Induced Apoptosis In H9c2 Cells Research Articles

Lead-induced cardiomyocytes apoptosis by inhibiting gap junction intercellular communication via modulating the PKCα/Cx43 signaling pathway

ObjectiveThe aim of this study was to investigate the regulatory mechanism of Pb regulates gap junction intercellular communication to induced apoptosis in H9c2 cells. MethodsH9c2 cell line is used as the research object in this study, and treated with different concentrations of Pb acetate. Subsequently, Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. The levels of lactate dehydrogenase (LDH), aspartate transaminase (AST) and creatine kinase-MB (CK-MB) in the supernatants were measured using respective commercial enzyme-linked immune sorbent assay (ELISA) kits. Western blot was used to detect the expression of apoptosis-related protein in H9c2 cells in each group. Quantitative RT-PCR Analysis Total RNA was extracted from frozen H9c2 cells using Trizol reagent, the PKCα and Cx43 in the supernatant of H9c2 cells was determined by the BCA protein detection kit. ResultsH9c2 cells increased release of cardiac enzymes (LDH, AST, and CK-MB) and decreased cell survival rate, and the Cx43, p-Cx43, PKCα and p-PKCα protein levels showed a dose-dependent decrease after Pb treatment. PKCα was activated with PMA, the relative expression level of Cx43 protein increased significantly, the expression of Bcl-2 increased and Bax and Cyt-c decreased compared with Pb exposure group, and the myocardial enzymes (LDH, AST, and CK-MB) in cell culture supernatant decreased compared with Pb exposure group, indicating that the degree of cell damage was alleviated. Results showed that Pb inhibited PKCα activity, decreased the expression of total Cx43 and P-Cx43 protein, and aggravated myocardial injury. ConclusionsPb decrease gap junction intercellular communication, which induce apoptosis in H9c2 cells by inhibiting the PKCα/Cx43 signaling pathway.

Apurinic/apyrimidinic endonuclease 1 regulates palmitic acid-mediated apoptosis in cardiomyocytes via endoplasmic reticulum stress

Cardiomyocyte apoptosis caused by fat metabolism disorder plays an essential role in the pathogenesis of diabetic cardiomyopathy (DCM). Apurinic/apyrimidinic endonuclease 1 (APE1) has multiple functions, including regulating redox and DNA repair. However, the role of APE1 in the pathogenesis of DCM remains unclear. To investigate the mechanism of APE1 on high-fat induced apoptosis in H9C2 cells, we treated H9C2 cells with palmitic acid (PA) as an apoptosis model caused by hyperlipidemia. We found that PA reduced the viability and increased apoptosis of H9C2 cells by inducing up-regulation of APE1 protein and endoplasmic reticulum (ER) stress. APE1 knockdown enhanced PA-induced apoptosis, and ER stress and overexpression of APE1 demonstrated the opposite effect. Furthermore, APE1 regulated PA-induced apoptosis via ER stress. The APE1 mutant (C65A, lack of redox regulation) loses its protective effect against ER stress and apoptosis. These findings indicate that APE1 protects PA-induced H9C2 cardiomyocyte apoptosis through ER stress via its redox-regulated function. This study provided new insights into the therapy for DCM.

Propofol postconditioning ameliorates hypoxia/reoxygenation induced H9c2 cell apoptosis and autophagy via upregulating forkhead transcription factors under hyperglycemia

BackgroundAdministration of propofol, an intravenous anesthetic with antioxidant property, immediately at the onset of post-ischemic reperfusion (propofol postconditioning, P-PostC) has been shown to confer cardioprotection against ischemia–reperfusion injury, while the underlying mechanism remains incompletely understood. The FoxO transcription factors are reported to play critical roles in activating cardiomyocyte survival signaling throughout the process of cellular injuries induced by oxidative stress and are also involved in hypoxic postconditioning mediated neuroprotection, however, the role of FoxO in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown.MethodsRat heart-derived H9c2 cells were exposed to high glucose (HG) for 48 h (h), then subjected to hypoxia/reoxygenation (H/R, composed of 8 h of hypoxia followed by 12 h of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol (P-PostC) at the onset of reoxygenation. After having identified the optical concentration of propofol, H9c2 cells were subjected to H/R and P-PostC in the absence or presence of FoxO1 or FoxO3a gene silencing to explore their roles in P-PostC mediated protection against apoptotic and autophagic cell deaths under hyperglycemia.ResultsThe results showed that HG with or without H/R decreased cell viability, increased lactate dehydrogenase (LDH) leakage and the production of reactive oxygen species (ROS) in H9c2 cells, all of which were significantly reversed by propofol (P-PostC), especially at the concentration of 25 µmol/L (P25) (all P < 0.05, NC vs. HG; HG vs. HG + HR; HG + HR + P12.5 or HG + HR + P25 or HG + HR + P50 vs. HG + HR). Moreover, we found that propofol (P25) decreased H9c2 cells apoptosis and autophagy that were concomitant with increased FoxO1 and FoxO3a expression (all P < 0.05, HG + HR + P25 vs. HG + HR). The protective effects of propofol (P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a (all P < 0.05, HG + HR + P25 vs. HG + HR + P25 + siRNA-1 or HG + HR + P25 + siRNA-5).ConclusionIt is concluded that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.

LncRNA XIST knockdown suppresses hypoxia/reoxygenation (H/R)-induced apoptosis of H9C2 cells by regulating miR-545-3p/G3BP2.

This study was aimed at determining the roles and functions of lncRNA XIST/miR-545-3p/G3BP2 axis during hypoxia/reoxygenation (H/R)-induced H9C2 cell apoptosis. H9C2 cells were distributed into two groups, the H/R injury and control groups. High-throughput lncRNA sequencing was applied in the determination of differentially expressed lncRNAs between H/R-induced H9C2 cells and normal H9C2 cells. Real-time polymerase chain reactions (RT-PCR) were used to confirm the expression levels of lncRNA XIST in H/R-induced H9C2 cells. H9C2 cells were then transfected with lncRNA XIST recombinant plasmid (lncRNA XIST), sh-LINC XIST, agomiR-545-3p, antagomiR-545-3p, pcDNA-G3BP2, sh-G3BP2, and a corresponding negative control (NC). Bioinformatic analyses revealed that MiR-545-3p was a target for lncRNA XIST. This finding was confirmed by dual-luciferase reporter assay. The degree of cell apoptosis was evaluated by a flow cytometer. RT-PCR and western blot were performed to assess the apoptotic-related proteins in each group. A total of 859 differentially expressed lncRNAs (up-regulated=502, down-regulated=357) were identified. LncRNA XIST was found to be down-regulated in H/R-induced H9C2 cells while miR-545-3p was distinctly up-regulated. miR-545-3p was established to be a direct target for LncRNA XIST. LncRNA XIST significantly enhanced the apoptotic rate, while its inhibition suppressed the apoptotic rate. AgomiR-545-3p partially blocked the lncRNA XIST and enhanced the apoptosis of H/R-induced H9C2 cells. Moreover, miR-545-3p was shown to be a direct target for G3BP2. The overexpression of G3BP2 partially reversed the apoptotic effects of miR-545-3p on H/R-induced H9C2 cells. lncRNA XIST/miR-545-3p/GBP2 was found to be an apoptotic regulator in H/R-induced H9C2 cells.

MicroRNA 379 Regulates Klotho Deficiency-Induced Cardiomyocyte Apoptosis Via Repression of Smurf1.

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Transcription factor Sp1 ameliorates sepsis-induced myocardial injury via ZFAS1/Notch signaling in H9C2 cells

PurposeTo investigate whether Sp1 can ameliorate sepsis-induced myocardial injury and explore the potential molecular mechanism. MethodsThe embryonic cardiomyocyte cell line H9C2 and primary cultured mouse neonatal cardiomyocytes (CMNCs) were treated with LPS or phosphate-buffered saline (PBS). A mouse model of LPS-induced sepsis was established using male C57BL/6J mice and their cardiomyocytes were collected. Real-time reverse transcription-PCR (qRT-PCR) assay was used to detect the expression levels of Sp1 and ZFAS1 in cardiomyocytes. Western blotting analysis was used to assess the protein expression levels of Sp1, apoptosis-associated proteins and Notch signaling pathway related proteins. Luciferase assay was used to detect the interaction between Sp1 and ZFAS1. Cell transfection was used to generate H9C2 cells with overexpressed or knocked down of Sp1 or ZFAS1. MTT assay and flow cytometry analysis were used to test the cell proliferation and cell apoptosis ratio. ResultsOur data revealed that the expressions of ZFAS1 and Sp1 were significantly reduced in LPS-treated H9C2 cells and primary CMNCs. The downregulation of ZFAS1 and Sp1 were also found in cardiomyocytes obtained from LPS-challenged mice. LPS induced H9C2 cell apoptosis and depressed cell proliferation was ameliorated by ZFAS1 overexpression and aggravated by ZFAS1 knockdown. Mechanistically, Luciferase assay indicated that Sp1 could bind to ZFAS1, and positively regulated ZFAS1 expression. Moreover, Notch signaling pathway participates in H9C2 cell apoptosis mediated by Sp1. ConclusionThe present study demonstrates that Sp1 regulates LPS-induced cardiomyocyte apoptosis via ZFAS1/Notch signaling pathway, which may serve as therapeutic targets for sepsis-induced myocardial injury.

H3K9ac modification was involved in doxorubicin induced apoptosis by regulating Pik3ca transcription in H9C2 cells

AimWe evaluated the effect of H3K9ac modification on the transcriptional level of Pik3ca and the apoptosis induction effects of Pik3ca in the PI3K/AKT pathway in rat H9C2 cells. Main methodsH9C2 cells were cultured with 1uM doxorubicin for 8 h. The acetylation level of histone H3K9 in the transcriptional initiation area of Pik3ca was determined by CHIP-seq. The enrichment of mRNA fragment of Pik3ca transcription initiation region by H3K9ac antibody was detected by CHIP-qPCR, and the expression of Pik3ca was detected by real-time Polymerase Chain Reaction(rt-PCR) and western blot. The transcription efficiency of Pik3ca siRNA was detected by immunofluorescence and western blot. Cell Counting Kit8(CCK8) was used to detect the cell activity and flow cytometry was used to detect the apoptosis rate. Western blot was applied to assess the protein expression level of PI3K, P-AKT, AKT, Bcl2, BAX and cleaved-caspase3 in H9C2 cells. Key findingsIn doxorubicin-inducedH9C2 cells, the acetylation levels of histone H3K9 in the Pik3ca transcriptional initiation region significantly increased and promoted the transcription of Pik3ca. After knockdown of Pik3ca, the PI3K/AKT signaling pathway was inhibited, and the protein expression of Bcl2 was increased, the protein expression of BAX and cleaved-caspase3 were decreased. PI3K/AKT pathway activator partially reversed the effect of silencing Pik3ca. SignificanceIn summary, the elevated H3K9ac level in the Pik3ca transcriptional initiation region promoted the transcription of Pik3ca, and Pik3ca promoted doxorubicin induced apoptosis in H9C2 cells by activating the PI3K/AKT pathway, providing a new theoretical basis for the study of doxorubicin cardiomyopathy.

Resveratrol Reduces Cardiomyocyte Apoptosis Induced by I-R Injury Through Upregulating DJ-1

The NF-E2 related factor 2 (Nrf2) involves in anti-oxidative stress. DJ-1 is a multifunctional protein widely distributed in various tissues that plays a role in enhancing Nrf2 stability and promoting Nrf2 protein expression. Resveratrol (Res) is an important polyphenolic substance with various pharmacological effects, such as anti-oxidation and protection of cardiovascular and cerebrovascular. Our study intends to assess Resveratrol?s role in regulating DJ-1/Nrf2 pathway activity and alleviating rat cardiomyocyte I-R injury. Rat cardiomyocyte I-R injury model was established to detect DJ-1 and Nrf2 expressions, caspase-3 activity, malondialdehyde (MDA), and superoxide dismutase (SOD). The I-R rats were assigned into I-R group, I-R+Res 20 (20 mg/Kg) group, and I-R+Res 40 (20 mg/Kg) group. Rat H9C2 cells were assigned into control group, I-R group, and I-R+Res group followed by analysis of cell apoptosis and reactive oxygen species (ROS) by flow cytometry. The MDA content and caspase-3 activity were significantly elevated, while the SOD activity was obviously reduced in the cardiomyocyte of the I-R model rats. DJ-1 and Nrf2 expressions were apparently upregulated in I-R model. MDA content and caspase-3 activity were significantly decreased, whereas SOD activity, DJ-1, and Nrf2 expression levels were obviously elevated in Res treatment group with dose dependence. I-R treatment induced H9C2 cell apoptosis and intracellular ROS production, and upregulated DJ-1 and Nrf2 expressions. Res treatment further enhanced DJ-1 and Nrf2 levels, attenuated ROS production, and reduced apoptotic rate in H9C2 cells under I-R treatment. Res attenuates brain cell apoptosis and neuronal damage after I-R by increasing DJ-1 level, enhancing DJ-1/Nrf2 pathway, and elevating the anti-oxidation level.

Effects of PGC1α on myocardial ischemia reperfusion injury and the underlying mechanisms.

Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) controls mitochondrial biogenesis, but its role in cardiovascular diseases is unclear. The purpose of this study is to explore the effect of PGC1α on myocardial ischemia-reperfusion injury and the underlying mechanisms. The transverse coronary artery of SD rat was ligated for 30 minutes followed by 2 hours of reperfusion. Triphenyltetrazolium chloride (TTC) staining was performed to measure the area of myocardial infarction. Immunohistochemistry and Western blotting were used to detect the PGC1α expression in myocardium. The rat cardiomyocyte H9C2 was subjected to hypoxia/reoxygenation (H/R) with the knockdown of PGC1α or hypoxia- inducible factor 1α (HIF-1α), or with treatment of metformin. Western blotting was used to detect the expression of PGC1α, HIF-1α, p21, BAX, and caspase-3. CCK-8 was performed to detect cell viability, and flow cytometry was used to detect apoptosis and mitochondrial superoxide (mitoSOX) release. RT-qPCR was used to detect the mRNA expression of PGC1α and HIF-1α. Besides, chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter gene assay were applied to detect the transcriptional regulation effect of HIF-1α on PGC1α. After I/R, the PGC1α expression was increased in infarcted myocardium. H/R induced H9C2 cell apoptosis (P<0.001). The release of mitoSOX (P<0.001) and protein expression of PGC1α, and apoptosis-related p21, BAX, and caspase-3 were increased. However, knockdown of PGC1α reduced apoptosis (P<0.01) and mitoSOX release (P<0.001), and decreased protein expression of apoptosis-related gene. HIF-1α is bound to the promoter region of PGC1α. Knockdown of HIF-1α inhibited the transcription and protein expression of PGC1α and further to reduce the apoptosis (all P<0.001) and mitoSOX release (P<0.01). While overexpression of PGC1α reversed the effects caused by HIF-1α knockdown (all P<0.001). Metformin effectively reduced H/R-induced apoptosis (P=0.013), mitoSOX release (P<0.001), HIF-1α, PGC1α and apoptosis-related protein expression, recovered the cell viability (P<0.001), and reduced myocardial infarction (P=0.003). After I/R, HIF-1α up-regulates the expression of PGC1α, leading to an increase in ROS production and aggravation of injury. Metformin can inhibit the accumulation of HIF-1α during hypoxia and effectively protect myocardium from ischemia/reperfusion injury.

MicroRNA-144 Regulates Cardiomyocyte Proliferation and Apoptosis by Targeting TBX1 through the JAK2/STAT1 Pathway

It is currently believed that the TBX1 gene is one of the core genes of congenital heart disease (CHD). However, there are few studies on the abnormal regulation of TBX1 gene expression. The purpose of this work was to investigate the role of miR-144 and TBX1 in cardiac development by studying the regulatory relationship and mechanism of miR-144 on TBX1/JAK2/STAT1 in cardiomyocytes. Cell proliferation was detected by MTT and clone formation assay and cell cycle and apoptosis by flow cytometry. The levels of miR-144 and TBX1 in H9c2 cells were assessed by qRT-PCR. Dual luciferase reporter assay was used to validate the direct targeting of TBX1 with miR-144. The protein expression levels of TBX1 and its downstream proteins were measured by Western blot analysis. miR-144 inhibited H9c2 cell proliferation by arresting cells in G1 phase. Furthermore, miR-144 induced H9c2 cell apoptosis and activated the JAK2/STAT1 signaling pathway. Bioinformatic predictions and luciferase reporter assay showed that miR-144 directly targets TBX1. Co-overexpression of miR-144 and TBX1 upregulated cell proliferation by accelerating G1 to S phase transition and downregulated cell apoptosis through inhibiting the JAK2/STAT1 signaling pathway. miR-144 acts as a proliferation inhibitor in cardiomyocytes via the TBX1/JAK2/STAT1 axis and is therefore a potential novel therapeutic target for CHD treatment.

The inhibition of miR‑101a‑3p alleviates H/R injury in H9C2 cells by regulating the JAK2/STAT3 pathway.

Hypoxia/reoxygenation (H/R) is used as an in vivo model of ischemia/reperfusion injury, and myocardial ischemia can lead to heart disease. Therefore, it is necessary to prevent myocardial H/R injury to avoid the risk of heart disease. The aim of the present study was to investigate whether inhibiting microRNA (miR)-101a-3p attenuated H9C2 cell H/R injury, apoptosis mechanisms and key target proteins. Cell viability and apoptosis were determined by Cell Counting Kit-8 assays and flow cytometry using a cell apoptosis kit, respectively. The contents of creatine kinase (CK) and lactate dehydrogenase (LDH) were detected using colorimetric assays. Dual luciferase assays were carried out to determine if miR-101a-3p inhibited Janus kinase (JAK)2. Western blot analysis and reverse transcription-quantitative PCR were used to determine proteins levels and mRNAs expression. It was found that the inhibition of miR-101a-3p increased the growth of H9C2 cells and decreased H9C2 cell apoptosis during H/R injury. The inhibition of miR-101a-3p reduced the amounts of CK and LDH in H/R model H9C2 cells. The inhibition of miR-101a-3p lowered the levels of Bax, interleukin-6 and tumor necrosis factor-α, but raised the levels of phosphorylated (p)-STAT3 and p-JAK2 in H9C2 cells subjected to H/R injury treatment. miR-101a-3p mimic was found to inhibit H9C2 cell viability, raise p-JAK2 level and slightly increase p-STAT3 during H/R injury. AG490 induced H9C2 cell apoptosis, and decreased the levels of p-JAK2 and p-STAT3 during H/R injury. The data indicated that inhibiting miR-101a-3p reduced H/R damage in H9C2 cells and decreased apoptosis via Bax/Bcl-2 signaling during H/R injury. In addition, it was suggested that the inhibition of miR-101a-3p decreased H/R injury in H9C2 cell by regulating the JAK2/STAT3 signaling pathway.

Postganglionic Parasympathetic but Not Postganglionic Sympathetic Neuron Derived Exosomes Inhibit Hyperglycemia Induced Apoptosis in H9c2 Cells

Diabetic cardiomyopathy involves the two main types of cell death: apoptosis and necrosis. It is unclear, however, on whether apoptosis induced by hyperglycemia in a cell culture model can be inhibited by postganglionic parasympathetic neuron derived exosomes (PPN‐Exos). Postganglionic parasympathetic and postganglionic sympathetic neurons were isolated, cell cultured, and exosomes were isolated. H9c2 cells were utilized to induce apoptosis via hyperglycemia, which were then divided into 4 groups: 1. Control, 2. H9c2 + Glucose, 3. H9c2 + Glucose + PPN‐Exos, 4. H9c2 + Glucose + postganglionic sympathetic neuron‐derived exosomes (PSN‐Exos). An MTT assay kit was utilized to determine cell proliferation and viability, while apoptosis was determined using TUNEL staining and cell death detection (CDD) ELISA kit. The H9c2 cells that were exposed to glucose displayed significantly (p&lt;0.05) reduced cell viability, and a significant (p&lt;0.05) increase in TUNEL staining and CDD ELISA was present in hyperglycemic H9c2 cells compared to controls and this reduced cell viability and increased apoptosis was reversed with PPN‐Exos but not with PSN‐Exos, suggesting a cell protecting components are present in these PPN‐Exos. Our data on pro‐apoptotic gene expression of caspase‐3 and BAX was significantly (p&lt;0.05) increased in glucose treated H9c2 cells compared with control. These pro‐apoptotic genes were attenuated with PPN‐Exos treatment. Anti‐apoptotic protein Bcl‐2 levels were significantly (p&lt;0.05) reduced in hyperglycemia group and this decrease in Bcl‐2 was improved following PPN‐Exos treatment. In conclusion, our data demonstrate that apoptosis is present in hyperglycemic H9c2 cells mediated through caspase‐3 and BAX pathway is inhibited with PPN‐Exos but not with PSN‐Exos. Our data also suggest that PPN‐Exos must be containing a unique set of protective factors that needs further investigation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Overexpression of DJ-1 expression protects cardiomyocyte apoptosis induced by ischemia reperfusion.

Phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway plays an important role in regulating cell survival, apoptosis and oxidative stress (OS). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) can negatively regulate PI3K/AKT signaling pathway. DJ-1 is also a key negative regulator of PTEN. DJ-1-PTEN/PI3K/AKT signaling pathway regulates ischemia reperfusion (I-R). This study investigated the role of DJ-1 in affecting myocardial I-R injury. The rat myocardial I-R injury model was established. Expression of DJ-1 and PTEN in myocardial tissue was detected. The reactive oxidative species (ROS) content was detected using flow cytometry. Caspase-3 activity, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activities were determined by ultraviolet spectrophotometry. Rat cardiomyocytes H9C2 were cultured in vitro and divided into control group, I-R group, I-R+pIRES2-NC group, and I-R+pIRES2-DJ-1 group. Levels of DJ-1, PTEN and phosphorylated AKT (p-AKT) were detected. Cell apoptosis and ROS content were evaluated using flow cytometry. Compared with sham group, caspase-3 activity, MDA content, and PTEN expression were significantly increased, while SOD activity and DJ-1 levels were significantly reduced in myocardial tissue of I-R group (p<0.05). Compared with the control, I-R treatment markedly induced H9C2 cell apoptosis, decreased DJ-1 and p-AKT expression, and enhanced ROS production and PTEN expression. DJ-1 overexpression apparently down-regulated PTEN expression, elevated p-AKT level, and attenuated apoptosis and ROS production in H9C2 cells (p<0.05). Abnormal expression of DJ-1 plays a regulatory role in the process of myocardial I-R injury. Over-expression of DJ-1 can reduce myocardial cell I-R damage sensitivity by inhibiting PTEN expression, enhancing the activity of PI3K/AKT signaling pathway, reducing ROS production, and alleviating apoptosis.

Suppression of Stim1 reduced intracellular calcium concentration and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cells.

Objective: Previous studies have demonstrated Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) contributes to intracellular Ca2+ accumulation. The present study aimed to investigate the expression of STIM1 and its downstream molecules Orai1/TRPC1 in the context of myocardial ischemia/reperfusion injury (MIRI) and the effect of STIM1 inhibition on Ca2+ accumulation and apoptosis in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R).Methods: Expression of STIM1/Orai1/TRPC1 was determined by RT-PCR and Western blot in mice subjected to MIRI and H9C2 cardiomyocytes subjected to H/R. To knock-down STIM1, H9C2 cardiomyocytes was transfected with Stealth SiRNA. Apoptosis was analyzed by both flow cytometry and TUNEL assay. Cell viability was measured by MTT assay. Intracellular Ca2+ concentration was detected by laser scanning confocal microscopy using Fluo-3/AM probe. Furthermore, the opening of mitochondrial permeability transition pore (mPTP) was assessed by coloading with calcein AM and CoCl2, while ROS generation was evaluated using the dye DCFH-DA in H9C2 cardiomyocytes.Results: Expression of STIM1/Orai1/TRPC1 significantly increased in transcript and translation level after MIRI in vivo and H/R in vitro. In H9C2 cardiomyocytes subjected to H/R, intracellular Ca2+ accumulation significantly increased compared with control group, along with enhanced mPTP opening and elevated ROS generation. However, suppression of STIM1 by SiRNA significantly decreased apoptosis and intracellular Ca2+ accumulation induced by H/R in H9C2 cardiomyocytes, accompanied by attenuated mPTP opening and decreased ROS generation. In addition, suppression of STIM1 increased the Bcl-2/Bax ratio, decreased Orai1/TRPC1, and cleaved caspase-3 expression.Conclusion: Suppression of STIM1 reduced intracellular calcium level and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cardiomyocytes. Our findings provide a new perspective in understanding STIM1-mediated calcium overload in the setting of MIRI.

Jujuboside A attenuates norepinephrine-induced apoptosis of H9c2 cardiomyocytes by modulating MAPK and AKT signaling pathways.

Cardiomyocyte apoptosis is closely associated with the pathogenesis of heart failure. Jujuboside A (JUA) is a type of saponin isolated from the seeds of Zizyphus jujuba. In traditional Chinese medicine, it is believed that JUA possesses multiple biological effects, including antianxiety, antioxidant and anti‑inflammatory activities. The present study aimed to evaluate the effects of JUA on norepinephrine (NE)‑induced apoptosis of H9c2 cells and to investigate its underlying mechanisms. Rat H9c2 cardiomyocytes were pretreated with JUA and were then exposed to NE as an invitro model of myocardial apoptosis. A cell viability assay, scanning electron microscopy, transmission electron microscopy, flow cytometry assay, acridine orange/ethidium bromide staining, reverse transcription‑quantitative polymerase chain reaction and western blotting, all revealed that NE induced H9c2 cell apoptosis. The results demonstrated that NE inhibited cell viability, and enhanced cell damage and apoptosis of H9c2 cells. Conversely, pretreatment with JUA was able to reverse NE‑induced decreased cell viability and increased apoptosis. Furthermore, JUA suppressed upregulation of the B‑cell lymphoma 2(Bcl‑2)‑associated X protein/Bcl‑2 ratio, and inhibited the increased protein expression levels of cleaved caspase‑3 and cleaved caspase‑9 following NE exposure. However, the protein expression levels of cleaved caspase‑12 and cleaved caspase‑8 were not significantly altered following exposure to NE or JUA pretreatment. In addition, in JUA‑pretreated cells, the protein expression levels of phosphorylated (p)‑p38 and p‑c‑Jun N‑terminal kinase were downregulated compared with in NE‑treated cells. Furthermore, JUA regulated the activation of extracellular signal‑regulated kinase (ERK) in NE‑treated cells and significantly increased the expression levels of p‑AKT. Taken together, these data suggested that JUA may protect against NE‑induced apoptosis of cardiomyocytes via modulation of the mitogen‑activated protein kinase and AKT signaling pathways. Therefore, JUA may be considered a potential therapeutic strategy for the treatment of heart disease.

Nicotine induces H9C2 cell apoptosis via Akt protein degradation.

Smoking is highly associated with cardiovascular diseases. However, the effect of nicotine, a key ingredient in smoking products, on cardiomyocyte apoptosis remains controversial. The present study aims to clarify the role of nicotine on cardiomyocyte cell apoptosis and to investigate the underlying mechanism. In the present study, H9C2 cells were exposed to nicotine at various concentrations (0, 10 and 100µM) for 48h. Cell Counting Kit‑8 and TUNEL assays were performed to assess cell viability and apoptosis, respectively, and reverse transcription‑quantitative polymerase chain reaction and western blot analysis were used to investigate the mRNA and protein expression. PYR‑41, a ubiquitin E1 inhibitor, was employed to investigate whether the ubiquitin‑proteasome system was involved in the downregulation of Akt. An Akt1 overexpression plasmid was used to demonstrate the role of Akt in H9C2 cells apoptosis. Tetratricopeptide repeat domain3 (TTC3) small interfering RNA (siRNA) was used to investigate the effect of TTC3 on Akt protein degradation. The results demonstrated that nicotine induced apoptosis in H9C2 cells compared with control cells (P<0.05). The protein level of Akt was downregulated by nicotine in a concentration‑dependent manner (P<0.05). PYR‑41 treatment restored the protein level of Akt. The cell viability was significantly improved by Akt overexpression when cells were exposed to nicotine at 10µM, compared with control cells. Nicotine also upregulated the level of TTC3 mRNA (P<0.05) and the protein level of Akt, and cell viability was recovered by TTC3 siRNA. In conclusion, the current study demonstrated that nicotine induced H9C2 cell apoptosis via Akt protein degradation, which may be mediated by TTC3.

Pim-2 protects H9c2 cardiomyocytes from hypoxia/reoxygenation-induced apoptosis via downregulation of Bim expression.

We know that silencing Bim, a pro-apoptosis protein, significantly attenuates glucose and oxygen-deprived induced apoptosis in cardiomyocytes. However, the mechanisms underlying the regulation of the Bim activation in the heart have remained unknown. Pim-2 is one of three Pim serine/threonine kinase family members thought to be involved in cell survival and proliferation. H9c2 cardiomyocytes were subjected to a hypoxia/reoxygenation (H/R) condition in vitro, mimicking ischemic/reperfusion injury in vivo. H/R augmented the expression of Bim, Cyt C, and Pim-2 and induced H9c2 cell apoptosis. Overexpression of Pim-2 attenuated apoptosis which induced by H/R in H9c2 cells, via downregulation of Bim and Cyt C expression. Silencing of Pim-2 promoted H/R-induced apoptosis via upregulation of Bim and Cyt C expression. Co-IP revealed the interaction between Pim-2 and Bim protein, with Bim Ser65 phosphorylated by Pim-2. Furthermore, blocking proteasome activity by MG132 prevented Bim degradation, and Bim S65A mutation could reverse the anti-apoptotic role of Pim-2 which induced by H/R. These data demonstrated that Pim-2 is a novel Bim-interacting protein, which negatively regulates Bim degradation and protects H9c2 cardiomyocytes from H/R-induced apoptosis.

Resveratrol-induced autophagy promotes survival and attenuates doxorubicin-induced cardiotoxicity

Resveratrol (RSV) has many biological effects, including antitumor and antiviral activities, and vascular protection. Recent studies have suggested that RSV exerts its antitumor effects through induction of autophagy by an unknown mechanism. Doxorubicin (DOX) is a wide spectrum antitumor drug, but its clinical application is limited by its cardiotoxicity. This study evaluated whether the manipulation of autophagy could attenuate the cardiotoxic effects of DOX in vitro as well as in a rat model of DOX-induced cardiotoxicity. We found that DOX induced H9C2 cell apoptosis by inhibiting AMPK activation and promoting pro-apoptotic protein expression through p38MAPK/p53 signaling. RSV-treated H9C2 cells showed increased autophagy through the AMPK/mTOR/Ulk1 pathway. When DOX and RSV were combined, apoptosis was decreased, despite a slight increase in the autophagy ratio. The same result was observed in the rat model of DOX-induced cardiotoxicity. Injection with DOX or RSV alone, or in combination, for a week, resulted in a reduced apoptotic ratio in the combination group compared with the DOX alone group. Our results strongly indicate that this co-treatment strategy with RSV can attenuate the cardiotoxic effects of DOX. Our findings may have important clinical implications.

Pachymic acid protects H9c2 cardiomyocytes from lipopolysaccharide-induced inflammation and apoptosis by inhibiting the extracellular signal-regulated kinase 1/2 and p38 pathways.

Pachymic acid (PA), a lanostane-type triterpenoid and the major component of Poria cocos alcoholic extracts, has various pharmacological effects, including anti-inflammatory, anti-oxidative and anti-apoptotic. However, few studies have investigated the effects of PA on lipopolysaccharide (LPS)-induced H9c2 cell apoptosis and inflammation, or identified the possible mechanisms underlying these effects. In the present study, H9c2 cardiomyocytes were stimulated by LPS and treated with or without PA. The increased mRNA expression levels of tumor necrosis factor-α, interleukin (IL)-1 and IL-6 induced by LPS were attenuated following treatment with PA. PA also attenuated LPS-induced apoptosis, as determined by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and regulated the LPS-induced protein expression levels of caspase 3, 8 and 9. Furthermore, the phosphorylations of extracellular-regulated kinase (Erk)1/2 and p38 in the LPS-treated H9c2 cells were inhibited by PA. These results suggested that treatment with PA prevented the LPS-induced inflammatory and apoptotic response in cardiomyocytes, which may be mediated by inhibition of the Erk1/2 and p38 pathways.

Ghrelin Protects H9c2 Cardiomyocytes From Angiotensin II–induced Apoptosis Through the Endoplasmic Reticulum Stress Pathway

Ghrelin, a gastric hormone, exerts cardioprotective function by increasing myocardial contractility and vasodilation. Previous studies have reported that angiotensin II (Ang II) production increased in heart failure, which can induce cardiomyocyte apoptosis. In this study, we investigated the effect of ghrelin on Ang II-induced H9c2 cardiomyocyte apoptosis. The results showed that Ang II inhibited H9c2 cell viability, which was blocked by ghrelin. By annexin V-propidium iodide dual staining and 2'-deoxyuridine 5'-triphosphate nick end-labeling analysis, we found that Ang II induced H9c2 cell apoptosis, whereas coincubation of ghrelin with Ang II significantly reduced H9c2 cell apoptosis induced by Ang II. Simultaneously, the results revealed that ghrelin regulated the Ang II-induced imbalance of Bax and Bcl-2 expression and reduced Ang II-induced caspase-3 expression. Moreover, mRNA expressions of endoplasmic reticulum stress-related molecules GRP78, caspase-12, and C/EBP homologous protein were significantly upregulated by Ang II. However, their expressions were significantly inhibited by ghrelin. In addition, we found that ghrelin markedly inhibited Ang II-induced Ang II type 1 receptor expression. These data suggest that ghrelin may play an antagonistic role in Ang II-induced cardiomyocyte apoptosis via decreasing Ang II type 1 receptor expression and inhibiting the activation of endoplasmic reticulum stress pathway.