Effects In H9c2 Cells Research Articles

Matrine induces cardiotoxicity by promoting ferroptosis through the Nrf2 antioxidant system in H9c2 cells

Matrine (MT) has shown promising efficacy in various cancers and chronic hepatitis; however, its clinical application is limited because of its side effects. Our previous studies have indicated that MT can induce severe hepatotoxicity and nephrotoxicity. The current study aimed to investigate its cardiotoxicity and potential underlying mechanisms in H9c2 cells. Our results showed that MT induced H9c2 cell death and disrupted the cellular membrane integrity. Moreover, MT decreased glutathione (GSH) and cysteine (Cys) levels, and increased Fe2+, lipid peroxidation, reactive oxygen species (ROS), and MDA levels, ultimately leading to ferroptosis. Interestingly, these phenomena were alleviated by the ferroptosis inhibitor Fer-1, whereas MT-induced ferroptosis was exacerbated by the ferroptosis agonist RSL3. In addition, MT significantly reduced FTH, Nrf2, xCT, GPX4, and FSP1 protein levels and inhibited the transcriptional activity of Nrf2 while increasing TFR1 protein levels. Supplementation with Nrf2 agonist (Dimethyl fumarate, DMF) or selenium (Sodium selenite, SS) and CoQ10 alleviated MT-induced cytotoxic effects in H9c2 cells. These results suggest that ferroptosis, which is mediated by an imbalance in the Nrf2 antioxidant system, is involved in MT-induced cardiac toxicity.

Scutellarin Protects against Myocardial Ischemia-reperfusion Injury by Enhancing Aerobic Glycolysis through miR-34c-5p/ALDOA Axis.

Aerobic glycolysis has recently demonstrated promising potential in mitigating the effects of ischemia-reperfusion (IR) injury. Scutellarin (Scu) possesses various cardioprotective properties that warrant investigation. To mimic IR injury in vitro, this study employed hypoxia/reoxygenation (H/R) injury. First, we conducted an assessment of the protective properties of Scu against HR in H9c2 cells, encompassing inflammation damage, apoptosis injury, and oxidative stress. Then, we verified the effects of Scu on the Warburg effect in H9c2 cells during HR injury. The findings indicated that Scu augmented aerobic glycolysis by upregulating p-PKM2/PKM2 levels. Following, we built a panel of six long noncoding RNAs and seventeen microRNAs that were reported to mediate the Warburg effect. Based on the results, miR-34c-5p was selected for further experiments. Then, we observed Scu could mitigate the HR-induced elevation of miR-34c-5p. Upregulation of miR-34c-5p could weaken the beneficial impacts of Scu in cellular viability, inflammatory damage, oxidative stress, and the facilitation of the Warburg effect. Subsequently, our investigation revealed a decrease in both ALDOA mRNA and protein levels following HR injury, which could be restored by Scu administration. Downregulation of ALDOA or Mimic of miR-34c-5p could reduce these effects induced by Scu. Scu provides cardioprotective effects against IR injury by upregulating the Warburg effect via miR-34c-5p/ALDOA.

Paeoniflorin Protects H9c2 Cardiomyocytes against Hypoxia/Reoxygenation Induced Injury via Regulating the AMPK/Nrf2 Signaling Pathway.

Myocardial ischemia/reperfusion (MIR) injury contributes to the exacerbation of heart disease by causing cardiac arrhythmias, myocardial infarction, and even sudden death. Studies have found that paeoniflorin (PF) has a protective effect on coronary artery disease (CAD). However, the mechanism of PF in MIR has not been fully investigated. The purpose of this study was to investigate the functional role of PF in H9c2 cells subjected to hypoxia/reoxygenation (H/R). Here, PF treatment enhanced cell viability in H/R-stimulated H9c2 cells. In H9c2 cells, PF treatment reduced the formation of reactive oxygen species (ROS) induced by H/R. In H/R-stimulated H9c2 cells, PF also increased the activity of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase. Furthermore, PF protected H9c2 cells against H/R-induced apoptosis, as demonstrated by increased Bcl-2 expression, decreased Bax expression, and decreased caspase-3 activity. Furthermore, PF increased the levels of p-AMPK and nuclear Nrf2 expression in response to H/R stimulation. AMPK inhibition, on the other hand, abolished the PF-mediated increase in Nrf2 signaling and the cardiac-protective effect in H9c2 cells exposed to H/R. These data suggest that PF protected H9c2 cells against H/R-induced oxidative stress and apoptosis through modulating the AMPK/Nrf2 signaling pathway. Our findings support the therapeutic potential of PF in myocardial I/R damage.

Propofol postconditioning alleviates diabetic myocardial ischemia‑reperfusion injury via the miR‑200c‑3p/AdipoR2/STAT3 signaling pathway.

Myocardial ischemia/reperfusion (MI/RI) syndrome is one of the leading causes of mortality and disability. Propofol postconditioning is known to improve myocardial ischemia/reperfusion injury (MI/RI). The present study aimed to explore the mechanism of propofol postconditioning in diabetic MI/RI. Diabetic MI/RI rat models were established and the rats were treated via propofol postconditioning. Staining with 2,3,5-triphenyl-2H-tetrazolium chloride, H&E staining, TUNEL staining and ELISA were applied to detect infarct size, pathological changes, apoptosis and oxidative stress-related factor and apoptotic factor levels, respectively. Subsequently, the effect of propofol on H9C2 cells was also assessed using the Cell Counting Kit-8 assay. High-glucose hypoxia/reperfusion (H/R) models of H9C2 cardiomyocytes were established. miR-200c-3p overexpression or AdipoR2 silencing combined with propofol postconditioning was performed in H/R-induced H9C2 cells and STAT3 protein expression levels were determined. Propofol postconditioning significantly reduced myocardial infarct size, oxidative stress and apoptosis in diabetic MI/RI models. Furthermore, propofol postconditioning significantly reduced the oxidative stress and apoptosis of H9C2 cells in high-glucose H/R models. Propofol postconditioning also significantly downregulated miR-200c-3p expression levels and promoted AdipoR2 expression levels. miR-200c-3p overexpression or AdipoR2 downregulation significantly reversed the effects of propofol postconditioning on its antioxidation and anti-apoptotic effects in H9C2 cells and on decreasing STAT3 phosphorylation levels. Together, the results of the present study demonstrated that propofol postconditioning inhibited miR-200c-3p, upregulated AdipoR2 and activated the STAT3 signaling pathway, thus alleviating diabetic MI/RI and therefore highlighting its potential as a treatment of diabetic MI/RI.

Anshen-Buxin-Liuwei pill, a Mongolian medicinal formula could alleviate cardiomyocyte hypoxia/reoxygenation injury via mitochondrion pathway.

Anshen Buxin Liuwei pill (ABLP) is a Mongolian medicinal formula that is composed of six medicinal materials: the Mongolian medicine Bos taurus domesticus Gmelin, Choerospondias axillaris (Roxb.) Burtt et Hill, Myristica fragrans Houtt., Eugenia caryophμllata Thunb., Aucklandia lappa Decne., and Liqui dambar formosana Hance. ABLP is considered to have a therapeutic effect on symptoms such as coronary heart disease, angina pectoris, arrhythmia, depression and irritability, palpitation, and shortness of breath. H9c2 cardiomyocytes were used to construct a hypoxia/reoxygenation (HR) injury model. CCK-8 assay and Annexin V-FITC cell apoptosis assays were used for cell viability and cell apoptosis determination. The LDH, SOD, MDA, CAT, CK, GSH-Px, Na+-K+-ATPase, and Ca2+-ATPase activities in cells were determined to assess the protective effects of ABLP. The mRNA levels of Sirtuin3 (Sirt3) and Cytochrome C (Cytc) in H9c2 cells were determined by quantitative real-time PCR. The results indicate that HR-treated cells began to shrink from the spindle in an irregular shape with some floated in the medium. By increasing the therapeutic dose of ABLP (5, 25, and 50μg/mL), the cells gradually reconverted in a concentration-dependent manner. The release of CK in HR-treated cells was significantly increased, indicating that ABLP exerts a protective effect in H9c2 cells against HR injury and can improve mitochondrial energy metabolism and mitochondrial function integrity. The present study scrutinized the cardioprotective effects of ABLP against HR-induced H9c2 cell injury through antioxidant and mitochondrial pathways. ABLP could be a promising therapeutic drug for the treatment of myocardial ischemic cardiovascular disease. The results will provide reasonable information for the clinical use of ABLP.

Tanshinone I Inhibits Oxidative Stress-Induced Cardiomyocyte Injury by Modulating Nrf2 Signaling.

Cardiovascular disease, a disease caused by many pathogenic factors, is one of the most common causes of death worldwide, and oxidative stress plays a major role in its pathophysiology. Tanshinone I (Tan I), a natural compound with cardiovascular protective effects, is one of the main active compounds extracted from Salvia miltiorrhiza. Here, we investigated whether Tan I could attenuate oxidative stress and oxidative stress–induced cardiomyocyte apoptosis through Nrf2/MAPK signaling in vivo and in vitro. We found that Tan I treatment protected cardiomyocytes against oxidative stress and oxidative stress–induced apoptosis, based on the detection of relevant oxidation indexes such as reactive oxygen species, superoxide dismutase, malondialdehyde, and apoptosis, including cell viability and apoptosis-related protein expression. We further examined the mechanisms underlying these effects, determining that Tan I activated nuclear factor erythroid 2 (NFE2)–related factor 2 (Nrf2) transcription into the nucleus and dose-dependently promoted the expression of Nrf2, while inhibiting MAPK signaling activation, including P38 MAPK, SAPK/JNK, and ERK1/2. Nrf2 inhibitors in H9C2 cells and Nrf2 knockout mice demonstrated aggravated oxidative stress and oxidative stress–induced cardiomyocyte injury; Tan I treatment suppressed these effects in H9C2 cells; however, its protective effect was inhibited in Nrf2 knockout mice. Additionally, the analysis of surface plasmon resonance demonstrated that Tan I could directly target Nrf2 and act as a potential Nrf2 agonist. Collectively, these data strongly indicated that Tan I might inhibit oxidative stress and oxidative stress–induced cardiomyocyte injury through modulation of Nrf2 signaling, thus supporting the potential therapeutic application of Tan I for oxidative stress–induced CVDs.

Baicalin Prevents Myocardial Ischemia/Reperfusion Injury Through Inhibiting ACSL4 Mediated Ferroptosis.

Baicalin is a natural flavonoid glycoside that confers protection against myocardial ischemia/reperfusion (I/R) injury. However, its mechanism has not been fully understood. This study focused on elucidating the role of ferroptosis in baicalin-generated protective effects on myocardial ischemia/reperfusion (I/R) injury by using the myocardial I/R rat model and oxygen–glucose deprivation/reoxygenation (OGD/R) H9c2 cells. Our results show that baicalin improved myocardial I/R challenge–induced ST segment elevation, coronary flow (CF), left ventricular systolic pressure , infarct area, and pathological changes and prevented OGD/R-triggered cell viability loss. In addition, enhanced lipid peroxidation and significant iron accumulation along with activated transferrin receptor protein 1 (TfR1) signal and nuclear receptor coactivator 4 (NCOA4)-medicated ferritinophagy were observed in in vivo and in vitro models, which were reversed by baicalin treatment. Furthermore, acyl-CoA synthetase long-chain family member 4 (ACSL4) overexpression compromised baicalin-generated protective effect in H9c2 cells. Taken together, our findings suggest that baicalin prevents against myocardial ischemia/reperfusion injury via suppressing ACSL4-controlled ferroptosis. This study provides a novel target for the prevention of myocardial ischemia/reperfusion injury.

Small Molecule Compound Nerolidol attenuates Hypertension induced hypertrophy in spontaneously hypertensive rats through modulation of Mel-18-IGF-IIR signalling

BackgroundCardiovascular diseases are caused by multitudes of stress factors like hypertension and their outcomes are associated with high mortality and morbidity worldwide. Nerolidol, a naturally occurring sesquiterpene found in several plant species, embodies various pharmacological benefits against numerous health disorders. However, their effects on hypertension induced cardiac complications are not completely understood. PurposeThe present study is to elucidate the efficacy of nerolidol against hypertension related cardiac hypertrophy in spontaneously hypertensive rats (SHRs). Study DesignFor preliminary in vitro studies, H9c2 cardiomyoblasts cells were challenged with 200 nM Angiotensin-II (AngII) for 12 h and were then treated with nerolidol for 24 h. The hypertrophic effect in H9c2 cells were analyzed by actin staining and the modulations in hypertrophic protein markers and mediators were determined by Western blotting analysis. For in vivo experiments, sixteen week-old male Wistar Kyoto (WKY) and SHRs were segregated into five groups (n = 9): Control WKY, hypertensive SHRs, SHRs with low dose (75 mg/kg b.w/day) nerolidol, SHRs with high dose (150 mg/kg b.w/day) nerolidol and SHR rats treated with an anti-hypertensive drug captopril (50 mg/kg b.w/day). Nerolidol treatment was given orally for 8 weeks and were analysed through Echocardiography. After euthanasia, hematoxylin and eosin staining, Immunohistochemical analysis and Western blotting was performed on left ventricle tissue. ResultsWestern blotting analysis revealed that nerolidol significantly attenuates AngII induced expression of hypertrophic markers ANP and BNP in H9c2 cardiomyoblasts. In addition, actin staining further ascertained the potential of nerolidol to ameliorate AngII induced cardiac hypertrophy. Moreover, nerolidol administration suppressed the hypertrophic signalling mediators like calcineurin, GATA4, Mel-18, HSF-2 and IGFIIR in a dose-dependent fashion. In silico studies also ascertained the role of Mel-18 in the ameliorative effects of nerolidol. Further, these intriguing in vitro results were further confirmed in in vivo SHR model. Oral neraolidol in SHRs efficiently reduced blood pressure and ameliorated hypertension induced cardiac hypertrophic effects by effectively reducing the levels of proteins involved in cardiac MeL-18-HSF2-IGF-IIR signalling. ConclusionCollectively, the data reveals that the cardioprotective effect of nerolidol against hypertension induced hypertrophy involves reduction in blood pressure and regulation of the cardiac Mel-18-IGFIIR signalling cascade.

MicroRNA-221/222 Mediates ADSC-Exosome-Induced Cardioprotection Against Ischemia/Reperfusion by Targeting PUMA and ETS-1.

Cardiovascular disease is a major health problem in industrialized and developing countries and is the leading cause of death and disability. Myocardial ischemia/reperfusion (I/R) causes cardiomyocyte damage such as apoptosis and hypertrophy. The purpose of this study was to investigate the effects of exosomes from adipose-derived stem cells (ADSC-Exo) on hearts from I/R mice and to explore the underlying mechanisms. ADSC-Exo significantly decreased I/R-induced cardiomyocyte apoptosis and hypertrophy, as detected by TdT-mediated dUTP nick end-labeling (TUNEL) and wheat germ agglutinin (WGA) staining, respectively. In addition, the expression of apoptosis-related proteins p-p53 and PUMA and hypertrophy-related proteins ETS-1 and ANP were significantly reduced in the cardiomyocytes of ADSC-Exo-treated I/R mice compared to those of control mice. Both PUMA and ETS-1 are reported to be target genes for miR-221/222. I/R operation significantly reduced miR-221/222 expression, while ADSC-Exo treatment increased miR-221/222 expression, as detected by RT-qPCR. We also observed that cardiac I/R operation markedly increased cell apoptosis and hypertrophy in miR-221/222 knockout (KO) mice, while ADSC-Exo reduced the effects of I/R operation. Furthermore, ADSC-Exo protected H9c2 cardiomyocytes from H2O2-induced damage by reducing apoptosis and hypertrophy in vitro. H2O2 treatment significantly reduced miR-221/222 expression, while ADSC-Exo treatment reversed this effect in H9c2 cells. ADSC-Exo treatment decreased H2O2-induced PUMA and ETS-1 expression. Compared with control treatment, I/R treatment significantly reduced p-AKT and increased p-p65, while ADSC-Exo and miR-221/222 mimics attenuated these effects. The AKT activator SC79 and p65 inhibitor Bay 11-7082 reduced H2O2-induced cell apoptosis and hypertrophy. Based on these findings, ADSC-Exo prevents cardiac I/R injury through the miR-221/miR-222/PUMA/ETS-1 pathway. Therefore, ADSC-Exo is an effective inhibitor of I/R-induced heart injury.

MiR-375-3p contributes to hypoxia-induced apoptosis by targeting forkhead box P1 (FOXP1) and Bcl2 like protein 2 (Bcl2l2) in rat cardiomyocyte h9c2 cells.

miRNAs have been pointed to play critical role in the development of congenital heart disease (CHD). miRNA-375-3p (miR-375-3p) was involved in cardiac dysfunction and cardiogenesis. However, no prior study had established a therapeutic role of miR-375-3p in CHD. We intended to investigate the effect and mechanism of miR-375-3p on apoptosis in hypoxic cardiomyocytes in vitro. Expression of miR-375-3p, forkhead box P1 (FOXP1) and Bcl2 like protein 2 (Bcl2l2) was detected using real-time quantitative PCR and western blot. Apoptosis was measured with MTT assay, flow cytometry and caspase-3 activity assay. The potential target binding between miR-375-3p and FOXP1/Bcl2l2 was predicted on DianaTools, and was validated by luciferase reporter assay and RNA pull-down assay. As a result, miR-375-3p was upregulated and FOXP1/Bcl2l2 was downregulated in maternal serum of women with fetal CHD and hypoxia-induced rat cardiomyocyte h9c2 cells. Hypoxia induced apoptosis rate elevation, caspase-3 activity promotion and viability inhibition in h9c2 cells; overexpression of miR-375-3p promoted, whereas knockdown of miR-375-3p antagonized hypoxia-induced effects in h9c2 cells. In addition, miR-375-3p was validated to negatively regulate FOXP1 and Bcl2l2 expression through target binding, and silencing of FOXP1 and Bcl2l2 could independently abate the anti-apoptosis role of miR-375-3p knockdown in hypoxic h9c2 cells. Collectively, blocking miR-375-3p suppressed hypoxia-evoked apoptosis of cardiomyocytes by targeting and upregulating FOXP1 and Bcl2l2. Our results might suggest maternal serum miR-375-3p as a potential biomarker for prenatal detection of fetal CHD.

Helix B Surface Peptide Protects Cardiomyocytes From Hypoxia/Reoxygenation-induced Autophagy Through the PI3K/Akt Pathway.

Helix B surface peptide (HBSP) is a newly discovered tissue-protective erythropoietin derivative that provides benefits after myocardial ischemia/reperfusion. This study explores the cardioprotective effects of HBSP in myocardial cells in response to hypoxia/reoxygenation injury and its potential mechanism. In this study, rat ventricular (H9c2) cell cultures were established and pretreated with HBSP. H9c2 cardiomyocytes were randomly assigned to the control, H/R, H/R + LY294002 (a PI3K inhibitor), HBSP + H/R, and HBSP + H/R + LY294002 groups. The pretreated cardiomyocytes underwent H/R, and the cardiomyocytes were monitored for viability through a CCK-8 assay, whereas flow cytometry was used to test cell apoptosis. Orgotein Superoxide Dismutase (SOD) and lactate dehydrogenase (LDH) expression were monitored by SOD and LDH kits, respectively. The expression of LC3 autophagosomes was determined by immunocytochemistry. The expression of LC3II/LC3I, p-Mammalian Target of Rapamycin (mTOR) mTOR, mTOR, Beclin 1, p-PI3K, PI3K p-Akt, and Akt was determined by Western blotting. HBSP increased cell viability and reduced SOD and LDH production, and it also reduced H/R-induced cell apoptosis. Moreover, the expression of the autophagy-related proteins (LC3II/LC3I) was inhibited by HBSP, whereas the expression of p-PI3K, p-Akt, and p-mTOR was enhanced. However, the PI3K inhibitor (LY294002) notably abolished these effects in H9c2 cells. HBSP inhibits excessive autophagy and apoptosis induced by H/R by activating the PI3K/Akt pathway. HBSP may potentially be a therapeutic intervention for myocardial ischemia/reperfusion injury.

Prostaglandin E1 protects cardiomyocytes against hypoxia-reperfusion induced injury via the miR-21-5p/FASLG axis.

Background: Prostaglandin-E1 (PGE1) is a potent vasodilator with anti-inflammatory and antiplatelet effects. However, the mechanism by which PGE1 contributes to the amelioration of cardiac injury remains unclear.Methods: The present study was designed to investigate how PGE1 protects against hypoxia/reoxygenation (H/R)-induced injuries by regulating microRNA-21-5p (miR-21-5p) and fas ligand (FASLG). Rat H9C2 cells and isolated primary cardiomyocytes were cultured under hypoxic conditions for 6 h (6H, hypoxia for 6 h), and reoxygenated for periods of 6 (6R, reoxygenation for 6 h), 12, and 24 h, respectively. Cells from the 6H/6R group were treated with various doses of PGE1; after which, their levels of viability and apoptosis were detected.Results: The 6H/6R treatment regimen induced the maximum level of H9C2 cell apoptosis, which was accompanied by the highest levels of Bcl-2-associated X protein (Bax) and cleaved-caspase-3 expression and the lowest level of B-cell lymphoma 2 (Bcl-2) expression. Treatment with PGE1 significantly diminished the cell cytotoxicity and apoptosis induced by the 6H/6R regimen, and also decreased expression of IL-2, IL-6, P-p65, TNF-α, and cleaved-caspase-3. In addition, we proved that PGE1 up-regulated miR-21-5p expression in rat cardiomyocytes exposed to conditions that produce H/R injury. FASLG was a direct target of miR-21-5p, and PGE1 reduced the ability of H/R-injured rat cardiomyocytes to undergo apoptosis by affecting the miR-21-5p/FASLG axis. In addition, we proved that PGE1 could protect primary cardiomyocytes against H/R-induced injuries.Conclusions: These results indicate that PGE1 exerts cardioprotective effects in H9C2 cells during H/R by regulating the miR-21-5p/FASLG axis.

Anti-hypertrophic and anti-apoptotic effects of short peptides of potato protein hydrolysate against hyperglycemic condition in cardiomyoblast cells

Cardiomyocyte hypertrophy is a critical pathological phenomenon observed in diabetic cardiomyopathy. Various molecular events including the Calcineurin/nuclear factor of activated T-cell (NFAT) mediated signaling contributes to the pathogenesis of cardiac hypertrophy. While different new therapeutic interventions are investigated in order to overcome pathological hypertrophic effects, recent studies on peptide hydrolysates from common foods have gained interest. In this study the cytoprotective efficiency of two short peptides DIKTNKPVIF (DF) and a dipeptide IF from a potato protein hydrolysate were evaluated for their anti-hypertrophic effects against high glucose (HG) challenge. Murine cardio myoblast (H9c2) cells were challenges with 33 mM of glucose and after 1 h were treated with DF or IF for 24 h. The results showed enlargement in cell size, elevated ANP and BNP expression induced by HG however the abnormalities were efficiently attenuated by IF and DF. Further, HG increased the levels of calcineurin and NFATC3 which was markedly suppressed by DF and IF in H9c2 cells. The results further showed that DF and IF suppresses the activation of p38 in a dose dependent manner with no notable effects on JNK activation. DF and IF also attenuated the HG induced apoptotic effects in H9c2 cells by suppressing the apoptotic proteins and by enhancing the survival and anti-apoptotic proteins. Further, it should be noted that administration of both the fragments showed similar effects in all the analysis. Our results therefore showed that DF and IF of potato protein hydrolysate possess efficient protective effects against HG-induced cardiomyocyte damages by ameliorating the apoptotic and hypertrophic effects.

Exosomes From Adipose-derived Mesenchymal Stem Cells Protect the Myocardium Against Ischemia/Reperfusion Injury Through Wnt/β-Catenin Signaling Pathway.

:Mesenchymal stem cells (MSCs) and their secreted exosomes exert a cardioprotective role in jeopardized myocardium. However, the specific effects and underlying mechanisms of exosomes derived from adipose-derived MSCs (ADMSCs) on myocardial ischemia/reperfusion (I/R) injury remain largely unclear. In this study, ADMSC-derived exosomes (ADMSCs-ex) were administrated into the rats subjected to I/R injury and H9c2 cells exposed to hypoxia/reoxygenation (H/R). Consequently, administration of ADMSCs-ex significantly reduced I/R-induced myocardial infarction, accompanied with a decrease in serum levels of creatine kinase-myocardial band, lactate dehydrogenase, and cardiac troponin I (cTnI). Simultaneously, ADMSCs-ex dramatically antagonized I/R-induced myocardial apoptosis, along with the upregulation of Bcl-2 and downregulation of Bax, and inhibition of Caspase 3 activity in rat myocardium. Similarly, ADMSCs-ex significantly reduced cell apoptosis and the expression of Bax, but markedly increased cell viability and the expression of Bcl-2 and Cyclin D1 under H/R. Furthermore, ADMSCs-ex observably induced the activation of Wnt/β-catenin signaling by attenuating I/R- and H/R-induced inhibition of Wnt3a, p-GSK-3β (Ser9), and β-catenin expression. Importantly, treatment with Wnt/β-catenin inhibitor XAV939 partly neutralized ADMSC-ex–induced antiapoptotic and prosurvival effects in H9c2 cells. In conclusion, we confirmed that ADMSCs-ex protect ischemic myocardium from I/R injury through the activation of Wnt/β-catenin signaling pathway.

Characterization of cytoprotective and toxic properties of iron chelator SIH, prochelator BSIH and their degradation products.

Free cellular iron catalyzes the formation of toxic hydroxyl radicals and therefore chelation of iron could be a promising therapeutic approach in pathological states associated with oxidative stress. Salicylaldehyde isonicotinoyl hydrazone (SIH) is a strong intracellular iron chelator with well documented potential to protect against oxidative damage both in vitro and in vivo. Due to the short biological half-life of SIH and risk of toxicity due to iron depletion, boronate prochelator BSIH has been designed. BSIH cannot bind iron until it is activated by certain reactive oxygen species to active chelator SIH. The aim of this study was to examine the toxicity and cytoprotective potential of BSIH, SIH, and their decomposition products against hydrogen peroxide-induced injury of H9c2 cardiomyoblast cells. Using HPLC, we observed that salicylaldehyde was the main decomposition products of SIH and BSIH, although a small amount of salicylic acid was also detected. In the case of BSIH, the concentration of formed salicylaldehyde consistently exceeded that of SIH. Isoniazid and salicylic acid were not toxic nor did they provide any antioxidant protective effect in H9c2 cells. In contrast, salicylaldehyde was able to chelate intracellular iron and significantly preserve cellular viability and mitochondrial inner membrane potential induced by hydrogen peroxide. However it was consistently less effective than SIH. The inherent toxicities of salicylaldehyde and SIH were similar. Hence, although SIH - the active chelating agent formed following the BSIH activation - undergoes rapid hydrolysis, its principal decomposition product salicylaldehyde accounts markedly for both cytoprotective and toxic properties.

Protective effect of Danggui (Radix Angelicae Sinensis) on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells.

BackgroundDanggui (Radix Angelicae Sinensis) is an herb often used in Traditional Chinese medicine. It is used to promote blood flow and has been used in the treatment of myocardial ischemia-reperfusion injury in animal models. Angiotensin II (Ang II) has been shown to play important roles in mediating cardiovascular diseases, and may cause cardiac hypertrophy and apoptosis. This study aimed to investigate whether Danggui has protective effects on Ang II-induced apoptosis in H9c2 cardiomyoblast cells and study the mechanisms involved.MethodsWe evaluated the effect of Danggui on Ang II-induced apoptosis in an in vitro model. H9c2 cardiomyoblast cells were cultured in serum-free medium for 4 hr, then treated with Danggui (50, 100 μg/ml) 1 hr pre- or post-Ang II treatment. After a further 23 hr of culture, cells were harvested for analyses with assays for apoptosis markers and cell signaling pathways.ResultsOur results showed that Ang II induced upregulation of pro-apoptotic Bad, instability of the mitochondria membrane potential, cytochrome c release, caspase-9 and caspase-3 activation and cardiomyocyte apoptosis. Pre- or post-treatment with Danggui reversed all of the above Ang II-induced apoptotic effects in H9c2 cells. Furthermore, the JNK (SP600125) inhibitor completely blocked Danggui inhibition of caspase-3 activation in Ang II-treated H9c2 cells.ConclusionsOur results showed that Danggui either pre-treatment or post-treatment highly attenuated the Ang II-induced apoptosis in cardiomyoblast cells. The findings demonstrated that the anti-apoptosis effect of Danggui is mediated by JNK and PI3k inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/1472-6882-14-358) contains supplementary material, which is available to authorized users.

MTORC2 phosphorylation of Akt1: a possible mechanism for hydrogen sulfide-induced cardioprotection.

Hydrogen sulfide (H2S) is known to have cardiac protective effects through Akt activation. Akt acts as a ‘central sensor’ for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i) normoxia, (ii) global ischemia and (iii) ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH) release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×103 vs. 10±3×103 mmHg/min, p<0.05) and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05) compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05). Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism. These data suggest mTORC2 phosphorylation of Akt is a key mediator of H2S-induced cardioprotection in I/R.

2,3,7,8-Tetrachlorodibenzo- p-dioxin and β-naphthoflavone induce cellular hypertrophy in H9c2 cells by an aryl hydrocarbon receptor-dependant mechanism

Cigarette smoke is a major risk factor for cardiovascular diseases. It contains thousands of compounds that activate the aryl hydrocarbon receptor (AhR). In addition, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), the most potent AhR ligand, has been shown to cause cardiotoxic effects in several in vivo models. Although induction of CYP1 family is the most important effect of AhR activation, the role of CYP1 induction in mediating the cardiotoxic effect of TCDD is usually overlooked. Therefore, we investigated whether AhR activation causes a hypertrophic effect in H9c2 cells and we related this effect to changes in CYP gene expression. In the current study, the cardiac derived H9c2 cells were treated with two AhR ligands, TCDD and β-naphthoflavone (BNF), for 24 and 48 h. The expression of the hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and several CYP genes were measured by real-time PCR. Treatment of H9c2 cells with TCDD or BNF for 24 h caused a significant induction of CYP1A1, CYP1B1, and CYP4A1; however, there was no change in the expression of other genes. On the other hand, treatment of the cells with TCDD or BNF for 48 h caused a significant induction of the hypertrophic markers, ANP and BNP, and several CYP genes such as CYP1A1, CYP1B1, CYP2E1, CYP2J3, and CYP4F4 parallel to a significant increase in the cell surface area. Neither TCDD nor BNF increased the oxidative stress in H9c2 cells at all concentrations tested. Interestingly, resveratrol, an AhR antagonist, protected the cells from TCDD-induced hypertrophy. In conclusion, AhR ligands caused a hypertrophic effect in H9c2 cells which was associated with induction of several CYP genes which can be prevented by resveratrol.

4-Guanidino-n-butyl Syringate (Leonurine, SCM 198) Protects H9c2 Rat Ventricular Cells From Hypoxia-induced Apoptosis

In the present study, we examined the ability of a chemically synthesized compound based on the structure of leonurine, a phytochemical component of Herba leonuri, to protect H9c2 rat ventricular cells from apoptosis induced by hypoxia and serum deprivation, as a model of ischemia. The results revealed a concentration-dependent increase in cell viability associated with leonurine treatment, accompanied by a consistent decline in lactate dehydrogenase leakage into the culture medium. The fraction of annexin V-fluorescein isothiocyanate-positive cells was increased by hypoxia but reduced by leonurine. These changes were associated with increased expression of the antiapoptotic gene, Bcl-2, and reduced expression of the proapoptotic gene, Bax. Leonurine also reduced the cytosolic Ca overload induced by hypoxia. These results suggest that leonurine elicits potent cardioprotective effects in H9c2 cells, and these effects may be mediated by inhibition of intracellular Ca overload and apoptosis during hypoxia.

KR-32570, a novel Na + / H + exchanger-1 inhibitor, attenuates hypoxia-induced cell death through inhibition of intracellular Ca 2+ overload and mitochondrial death pathway in H9c2 cells

A novel Na + / H + exchanger-1 (NHE-1) inhibitor [5-(2-methoxy-5-chloro-5-phenyl)furan-2-ylcarbonyl]guanidine (KR-32570) has been previously demonstrated to elicit cardioprotective effect against ischemic injury in rat heart. In the present study, we examined the effects of KR-32570 on cell death induced by hypoxic insult in heart-derived H9c2 cells. Treatment with KR-32570 (1–10 μM) significantly reduced hypoxia-induced necrotic cell death (lactate dehydrogenase release) and apoptotic cell death (TUNEL-positivity, caspase-3 activity). KR-32570 also decreased the cytosolic and mitochondrial Ca 2+ overload induced by hypoxia. Inhibition of mitochondrial Ca 2+ overload by ruthenium red mimicked the anti-apoptotic effect of KR-32570. In addition, KR-32570 significantly recovered the large reduction in mitochondrial membrane potential (Δ Ψ m) and cytochrome c release induced by hypoxia. Taken together, our results suggest that a new NHE-1 inhibitor KR-32570 elicits potent cardioprotective effects in H9c2 cells, and its effects may be mediated by inhibition of intracellular Ca 2+ overload and mitochondrial death pathway during hypoxia.