H9c2 Myocardial Cells Research Articles

Protective Effect of miR-204 on Doxorubicin-Induced Cardiomyocyte Injury via HMGB1.

The toxicity of doxorubicin (DOX) limits its clinical application. Nevertheless, at present, there is no effective drug to prevent DOX-induced cardiac injury. miR-204 is a newly discovered miRNA with many protective effects on cardiovascular diseases. However, little research has been done on the effects of miR-204 on DOX-induced cardiac injury. Our study is aimed at investigating the effect of miR-204 on DOX-induced myocardial injury. An adenoassociated virus system was used to achieve cardiac-specific overexpression of miR-204. Two weeks later, the mice were intraperitoneally injected with DOX (15 mg/kg) to induce cardiac injury. H9c2 myocardial cells were used to validate the role of miR-204 in vitro. Our study showed that miR-204 expression was decreased in DOX-treated hearts. miR-204 overexpression improved cardiac function and alleviated cardiac inflammation, apoptosis, and autophagy induced by DOX. In addition, our results showed that miR-204 prevented DOX-induced injury in cardiomyocytes by directly decreasing HMGB1 expression. Moreover, the overexpression of HMGB1 could offset the protective effects of miR-204 against DOX-induced cardiac injury. In summary, our study showed that miR-204 protected against DOX-induced cardiac injury via the inhibition of HMGB1, and increasing miR-204 expression may be a new treatment option for patients with DOX-induced cardiac injury.

MiR-133b inhibits myocardial ischemia-reperfusion-induced cardiomyocyte apoptosis and accumulation of reactive oxygen species in rats by targeting YES1

To investigate the effect of miR-133b on cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion (I/R) and explore the mechanism. Thirty-six adult SD rats were randomized into sham-operated group, I/R group, AdmiR-NC group and AdmiR-133b group, and rat models of myocardial I/R were established in the latter 3 groups with myocardial injections of saline or recombinant adenoviruses in the left ventricle. The expression of MiR-133b was detected using RT-qPCR, and cardiac function of the rats was determined using FDP 1 HRV and BRS analysis system. Serum CK-MB and cTnI levels were determined by ELISA, myocardial injury was evaluated with HE staining, cardiomocyte apoptosis was detected by flow cytometry, and ROS content was determined using a DCFH-DA probe. In the in vitro experiment, H9C2 myocardial cells with hypoxia/reoxygenation (H/R) treatment were transfected with Mir-NC or MiR-133b mimic, and the cellular expression of MiR-133b, cell apoptosis, and ROS content were determined. Dual luciferase reporter assay was performed to verify the targeting relationship between miR-133b and YES1. The effects of pc-YES1 or miR-133b mimic transfection on YES1 expression, apoptosis, and ROS content in H9C2 cells were evaluated. Compared with those in I/R group, miR-133b expression was obviously up-regulated, LVEDP, cTnI and CK-MB levels were significantly decreased, and LVSP, +dp/dt, -dp/dt, HR and CF levels were increased in admiR-133b group (P < 0.01). The rats in admiR-133b group showed obviously reduced pathological damage, cell apoptosis and ROS content compared with those in I/ R group (P < 0.01). In H9C2 cells exposed to H/R, transfection with miR-133b mimic significantly up-regulated miR-133b expression and decreased cell apoptosis and ROS content (P < 0.01). The results of dual luciferase reporter assay suggested a direct targeting relationship between miR-133b and YES1, and MiR-133b mimic transfection significantly down-regulated YES1 protein expression in cells with H/R exposure (P < 0.01). Co-transfection with pc-YES1 reversed the effect of miR-133b overexpression on myocardial cell apoptosis and ROS accumulation. miR-133b can inhibit I/R-induced myocardial cell apoptosis and ROS accumulation by targeting YES1 to reduce myocardial I/R injury in rats.

The MicroRNA-210/Casp8ap2 Pathway Alleviates Hypoxia-Induced Injury in Myocardial Cells by Regulating Apoptosis and Autophagy.

This study was conducted to investigate the role of the miR-210/Caspase8ap2 pathway in apoptosis and autophagy in hypoxic myocardial cells. The miR-control, miR-210 mimic, and miR-210 inhibitor were transfected into rat myocardial H9C2 cells. The transfection efficiency of exogenous miR-210 was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). H9C2 cells were then treated with CoCl2 for 24, 48, and 72 h to generate a myocardial injury model. The apoptosis of H9C2 cells was assessed by flow cytometry. Additionally, a western blot assay was used to determine the expression of the autophagy-associated proteins light chain 3 (LC3), p62 and Beclin-1, and apoptosis-associated proteins Caspase8ap2, cleaved caspase 8, and cleaved caspase 3. We determined that a 48 h hypoxia treatment duration in H9C2 cardiomyocytes induced myocardial injury. Additionally, the overexpression of miR-210 significantly inhibited cell apoptosis. MiR-210 suppressed autophagy by upregulating p62 and downregulating LC3II/I in hypoxic H9C2 cells. Caspase8ap2 was a putative target of miR-210, miR-210 mediated apoptosis, and autophagy of H9C2 cells via suppressing Caspase8ap2. Furthermore, the expression of caspase 8, caspase 3, and Beclin-1 were decreased in response to miR-210. miR-210 exhibits anti-apoptosis and anti-autophagy effects, which alleviate myocardial injury in response to hypoxia.

Heat shock protein 70 expression protects against sepsis-associated cardiomyopathy by inhibiting autophagy.

Increasing evidence suggests that heat shock protein 70 (Hsp70) has a protective effect in sepsis-induced cardiomyopathy; however, the protective mechanism remains unclear. Previous studies have also implicated autophagy in sepsis-induced cardiomyopathy. The aim of the current study was to reveal the protective mechanisms of Hsp70 in sepsis-induced cardiomyopathy using a cecal ligation and puncture (CLP) rat sepsis model. The roles of Hsp70 and autophagy in sepsis-induced cardiomyopathy were investigated by pretreating rats with the Hsp70 inhibitor quercetin or the autophagy inhibitor 3-methyladenine (3-Ma) before CLP. We also investigated the protective mechanisms of Hsp70 and the relationship between Hsp70 and autophagy in vitro by stimulating H9c2 cells with lipopolysaccharide (LPS) to simulate sepsis. The result show that inhibition of Hsp70 promoted sepsis-induced death in rats, while inhibition of autophagy inhibited sepsis-induced death. These results suggested that both Hsp70 and autophagy were involved in sepsis-induced cardiomyopathy. Overexpression of Hsp70 in H9c2 myocardial cells in vitro suppressed LPS-induced apoptosis, while inhibition of autophagy with 3-Ma also decreased LPS-induced H9c2 cell apoptosis, suggesting that the protective effect of Hsp70 in sepsis-induced cardiomyopathy was related to autophagy regulation. Overall, these results suggested that Hsp70 protected against sepsis-induced cardiac impairment by attenuating sepsis-induced autophagy.

Attenuation of the Na/K‑ATPase/Src/ROS amplification signaling pathway by astaxanthin ameliorates myocardial cell oxidative stress injury.

The 3S, 3′S-ASTaxanthin (3S, 3′S-AST) isomer has strong antioxidant activity; however, its protective roles and potential mechanisms against oxidative stress damage in cardiomyocytes have not been investigated. Na+/K+-ATPase (NKA)/Src signal activation has an important role in increasing reactive oxygen species (ROS) production. The aim of the present study was to investigate the protective effects and mechanism of 3S, 3′S-AST on hydrogen peroxide (H2O2)-induced oxidative stress injury in H9c2 myocardial cells. The protective effects of 3S, 3′S-AST on H2O2-induced H9c2 cell injury was observed by measuring lactate dehydrogenase and creatine kinase myocardial band content, cell viability and nuclear morphology. The antioxidant effect was investigated by analyzing ROS accumulation and malondialdehyde, glutathione (GSH) peroxidase, GSH and glutathione reductase activity levels. The protein expression levels of Bax, Bcl-2, caspase-3 and cleaved caspase-3 were analyzed using western blotting to determine cardiomyocyte apoptosis. Western blot analysis of the phosphorylation levels of Src and Erk1/2 were also performed to elucidate the molecular mechanism involved. The results showed that 3S, 3′S-AST reduced the release of LDH and promoted cell viability, and attenuated ROS accumulation and cell apoptosis induced by H2O2. Furthermore, 3S, 3′S-AST also restored apoptosis-related Bax and Bcl-2 protein expression levels in H2O2-treated H9c2 cells. The phosphorylation levels of Src and Erk1/2 were significantly higher in the H2O2 treatment group, whereas 3S, 3′S-AST pretreatment significantly decreased the levels of phosphorylated (p)-Src and p-ERK1/2. The results provided evidence that 3S, 3′S-AST exhibited a cardioprotective effect against oxidative stress injury by attenuating NKA/Src/Erk1/2-modulated ROS amplification.

Visfatin relieves myocardial ischemia-reperfusion injury through activation of PI3K/Akt/HSP70 signaling axis.

Myocardial ischemia-reperfusion injury (MIRI) is the most common complication of ischemic cardiomyopathy, which severely affects the prognosis of patients. The purpose of this study was to investigate the protective effects of visfatin on the myocardium after ischemia-reperfusion (I/R) and its mechanism. Sprague Dawley rats were used to construct the MIRI model and visfatin was administrated intraperitoneally in rats to determine the protective effect of visfatin on myocardium after I/R. In addition, visfatin was used to treat rat myocardial cell line H9c2 cells and detect its effect on H9c2 cells. The effect of visfatin on the PI3K/Akt/HSP70 signaling axis in H9c2 cells was also detected to determine the mechanism of the myocardial protection of visfatin. The damage of cardiomyocytes in MIRI rats pretreated with visfatin was significantly improved compared with untreated MIRI rats. Visfatin also reduced the level of inflammation and apoptosis of cardiomyocytes in MIRI rats, reduced myocardial injury markers, and improved cardiac function. In vitro, visfatin also reduced inflammatory and apoptotic factors in H9c2 cells. In addition, visfatin also promoted the activity of the PI3K/Akt signaling pathway and increased HSP70 expression in H9c2 cells. The inhibition of the PI3K/Akt signaling pathway was found to attenuate the promotion of HSP70 by visfatin. SiRNA-HSP70 also attenuated the protective effect of visfatin on H9c2 cells. Visfatin reduces the inflammation and apoptosis levels of myocardial cells through the PI3K/Akt/HSP70 signaling axis, thereby reducing I/R-induced myocardial injury.

Clinical Significance of Serum PGC-1 Alpha Levels in Diabetes Mellitus with Myocardial Infarction Patients and Reduced ROS-Oxidative Stress in Diabetes Mellitus with Myocardial Infarction Model.

BackgroundIn this study, we explored the clinical significance of serum peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1) alpha levels in diabetes mellitus with myocardial infarction (DMMI) patients and investigated the possible mechanism.Materials and MethodsSerum samples were obtained from patients with DMMI or normal volunteer in Baoding First Center Hospital. C57BL/6 mice were induced by a single intraperitoneal (i.p.) injection of 100 mg/kg STZ (streptozocin) for in vivo model. Human myocardial cell lines H9C2 cells were induced with high glucose medium (33 mmol/L glucose) for in vitro model. Western blot was used to analyze the protein expressions in this study.ResultsSerum PGC-1 alpha levels were down-regulated in patients with DMMI. There was negative correlation between serum PGC-1 alpha levels and glycated hemoglobin, blood glucose or glucagon in DMMI patients. Recombination of PGC-1 alpha protein decreased the levels of glycated hemoglobin, blood glucose and glucagon, and inhibited oxidative stress and myocardial damage in mice of DMMI. Over-expression of PGC-1 alpha reduced reactive oxygen species (ROS)-oxidative stress, while down-regulation of PGC-1 alpha promoted ROS-oxidative stress via regulation of hemeoxygenase−1 (HO-1) expression in in vitro model of DMMI. The inhibition of HO-1 expression attenuated the anti-oxidation effects of PGC-1 alpha in vitro.ConclusionPGC-1 alpha attenuated ROS-oxidative stress in diabetic cardiomyopathy model, and PGC-1 alpha served as a potential intervention to alleviate DMMI in clinical applications.

6-Gingerol Pretreatment Alleviates Hypoxia/Reoxygenation-induced H9C2 Cardiomyocyte Injury by Inhibiting Oxidative Stress and Inflammation

To observe the effect of 6-gingerol (6-G) pretreatment on hypoxia/reoxygenation (H/R) induced injury in H9C2 myocardial cell and investigate its related mechanism. The H/R in vitro model of cardiomyocytes was prepared by conventional methods. In detail, H9C2 cells were added with the nitrogen-saturated hypoxic liquid, and placed in an incubator, mixed with gas (1% O 2, 5% CO 2, 94% N 2) applying for 15 min. After culturing for 3 h, the cells were taken out and placed in an incubator (37℃, 5% CO 2) for 1 h. Before establishing the cell model, the cells were pretreated with 6-G, and the cell viability was measured by MTT method to observe the protective effect of different concentrations of 6-G on H/R-induced cell damage. The 6-G mass concentration for pretreatment that led to the highest cell viability was used for follow-up experiments. DCFH-DA fluorescent probe was used to detect the effect of 6-G pretreatment on H9C2 oxidative stress level, and the intracellular oxidative stress was observed with fluorescence microscope and flow cytometry. Western blot method was used to detect the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in H/R-induced cell inflammatory responses. Compared with the H/R group, the cell viability of the 6-G+H/R group began to increase when the concentration of 6-G promoted to50 μg/mL. The cell viability was the highest after pretreated with 200 μg/mL 6-G. Therefore, 200 μg/mL was considered as the best 6-G intervention concentration for subsequent experiment. The content of reactive oxygen species (ROS) in the 200 μg/mL 6-G group had no significant changes compared with the control group (P>0.05), and the ROS fluorescence peak did not migrate significantly. However the ROS content in the H/R group increased significantly compared with the control (P<0.05), and the ROS fluorescence peak shifted to the right. Compared with the H/R group, the ROS content of the 6-G+H/R group decreased (P<0.05), and the ROS fluorescence peak shifted to the left. Compared with the control group, the expressions of TNF-α, IL-6, IL-1β in the 6-G group had no significant changes (P>0.05); the expressions of TNF-α, IL-6, IL-1β in the H/R group increased (P<0.05). Compared with H/R group, the expressions of TNF-α, IL-6 and IL-1β in 6-G+H/R group decreased (P<0.05). 6-G pretreatment can alleviate H/R-induced H9C2 myocardial injury, which may be related to the inhibition of oxidative stress and inflammatory responses.

Protective Effect of Pravastatin on Myocardial Ischemia Reperfusion Injury by Regulation of the miR-93/Nrf2/ARE Signal Pathway.

PurposeThis research intended to study the mechanism of pravastatin in myocardial ischemia reperfusion (I/R) injury.Patients and MethodsAltogether 70 male rats were selected and grouped into Sham operation group (Sham group), ischemia reperfusion group (I/R group), pravastatin pretreatment group (I/R+P group), I/R+miR-93-mimics, I/R+P+miR-93-mimics, I/R+Nrf2 siRNA, and I/R+P+Nrf2 siRNA group. The myocardial function of each group was detected.ResultsMyocardial I/R injury could lead to abnormal myocardial enzyme activity, inflammatory reaction and oxidative stress. However, pravastatin could significantly inhibit the activity of myocardial enzymes, alleviate inflammatory reaction and inhibit oxidative stress reaction, thus playing a protective role. Furthermore, cell experiments showed that pravastatin can alleviate the injury of H9C2 myocardial cells caused by I/R, inhibit the apoptosis of myocardial cells, and lead to a significant reduction in pro-apoptotic genes Bax, caspase-3 and caspase-9 transcription levels, an obvious increase in anti-apoptotic gene Bcl-2, and an increase in cell activity. After I/R induced injury, miR-93 level was significantly up-regulated and Nrf2 level was down-regulated. Over-expression of miR-93 or inhibition of Nrf2 expression would lead to further aggravation of I/R myocardial injury, increase the apoptosis rate of cells and decrease the activity of myocardial cells. Pravastatin administration could inhibit miR-93, activate and promote Nrf2 in myocardial tissue, and promote protein expression of downstream regulatory genes HO-1 and NQO1. In the I/R model, pravastatin was given. Over-expression of miR-93 or silencing Nrf2 could reverse the therapeutic effect of pravastatin on I/R.ConclusionPravastatin acts as a protector on myocardial ischemia reperfusion injury by regulating miR-93/Nrf2/ARE signaling pathway.

USP47 promotes apoptosis in rat myocardial cells after ischemia/reperfusion injury via NF-κB activation.

An increasing number of studies have associated nuclear factor-κB (NF-κB) activation to the progression of myocardial infarction (MI). Multiple members of the ubiquitin-specific protease (USP) family regulate the NF-κB signaling pathway. This study attempted to investigate the function and underlying mechanism of USP family members in MI. Monocytes were isolated from peripheral blood samples of MI and healthy control, and the expression of several USP family members and NF-κB was examined. After USP47 knockdown or overexpression, cell apoptosis, related protein levels, and NF-κB promoter activity were measured. We found that USP47 and NF-κB expression was significantly increased in MI, and USP47 was positively correlated with NF-κB. In a rat H9c2 myocardial cell ischemia/reperfusion (I/R) injury model, USP47 expression was increased in a time-dependent manner. USP47 knockdown decreased I/R injury-induced apoptosis, with increased survivin and cytoplasmic NF-κB and decreased cleaved caspase-3 and nuclear NF-κB. USP47-induced apoptosis was potently counteracted by the NF-κB inhibitor, and cytoplasmic NF-κB was increased and nuclear NF-κB was decreased. Luciferase reporter showed that USP47 promotes NF-κB promoter activity. These results suggested that USP47 expression may be correlated with MI progression. USP47 knockdown attenuated myocardial cell I/R injury by inhibiting apoptosis.

Protective Effects on Hypoxia Reoxygenation Cardiomyocytes by GLP-1R Agonists via PI3K/AKT Signaling Pathway

Objective: The aim of the current study was to observe the protective effect of GLP-1R agonist on hypoxia reoxygenation cardiomyocytes and to explore the role of GLP-1R/PI3K/AKT signaling pathway. Methods: H9c2 myocardial cells were randomly divided into four groups: control group, hypoxia reoxygenation model group (H/R), Exenatide group and PI3K/AKT signaling pathway inhibitor group. CCK-8 method was used to detect myocardial enzymes, and the expression of Caspase-3, Bcl-2 and Bax proteins was detected by Western blot. Results: The apoptosis rate in exenatide group was decreased than the H/R group. The levels of LDH and CK-MB in H/R group were higher than control group, which compared with the H/R group, exenatide group were decreased, and it in PI3K/AKT pathway inhibitor group were increased than exenatide group. The expression of Caspase-3 and Bax in H/R group was higher than that in control group group, and the level of Bcl-2 was decreased. Compared with H/R group, the levels of Caspase-3 and Bax in exenatide group were decreased, and the level of Bcl-2 was increased. Compared with exenatide group, the levels of Caspase-3 and Bax were significantly increased in the PI3K/AKT pathway inhibitor group, and the level of Bcl-2 was decreased. Conclusion: Exenatide has protective effects on the cardiomyocytes during hypoxia reoxygenation injury. The GLP-1R/PI3K/AKT signaling pathway may be involved in the process that exenatide inhibiting cardiomyocyte apoptosis.

The MicroRNA-210/Casp8ap2 Pathway Alleviates Hypoxia-Induced Injury in Myocardial Cells by Regulating Apoptosis and Autophagy

Aim: This study was conducted to investigate the role of the miR-210/Caspase8ap2 pathway in apoptosis and autophagy in hypoxic myocardial cells. Methods: The miR-control, miR-210 mimic and miR-210 inhibitor were transfected into rat myocardial H9C2 cells. The transfection efficiency of exogenous miR-210 was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). H9C2 cells were then treated with CoCl2 for 24, 48 and 72 h to generate a myocardial injury model. The apoptosis of H9C2 cells was assessed by flow cytometry. Additionally, a western blot assay was used to determine the expression of the autophagy-associated proteins light chain 3 (LC3), p62 and Beclin-1, and apoptosis-associated proteins Caspase8ap2, cleaved caspase 8 and cleaved caspase 3. Results: We determined that a 48 h hypoxia treatment duration in H9C2 cardiomyocytes induced myocardial injury. Additionally, the overexpression of miR-210 significantly inhibited cell apoptosis. MiR-210 suppressed autophagy by upregulating p62 and downregulating LC3II/I in hypoxic H9C2 cells. Since Caspase8ap2 was a putative target of miR-210, miR-210 mediated apoptosis and autophagy of H9C2 cells via suppressing Caspase8ap2. Furthermore, the expression of caspase 8, caspase 3 and Beclin-1 were decreased in response to miR-210. Conclusions:miR-210 exhibits anti-apoptosis and anti-autophagy effects, which alleviate myocardial injury in response to hypoxia.

FOXO3a-mediated long non-coding RNA LINC00261 resists cardiomyocyte hypoxia/reoxygenation injury via targeting miR23b-3p/NRF2 axis.

Ischemia/reperfusion (I/R)‐mediated acute myocardial infarction (AMI) is a major pathological factor implicated in the progression of ischemic heart disease (IHD). Long non‐coding RNA plays an important role in regulating the occurrence and development of cardiovascular disease. The aim of this study was to investigate the regulating role of LINC00261 in hypoxia/reoxygenation (H/R)‐induced cardiomyocyte apoptosis. The relative expression of LINC00261, miR‐23b‐3p and NRF2 were determined in rats I/R myocardial tissues and H/R‐induced cardiomyocytes. The rat model and cell model of LINC00261 overexpression were established to investigate the biological function of LINC00261 on H9C2 cell. The interaction between LINC00261, miR‐23b‐3p, NRF2 and FOXO3a was identified using bioinformatics analysis, luciferase reporter assay, RNA immunoprecipitation (RIP) assay, chromatin immunoprecipitation (CHIP) assay and qRT‐PCR. The expression of LINC00261 was significantly down‐regulated in myocardial tissues and H9C2 cell. Overexpression of LINC00261 improves cardiac function and reduces myocardium apoptosis. Interestingly, transcription factor FOXO3a was found to promote LINC00261 transcription. Moreover, LINC00261 was confirmed as a spong of miR23b‐3p and thereby positively regulates NRF2 expression in cardiomyocytes. Our findings reveal a novel role for LINC00261 in regulating H/R cardiomyocyte apoptosis and the potency of the LINC00261/miR‐23b‐3p/NRF2 axis as a therapeutic target for the treatment of MIRI.

Oxypaeoniflorin improves myocardial ischemia/reperfusion injury by activating the Sirt1/Foxo1 signaling pathway.

Myocardial ischemia/reperfusion (MI/R) injury is a leading cause of damage to cardiac tissues and is associated with high mortality and disability rates worldwide. Oxypaeoniflorin (OPA) has been found to be the main constituent of Paeonia veitchii Lynch. This study was conducted to explore the effect of OPA on MI/R injury and its potential mechanism. An in vivo MI/R injury model was established by transient coronary ligation in BALB/c mice, and an in vitro hypoxia/reoxygenation (H/R) injury model was established with rat cardiomyocyte H9c2 cells. Echocardiographic assessments demonstrated that OPA significantly reduced disruption of cardiac function and improved the indicators of ejection fraction (EF) and fractional shortening (FS). The enzyme-linked immunosorbent assay (ELISA) results suggested that OPA significantly reduced the release of myocardial infarction-related factors, such as the creatine kinase (CK-MB), cardiac troponin I (cTnI) and cardiac troponin T (cTnT). Additionally, hematoxylin-eosin (HandE) staining demonstrated that OPA markedly inhibited the myocardial apoptosis and necrosis caused by MI/R. Consistently, the results obtained from the cell counting kit-8 (CCK-8) and flow cytometry assays revealed that OPA obviously reversed the H/R-induced decrease in cell activity and increase in apoptosis of H9c2 cells. Furthermore, western blot assays indicated that OPA inhibited apoptosis by activating the Sirt1 (silent information regulator factor 2 related enzyme 1)/Foxo1(forkhead transcription factor FKHR) signaling pathway in myocardial tissues and H9c2 cells. Collectively, these novel findings are the first to provide strong evidence that OPA attenuates MI/R injury by activating the Sirt1 (silent information regulator factor 2 related enzyme 1)/Foxo1(forkhead transcription factor FKHR) signaling-mediated anti-apoptotic pathway.

TRAP1 attenuates H9C2 myocardial cell injury induced byextracellular acidification via the inhibition of MPTP opening.

Extracellular acidification leads to cardiac dysfunction in numerous diseases. Mitochondrial dysfunction plays an important role in this process. However, the mechanisms through which extracellular acidification induces mitochondrial dysfunction remain unclear. Tumor necrosis factor receptor-associated protein 1 (TRAP1) maintains mitochondrial function and cell viability in tumor and non-tumor cells. In the present study, extracellular acidification was found to induce H9C2 cell apoptosis, mitochondrial dysfunction and TRAP1 expression. The overexpression of TRAP1 attenuated H9C2 cell injury, while the silencing of TRAP1 exacerbated it. Moreover, mitochondrial permeability transition pore (MPTP) opening, which is associated with the mitochondrial apoptotic pathway and cell death, was also increased in acidic medium. The overexpression of TRAP1 inhibited MPTP opening, while the silencing of TRAP1 promoted it. The protective effect of TRAP1 on cardiomyocytes was abolished by the addition of a specific MPTP opening promoter. Similarly, a specific MPTP opening inhibitor reversed cell injury by silencing TRAP1. Taken together, the findings of the present study demonstrate that TRAP1 attenuates H9C2 cell injury induced by extracellular acidification by inhibiting MPTP opening.

Mechanism of N-acetylcysteine in alleviating diabetic myocardial ischemia reperfusion injury by regulating PTEN/Akt pathway through promoting DJ-1.

Ischemic heart disease is the main cardiovascular complication of diabetes patients which is mainly caused by oxidative stress. DJ-1 is the key regulator for myocardial protection through inhibiting phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and activating Akt (also known as PKB or protein kinase B). This research is to investigate whether the antioxidant N-acetylcysteine (NAC) could alleviate diabetic myocardial ischemia/reperfusion (I/R) injury by the protective molecule DJ-1. DJ-1 in rat myocardial H9c2 cells and cardiac tissue was respectively knocked down by siRNA and adeno-associated virus (AAV). From the present study, it could be found that compared with high glucose (HG)-normal (N)/DM group, hypoxia/reoxygenation (H/R) or I/R injury can aggravate oxidative stress injury and apoptosis rate of myocardial cells, inhibit the expression of Bcl-2, activate the BAX and cleaved caspase-3 (c-caspase-3) protein and PTEN/Akt pathway. However, in the groups of HG-N, DM, HG-N+I/R and DM+I/R, NAC can significantly reduce oxidative stress injury and apoptosis rate of myocytes, promote the Bcl-2 and DJ-1 molecules, inhibit BAX and c-caspase-3 protein and PTEN/Akt pathway. Compared with HG-N+I/R+NAC and DM+I/R+NAC groups, the oxidative stress injury, apoptosis rate of myocardial cells and heart tissues increased after the knockdown of DJ-1, the expression of Bcl-2 and DJ-1 were inhibited, the BAX and c-caspase-3 expression was increased, and PTEN/Akt pathway was activated. Taken together, the findings suggest that NAC can reduce I/R injury in diabetic myocardium by up-regulating the PTEN/Akt pathway through the level of DJ-1.

L-Carnitine Reduces Myocardial Oxidative Stress and Alleviates Myocardial Ischemia-Reperfusion Injury by Activating Nuclear Transcription-Related Factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) Signaling Pathway.

BackgroundMyocardial ischemia-reperfusion injury (IRI) is an important injury mechanism of myocardial infarction. The purpose of this study was to explore the effects of L-carnitine (LC) on myocardial IRI and its mechanism.Material/MethodsThe IRI model was made by ligating the left anterior descending coronary artery. Then, we injected LC intraperitoneally into the rats of the experimental group to assess the effect of LC on IRI rats by use of serum markers, Western blot, and qRT-PCR. H9c2 cells were cultured and then treated with hypoxia-reoxygenation. The effect of LC on oxidative stress, apoptosis, and nuclear transcription-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway of H9c2 cells were detected by Western blot, RT-PCR, and flow cytometry.ResultsLC significantly reduced malondialdehyde (MDA), creatine kinase (CK), and lactate dehydrogenase (LDH) levels in rat myocardial tissue and increased superoxide dismutase (SOD) expression. LC also increased the expression of SOD1/2 and Bcl-2 in rat myocardial tissue and H9c2 cells and decreased the expression of caspase3/8 and Bax. In addition, LC increased the expression of Nrf2/HO-1 signaling pathway-related molecules in H9c2 cells and increased the activity of the Nrf2/HO-1 signaling pathway. Moreover, inhibition of the Nrf2/HO-1 signaling pathway attenuated the protective effect of LC on H9c2 cells.ConclusionsLC can activate the Nrf2/HO-1 signaling pathway and reduce oxidative stress and apoptosis in cardiomyocytes, thereby reducing myocardial IRI.

SAT-562 Angiotensin II Induces Aldosterone Synthesis in the Rat Heart Stressed by Angiotensin II

Aldosterone (Aldo) causes myocardial injury and fibrosis. While most Aldo is made by the adrenal zona glomerulosa; there have been controversial reports that Aldo is also synthesized in the heart; such myocardial synthesis of Aldo might contribute to myocardial injury. We induced cardiac fibrosis in rats by infusing angiotensin II (AngII) @ 500 ng/kg/min via subcutaneous pumps. After 4 weeks, circulating corticosterone increased about 400-fold from ~29 nM to ~11 μM. Aldo synthesis in isolated mitochondria (mito) was assessed by conversion of tritiated deoxycorticosterone to Aldo; AngII infusion doubled Aldo synthesis, and this augmented synthesis was inhibited in mito from rats receiving AngII + telmisartan, which inhibits the binding of AngII to the AT1 receptor. Western blotting showed P450c11AS (Aldo synthase) was also stimulated by AngII and inhibited by telmisartan in both rat heart and H9c2 myocardial cells. 2-dimentional native PAGE and mass spectrometry showed that a 290-kDa complex on the inner mitochondrial membrane (IMM) contained P450c11AS, Tom22 (a translocase associated with the outer mitochondrial membrane, OMM), and StAR (the steroidogenic acute regulatory protein). Immunocytochemistry and transmission electron microscopy monitoring of immune-gold particles confirmed that P450c11AS, Tom22, and StAR were associated with the mito, that P450c11AS and StAR were associated with the IMM and that P450c11AS and StAR, but not Tom22, were increased by AngII. Cardiac Aldo synthesis required myocardial expression of P450c11AS, but expression of P450scc, the initial steroidogenic enzyme that converts cholesterol to pregnenolone, was undetectable, indicating the heart cannot make Aldo de novo from cholesterol. The only known action of StAR is to promote the movement of cholesterol from the OMM to IMM; nevertheless, we found that intramitochondrial StAR is required for Aldo synthesis; protein crosslinking with BS3 showed that Tom22 forms a bridge between StAR and P450c11AS. This is the first activity ascribed to intramitochondrial StAR, but the manner by which StAR promotes P450c11AS activity is unclear. As P450scc was undetectable, and circulating concentrations of corticosterone approached the Km (~28 μM) for the use of corticosterone as a substrate for P450c11AS, we suggest that cardiac P450c11AS uses circulating steroids for substrate. Thus the stressed heart produces aldosterone using a previously undescribed intramitochondrial mechanism that involves P450c11AS, Tom22 and StAR

TRIM59 attenuates inflammation and apoptosis caused by myocardial ischemia reperfusion injury by activating the PI3K/Akt signaling pathway.

Myocardial ischemia reperfusion injury (MIRI) is a common factor in heart-related diseases. The aim of this study was to explore the effect of TRIM59 gene on MIRI and its mechanism. Rats were used to construct MIRI models, and TRIM59 gene was overexpressed in myocardium by Entranster technique to detect the effects of TRIM59 on myocardial oxidative stress, myocardial injury, and ATPase. In addition, rat myocardial H9c2 cells were cultured, and a hypoxia-reoxygenation model of H9c2 cells was constructed to detect the effect of TRIM59 overexpression on the inflammation and apoptosis of H9c2 cells. Finally, the PI3K/Akt signaling pathway inhibitor LY294002 was used to study the effect of TRIM59 on the PI3K/Akt signaling pathway. Overexpression of TRIM59 in vivo effectively reduced the expressions of MDA, CK, and LDH, and increased the expression of SOD and the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase. In addition, overexpression of TRIM59 in H9c2 cells significantly reduced the expression of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and oxidative stress (ROS) levels. TRIM59 also significantly increased the activity of PI3K/Akt signaling pathway and promoted the phosphorylation of Akt. TRIM59 reduces the level of inflammation and apoptosis of myocardial cells caused by MIRI by activating the PI3K/Akt signaling pathway, thereby reducing myocardial injury.

Effects of hypoxia/reoxygenation on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes

Objective To investigate the effects of hypoxia/reoxygenation (HR) on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes. Methods The cultured H9C2 cardiomyocytes were divided into two groups according to the random number table method: a normal control (Ctrl) group and an HR group. The H9C2 cardiomyocytes in the HR group were deprived of oxygen and glucose for 8 h in a hypoxic incubator, followed by reoxygenation for 12 h. Cell damage was assessed through detection of lactate dehy-drogenase (LDH) content in culture medium and determination of cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method (n=6). Apoptosis related proteins [cysteinyl aspartate-specific proteinase (caspase)-3, B-cell lymphoma/leuke-mia 2 (Bcl-2) and Bcl-associate x protein (Bax)], autophagy related proteins [(light chain 3 (LC3) Ⅱ/Ⅰ , p62, Beclin-1, and phosphory-lated mammalian target of rapamycin (p-mTOR)]and pyroptosis related proteins [(nod-like receptor pyrin domain 3 (NLRP3), apoptosis associated speck-like protein (ASC), caspase-1p20, interleukin (IL)-1β, and IL-18]were detected by Western blot (n=9). Cell apopto-sis, autophagy and pyroptosis were further assessed by immunofluorescence staining (n=6) Results Compared with the Ctrl group, the HR group presented reduced viability of H9C2 cardiomyocytes (P<0.05), increased LDH release (P<0.05), and up-regulated expres-sion of activated caspase-3 and Bax (P<0.05), as well as decreased expression of Bcl-2 (P<0.05). TUNEL staining showed that apoptosis significantly was enhanced in the cells after HR (P<0.05). These results indicated that apoptosis and cell damage were enhanced after HR. In contrast, the expression of p-mTOR and p62 increased (P<0.05), but the expression of LC3 Ⅱ/Ⅰ and Beclin-1 protein decreased(P<0.05). The immunofluorescence staining of LC3 Ⅱ/Ⅰ showed that the number of autophagosome within the cells increased after HR(P<0.05), indicating that autophagy in H9C2 myocardial cells was significantly inhibited. Meanwhile, the expression of NLRP3 inflamma-some, ASC and caspase-1p20 protein was remarkably up-regulated (P<0.05), and the expression of activated inflammatory cellular factors IL-1β and IL-18 increased (P<0.05), suggesting that NLRP3 inflammasomes were activated and pyroptosis enhanced after HR. Conclusions Autophagy is inhibited in H9C2 cardiomyocytes during HR injury, but pyroptosis and apoptosis are enhanced. Key words: Autophagy; Apoptosis; Pyroptosis; Cardiomyocytes; Hypoxia-reoxygenation injury