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.     

Calcineurin suppresses AMPK-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress

Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H₂O₂) treatment. However, prolonged (12 h) H₂O₂ treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H₂O₂ treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.     

Effects of resveratrol on H2O2‐induced apoptosis and expression of SIRTs in H9c2 cells

Resveratrol, a polyphenol found in fruits, has been demonstrated to activate Sir2. Though many studies have demonstrated that resveratrol can activate SIRT1, whether it has effect on other sirtuins (SIRT2–7) are unknown. The present study shows that exposure of H9c2 cells to 50 µM H₂O₂ for 6 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry (FCM), but pretreatment of resveratrol (20 µM) eliminated H₂O₂‐induced apoptosis. Resveratrol also prevented H₂O₂‐induced caspase‐3 activation. Exposure of cells to resveratrol caused rapid activation of SIRT1,3,4,7. Sirtuin inhibitor, nicotinamide (20 mM) attenuated resveratrol's inhibitory effect on cell apoptosis and caspase‐3 activity. These results suggest that resveratrol protects cardiomyocytes from H₂O₂‐induced apoptosis by activating SIRT1,3,4,7. J. Cell. Biochem. 107: 741–747, 2009. © 2009 Wiley‐Liss, Inc.     

Tissue transglutaminase 2 promotes apoptosis of rat neonatal cardiomyocytes under oxidative stress

The role of tissue transglutaminase 2 (TG2) in cardiac myocyte apoptosis under oxidative stress induced by ischemic injury remains unclear. Here, we investigated the effects of TG2 on apoptosis of cardiomyocytes under oxidative stress. Ectopic expression of TG2 increased caspase-3 activity and calcium overload in cardiomyocytes. Expression levels of TG2 were significantly increased in H₂O₂-treated cardiomyocytes. Caspase-3 activity assay demonstrated its considerable correlation with TG2 expression, which supported that caspase-3 inhibitor inhibited the apoptosis induced by the ectopic overexpression of TG2. In addition, the other apoptotic signals, such as caspase-8, cytochrome c, and Bax, were increased dependent with TG2 expression in H₂O₂-treated cardiomyocytes. These results indicated that apoptotic signals had a positive correlation with TG2 expression. The decreased expression of phospholipase C (PLC)-δ1 and phospho-PKC in H₂O₂-treated cardiomyocytes were rescued by TG2 silencing. Together, our data strongly suggest that oxidative stress up-regulates TG2 expression in cardiomyocytes, leading to apoptosis.     

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.     

Acute activation of AMP-activated protein kinase prevents H2O2-induced premature senescence in primary human keratinocytes

We investigated the effects of AMPK on H₂O₂-induced premature senescence in primary human keratinocytes. Incubation with 50 µM H₂O₂ for 2 h resulted in premature senescence with characteristic increases in senescence-associated ß-galactosidase (SA-gal) staining 3 days later and no changes in AMPK or p38 MAPK activity. The increase in SA-gal staining was preceded by increases in both p53 phosphorylation (S15) (1 h) and transactivation (6 h) and the abundance of the cyclin inhibitor p21^CIP1 (16 h). Incubation with AICAR or resveratrol, both of which activated AMPK, prevented the H₂O₂-induced increases in both SA-Gal staining and p21 abundance. In addition, AICAR diminished the increase in p53 transactivation. The decreases in SA-Gal expression induced by resveratrol and AICAR were prevented by the pharmacological AMPK inhibitor Compound C, expression of a DN-AMPK or AMPK knock-down with shRNA. Likewise, both knockdown of AMPK and expression of DN-AMPK were sufficient to induce senescence, even in the absence of exogenous H₂O₂. As reported by others, we found that AMPK activation by itself increased p53 phosphorylation at S15 in embryonic fibroblasts (MEF), whereas under the same conditions it decreased p53 phosphorylation in the keratinocytes, human aortic endothelial cells, and human HT1080 fibrosarcoma cells. In conclusion, the results indicate that H₂O₂ at low concentrations causes premature senescence in human keratinocytes by activating p53-p21^CIP1 signaling and that these effects can be prevented by acute AMPK activation and enhanced by AMPK downregulation. They also suggest that this action of AMPK may be cell or context-specific.     

Protectin DX prevents H2O2-mediated oxidative stress in vascular endothelial cells via an AMPK-dependent mechanism

Protectin DX (PDX), which is a novel regulator of 5′ adenosine monophosphate-activated protein kinase (AMPK), has recently gained attention for its ability to improve several metabolic diseases. However, the function of PDX in vascular endothelial cells remains unclear. To confirm the protective effects of PDX on endothelial oxidative stress, human umbilical vein endothelial cells (HUVECs) were treated with hydroperoxide (H₂O₂) and PDX. PDX treatment significantly increased the level of AMPK phosphorylation, and this elevation was attenuated after treatment with G-protein coupled receptor 120 (GPR120) antagonist or GPR120 knockdown. Expressions and activities of antioxidant proteins, including catalase and superoxide dismutase 2 (SOD2), were elevated by PDX and were inhibited by treatment with AMPK inhibitor or with GPR120 antagonist. Production of H₂O₂-induced reactive oxygen species (ROS), the Bax/Bcl-2 ratio, and the loss of mitochondrial membrane potential were all reversed by PDX, leading to improved cell viability and reduced release of lactate dehydrogenase (LDH). Using flow cytometry, we also found that PDX significantly reduced the H₂O₂-induced apoptotic population of cells. These protective effects of PDX were all reversed after treatment with AMPK inhibitor or GRP120 antagonist. These results show that the PDX-AMPK axis has a protective role against H₂O₂-induced oxidative stress in vascular endothelial cells.     

Neuroprotective effects of corn silk maysin via inhibition of H2O2-induced apoptotic cell death in SK-N-MC cells

Aims

Neuroprotective effects of maysin, which is a flavone glycoside that was isolated from the corn silk (CS, Zea mays L.) of a Korean hybrid corn Kwangpyeongok, against oxidative stress (H₂O₂)-induced apoptotic cell death of human neuroblastoma SK-N-MC cells were investigated.

Main methods

Maysin cytotoxicity was determined by measuring cell viability using MTT and lactate dehydrogenase (LDH) assays. Intracellular reactive oxygen species (ROS) were measured using a 2,7-dichlorofluorescein diacetate (DCF-DA) assay. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by a TUNEL assay. Antioxidant enzyme mRNA levels were determined by real-time PCR. The cleavage of poly (ADP-ribose) polymerase (PARP) was measured by western blotting.

Key findings

Maysin pretreatment reduced the cytotoxic effect of H₂O₂ on SK-N-MC cells, as shown by the increase in cell viability and by reduced LDH release. Maysin pretreatment also dose-dependently reduced the intracellular ROS level and inhibited PARP cleavage. In addition, DNA damage and H₂O₂-induced apoptotic cell death were significantly attenuated by maysin pretreatment. Moreover, maysin pretreatment (5–50 μg/ml) for 2 h significantly and dose-dependently increased the mRNA levels of antioxidant enzymes (CAT, GPx-1, SOD-1, SOD-2 and HO-1) in H₂O₂ (200 μM)-insulted cells.

Significance

These results suggest that CS maysin has neuroprotective effects against oxidative stress (H₂O₂)-induced apoptotic death of human brain SK-N-MC cells through its antioxidative action. This report is the first regarding neuroprotective health benefits of corn silk maysin by its anti-apoptotic action and by triggering the expression of intracellular antioxidant enzyme systems in SK-N-MC cells.     

Inhibition of bromodomain‐containing protein 4 ameliorates oxidative stress–mediated apoptosis and cartilage matrix degeneration through activation of NF‐E2–related factor 2‐heme oxygenase‐1 signaling in rat chondrocytes

During the progression of osteoarthritis, dysregulation of extracellular matrix (ECM) anabolism, abnormal generation of reactive oxygen species, and proteolytic enzymes have been shown to accelerate the degradation process of cartilage. The purpose of the current study was to investigate the functional role of bromodomain‐containing protein 4 (BRD4) in hydrogen peroxide (H₂O₂)–stimulated chondrocyte injury and delineate the underlying molecular mechanisms. We observed that the expression BRD4 was markedly elevated in rat chondrocytes after H₂O₂ stimulation. Additionally, inhibition of BRD4 using small interfering RNA or JQ1 (a selective potent chemical inhibitor) led to repression of H₂O₂‐induced oxidative stress, as revealed by a decrease in the reactive oxygen species production accompanied by a decreased malondialdehyde content, along with increased activities of antioxidant markers superoxide dismutase, catalase, and glutathione peroxidase on exposure of chondrocytes to H₂O₂. Meanwhile, depletion of BRD4 led to repress the oxidative stress–induced apoptosis of chondrocytes triggered by H₂O₂ accompanied by an increase in the expression of anti‐apoptotic Bcl‐2 and a decrease in the expression of pro‐apoptotic Bax and caspase 3 as well as attenuated caspase 3 activity. Moreover, knockdown of BRD4 or treatment with JQ1 markedly attenuated ECM deposition, reflected in a marked upregulation of proteoglycans collagen type II and aggrecan as well as downregulation of ECM–degrading enzymes matrix metalloproteinase 13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‐5). More importantly, inhibition of BRD4‐activated NF‐E2–related factor 2 (Nrf2)–heme oxygenase‐1 signaling. Mechanistically, the protective effect of BRD4 inhibition on H₂O₂‐stimulated apoptosis and cartilage matrix degeneration was markedly abrogated by Nrf2 depletion. Altogether, we concluded that the protective effect of BRD4 inhibition against oxidative stress–mediated apoptosis and cartilage matrix degeneration occurred through Nrf2–heme oxygenase‐1 signaling, implying that BRD4 inhibition may be a more effective therapeutic strategy against osteoarthritis.     

Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells

Reactive oxygen species including H₂O₂ lead vascular endothelial cells (EC) to undergo apoptosis. Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid mediator that elicits various EC responses. We aimed to explore whether and how S1P modulates EC apoptosis induced by H₂O₂. Treatment of cultured bovine aortic EC (BAEC) with H₂O₂ (750 μM for 6 h) led to DNA fragmentation (ELISA), DNA nick formation (TUNEL staining), and cleavage of caspase-3, key features of EC apoptosis. These responses elicited by H₂O₂ were alike markedly attenuated by pretreatment with S1P (1 μM, 30 min). H₂O₂ induced robust phosphorylation of both p38 and JNK MAP kinases. However, pretreatment with S1P decreased phosphorylation of only p38 MAP kinase, but not that of JNK; conversely, an inhibitor of p38 MAP kinase, but not that of JNK, attenuated H₂O₂-induced caspase-3 activation. Thus S1P attenuates H₂O₂-induced apoptosis of cultured BAEC, involving p38 MAP kinase.     

Metformin protects PC12 cells and hippocampal neurons from H2O 2-induced oxidative damage through activation of AMPK pathway

Metformin, a first line anti type 2 diabetes drug, has recently been shown to extend lifespan in various species, and therefore, became the first antiaging drug in clinical trial. Oxidative stress due to excess reactive oxygen species (ROS) is considered to be an important factor in aging and related disease, such as Alzheimer's disease (AD). However, the antioxidative effects of metformin and its underlying mechanisms in neuronal cells is not known. In the present study, we showed that metformin, in clinically relevant concentrations, protected neuronal PC12 cells from H₂O₂-induced cell death. Metformin significantly ameliorated cell death due to H₂O₂ insult by restoring abnormal changes in nuclear morphology, intracellular ROS, lactate dehydrogenase, and mitochondrial membrane potential induced by H₂O₂. Hoechst staining assay and flow cytometry analysis revealed that metformin significantly reduced the apoptosis in PC12 cells exposed to H₂O₂. Western blot analysis further demonstrated that metformin stimulated the phosphorylation and activation of AMP-activated protein kinase (AMPK) in PC12 cells, while application of AMPK inhibitor compound C, or knockdown of the expression of AMPK by specific small interfering RNA or short hairpin RNA blocked the protective effect of metformin. Similar results were obtained in primary cultured hippocampal neurons. Taken together, these results indicated that metformin is able to protect neuronal cells from oxidative injury, at least in part, via the activation of AMPK. As metformin is comparatively cheaper with much less side effects in clinic, our findings support its potential to be a drug for prevention and treatment of aging and aging-related diseases.     

Suppression of Sox4 protects against myocardial ischemic injury by reduction of cardiac apoptosis in mice

Sox4 participates in the progression of embryo development and regulation of apoptosis in tumors. However, the effect and mechanism of Sox4 in myocardial infarction (MI) remains unclear. Therefore, we aimed at examining the role and molecular mechanism of Sox4 in the process of cardiomyocytes apoptosis during MI. The expression of Sox4 were obviously increased both in MI mice and in neonatal mouse cardiomyocytes treated with H₂O₂. Overexpression of Sox4 promoted cardiomyocyte apoptosis with or without H₂O₂, whereas knocking down of Sox4 alleviated H₂O₂‐induced apoptosis in cardiomyocytes. Furthermore, silencing Sox4 by AAV‐9 carried short hairpin RNA targeting Sox4 (AAV‐9‐sh‐Sox4) markedly decreased cardiac infarct area, imprfoved cardiac dysfunction, and reversed apoptosis in MI mice. Mechanistically, there is a potential Sox4‐binding site in the promoter region of Bim, and forced expression of Sox4 significantly promoted Bim expression in cultured cardiomyocytes with or without H₂O₂, whereas knocking down of Sox4 inhibited the expression of Bim. Further studies showed that silencing Bim attenuated Sox4‐induced apoptosis in cardiomyocytes, indicating that Sox4 promoted cardiomyocytes apoptosis through regulation of Bim expression, which can be used as a potential therapeutic target for MI.     

Pretreatment with H(2) O(2) alleviates aluminum-induced oxidative stress in wheat seedlings

Hydrogen peroxide (H₂O₂) is a key reactive oxygen species (ROS) in signal transduction pathways leading to activation of plant defenses against biotic and abiotic stresses. In this study, we investigated the effects of H₂O₂ pretreatment on aluminum (Al) induced antioxidant responses in root tips of two wheat (Triticum aestivum L.) genotypes, Yangmai‐5 (Al‐sensitive) and Jian‐864 (Al‐tolerant). Al increased accumulation of H₂O₂ and O₂^?? leading to more predominant lipid peroxidation, programmed cell death and root elongation inhibition in Yangmai‐5 than in Jian‐864. However, H₂O₂ pretreatment alleviated Al‐induced deleterious effects in both genotypes. Under Al stress, H₂O₂ pretreatment increased the activities of superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and monodehydroascorbate reductase, glutathione reductase and glutathione peroxidase as well as the levels of ascorbate and glutathione more significantly in Yangmai‐5 than in Jian‐864. Furthermore, H₂O₂ pretreatment also increased the total antioxidant capacity evaluated as the 2, 2‐diphenyl‐1‐picrylhydrazyl‐radical scavenging activity and the ferric reducing/antioxidant power more significantly in Yangmai‐5 than in Jian‐864. Therefore, we conclude that H₂O₂ pretreatment improves wheat Al acclimation during subsequent Al exposure by enhancing the antioxidant defense capacity, which prevents ROS accumulation, and that the enhancement is greater in the Al‐sensitive genotype than in the Al‐tolerant genotype.     

Mitigation of H2O2-induced autophagic cell death by propofol in H9c2 cardiomyocytes

Autophagy, a self-eating process, is responsible for degradation of long-lived proteins and damaged cellular proteins/organelles. Double-membrane autophagosomes, formed during the process, engulf proteins/organelles and fuse with lysosomes to degrade the contents. It is important to maintain cell homeostasis and many physiological processes including cellular responses to oxidative stress. Oxidative stress induced by myocardial infarction is a major factor of heart failures. In this study, we examined how propofol modulates hydrogen peroxide (H₂O₂)-induced autophagic cell death in H9c2 cardiomyocytes. H₂O₂ dramatically induced cell death, which was similarly reduced in the presence of either propofol or autophagy inhibitors (e.g., wortmannin), suggesting that propofol has a protective effect in H₂O₂-induced autophagic cell death. Acidic autophagic vacuoles were elevated in H₂O₂-treated H9c2 cells, but they were largely decreased in the presence of propofol. Furthermore, many autophagy-related proteins such as LC3-II, ATG proteins, p62, AMPK, and JNK were activated in H₂O₂-treated H9c2 cells and were significantly deactivated in the presence of propofol. These results show that propofol regulates oxidative stress-induced autophagic cell death in cardiomyocytes. We further suggest that propofol can act as a cardioprotectant in heart diseases.     

Estradiol protects adipose tissue-derived stem cells against H(2) O(2) -induced toxicity

Oxidative stress is associated with various pathophysiological processes, including cell survival, adhesion, apoptosis, and cancer. In the present study, we aimed to evaluate the effects of H₂O₂‐induced toxicity on adipose tissue–derived stem cells (ADSCs) and whether 17β‐estradiol (E₂) has protective effects on these cells. ADSCs derived from adult Sprague–Dawley rats were pretreated with different doses of E₂ for 24 h and then exposed to 200 µM H₂O₂ for 4 h. Incubation of ADSCs with H₂O₂‐decreased cell viability in a concentration‐dependent fashion (p < 0.0001), whereas pretreatment of these cells with E₂ significantly reversed toxicity (p < 0.05), inhibited apoptotic changes, and decreased lipid peroxidation (p < 0.0005). Our findings suggest that E₂ protects ADSCs from oxidative‐induced cell death, and therefore, it may be used to improve the survival rate and regenerative capacity of stem cells. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:301–307, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21421     

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.