Oxidative Stress In H9c2 Cardiomyocytes Research Articles

Cardioprotective Potential of Cardiboost, a Standardized Extract from Terminalia arjuna Bark against H2O2 Induced Oxidative Stress in H9c2 Cardiomyocytes

Cardioprotective Potential of Cardiboost, a Standardized Extract from Terminalia arjuna Bark against H2O2 Induced Oxidative Stress in H9c2 Cardiomyocytes

Effect of Syzygium cumini on Oxidative Stress Induced Cardiac Cellular Anomalies.

Doxorubicin (Dox), an antineoplastic agent is used as a primary anticancerous drug against various types of cancers. However, its associated toxicity to the cardiovascular system is major. Literature has recorded the cases of mortality due to poor validation and lack of prediagnosis of Dox-induced cardiotoxicity. Therapeutic interventions using natural products having cardioprotective properties with low toxic outcomes hold therapeutic potential for future cardio-oncological therapies. Syzygium cumini (Black berry), a traditional Indian herbal plant, has been researched and found to exert cardioprotective, anti-inflammatory, and antioxidant activities, which have been credited due to the presence of polyphenols, flavonoids, and tannins. In the current research, we investigated the cardioprotective potential of Syzygium cumini against Doxorubicin-induced cardiotoxicity (DIC) in H9C2 cardiomyocytes. Methanolic seed extract preparation of Syzygium cumini was performed using the Soxhlet apparatus. Cell viability and cell death assays were performed to determine the cardiotoxic doses of Doxorubicin. Furthermore, the cardioprotective potential of Syzygium cumini extract against DIC was studied. Morphological and nuclear alterations in H9C2 cells were studied by microscopic assays using Giemsa, Haematoxylin-Eosin stain, and PI. The intracellular stress level and ROS production were studied using DCFH-DA followed by mitochondrial integrity analysis using fluorescent microscopic methods. In the results, we investigated that Dox exerted a dose and time-dependent cardiotoxicity on H9C2 cardiomyocytes. Moreover, we observed that morphological and nuclear alterations caused by doxorubicin in dose-dependent manner were prevented by supplementing with Syzygium cumini polyphenols and it attenuated the oxidative stress in H9C2 cardiomyocytes effectively. Conclusively, Syzygium cumini possesses cardioprotective potential in H9C2 cardiomyocytes in dox-induced cardiotoxicity.

Mitochondria-targeting EGCG derivatives protect H9c2 cardiomyocytes from H2O2-induced apoptosis: design, synthesis and biological evaluation.

Pathologies related to cardiovascular diseases mostly emerge as a result of oxidative stress buildup in cardiomyocytes. The heavy load of mitochondrial oxidative phosphorylation in cardiac tissues corresponds to a surge in oxidative stress leading to mitochondrial dysfunction and cellular apoptosis. Thus, scavenging the reactive oxygen species (ROS) linked to mitochondria can significantly improve cardio-protection. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea has been extensively studied for its profound health-beneficial activities. Herein, we designed and synthesized a series of mitochondrial-targeting EGCG derivatives, namely MitoEGCGn (n = 4, 6, 8) by incorporating triphenylphosphonium ion onto it using different linkers. MitoEGCGn were found to be non-toxic to H9c2 rat cardiomyocyte cells even at higher doses in comparison to its parent molecule EGCG. Interestingly, MitoEGCG4 and MitoEGCG6 protected the H9c2 cardiomyocyte cells from the oxidative damage induced by H2O2 whereas EGCG was found to be toxic and ineffective in protecting the cells from H2O2 damage. MitoEGCG4 and MitoEGCG6 also protected the cells from the H2O2-induced disruption of mitochondrial membrane potential as well as activation of apoptosis as revealed by pro-caspase 3 expression profile, DNA fragmentation assay, and AO/EtBr staining. Taken together, our study shows that the mitochondria targeting EGCG derivatives were able to effectively combat the H2O2-induced oxidative stress in H9c2 cardiomyocytes. They eventually augmented the mitochondrial health of cardiomyocytes by maintaining the mitochondrial function and attenuating apoptosis. Overall, MitoEGCG4 and MitoEGCG6 could provision a cardioprotective role to H9c2 cardiomyocytes at the time of oxidative insults related to mitochondrial dysfunction-associated injuries.

Forsythiaside Protected H9c2 Cardiomyocytes from H2O2-Induced Oxidative Stress and Apoptosis via Activating Nrf2/HO-1 Signaling Pathway.

Forsythiaside, one of the main bioactive components of Chinese medicine Lian Qiao, exerts antioxidant, anti-bacterial, and anti-inflammatory effects. To date, the mechanism of Forsythiaside in cardiomyocyte injury remains unclear. However, the antioxidant effects of Forsythiaside on cardiac cells are currently unknown. This study investigated the effect and mechanism of Forsythiaside on oxidative stress in H9c2 cardiomyocytes. H9c2 cells were treated with H2O2 and Forsythiaside and then transfected with small-interfering RNA against nuclear factor erythroid 2-related factor 2 (siNrf2). Cell viability, apoptosis, accumulation of reactive oxygen species (ROS), and mitochondrial membrane potential were measured using methyl thiazolyl tetrazolium (MTT), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123, respectively. The levels of oxidative stress-related markers were determined using their respective detection kits. Furthermore, the levels of apoptosis- and Nrf2 pathway-related molecules were determined via Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Forsythiaside had no obvious toxicity on H9c2 cells. H2O2 suppressed the viability, and reduced the levels of mitochondrial membrane potential, B-cell lymphoma-2 (Bcl-2), glutathione peroxidase (GSH-Px) and catalase (CAT) and superoxide dismutase (SOD), while promoted apoptosis, ROS accumulation, and elevated the levels of cleaved caspase 3, BCL2-Associated X (Bax) and malondialdehyde (MDA) in H9c2 cells. Contrarily, Forsythiaside reversed the aforementioned effects. H2O2 advanced the levels of cytoplasm Nrf2, heme oxygenase-1 (HO-1), and nucleus Nrf2 in H9c2 cells, whereas Forsythiaside enhanced these effects. SiNrf2 reversed the functions of H2O2 or Forsythiaside in cell viability, MDA, SOD, GSH-Px, CAT, Nrf2, and HO-1 in H9c2 cells, whereas Forsythiaside reversed the aforementioned effects of siNrf2. In sum, Forsythiaside protected H9c2 cells from oxidative stress and apoptosis induced by H2O2 by activating the Nrf2/HO-1 pathway.

Effects of protein disulfide isomerase on hyperglycemia and hypoxia/reoxygenation injury in H9c2 cardiomyocytes

Objective: To explore the effect of protein disulfide isomerase (PDI) in diabetic ischemic heart disease. Methods: We established an in vitro model of high glucose and hypoxia/reoxygenation in H9c2 rat myocardial cells. Cultured cells were divided into four groups: Control, high glucose (HG), hypoxia/reoxygenation (H/R) and HG+H/R. Changes in PDI expression mediated by PDI adenovirus(Ad-PDI) infection and siRNA(PDI-siRNA) transfection in myocardial cells were observed by inverted fluorescence microscopy. We also measured lactate dehydrogenase(LDH) activity and malondialdehyde(MDA) and high molecular weight(HMW)-APN concentrations. PDI, APN, cleaved caspase-3, and glucose regulated protein 78 (Grp78) protein expression were detected. Results: PDI expression was significantly decreased in the HG, H/R and HG+H/R groups compared to the Control group; however, LDH activity[(179.7±10.4) U/L、(218.4±18.4) U/L、(328.2±5.3) U/L vs (91.0±11.0) U/L], MDA concentration[(7.0±0.4) μmol/L、(10.0±1.0) μmol/L、(11.7±1.0) μmol/L vs (4.2±1.8) μmol/L], cleaved caspase-3, and Grp78 expression were increased. Interestingly, APN and HMW-APN expression were decreased [(2.01±0.21) μg/L、(1.64±0.27) μg/L、(1.20±0.14) μg/L vs (2.62±0.12) μg/L, all P<0.05]. Over expression of PDI attenuated high glucose and hypoxia/reoxygenation induced apoptosis and oxidative stress in H9c2 cardiomyocytes(all P<0.05), and simultaneously increased APN and HMW-APN expression [(2.86±0.03) μg/L vs (3.03±0.10) μg/L、(2.06±0.05) μg/L vs (2.31±0.06) μg/L、(1.83±0.07) μg/L vs (1.96±0.11) μg/L、(1.20±0.06) μg/L vs (1.39±0.09) μg/L]. PDI-siRNA transfection increased LDH activity, MDA concentration, and cleaved caspase-3 and Grp78 expression, and decreased APN and HMW-APN expression [(0.75±0.09) μg/L vs (0.59±0.09) μg/L、(0.62±0.04) μg/L vs (0.53±0.05) μg/L、(0.55±0.14) μg/L vs (0.51±0.12) μg/L、(0.48±0.12) μg/L vs (0.35±0.08) μg/L] in response to different treatments in cultured H9c2 cardiomyocytes (all P<0.05). Conclusion: PDI may regulate the expression of APN and HMW-APN, and play an important role in the function of diabetic ischemia-reperfusion cardiomyocytes.

Muscone alleviates myocardial ischemia-reperfusion injury via inhibition of oxidative stress and enhancement of SIRT3.

This study aimed at probing into the function of muscone in ameliorating myocardial ischemiareperfusion (I/R) injury and exploring the underlying mechanism. To analyze the function of muscone, H9c2 cardiomyocytes were treated with hypoxia/reoxygenation (H/R) and Sprague-Dawley (SD) rats were treated with left anterior descending (LAD) of the coronary artery ligation for 30 min and reperfusion for 2 h to induce myocardial I/R injury. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of SIRT3. MTT assay and TUNEL assay were performed to investigate H9c2 viability and apoptosis, respectively. ELISA was employed to determine the expressions of inflammatory cytokines TNF-α, IL-6 and IL-1β, and myocardial injury markers CK and LDH. Oxidative stress markers MDA and SOD, and ROS expression levels were also detected. SIRT3 inhibitor 3-TYP was used to further confirm whether muscone worked via the augmentation of SIRT3. Herein, we found that muscone significantly inhibited inflammation and oxidative stress in H9c2 cardiomyocytes in a dose-dependent manner. H9c2 viability was promoted by muscone while apoptosis was inhibited. In SD rats, pre-treatment of muscone alleviated I/R injury-induced cardiac function dysregulation and left ventricle remolding. Furthermore, muscone increased SIRT3 expression at both mRNA and protein levels. With 3-TYP inhibiting SIRT3, the protective effects of muscone in H9c2 cardiomyocytes and SD rats were all significantly alleviated. In summary, muscone can attenuate inflammation, oxidative stress and cardiomyocytes injury in H9c2 cells treated with H/R and alleviate myocardial I/R injury of SD rats, which are dependent on SIRT3.

Mitochondrial Translocation of DJ-1 Is Mediated by Grp75: Implication in Cardioprotection of Resveratrol Against Hypoxia/Reoxygenation-Induced Oxidative Stress.

Resveratrol (Res) was recently reported to ameliorate hypoxia/reoxygenation (H/R)-caused oxidative stress in H9c2 cardiomyocytes through promoting the mitochondrial translocation of DJ-1 protein and subsequently preserving the activity of mitochondrial complex I. However, it is noteworthy that DJ-1 possesses no mitochondria-targeting sequence. Therefore, how Res induces DJ-1 mitochondrial translocation is an important and interesting question for further exploration. Glucose-regulated protein 75 (Grp75), whose N-terminus contains a 51-amino acid long mitochondrial-targeting signal peptide, is a cytoprotective chaperone that partakes in mitochondrial import of several proteins. Here, the contribution of Grp75 to mitochondrial import of DJ-1 by Res was investigated in a cellular model of H/R. Our results showed that Res upregulated the expression of DJ-1 protein, enhanced the interaction of DJ-1 and Grp75, and promoted DJ-1 translocation to mitochondria from cytosol in H9c2 cardiomyocytes undergoing H/R. Importantly, knockdown of Grp75 markedly reduced the interaction of DJ-1 with Grp75 and subsequent DJ-1 mitochondrial translocation induced by Res. Furthermore, Res pretreatment promoted the association of DJ-1 with ND1 and NDUFA4 subunits of complex I, preserved the activity of complex I, decreased mitochondria-derived reactive oxygen species production, and eventually ameliorated H/R-caused oxidative stress damage. Intriguingly, these effects were largely prevented also by small interfering RNA targeting Grp75. Overall, these results suggested that Grp75 interacts with DJ-1 to facilitate its translocation from cytosol to mitochondria, which is required for Res-mediated preservation of mitochondria complex I and cardioprotection from H/R-caused oxidative stress injury.

Prokineticin 2 relieves hypoxia/reoxygenation-induced injury through activation of Akt/mTOR pathway in H9c2 cardiomyocytes

Prokineticin 2 (PK2) was reported to be decreased in the hearts of end-state heart failure patients. Our study aimed to explore the effects of PK2 on hypoxia/reoxygenation (H/R) injury and the underlying mechanism. H9c2 cardiomyocytes were treated with 5 nM PK2 in the presence or absence of 5 mM dual phosphatidylinositol 3-kinase (PI3K)/the mammalian target of rapamycin (mTOR) inhibitor (BEZ235) for 24 h and then subjected to H/R treatment. Cell viability and lactate dehydrogenase (LDH) release were evaluated by CCK-8 and LDH release assays, respectively. Apoptosis was determined by flow cytometry analysis. Oxidative stress was assessed by measuring superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content. Results showed that H/R treatment decreased PK2 expression and inactivated the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 treatment activated the Akt/mTOR pathway in H/R-exposed H9c2 cardiomyocytes. H/R stimulation suppressed cell viability, increased LDH release, induced apoptosis and oxidative stress in H9c2 cardiomyocytes, while these effects were neutralised by treatment with PK2. However, the inhibitory effects of PK2 on H/R-induced injury in H9c2 cardiomyocytes were abolished by the addition of BEZ235. In conclusion, PK2 relieved H/R-induced injury in H9c2 cardiomyocytes by activation of the Akt/mTOR pathway.

Modulation of NOX4 and MAPK Signaling Pathways by Parkia speciosa Empty Pods in H9c2 Cardiomyocytes Exposed to H2O2

Parkia speciosa, a plant that grows abundantly in Southeast Asia region, has been reported to exhibit antioxidant property. This study aimed to investigate the mechanism of antioxidant effects of empty pod extract of Parkia speciosa on hydrogen peroxide-induced oxidative stress in H9c2 cardiomyocytes. There were 4 groups of cardiomyocytes, the negative control, hydrogen peroxide, extract of Parkia speciosa+hydrogen peroxide and quercetin+hydrogen peroxide. Cardiomyocytes of groups 3 and 4 were preincubated for 1 h with 500 µg/ml of the ethyl acetate fraction of the Parkia speciosa extract or 1000 µM quercetin prior to exposure to 500 μM hydrogen peroxide for 1 h. Hydrogen peroxide significantly increased the protein expression of NADPH oxidase 4, superoxide dismutase 1 and p38 mitogen-activated protein kinase, in comparison to the negative control. NADPH oxidase 4 and superoxide dismutase 1 activities as well as reactive oxygen species level were also elevated. These elevations were significantly decreased in the pretreated groups. The Parkia speciosa extract group had comparable effects to that of quercetin. Parkia speciosa extract showed antioxidant property against hydrogen peroxide-induced oxidative stress in H9c2 cardiomyocytes by regulating mitogen-activated protein kinase and NADPH oxidase 4 signaling pathways.

Cucurbita argyrosperma seed extract ameliorating oxidative stress in H9c2 cardiomyocytes through suppression of intracellular reactive oxygen species production

Background: Oxidative stress induced by the excessive reactive oxygen species (ROS) generation is involved in the pathogenesis of various diseases. Methanol extract from Cucurbita argyrosperma (CM) was used to test for antioxidant and antioxidative stress. Materials and Methods: The radical scavenging potential was determined using eight different in vitro assays: 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical, β-carotene-linoleic acid, chelating activity, ferric reducing antioxidant power (FRAP), hydroxyl radical, nitric oxide (NO) radical, and superoxide anion radicals, palmitate (PA) on H9c2 cells, and H2O2-induced cell damage on H9c2 cardiomyocytes. Results: CM showed antioxidant activity with DPPH radical values of EC507.6 ± 5.38 mg/mL, β-carotene-linoleic acid EC50 of 279.4 ± 36.32 mg/ml, chelating activity EC50 of 301.2 ± 9.78 mg/ml, FRAP EC50 of 101.3 ± 3.10 mg/ml, hydroxyl EC50 of 42.6 ± 8.6 mg/mL, superoxide EC50 of 50.6 ± 8.66 mg/ml, NO EC50 of 101.2 ± 19.74 mg/ml, and ABTS EC50 of 13.2 ± 4.9 mg/ml. The methanol extract preincubation decreases the intracellular ROS induced by H2O2 and PA in H9c2 cells in a concentration-dependent manner. The methanol extract could also protect H9c2 cells and inhibit the activity of xanthine oxidase, malondialdehyde, and lactate dehydrogenase which increases cell viability, heme oxygenase-1, glutathione/oxidized glutathione, superoxide dismutase-1, catalase, and GSH-Px? levels. Conclusions: The finding suggests that methanol extract can protect the oxidative stress-induced cardiomyocyte damage through ROS regulation and can be used as a therapeutic agent for the improvement of oxidative stress in various diseases. Abbreviations used: ABTS radical: 2,2'-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; CAT: Catalase; DCFH-DA: 2',7'-dichlorodihydrofluorescein diacetate; DMSO: Dimethyl sulfoxide; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DPPH: 1,1-diphenyl-2-picrylhydrazyl; DCFH-DA: 2',7'-dichlorofluorescin diacetate; FRAP: Ferric reducing antioxidant power; GSH: Glutathione; GSH-Px: Glutathione peroxidase; HO-1: Heme oxygenase-1; OH−: Hydroxyl; LDH: Lactate dehydrogenase; LOOH: Lipid peroxide; LOO•: Lipid peroxyl; MDA: Malondialdehyde; NO: Nitric oxide; GSSG: Oxidized glutathione; O2−1: Oxygen; O3 Ozone; PA: Palmitate; RO2−: Peroxyl; PBS: Phosphate-buffered saline; ROS: Reactive oxygen species; O2•: Superoxide; SOD: Superoxide dismutase; TFs: Total flavonoids; TP: Total phenolic; TCA: Trichloroacetic acid; XO: Xanthine oxidase.

Luteolin protects against diabetic cardiomyopathy by inhibiting NF-κB-mediated inflammation and activating the Nrf2-mediated antioxidant responses

BackgroundDiabetes mellitus is a well-known risk factor for the development of heart failure. Inflammation and oxidative stress play a key role in the development of diabetic cardiomyopathy (DCM), and this nexus represents an attractive target to combat this disease. Naturally occurring flavonoid luteolin exhibits both anti-inflammatory and antioxidant activities in various systems. Hypothesis/PurposeIn this study, we aimed to investigate potential cardioprotective effects of luteolin in cultured cardiomyocytes and in mice with type 1 diabetes. MethodsC57BL/6 mice were intraperitoneal injection of streptozotocin (STZ) to induce DCM. High glucose (HG) was used to induce H9C2 cells injury in vitro. Cardiac fibrosis, hypertrophy, inflammation and oxidative stress were studied both in vitro and in vivo. ResultsOur studies show that luteolin significantly reduces HG-induced inflammatory phenotype and oxidative stress in H9C2 cardiomyocytes. We found that the mechanisms involved inhibition of nuclear factor-kappa B (NF-κB) pathway and the activation of antioxidant nuclear factor-erythroid 2 related factor 2 (Nrf2) signaling pathway. Modulation of these pathways resulted in reduced expression of matrix proteins and cellular hypertrophy. Luteolin also prevented cardiac fibrosis, hypertrophy, and dysfunction in STZ-induced diabetic mice. These readouts were also associated with reduced levels of inflammatory cytokines and oxidative stress biomarkers. ConclusionOur results indicate that luteolin protects heart tissues in STZ-induced diabetic mice through modulating Nrf2-mediated oxidative stress and NF-κB-mediated inflammatory responses. These findings suggest that luteolin may be a potential therapeutic agent for DCM.

Isorhamnetin protects against hypoxia/reoxygenation-induced injure by attenuating apoptosis and oxidative stress in H9c2 cardiomyocytes

To unveil the possible protective role of isorhamnetin, an immediate 3′-O-methylated metabolite of quercetin, in cardiomyocyte under hypoxia/reoxygenation (H/R) condition and the underlying mechanisms involved, H9c2 cardiomyocytes were exposed to the vehicle or H/R for 6 h (2 h of hypoxia following by 4 h of reoxygenation) with isorhamnetin (0, 3, 6, 12, 25, 50 μM for 4 h prior to H/R exposure). Apoptosis was evaluated by TUNEL staining, flow cytometry analysis and western blot assay for cleaved caspase-3. Myocardial injure in vivo was determined by infarct size using TTC staining, histological damage using H&E staining and myocardial apoptosis. Here, we found that isorhamnetin dose-dependently protected H9c2 cardiomyocytes against H/R-induced injure, as evidenced by the reduction in lactate dehydrogenase (LDH) levels, increases in cell viability, superoxide dismutase (SOD) and catalase (CAT) activity, with the maximal effects at 25 μΜ. In addition, isorhamnetin treatment significantly inhibited apoptosis in H/R-induced H9c2 cardiomyocytes and ameliorated H/R-induced myocardial injure in vivo, concomitant with the upregulation of sirtuin 1 (SIRT1) expression. Mechanism studies demonstrated that isorhamnetin pretreatment remarkably abolished H/R-induced downregulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expressions and upregulation of NADPH oxidase-2/4 (NOX-2/4) expressions in cardiomyocytes. However, SIRT1 inhibition (Sirtinol) not only inhibited isorhamnetin-induced Nrf2/HO-1 upregulation and NOX-2/4 downregulation, but also alleviated its anti-apoptotic effects. Taken together, these data indicate that isorhamnetin can exhibit positive effect on H/R-induced injure by attenuating apoptosis and oxidative stress in H9c2 cardiomyocytes, which is partly attributable to the upregulation of SIRT1 and Nrf2/HO-1-mediated antioxidant signaling pathway.

Engineering Bioinspired Antioxidant Materials Promoting Cardiomyocyte Functionality and Maturation for Tissue Engineering Application.

Oxidative stress plays an important role in various pathological conditions, such as wound healing, inflammation, myocardial infarction, and biocompatibility of the materials. Antioxidant polymers to attenuate oxidative stress is an emerging field of biomaterial research with a huge impact in the field of tissue engineering and regenerative medicine. We describe here the fabrication and evaluation of an elastomeric antioxidant polyurethane (PUAO) for tissue engineering applications. Uniaxial and cyclic tensile testing, thermal analysis, degradation, cytotoxicity and antioxidant analysis was carried out. An in vitro oxidative stress model demonstrated that PUAO reduced intracellular oxidative stress in H9C2 cardiomyocytes (p < 0.05) and attenuated reactive oxygen species (ROS) induced cell death (p < 0.001). Under simulated ischemic reperfusion, PUAO could rescue hypoxia induced cell death. Further as a proof of concept, neonatal rat cardiomyocytes cultured on PUAO film displayed synchronous beating with mature phenotype showing expression of cardiac specific α-actinin, troponin-T, and connexin-43 proteins. Intracellular calcium transients established the functionality of cultured cardiomyocytes on PUAO film. Our study demonstrated the potential of this biomaterial to be developed into tissue engineered scaffold to attenuate oxidative stress for treatment of diseased conditions with increased oxidative stress, such as cardiovascular diseases, chronic wound healing, and myocardial infarction.

L-ascorbic acid and α-tocopherol attenuate arsenic trioxide-induced toxicity in H9c2 cardiomyocytes by the activation of Nrf2 and Bcl2 transcription factors

Arsenic trioxide (As2O3) is a potent drug for the treatment of acute promyelocytic leukemia (APL) and has achieved remarkable remissions in patients. Unfortunately, clinical reports have shown that the treatment is associated with cardiotoxicity. Many efforts have been made to mitigate drug-mediated cardiac damage using naturally occurring antioxidant compounds possessing free radical scavenging activity. The present investigation aims to explore protective role of L-ascorbic acid (L-AA) and α-tocopherol (α-TOC) from As2O3-induced oxidative stress in H9c2 cardiomyocytes through the evaluation of Nrf2 (nuclear factor erythroid 2-related factor 2) and Bcl-2 (B-cell lymphoma 2) transcription factors. The in vitro study was conducted using H9c2 cardiomyocytes. The evaluation of total antioxidant capacity, mitochondrial membrane potential, cellular calcium concentration and reactive oxygen species generation was performed. Oxidative stress (Nrf2) and anti-apoptotic (Bcl2) signaling indicators were measured by reverse transcriptase polymerase chain reaction. A depletion of the total antioxidant capacity and mitochondrial transmembrane potential were observed in As2O3-treated cardiomyocytes. In addition, the cellular calcium concentration and ROS generation were found to be increased on treatment with As2O3 with the alterations in the activity of transcription factors, Nrf2 and Bcl2. Co-treatment of antioxidant vitamins with As2O3 resulted in a significant reversal of oxidative stress and alteration on the antioxidant defense through the activation of Nrf2 and Bcl2. L-AA and α-TOC alleviates As2O3-induced oxidative stress in cardiac cells by activating Nrf2 and Bcl2 transcription factors that results in increased cell survival and prevents apoptosis.

Microvesicles derived from hypoxia/reoxygenation-treated human umbilical vein endothelial cells promote apoptosis and oxidative stress in H9c2 cardiomyocytes.

BackgroundVascular endothelial dysfunction is the closely related determinant of ischemic heart disease (IHD). Endothelial dysfunction and ischemia/reperfusion injury (IRI) have been associated with an increase in microvesicles (MVs) in vivo. However, the potential contribution of endothelial microvesicles (EMVs) to myocardial damage is unclear. Here we aimed to investigate the role of EMVs derived from hypoxia/reoxygenation (H/R) -treated human umbilical vein endothelial cells (HUVECs) on cultured H9c2 cardiomyocytes.ResultsH/R injury model was established to induce HUVECs to release H/R-EMVs. The H/R-EMVs from HUVECs were isolated from the conditioned culture medium and characterized. H9c2 cardiomyocytes were then incubated with 10, 30, 60 μg/mL H/R-EMVs for 6 h. We found that H9c2 cells treated by H/R-EMVs exhibited reduced cell viability, increased cell apoptosis and reactive oxygen species (ROS) production. Moreover mechanism studies demonstrated that H/R-EMVs could induce the phosphorylation of p38 and JNK1/2 in H9c2 cells in a dose-dependent manner. In addition, H/R-EMVs contained significantly higher level of ROS than EMVs generated from untreated HUVECs, which might be a direct source to trigger a cascade of myocardial damage.ConclusionWe showed that EMVs released during H/R injury are pro-apoptotic, pro-oxidative and directly pathogenic to cardiomyocytes in vitro. EMVs carry ROS and they may impair myocardium by promoting apoptosis and oxidative stress. These findings provide new insights into the pathogenesis of IRI.

The Protective Role of the TOPK/PBK Pathway in Myocardial Ischemia/Reperfusion and H₂O₂-Induced Injury in H9C2 Cardiomyocytes.

T-LAK-cell-originated protein kinase (TOPK) is a PDZ-binding kinase (PBK) that was recently identified as a novel member of the mitogen-activated protein kinase (MAPK) family. It has been shown to play an important role in many cellular functions. However, its role in cardiac function remains unclear. Thus, we have herein explored the biological function of TOPK in myocardial ischemia/reperfusion (I/R) and oxidative stress injury in H9C2 cardiomyocytes. I/R and ischemic preconditioning (IPC) were induced in rats by 3-hour reperfusion after 30-min occlusion of the left anterior descending coronary artery and by 3 cycles of 5-min I/R. Hydrogen peroxide (H2O2) was used to induce oxidative stress in H9C2 cardiomyocytes. TOPK expression was analyzed by western blotting, RT-PCR, immunohistochemical staining, and immunofluorescence imaging studies. The effects of TOPK gene overexpression and its inhibition via its inhibitor HI-TOPK-032 on cell viability and Bcl-2, Bax, ERK1/2, and p-ERK1/2 protein expression were analyzed by MTS assay and western blotting, respectively. The results showed that IPC alleviated myocardial I/R injury and induced TOPK activation. Furthermore, H2O2 induced TOPK phosphorylation in a time-dependent manner. Interestingly, TOPK inhibition aggravated the H2O2-induced oxidative stress injury in myocardiocytes, whereas overexpression relieved it. In addition, the ERK pathway was positively regulated by TOPK signaling. In conclusion, our results indicate that TOPK might mediate a novel survival signal in myocardial I/R, and that its effect on anti-oxidative stress involves the ERK signaling pathway.

Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes

Oxidative stress plays a great role in the pathogenesis of heart failure (HF). Oxidative stress results in apoptosis, which can cause the damage of cardiomyocytes. Hydrogen sulfide (H2S), the third gasotransmitter, is a good reactive oxygen species (ROS) scavenger, which has protective effect against HF. Sirtuin-1 (SIRT1) is a highly conserved nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase that plays a critical role in promoting cell survival under oxidative stress. The purpose of this article is to investigate the interaction between H2S and SIRT1 under oxidative stress in H9c2 cardiomyocytes. Oxidative stress was induced by hydrogen peroxide (H2O2). Treatment with NaSH (25–100 µmol/L) dose-dependently increased the cell viability and improved the cell apoptosis induced by H2O2 in H9c2 cardiomyocytes. The protective effect of NaSH against the apoptosis could be attenuated by SIRT1 inhibitor Ex 527 (10 µmol/L). Treatment with NaSH (100 µmol/L) could increase the expression of SIRT1 in time dependent manner, which decreased by different concentration of H2O2. NaSH (100 µmol/L) increased the cellular ATP level and the expression of ATPase. These effects were attenuated by Ex 527 (10 µmol/L). After NaSH (100 µmol/L) treatment, the decrease in ROS production and the enhancement in SOD, GPx and GST expression were observed. Ex 527 (10 µmol/L) reversed these effects. In conclusion, for the first time, this article can identify antioxidative effects of H2S under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes.

L-Ascorbic acid and α-Tocopherol to protect against arsenic trioxide induced oxidative stress in H9c2 cardiomyocytes

Arsenic has been used as a therapeutic agent against various diseases right from the historical periods. Arsenic trioxide (As2O3), a first line chemotherapeutic against Acute Promyelocytic Leukemia (APL), is well known for its cardiotoxicity. We hypothesized that combination of drug and protective molecules can be used for reducing the side effects of chemotherapeutics. The present study evaluates the protective role of L- ascorbic acid (L-AA) and α-Tocopherol (α-TOC) against oxidative stress in H9c2 cardiomyocytes induced by As2O3. Clinically relevant concentration of arsenic trioxide induces cytotoxicity in cardiac cells. Treatment with antioxidant vitamins significantly reduced the cell death and apoptosis in H9c2 cells. The activity of antioxidant enzymes such as catalase and the concentration of reduced glutathione, which was lowered due to As 2O3 treatment, were found to be increased in cells co treated with L-AA and α-TOC. The treatment with antioxidant vitamins also reduced lactate dehydrogenase release in cells. The overall results clearly indicate the protective potential of L-AA and α-TOC against arsenic induced chemotherapeutic stress on cardiomyocytes. Keywords: Arsenic trioxide, Cardiotoxicity, H9c2 cardiomyocytes, L-Ascorbic acid, Oxidative stress, α- Tocopherol