H9c2 Cardiac Muscle Cells Research Articles

Inhibition of mitochondrial reactive oxygen species reduces high glucose-induced pyroptosis and ferroptosis in H9C2 cardiac myocytes

To observe the effect of inhibiting mitochondrial oxidative stress and NLRP3 inflammasomes on high glucose (HG)-induced pyroptosis and ferroptosis in H9C2 cardiac muscle cells and explore the possible interactions between mitochondrial reactive oxygen species (ROS) and inflammasomes. H9C2 cells exposed to high glucose (35 mmol/L) were treated with the mitochondrial antioxidant mitoquinone (MitoQ), the NLRP3 antagonist MCC950, or both MCC950 and rotenone (a mitochondrial electron transport antagonist), and the cell viability was measured with CCK-8 assay. The cellular and mitochondrial ROS levels were measured using CellRox and Mitosox fluorescent probes, respectively. The cellular NLRP3 inflammasome level was detected with immunofluorescence assay, and the expressions of the key proteins related with pyroptosis and ferroptosis were determined with Western blotting. HG exposure significantly lowered the viability of H9C2 cells (P < 0.01), reduced the expression of GPX4 protein (a key protein related with ferroptosis) (P < 0.01), and increased the fluorescence intensities of NLRP3 (P < 0.01) and ROS (at both the cellular and mitochondrial levels, P < 0.01) and the protein expressions of NLRP3 and GSDMD-NT (P < 0.01). Treatment with either MitoQ or MCC950 significantly increased the viability of HG-exposed cells (P < 0.01), increased GPX4 expression (P < 0.01), and reduced the fluorescence intensities of NLRP3 (P < 0.01) and cellular and mitochondrial ROS (P < 0.01) and the protein expressions of NLRP3 and GSDMD-NT (P < 0.05). Compared with MCC950 treatment, treatment with both MCC950 and rotenone significantly reduced the viability of HG-exposed cells (P < 0.01), lowered GPX4 expression (P < 0.01), and increased the fluorescence intensities of ROS and NLRP3 (P < 0.01) and the protein levels of NLRP3 and GSDMD-NT (P < 0.05). MitoQ inhibits mitochondrial ROS production to reduce HGinduced NLRP3 inflammasome activation and thus suppress pyroptosis and ferroptosis of cardiac muscle cells. There may be an interaction between mitochondrial ROS and NLRP3 inflammasomes.

Protective Effect of Two Alkaloids from Hippophae rhamnoides Linn. against Doxorubicin-Induced Toxicity in H9c2 Cardiomyoblasts.

Background: Doxorubicin (Dox) is one of the most frequently prescribed anti-cancer drugs. However, clinical application with Dox is limited due to its potentially fatal cumulative cardiotoxicity. N-p-coumaroyl-4-aminobutan-1-ol (alk-A), an organic amide alkaloid and hippophamide (alk-B), a rare pyridoindole alkaloid were successfully obtained by purification and separation of seabuckthorn seed residue in our previous research. This study was undertaken to investigate the protective effect of alk-A and alk-B against Dox-induced embryonic rat cardiac cells (H9c2 cells) apoptosis. Methods: H9c2 cells were treated with Dox (2.5 µM) in the presence of alk-A and alk-B (10, 20, and 40 µM) and incubated for 24 h. Results: It was shown that pretreatment of the H9c2 cells with alk-A and alk-B significantly reduced Dox-induced apoptosis. Alk-A and alk-B both inhibited reactive oxygen species (ROS) production and suppressed cleaved-caspase-3 protein expression and the activation of JNK (Jun N-terminal kinases), as well as increasing ATP levels, favoring mitochondrial mitofusin protein expression, and relieving damage to mitochondrial DNA. Conclusions: These results suggest that alk-A and alk-B can inhibit Dox-induced apoptosis in H9C2 cardiac muscle cells via inhibition of cell apoptosis and improvement of mitochondrial function, while alk-B showed more protection. Alk-B could be a potential candidate agent for protecting against cardiotoxicity in Dox-exposed patients.

Upregulation of long noncoding RNA RP4 exacerbates hypoxia injury in cardiomyocytes through regulating miR-939/Bnip3/Wnt/β-catenin pathway

This research planned to dig the impacts and potential principles of long noncoding RNA RP4 onH9c2 cell injury induced by hypoxia. The H9c2 cardiac muscle cells were cultured under 3% O2 concentration to induce hypoxia injury, followed by detection of RP4 expression. RP4 was then overexpressed and silenced to investigate its effects on cell injury induced by hypoxia. The potential correlation between RP4 and miR-939, between miR-939 and Bnip3, and between RP4/miR-939/Bnip3 axis and Wnt/β-catenin pathway activation were explored. Biological processes (suppressed cell viability, migration and invasion, but enhanced cell apoptosis) were changed by hypoxia. Upregulation of RP4 enhanced hypoxia-produced damage in H9c2 cells. Additionally, miR-939 expression was opposite regulated by RP4, and miR-939 mimic abrogated the influences of pc-RP4 on enhanced hypoxia damage in H9c2 cells. Moreover, Bnip3 was targeted by miR-939 and their correlation is negative. Furthermore, upregulation of RP4 exacerbated hypoxia-produced injury in H9c2 cells by sensitizing Wnt/β-catenin signals in H9c2 cells, which was regulated by miR-939/Bnip3 axis. Our findings reveal that RP4 is highly expressed in the hypoxia-resulted H9c2 cells. Enhanced expression of RP4 may exacerbate hypoxia injury in cardiomyocytes through regulating miR-939/Bnip3 axis-mediated briskness of Wnt/β-catenin signals. Our study will offer a fresh theoretical basis for the treatment of ischemic myocardial injury.

Ginsenoside Re enhances the survival of H9c2 cardiac muscle cells through regulation of autophagy

We examined the effect of ginsenoside Re (G-Re) on autophagy in H9c2 cardiomyocytes cultured in glucose deprivation (GD). Levels of the membrane-bound autophagy-related microtubule-associated protein 1A/1B-light chain 3 (LC3) B-2 were measured via immunoblotting and immunofluorescence was conducted to assess autophagosome formation. GD H9c2 cells were treated with 100 μmol/l G-Re. Cell viability was determined in culture medium. Phosphorylated 5′ AMP-activated protein kinase (AMPK)-α and mammalian target of rapamycin (mTOR) levels were measured to explore the mechanisms underlying the effects of G-Re on autophagy in GD cells. G-Re treatment inhibited autophagosome formation and may be beneficial to GD cardiomyocytes.

Muscle‐derived exosome/miRNA‐26a attenuates skeletal muscle wasting and cardiomyopathy in chronic kidney disease (CKD) mice

IntroductionUremic cardiomyopathy and muscle atrophy contribute to CKD‐induced morbidity and mortality. Exosomes, natural carriers of many signal molecules including microRNA (miR), mediate organ to organ communication. We hypothesized that miR‐26 would benefit both CKD‐induced muscle wasting and cardiomyopathy through exosome‐mediated muscle‐heart crosstalk.MethodsWe used an engineered exosome vector which contains an exosomal membrane protein gene Lamp2b fused with muscle specific surface peptide for targeting delivery. Exosome encapsulated miR‐26a precursor RNA (Exo/miR26) were generated in muscle satellite cells and injected into the tibialis anterior (TA) muscle of CKD mice (5/6 subtotal nephrectomy) for 12 weeks. NanoSight instrument was used to quantify exosomes. A miR deep sequencing assay and qPCR were used to identify microRNA expression. Cardiac ultrasound was used to detect heart size and function. In‐Vivo Xtreme camera system was used to detect exosome in vivo.ResultsmiR‐26a was decreased in skeletal muscle and heart of CKD mice. Uremic serum enhanced secretion of miR‐26a exosomes in cultured C2C12 skeletal and H9C2 cardiac muscle cells. The intervention of Exo/miR26a increased the expression of miR‐26a in skeletal muscle and heart, as well as increased muscle cross‐section area and decreased CKD‐induced upregulation of atrogin‐1 and MuRF1. Curiously, cardiac fibrosis lesion was partially depressed, and FoxO1, α‐SMA, connect tissue growth factor (CTGF), fibronectin and collagen1α were decreased in CKD mice with intramuscular injection of Exo/miR‐26a. Echocardiography showed that the percentage of ejection fraction was increased in CKD mice treated with Exo/miR26a. Using fluorescence dye labeled Exo/miR26a, we found that the fluorescence intensity in heart was correlated with skeletal muscle, examined by linear regression. We found that miR‐26a directly inhibits FoxO1 and CTGF, which provided mechanism for inhibition of muscle atrophy and cardiac fibrosis by Exo/miR26a.ConclusionsOverexpression of miR‐26a in muscle prevents CKD‐induced muscle loss and attenuates cardiac fibrosis via exosome‐mediated muscle‐heart crosstalk.Support or Funding InformationNational Natural Science Foundation of China (81700618), National Key Research Program (2018YFC130046, 2018YFC1314000). imageThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mdivi‑1 attenuates sodium azide‑induced apoptosis in H9c2 cardiac muscle cells.

The aim of the current study was to investigate the effect of mitochondrial division inhibitor 1 (Mdivi-1) in sodium azide-induced cell death in H9c2 cardiac muscle cells. Mdivi-1 is a key inhibitor of the mitochondrial division protein dynamin-related protein 1 (Drp1). Mdivi-1 was added to H9c2 cells for 3 h, after which, the cells were treated with sodium azide for 24 h. Cell viability was measured by Cell Counting kit-8 assay. DAPI staining was used to observe nuclear morphology changes by microscopy. To further investigate the role of mitochondria in sodium azide-induced cell death, mitochondrial membrane potential (ΔΨm) and the cellular ATP content were determined by JC-1 staining and ATP-dependent bioluminescence assay, respectively. Reactive oxygen species (ROS) production was also assessed by use of the specific probe 2′,7′-dichlorodihydrofluorescein diacetate. In addition, the expression of Drp1 and of the apoptosis-related proteins BCL2 apoptosis regulator (Bcl-2), and BCL2 associated X (Bax) was determined by western blotting. The present findings demonstrated that pretreatment with Mdivi-1 attenuated sodium azide-induced H9c2 cell death. Mdivi-1 pretreatment also inhibited the sodium azide-induced downregulation of Bcl-2 expression and upregulation of Bax and Drp1 expression. In addition, the mitochondrion was revealed to be the target organelle of sodium azide-induced toxicity in H9c2 cells. Mdivi-1 pretreatment moderated the dissipation of ΔΨm, preserved the cellular ATP contents and suppressed the production of ROS. The results suggested that the mechanism of sodium azide-induced cell death in H9c2 cells may involve the mitochondria-dependent apoptotic pathway. The present results indicated that Mdivi-1 may have a cardioprotective effect against sodium azide-induced apoptosis in H9c2 cardiac muscle cells.

Protective effect of total saponins of Panax notoginseng combined with total flavonoids of epimedium on D-galactose-incuced senescence of H9c2 cell

To investigate the protective effect of Panax notoginseng saponins combined with total flavonoids of epimedium on D-gal-induced senescence of H9c2 cells and explore its underlying mechanisms. The 50 mol•L⁻¹ D-gal was used to induce H9c2 cells senescence. Different concentrations of TPNS, TFE, and TPNS combined with TFE were used for 4 hours for pre-treatment. D-gal was used to stimulate H9c2 cardiac muscle cells for 24 h. Then in order to determine the best combined scheme, MTT was used to detect cell viability. Cell senescence was identified by β-galactosidase staining. Levels of reactive oxygen species(ROS) was observed by DCFH-DA detection. The changes of mitochondrial membrane potential were identified by JC-1 detection. Protein levels of silentmating type information regulation 2 Homolog-1(SIRT1), peroxisomal proliferator-activated receptor-coactivator 1α(PGC-1α) and silentmating type information regulation 2 Homolog-3(SIRT3) were detected by western blot analysis. The results showed that TPNS(5 mg•L⁻¹) combined with TFE(5 mg•L⁻¹) had significant synergistic effect on H9c2 myocardial cell proliferation(Q=1.154), so 5 mg•L-1TPNS combined with 5 mg•L⁻¹ TFE was determined as the best scheme. The quantity of β-galactosidase staining and the fluorescence intensity of ROS were apparently decreased in 5 mg•L⁻¹ TPNS combined with 5 mg•L⁻¹ TFE scheme. Meanwhile, it markedly increased the florescence intensity of mitochondrial membrane potential and enhanced the protein expression of SIRT1, PGC-1α and SIRT3. TPNS combined with TFE could protect H9c2 cells from D-gal-induced senescence. The mechanism might be related to adjusting the signal pathways of SIRT1/PGC-1α, SIRT3, adjusting the structure and function of mitochondria and reducing oxidative stress injury.

Protective effects of deep sea water against doxorubicin‑induced cardiotoxicity in H9c2 cardiac muscle cells.

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents in the treatment of a variety of tumors. However, its clinical use has been compromised by the risk of cardiotoxicity. Thus, many efforts have been focused on exploring new strategies to prevent or reverse DOX-induced cardiotoxicity. Recently, deep sea water (DSW) has drawn much scientific interest for therapeutic intervention due to its enrichment in nutrients and minerals. In this study, we investigated whether DSW has protective effects against DOX-induced cardiotoxicity. Pre-treatment with DSW significantly increased the viability of DOX-treated rat H9c2 cardiac muscle cells. This protective effect of DSW appears to be mediated through the inhibition of DNA damage rather than suppression of reactive oxygen species (ROS) production in DOX‑treated H9c2 cardiac muscle cells. The inhibitory effect of DSW on DOX-induced DNA damage subsequently attenuated apoptotic signaling such as activation of cysteine-aspartic acid protease-3 (caspase-3) and fragmentation of poly(ADP-ribose) polymerase (PARP), whereas the expression of anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL) was increased. Moreover, DSW treatment rescued the activation of protein kinase B (Akt) to protect cells from DOX-triggered apoptosis. Taken together, our data showed that DSW has protective effects against DOX-induced cardiotoxicity, suggesting that DSW has some promise as a novel protective supplement for promoting the successful use of DOX in clinical regimen.

Manipulation of Cardiac Phosphatidylinositol 3-Kinase (PI3K)/Akt Signaling by Apoptosis Regulator through Modulating IAP Expression (ARIA) Regulates Cardiomyocyte Death during Doxorubicin-induced Cardiomyopathy

PI3K/Akt signaling plays an important role in the regulation of cardiomyocyte death machinery, which can cause stress-induced cardiac dysfunction. Here, we report that apoptosis regulator through modulating IAP expression (ARIA), a recently identified transmembrane protein, regulates the cardiac PI3K/Akt signaling and thus modifies the progression of doxorubicin (DOX)-induced cardiomyopathy. ARIA is highly expressed in the mouse heart relative to other tissues, and it is also expressed in isolated rat cardiomyocytes. The stable expression of ARIA in H9c2 cardiac muscle cells increased the levels of membrane-associated PTEN and subsequently reduced the PI3K/Akt signaling and the downstream phosphorylation of Bad, a proapoptotic BH3-only protein. When challenged with DOX, ARIA-expressing H9c2 cells exhibited enhanced apoptosis, which was reversed by the siRNA-mediated silencing of Bad. ARIA-deficient mice exhibited normal heart morphology and function. However, DOX-induced cardiac dysfunction was significantly ameliorated in conjunction with reduced cardiomyocyte death and cardiac fibrosis in ARIA-deficient mice. Phosphorylation of Akt and Bad was substantially enhanced in the heart of ARIA-deficient mice even after treatment with DOX. Moreover, repressing the PI3K by cardiomyocyte-specific expression of dominant-negative PI3K (p110α) abolished the cardioprotective effects of ARIA deletion. Notably, targeted activation of ARIA in cardiomyocytes but not in endothelial cells reduced the cardiac PI3K/Akt signaling and exacerbated the DOX-induced cardiac dysfunction. These studies, therefore, revealed a previously undescribed mode of manipulating cardiac PI3K/Akt signaling by ARIA, thus identifying ARIA as an attractive new target for the prevention of stress-induced myocardial dysfunction.

Synthetic liver X receptor agonist T0901317 attenuates high glucose-induced oxidative stress, mitochondrial damage and apoptosis in cardiomyocytes

The aim of the present study was to investigate the protective effects of T0901317 (T0), a potent agonist of liver X receptors (LXRs), on high glucose-induced oxidative stress and apoptosis in H9c2 cardiac cells. Exposure of H9c2 cells to high glucose alone, not only caused a significant increase in apoptosis and reactive oxygen species (ROS) generation, but also led to a decrease in mitochondrial membrane potential (ΔΨm), release of cytochrome c, decrease in Bcl-2, increase in Bax expression and the activation of caspase-3, caspase-9, poly (ADP-ribose) polymerase (PARP) and nuclear factor (NF)-κB. However, pretreatment with T0 effectively decreased apoptosis, reduced the levels of ROS, abrogated ΔΨm, inhibited cytochrome c release and NF-κB activation, increased Bcl-2 and decreased Bax expression. In conclusion, our data suggest that T0 exerts protective effects against high glucose-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS production, mitochondrial death and NF-κB activation.

Extracts ofArtemisia ciniformisProtect Cytotoxicity Induced by Hydrogen Peroxide in H9c2 Cardiac Muscle Cells through the Inhibition of Reactive Oxygen Species

Objective. Artemisia ciniformis (Asteraceae) and A. biennis are two of 34 Artemisia species growing naturally in Iran. In this study we investigated whether different extracts of A. ciniformis and A. biennis have protective effect against hydrogen peroxide-induced cytotoxicity in rat cardiomyoblast cells (H9c2). Method. The dried and ground aerial parts of these two species were extracted successively using petroleum ether (40–60), dichloromethane, ethyl acetate (EA), ethanol (EtOH) and ethanol : water (1 : 1) by maceration method. To evaluate whether different extracts of A. ciniformis and A. biennis protect cardiomyoblast H9c2 cells from H2O2 cytotoxicity, we examined the direct cytotoxic effect of H2O2 on H9c2 cells in the presence and absence of different extracts. After then, cell viability was measured by MTT assay. Results. H2O2 induced cytotoxicity in a concentration dependent manner. The IC50 value was 62.5 μM for 24 h exposure. However, pretreatment of cells with various concentrations of EA, EtOH, and EtOH/wt extract of A. ciniformis protected cells from H2O2-induced cytotoxicity. Moreover, pretreatment with EA, EtOH and EtOH/wt extracts of A. ciniformis lead to a decrease in the reactive oxygen species (ROS) generation. Taken together our observation indicated that nontoxic concentration of different extracts of A. ciniformis has protective effect on H2O2-induced cytotoxicity in H9c2 cells.

Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species

Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species

Development of fluorescence imaging-based assay for screening cardioprotective compounds from medicinal plants

Medicinal plants have been widely recognized as a renewable resource for the discovery of novel leads and drug. In this study, an approach for screening and identification compounds with cardioprotective activity from medicinal plant extracts by cellular-fluorescence imaging technique was developed. It is a cell-based assay for measuring mitochondrial membrane potential changes in H9c2 cardiac muscle cells exposed to H 2O 2 by using a fluorescence automatic microscopy screening platform. Rhodamine 123 was used as the fluorescent dye to indicate the change of mitochondrial membrane potential. The sensitivity and linear range of the proposed approach were evaluated and validated using vitamin C, an antioxidative compound. The method was applied to screen active components with potent cardioprotective effects from a traditional Chinese formula. The potential cardioprotective components were identified by liquid chromatography coupled with mass spectrometry (LC/MS). Moreover, the utility of the proposed approach was further validated by three compounds (salvianolic acid B, protocatechuic aldehyde, and tanshinone II A) identified from the formula which showed cardioprotective effects in a dose-dependent manner. These applications suggested that the proposed rapid and sensitive screening approach offers an efficient way to discover active components or compounds from medicinal plants.

Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species

Doxorubicin (DOX) is a widely used chemotherapy agent. The major adverse effect of DOX treatment in cancer patients is the onset of cardiomyopathy and heart failure. Reactive oxygen species (ROS) are proposed to be responsible for DOX cardiotoxicity. Curcumin, a natural compound extracted from Curcuma Longa L., is known for its anti-oxidant properties. It has been identified as increased apoptosis in several cancer cell lines in combination with doxorubicin, but there are few studies about the effect of curcumin and doxorubicin on normal cardiac cells. Therefore, we evaluated the effects of curcumin on apoptosis induced by DOX in cardiac muscle cells. Preteatment with curcumin significantly increased DOX-induced apoptosis of cardiac muscle cells through down regulation of Bcl-2, up-regulation of caspase-8 and caspase-9. The Bax/Bcl-2 ratio increased significantly after 1h pretreatment with curcumin. As well, curcumin increases ROS generation by DOX. In response to DOX, NF-κB was activated. However, curcumin was able to inhibit NF-κB activation. In conclusion, our results indicated that pretreatment with nontoxic concentrations of curcumin sensitized H9c2 cells to DOX-mediated apoptosis by generation of ROS.

Autophagy plays an important role in Sunitinib-mediated cell death in H9c2 cardiac muscle cells

Sunitinib, which is a multitargeted tyrosine-kinase inhibitor, exhibits antiangiogenic and antitumor activity, and extends survival of patients with metastatic renal-cell carcinoma (mRCC) and gastrointestinal stromal tumors (GIST). This molecule has also been reported to be associated with cardiotoxicity at a high frequency, but the mechanism is still unknown. In the present study, we observed that Sunitinib showed high anti-proliferative effect on H9c2 cardiac muscle cells measured by PI staining and the MTT assay. But apoptotic markers (PARP cleavage, caspase 3 cleavage and chromatin condensation) were uniformly negative in H9c2 cells after Sunitinib treatment for 48 h, indicating that another cell death pathway may be involved in Sunitinib-induced cardiotoxicity. Here we found Sunitinib dramatically increased autophagic flux in H9c2 cells. Acidic vesicle fluorescence and high expression of LC3-II in H9c2 cells identified autophagy as a Sunitinib-induced process that might be associated with cytotoxicity. Furthermore, knocking down Beclin 1 by RNA-interference to block autophagy in H9c2 cells revealed that the death rate was decreased when treated with Sunitinib in comparison to control cells. These results confirmed that autophagy plays an important role in Sunitinib-mediated H9c2 cells cytotoxicity. Taken together, the data presented here strongly suggest that autophagy is associated with Sunitinib-induced cardiotoxicity, and that inhibition of autophagy constitutes a viable strategy for reducing Sunitinib-induced cardiomyocyte death thereby alleviating Sunitinib cardiotoxicity.

N-acetylcysteine prevents glucose/glucose oxidase-induced oxidative stress, mitochondrial damage and apoptosis in H9c2 cells

Aims High blood glucose may auto-oxidize and generate free radicals, which are proposed to induce apoptosis in cardiac cells. The aim of the present study was to investigate the cell damage induced by glucose/glucose oxidase-dependent oxidative stress and the protective effect of N-acetylcysteine (NAC) on H9c2 cardiac muscle cells. Main methods H9c2 cells were exposed to 33 mM glucose (G) + 1.6 milliunits (mU) of glucose oxidase (GO) and termed G/GO. Cell apoptosis, generation of reactive oxygen species (ROS-super oxide anion and hydrogen peroxide) and reactive nitrogen species (RNS-peroxinitrite), and the change in mitochondrial membrane potential (Δ Ψm) was studied using flow cytometry and confocal microscopy, and cytochrome c release was measured using confocal microscopy. The expression of Bcl-2, Bax and the activation of procaspase-9 was studied by western blot. Key findings Exposure of H9c2 cells to G/GO resulted in a significant increase in cellular apoptosis ( P < 0.05) and the generation of ROS and RNS ( P < 0.001). Further, G/GO treatment led to a decrease in Δ Ψm, release of cytochrome c, decrease in Bcl-2, increase in Bax expression and the activation of procaspase-9. Treatment with NAC significantly decreased apoptosis ( P < 0.05) and reduced the levels of ROS and RNS ( P < 0.001). NAC was also able to normalize Δ Ψm, inhibit cytochrome c release, increase Bcl-2 and decrease Bax expression and procaspase-9 activation. Significance Our studies suggest that NAC has antioxidative and antiapoptotic activity against G/GO-induced oxidative stress through the inhibition of mitochondrial damage in H9c2 cells.

Role of NADPH oxidase in H9c2 cardiac muscle cells exposed to simulated ischaemia‐reperfusion

Oxidative stress is associated with several cardiovascular pathologies, including hypertension, cardiac hypertrophy and heart failure. Although oxidative stress is also increased after ischaemia-reperfusion (I/R), little is known about the role and the activation mechanisms, in cardiac myocytes under these conditions, of NADPH oxidase, a superoxide-producing enzyme. We found that rat cardiac muscle cells (H9c2) subjected to an in vitro simulated ischaemia (substrate-free medium plus hypoxia) followed by 'reperfusion', displayed increased reactive oxygen species (ROS) production attributable to a parallel increase of NADPH oxidase activity. Our investigation on mechanisms responsible for NADPH oxidase activation showed a contribution of both the increase of NOX2 expression and p47(phox) translocation to the membrane. We also found that the increase of NADPH oxidase activity was associated with higher levels of lipid peroxidation, the activation of redox-sensitive kinases, in particular ERK and JNK, and with cell death. Diphenyleneiodonium (DPI), a flavoprotein inhibitor used as NADPH oxidase inhibitor, prevented I/R-induced ROS formation in treated cells, together with the related lipoperoxidative damage, and JNK phosphorylation without affecting ERK activation, resulting in protection against cell death. Our results provide evidence that NADPH oxidase is a key enzyme involved in I/R-induced oxidant generation and suggest it can be a possible target in cardioprotective strategies against I/R injury, a condition of great importance in human pathology.

Kaempferol Protects Ischemia/Reperfusion-Induced Cardiac Damage Through the Regulation of Endoplasmic Reticulum Stress

This study examined whether or not the ER stress and Bcl-2 proteins are linked to the protective effect of kaempferol, a phytoestrogen, on ischemia-reperfusion (I/R)-induced cardiac damage. In order to determine if kaempferol modifies the I/R-induced response in H9c2 cardiac muscle cells, the cells were exposed to kaempferol followed by ischemia 12h/reperfusion 4h. kaempferol had a protective effect on the apoptosis induced by I/R in the cardiac muscle cells. The Kaempferol treatment significantly increased the expression level of the anti-apoptotic protein, Bcl-2, but decreased the level of the pro-apoptotic protein, bax. Kaempferol down-regulated the expressions of the endoplasmic reticulum (ER) stress proteins, GRP78, ATF-6alpha, XBP-2, IRE1-alpha, phosphor-eIF-2alpha and CHOP. In ex vivo-Langendorff experiment, the kaempferol treatment regulated the expression of ER stress proteins-CHOP and GRP78. The kaempferol also improved the post-ischemic LVEDP and LVDP significantly after 20, 30, 40 and 50 min of reperfusion compared with the untreated control hearts, which shows that kaempferol offers protection against I/R-associated cardiac dysfunction.

Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells.

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.

Plantainoside D protects adriamycin-induced apoptosis in H9c2 cardiac muscle cells via the inhibition of ROS generation and NF-κB activation

Plantainoside D (PD), was isolated from the leaves of Picrorhiza scrophulariiflora (Scrophulariaceae). The anti-oxidative activity of PD was evaluated based on scavenging effects on hydroxyl radicals and superoxide anion radicals. Adriamycin (ADR) is a potent anti-tumor drug known to cause severe cardiotoxicity. Although ADR generates free radicals, the role of free radicals in the development of cardiac toxicity has not been understood. This study was undertaken to investigate the protective effect of PD against ADR-induced apoptosis. In vitro, ADR caused dose-dependent toxicity in H9c2 cardiac muscle cells. Pre-treatment of the cardiac muscle cells with PD significantly reduced ADR-induced apoptosis of cardiac muscle cells. PD inhibited the ROS produced by ADR in the cardiac muscle cells. As well, PD increased GSH(glutathione), compared with ADR. In response to ADR, NF-κB was activated in H9c2 cells. However the treatment of PD reduced the activation of NF-κB. We also observed that the NF-κB inhibitor, PDTC, inhibited the cytotoxic effect on ADR-induced apoptosis in cardiac muscle cells. In parallel, IκBα-dominant negative plasmid-overexpression abrogated ADR-induced apoptosis in H9c2 cardiac muscle cells. In conclusion, these results suggest that Plantaionoside D can inhibit ADR-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS generation and NF-κB activation. The pure compound PD can be a potential candidate agent which protects cardiotoxicity in ADR-exposed patients.