Oxidative modulation of marcaine and lekoptin in H9C2 rat myoblasts.
Ontology highlight
ABSTRACT:
SUBMITTER: Kulbacka J
PROVIDER: S-EPMC4002470 | biostudies-other | 2009 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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 ?M H2O2 exposure for 30 min with and/or without 10 to 20 ?M 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, ?-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-3? (GSK-3?)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3? inhibitor (SB 216763) significantly improved the H2O2-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3? (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-3? mediated cardiac cell injury.
| S-EPMC4070642 | biostudies-literature
Project description:The present study aimed to evaluate the effect of the ethyl acetate fraction of P. reptans root (PEF) preconditioning on expressions of lncRNAs H19 and MIAT in H9C2 myoblasts I/R injury.H9C2 cells were treated with different concentrations ranging from (10-400 µg/ml) of PEF for 24 h, followed by simulation of I/R condition. For I/R experiments, H9C2 cells were subjected with the oxygen and glucose deprivation for 2 h.H9C2 cell viability was significantly enhanced by PEF preconditioning under I/R condition in a concentration-dependent manner up to 200 µg/ml as a EC50. The PEF significantly diminished the expression of lncRNA MIAT and rate of apoptosis against the I/R group. In addition, PEF pretreated before stimulation I/R condition increased H19 expression compared to the normal PEF group with no statistically significant differences between groups. Hence, the results suggest that PEF can protect cardiomyocytes during hypoxia-induced myocardial cell injury by targeting specific involved genes.
| S-EPMC10388489 | biostudies-literature
Project description:The method presented in this article are related to the research article entitled as "Role of the TRPM4 channel in mitochondrial function, calcium release, and ROS generation in oxidative stress" [1]. TRPM4, a non-selective monovalent cation channel, is not only involved in the generation of the action potential in cardiomyocytes, but also thought to be a key molecule in the development of the ischemia-reperfusion injury of the brain and the heart [2], [3], [4], [5]. However, existing pharmacological inhibitors for the TRPM4 channel have problems of non-specificity [6]. This article describes methods used for targeted genomic deletion in the rat cardiomyocyte H9c2 using the CRISPR-Cas9 genome editing system in order to suppress TRPM4 protein expression. Confocal microscopy, flow cytometry, Sanger sequencing, and western blotting are performed to confirm vector transfection and the subsequent knockout of the TRPM4 protein.•These data provide information on the comprehensive analyses for knocking out the rat TRPM4 channel using CRISPR/Cas9. The analyses include confocal microscopy, flow cytometry, Sanger sequencing, and western blotting.•This dataset will benefit biological and medical researchers studying the function of TRPM4-expressing cells including neurons, cardiomyocytes, and vascular endothelial cells. It is also useful to study the involvement of the TRPM4 channel in pathological processes such as cardiac arrhythmia and ischemia-reperfusion injury.•The dataset can be used to guide the experiment of knocking out the TRPM4 gene and its subsequent application to the study of disease process caused by the gene.
| S-EPMC8374525 | biostudies-literature
Project description:Trehalose, a naturally occurring non-toxic disaccharide, has attracted considerable attention for its potential in alleviating oxidative stress in skeletal muscle. In this study, our aim was to elucidate the metabolic mechanisms underlying the protective effects of trehalose against hydrogen peroxide (H2O2)-induced oxidative stress in C2C12 myoblasts. Our results show that both trehalose treatment and pretreatment effectively alleviate the H2O2-induced decrease in cell viability, reduce intracellular reactive oxygen species (ROS), and attenuate lipid peroxidation. Furthermore, using NMR-based metabolomics analysis, we observed that trehalose treatment and pretreatment modulate the metabolic profile of myoblasts, specifically regulating oxidant metabolism and amino acid metabolism, contributing to their protective effects against oxidative stress. Importantly, our results reveal that trehalose treatment and pretreatment upregulate the expression levels of P62 and Nrf2 proteins, thereby activating the Nrf2-NQO1 axis and effectively reducing oxidative stress. These significant findings highlight the potential of trehalose supplementation as a promising and effective strategy for alleviating oxidative stress in skeletal muscle and provide valuable insights into its potential therapeutic applications.
| S-EPMC10488301 | biostudies-literature
Project description:Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse? transcription PCR (RT?PCR) and quantitative real time?PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae?infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose?dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy?dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction.
| S-EPMC4592223 | biostudies-literature
Project description:Despite recent advances, the role of ROS in mediating hypertrophic and apoptotic responses in cardiac myocytes elicited by norepinephrine (NE) is rather poorly understood. We demonstrate through our experiments that H9c2 cardiac myoblasts treated with 2 µM NE (hypertrophic dose) generate DCFH-DA positive ROS only for 2h; while those treated with 100 µM NE (apoptotic dose) sustains generation for 48 h, followed by apoptosis. Though the levels of DCFH fluorescence were comparable at early time points in the two treatment sets, its quenching by DPI, catalase and MnTmPyP suggested the existence of a different repertoire of ROS. Both doses of NE also induced moderate levels of H2O2 but with different kinetics. Sustained but intermittent generation of highly reactive species detectable by HPF was seen in both treatment sets but no peroxynitrite was generated in either conditions. Sustained generation of hydroxyl radicals with no appreciable differences were noticed in both treatment sets. Nevertheless, despite similar profile of ROS generation between the two conditions, extensive DNA damage as evident from the increase in 8-OH-dG content, formation of γ-H2AX and PARP cleavage was seen only in cells treated with the higher dose of NE. We therefore conclude that hypertrophic and apoptotic doses of NE generate distinct but comparable repertoire of ROS/RNS leading to two very distinct downstream responses.
| S-EPMC4477046 | biostudies-literature
Project description:Safranal (SFR), an active ingredient extracted from saffron, exhibits a protective effect on the cardiovascular system. However, the mechanism of SFR against hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury has previously not been investigated in vitro. The aim of the present study was therefore to observe the protective effects of SFR on H/R-induced cardiomyocyte injury and to explore its mechanisms. A H/R injury model of H9c2 cardiac myoblasts was established by administering 800 µmol/l CoCl2 to H9c2 cells for 24 h and reoxygenating the cells for 4 h to induce hypoxia. H9c2 cardiac myoblasts were pretreated with SFR for 12 h to evaluate the associated protective effects. A Cell Counting Kit-8 assay was used for cell viability detection, and the expression levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), glutathione peroxidase (GSH-px), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and caspase-3, and the intracellular Ca2+ concentration were measured using the corresponding commercial kits. Levels of reactive oxygen species (ROS) in the cells were detected using 2,7-dichlorodihydrofluorescein diacetate. Flow cytometry was used to determine the degree of apoptosis and the level of mitochondrial membrane potential (MMP). Moreover, the expression levels of phosphorylated (p-)PI3K, AKT, p-AKT, glycogen synthase kinase 3β (GSK3β), p-GSK3β, Bcl-2, Bax, caspase-3 and cleaved caspase-3 were measured using western blot analysis. Results of the present study demonstrated that the H9c2 cardiac myoblasts treated with SFR exhibited significantly improved levels of viability and significantly reduced levels of ROS, compared with the H/R group. Furthermore, compared with the H/R group, SFR treatment significantly increased the MMP levels and antioxidant enzyme levels, including CAT, SOD and GSH-px; whereas the levels of CK-MB, LDH, MDA and intracellular Ca2+ concentration were significantly decreased. Moreover, the results of the present study demonstrated that SFR significantly reduced caspase-3, cleaved caspase-3 and Bax protein expression levels, but upregulated the Bcl-2 protein expression levels. SFR also increased the protein expressions of PI3K/AKT/GSK3β. In summary, the results suggested that SFR may exert a protective effect against H/R-induced cardiomyocyte injury, which occurs in connection with the inhibition of oxidative stress and apoptosis via regulation of the PI3K/AKT/GSK3β signaling pathway.
| S-EPMC8524664 | biostudies-literature
Project description:High levels of reactive oxygen species (ROS) contribute to muscle cell death in aging and disuse. We have previously found that resveratrol can reduce oxidative stress in response to aging and hindlimb unloading in rodents in vivo, but it was not known if resveratrol would protect muscle stem cells during repair or regeneration when oxidative stress is high. To test the protective role of resveratrol on muscle stem cells directly, we treated the C2C12 mouse myoblast cell line with moderate (100 ?M) or very high (1 mM) levels of H2O2 in the presence or absence of resveratrol. The p21 promoter activity declined in myoblasts in response to high ROS, and this was accompanied a greater nuclear to cytoplasmic translocation of p21 in a dose-dependent matter in myoblasts as compared to myotubes. Apoptosis, as indicated by TdT-mediated dUTP nick-end labeling, was greater in C2C12 myoblasts as compared to myotubes (P<.05) after treatment with H2O2. Caspase-9, -8 and -3 activities were elevated significantly (P<.05) in myoblasts treated with H2O2. Myoblasts were more susceptible to ROS-induced oxidative stress than myotubes. We treated C2C12 myoblasts with 50 ?M of resveratrol for periods up to 48 h to determine if myoblasts could be rescued from high-ROS-induced apoptosis by resveratrol. Resveratrol reduced the apoptotic index and significantly reduced the ROS-induced caspase-9, -8 and -3 activity in myoblasts. Furthermore, Bcl-2 and the Bax/Bcl-2 ratio were partially rescued in myoblasts by resveratrol treatment. Similarly, muscle stem cells isolated from mouse skeletal muscles showed reduced Sirt1 protein abundance with H2O2 treatment, but this could be reversed by resveratrol. Reduced apoptotic susceptibility in myoblasts as compared to myotubes to ROS is regulated, at least in part, by enhanced p21 promoter activity and nuclear p21 location in myotubes. Resveratrol confers further protection against ROS by improving Sirt1 levels and increasing antioxidant production, which reduces mitochondrial associated apoptotic signaling, and cell death in myoblasts.
| S-EPMC5694367 | biostudies-literature
Project description:MicroRNAs are a novel class of powerful endogenous regulators of gene expression. MiR-378 and miR-378* are localized in the first intron of the Ppargc1b gene that codes the transcriptional co-activator PGC-1?. The latter regulates energy expenditure as well as mitochondrial biogenesis. The miR-378:miR-378* hairpin is highly expressed in cardiac cells. To better assess their role in cardiomyocytes, we identified miR-378 and miR-378* targets via a proteomic screen. We established H9c2 cellular models of overexpression of miR-378 and miR-378* and identified a total of 86 down-regulated proteins in the presence of either one of these miRs. Functional annotation clustering showed that miR-378 and miR-378* regulate related pathways in cardiomyocytes, including energy metabolism, notably glycolysis, cytoskeleton, notably actin filaments and muscle contraction. Using bioinformatics algorithms we found that 20 proteins were predicted as direct targets of the miRs. We validated eight of these targets by quantitative RT-PCR and luciferase reporter assay. We found that miR-378 targets lactate dehydrogenase A and impacts on cell proliferation and survival whereas miR-378* targets cytoskeleton proteins actin and vimentin. Proteins involved in endoplasmic reticulum stress response such as chaperone and/or calcium buffering proteins GRP78, PPIA (cyclophilin A), calumenin, and GMMPA involved in glycosylation are repressed by these miRs. Our results show that the miR-378/378* hairpin establishes a connection among energy metabolism, cytoskeleton remodeling, and endoplasmic reticulum function through post-transcriptional regulation of key proteins involved in theses pathways.
| S-EPMC3879613 | biostudies-literature
Project description:Trazodone (TRZ) is an antidepressant drug which widely used to treat insomnia, but it has a cardiotoxic effect which considered one of the TRZ limitations. The aim of this study was to investigate the protective role of L-carnitine in rats against TRZ-induced cardiotoxicity, as well as to look into the molecular mechanisms underlying its cardioprotective effects via autophagy-mediated cell death and oxidative stress. Male albino rats were randomized into four experimental groups (n = 8): normal control, TRZ group (TRZ, 20 mg/kg/day), L-carnitine group (LC, 200 mg/kg/day), and Co-treated group (L-carnitine and TRZ). All treatments were administered via oral gavage for 4 weeks. Cardiac enzymes (AST & CK-MB) and serum cardiac troponin T(cTnI) were assessed. Oxidative stress biomarkers in heart tissue (malondialdehyde; MDA, total thiol, and catalase activity) were measured. Autophagy related-genes (ATG-5 and Beclin-1), P62, and TNF-α were quantified. AST and CK-MB and cTnI significantly (p < 0.001) were increased with enhanced autophagy as well as severe histopathological changes which were manifested as scattered chronic inflammatory cells with focal fragmentation of myocardial fibers and loss of nuclei in TRZ-treated group. However, daily administration of L-carnitine (200 mg/kg) for 28 days completely reversed TRZ-induced the increased cardiac enzymes, autophagy, and myocardial inflammatory processes to the normal values. TRZ administration might have the potential to cause cardiotoxic effects that can be treated with L-carnitine administration.
| S-EPMC9381465 | biostudies-literature