenuation of apoptosis by Salvigenin promote survival of SH-SY5Y cells following treatment with H2O2

Oxidative stress is a major component of harmful cascades activated in neurodegenerative disorders. Here, we tried to elucidate the possible neuroprotective effect of Salvigenin, a natural polyphenolic compound, on oxidative stress-induced apoptosis and autophagy in human neuroblastoma SH-SY5Y cells. We measured cell viability by MTT test and found that 25?μM is the best protective concentration of Salvigenin. GSH and SOD assays suggested that Salvigenin activates antioxidant factors. At the same time, measurement of ER stress-associated proteins including calpain and caspase-12 showed the ability of Salvigenin to decrease ER stress. We found that Salvigenin could decrease the apoptotic factors. Salvigenin inhibited H₂O₂-induced caspase-3 which is a hallmark of apoptosis in addition to reducing Bax\Bcl-2 ratio by 1.45 fold. Additionally, Salvigenin increased the levels of autophagic factors. Our results showed an increase in LC3-II/LC3-I ratio, Atg7, and Atg12 in the presence of 25?μM of Salvigenin by about 1.28, 1.25, and 1.54 folds, respectively, compared to H₂O₂-treated cells. So it seems that H₂O₂ cytotoxicity mainly results from apoptosis. Besides, Salvigenin helps cells to survive by inhibiting apoptosis and enhancing autophagy that opens a new horizon for the future experiments.     

Autophagy induction is a survival response against oxidative stress in bone marrow–derived mesenchymal stromal cells

Background aimsBone marrow–derived mesenchymal stromal cells (BMSCs) are being extensively investigated as cellular therapeutics for many diseases, including cardiovascular diseases. Although preclinical studies indicated that BMSC transplantation into infarcted hearts improved heart function, there are problems to be resolved, such as the low survival rate of BMSCs during the transplantation process and in the ischemic region with extreme oxidative stress. Autophagy plays pivotal roles in maintaining cellular homeostasis and defending against environmental stresses. However, the precise roles of autophagy in BMSCs under oxidative stress remain largely uncharacterized.MethodsBMSCs were treated with H₂O₂, and autophagic flux was examined by means of microtubule-associated protein 1A/1B-light chain 3 II/I ratio (LC3 II/I), autophagosome formation and p62 expression. Cytotoxicity and cell death assays were performed after co-treatment of BMSCs by autophagy inhibitor (3-methyladenine) or autophagy activator (rapamycin) together with H₂O₂.ResultsWe show that short exposure (1 h) of BMSCs to H₂O₂ dramatically elevates autophagic flux (2- to 4-fold), whereas 6-h prolonged oxidative treatment reduces autophagy but enhances caspase-3 and caspase-6–associated apoptosis. Furthermore, we show that pre- and co-treatment with rapamycin ameliorates H₂O₂-induced caspase-3 and caspase-6 activation and cell toxicity but that 3-methyladenine exacerbates H₂O₂-induced cell apoptotic cell death.ConclusionsOur results demonstrate that autophagy is critical for the survival of BMSCs under oxidative conditions. Importantly, we also suggest that the early induction of autophagic flux is possibly a self-defensive mechanism common in oxidant-tolerant cells.     

MnSOD Upregulation Induces Autophagic Programmed Cell Death in Senescent Keratinocytes

Senescence is a state of growth arrest resulting mainly from telomere attrition and oxidative stress. It ultimately leads to cell death. We have previously shown that, in keratinocytes, senescence is induced by NF-kappaB activation, MnSOD upregulation and H₂O₂ overproduction. We have also shown that senescent keratinocytes do not die by apoptosis but as a result of high macroautophagic activity that targets the primary vital cell components. Here, we investigated the mechanisms that activate this autophagic cell death program. We show that corpses occurring at the senescence plateau display oxidatively-damaged mitochondria and nucleus that colocalize with autophagic vacuoles. The occurrence of such corpses was decreased by specifically reducing the H₂O₂ level with catalase, and, conversely, reproduced by overexpressing MnSOD or applying subtoxic doses of H₂O₂. This H₂O₂-induced cell death did occur through autophagy since it was accompanied by an accumulation of autophagic vesicles as evidenced by Lysotracker staining, LC3 vesiculation and transmission electron microscopy. Most importantly, it was partly abolished by 3-methyladenine, the specific inhibitor of autophagosome formation, and by anti-Atg5 siRNAs. Taken together these results suggest that autophagic cell death is activated in senescent keratinocytes because of the upregulation of MnSOD and the resulting accumulation of oxidative damages to nucleus and mitochondria.     

Induced pluripotent stem cell‐conditional medium inhibits H9C2 cardiomyocytes apoptosis via autophagy flux and Wnt/β‐catenin pathway

Induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) could improve cell viability in many types of cells and may be a better alternative for the treatment of myocardial infarction. This study aimed to examine the influence of iPS‐CM on anti‐apoptosis and the proliferation of H9C2 cardiomyocytes and investigate the underlying mechanisms. H9C2 cardiomyocytes were exposed to 200 μmol/L hydrogen peroxide (H₂O₂) for 24 hours with or without pre‐treatment with iPS‐CM. The ratio of apoptotic cells, the loss of mitochondrial membrane potential (△Ψm) and the levels of intracellular reactive oxygen species were analysed by flow cytometric analysis. The expression levels of BCL‐2 and BAX proteins were analysed by Western blot. Cell proliferation was assessed using cell cycle and EdU staining assays. To study cell senescence, senescence‐associated β‐galactosidase (SA‐β‐gal) staining was conducted. The levels of malondialdehyde, superoxide dismutase and glutathione were also quantified using commercially available enzymatic kits. The results showed that iPS‐CM containing basic fibroblast growth factor significantly reduced H₂O₂‐induced H9C2 cardiomyocyte apoptosis by activating the autophagy flux pathway, promoted cardiomyocyte proliferation by up‐regulating the Wnt/β‐catenin pathway and inhibited oxidative stress and cell senescence. In conclusion, iPS‐CM effectively enhanced the cell viability of H9C2 cardiomyocytes and could potentially be used to inhibit cardiomyocytes apoptosis to treat myocardial infarction in the future.