Transfected H9c2 Cells Research Articles

MiR-486 alleviates hypoxia/reoxygenation-induced H9c2 cell injury by regulating forkhead box D3.

Myocardial ischemia-reperfusion injury (IRI) is common in myocardial infarction and is the leading cause of death. Therefore, we investigated the effect of miR-486 on myocardial IRI to explore new targets for clinical treatment of IRI. We made a rat myocardial IRI model by obstructing the coronary arteries and detected the change of miR-486 expression in rat myocardial tissue. In addition, we induced injury of rat cardiomyocytes (H9c2 cells) by hypoxia/reoxygenation and transfected H9c2 cells with agomir-miR-486 and antagomir-miR-486 to detect the effects of miR-486 on the viability, inflammation and apoptosis of cardiomyocytes. We also used the Targetscan system to predict the direct target of miR-486 and verified the effect of miR-486 on downstream targets through the Dual-Luciferase reporter assay. HE staining and the detection of myocardial injury markers and inflammatory factors verified the effectiveness of IRI rat model. The expression of miR-486 in myocardium of IRI rats was significantly lower than that of the control group. The overexpression of miR-486 in H9c2 cells increased the viability of H9c2 cells and reduced the levels of inflammation and apoptosis. MiR-486 is predicted to have a potential binding site to forkhead box D3 (FOXD3). The Dual-Luciferase reporter assay proved that miR-486 can bind and degrade FOXD3 mRNA. In addition, the overexpression of FOXD3 was found to attenuate the protective effect of miR-486 on H9c2 cells. MiR-486 protects cardiomyocytes and reduces the levels of inflammation and apoptosis by binding and inhibiting FOXD3 activity. Therefore, miR-486 may become a new target for myocardial IRI therapy.

CD14 Involvement in Third-degree Skin Burn-induced Myocardial Injury via the MAPK Signaling Pathway.

This study investigated the potential genes and related pathways in burn-induced myocardial injury. Rat myocardial injury induced by third-degree burn and the histopathological structures, apoptosis, and cardiac injury markers were then identified using hematoxylin & eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, and enzyme-linked immunosorbent assay. Next, differentially expressed mRNAs were screened through next-generation sequencing (NGS), followed by functional annotation and key gene validation through quantitative reverse transcription-polymerase chain reaction. Subsequently, CD14 was screened out, and small interfering RNAs against CD14 were transfected to H9C2 cells to further verify the role of CD14 in burn-induced injury. The results showed that third-degree burn could markedly damage the structure of myocardial tissue, induce the apoptosis of myocardial cells, and increase the levels of myocardial injury-related markers, suggesting that burns could induce myocardial injury in rats. Besides, NGS data discovered that third-degree burn could result in 416 differentially upregulated mRNAs and 285 differentially downregulated mRNAs in myocardial tissue. It was also disclosed that differentially expressed mRNAs were mainly enriched in the phosphatidylinositol 3-kinase/Akt, mitogen-activated protein kinase (MAPK), and tumor necrosis factor signaling pathways. Furthermore, cell viability was significantly decreased in H9C2 cells treated with 10% rat burn serum. CD14 was significantly differentially expressed and screened out for further studies. Treatment with burn serum can significantly upregulate the phosphorylation level of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase and the expression of cleaved caspase-3 and downregulate the expression of Bcl2 when compared with those in negative control of small interfering RNA transfected H9C2 cells, whereas interfering with CD14 expression reversed the effects of burn serum. The study demonstrated that burn serum treatment could activate the MAPK signaling pathway to promote cell apoptosis, and it can be reversed by interfering with the expression of CD14.

Protective effects of dioscin against isoproterenol-induced cardiac hypertrophy via adjusting PKCε/ERK-mediated oxidative stress

Cardiac hypertrophy (CH) plays a central role in cardiac remodeling and is an independent risk factor for cardiac events. It is imperative to find drugs with protective effect on CH. Dioscin, one natural product, shows various pharmacological activities, and PKCepsilon (PKCε) plays an important role in the physiological hypertrophic responses. Thus, we aimed to investigate the possible protective effect of dioscin on CH through PKCε. In the present study, the isoproterenol (ISO)-induced H9C2 cells and primary cardiomyocytes models, and the ISO-induced rat model were established, and the pharmacodynamics and mechanism of dioscin were investigated. In vitro results prompted that, dioscin significantly improved ISO-induced cardiomyocyte hypertrophy, decreased the levels of cell size, protein content of single cell, reactive oxygen species, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), beta-myosin heavy chain (β-MHC). Moreover, in vivo, changes in histopathological of the animals caused by ISO are improved by dioscin. And dioscin decreased the index of CH and the levels of CK, MDA, LDH, and increased the levels of GSH, SOD and GSH-Px. Mechanism research showed that dioscin inhibited the expression levels of PKCε, and affected the expression levels of p-MEK, p-ERK, Nrf2, Keap1 and HO-1 to inhibit oxidative stress. In addition, the results of ISO-induced CH in PKCε siRNA transfected H9C2 cells and C57BL/6 mice further showed that the protective effect of dioscin on CH, which was mediated by inhibition of PKCε/ERK signal pathway. In summary, dioscin can effectively inhibit CH by regulating PKCε-mediated oxidative stress, which should be considered as one potent candidate for new drug research and development to treat CH in the future.

Inhibition of Autophagy Prevents Panax Notoginseng Saponins (PNS) Protection on Cardiac Myocytes Against Endoplasmic Reticulum (ER) Stress-Induced Mitochondrial Injury, Ca2+ Homeostasis and Associated Apoptosis

Endoplasmic reticulum (ER) stress is often closely linked to autophagy, hypoxia signaling, mitochondrial biogenesis and reactive oxygen species (ROS) responses. Understanding the interaction between ER stress, mitochondrial function and autophagy is of great importance to provide new mechanisms for the pathology, prevention and treatment of cardiovascular diseases. Our previous study has reported that Panax notoginseng saponins (PNS) protection against thapsigargin (TG)-induced ER stress response and associated cell apoptosis in cardiac myocytes is calcium dependent and mediated by ER Ca2+ release through RyR2. However, whether its protection upon ER stress and associated apoptosis is related to mitochondrial function and autophagy remains largely unknown. Here, we investigated the roles of PNS played in TG-induced mitochondrial function, ROS accumulation and autophagy. We also assessed its effects on Ca2+ homeostasis, ER stress response and associated cell death in the presence of autophagy inhibition. PNS-pretreated primary cultured neonatal rat cardiomyocytes were stimulated with TG to induce ER stress response. Mitochondrial potential (Δψm) was measured by JC-1. The general and mitochondrial ROS were measured by DCFH-DA and MitoSOX Red, respectively. Autophagy was evaluated by immunofluorescence of LC3, and immunoblots of LC3, p62, ATG7 and PINK1. In addition, mRFP-GFP-LC3 labeling was used to assess the autophagic influx. SiATG7 transfected H9c2 cells were generated to inhibit autophagy. Cytosolic and ER Ca2+ dynamics were investigated by calcium imaging. RyR2 oxidation was tested by oxyblot. Cell viability was examined by TUNEL assay. ER stress response and cell apoptosis were detected by immunoblots of BiP, CHOP, Cleaved Caspase-3 and Caspase-12. The results demonstrated that firstly, PNS protects against TG-induced mitochondrial injury and ROS accumulation. Secondly, PNS enhances autophagy in TG-induced cardiac myocytes. Thirdly, inhibition of autophagy diminishes PNS prevention of TG-induced mitochondrial injury, ROS accumulation and disruption of Ca2+ homeostasis. Last but not least, inhibition of autophagy abolishes PNS protection against TG-induced ER stress response and associated apoptosis. In summary, PNS protection against ER stress response and associated apoptosis is related to the regulation of mitochondrial injury and ROS overproduction via modulation of autophagy. These data provide new insights for molecular mechanisms of PNS as a potential preventive approach to the management of cardiovascular diseases.

CREG improves cardiac function by regulating cardiomyocytes' autophagy in diabetic myocardial infarction rats.

We aimed to observe the changes of cardiac function, cell morphology, cellular repressor of E1A-stimulated genes (CREG) and LC3-II after myocardial infarction (MI) in non-diabetic and diabetic rats, and to explore the relationship between myocardial damage and CREG and autophagy in diabetic rats. Diabetic rat models were prepared by intraperitoneal injection of low concentration (50 mg/kg) streptozotocin (STZ). MI models were established in normal rats and diabetic rats. The cardiac function of each group was detected by echocardiography. The pathological results of myocardial tissue in the infarcted area were observed under light microscope. The expression of CREG was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Myocardial protein CREG LC3-II expression was measured by Western blot. Autophagy levels were also detected at the cellular level. Construction of CREG overexpressing adenovirus transfected H9c2 cells, and injection of rats with AAV-CREG to achieve the purpose of overexpressing CREG. In vitro and in vivo experiments were conducted to explore the effects of CREG on autophagy and cardiac function in a diabetic MI model. Compared with the non-diabetic sham (NS) group, there were no marked differences in cardiac function and CREG levels in the diabetic sham (DS) group. Compared with the NS group, the cardiac function of the non-diabetic myocardial infarction (NI) group and the diabetic infarction myocardial (DI) group were reduced, and the levels of autophagy were increased. However, the cardiac function of the DI group was worse than that of the NI group, and the autophagy level of the DI group was lower than the NI group. The results at the cellular level were similar to the experiments in vivo. The overexpression of CREG in vivo or in vitro can increase autophagy levels and improve cardiac function. The exacerbation of myocardial injury after MI in diabetic rats may be related to the inhibition of CREG in myocardial cells of infarcted area by diabetes.

The effect of cell‐permeable peptides to limit TRPV‐1 activation during myocardial reperfusion injury

Cardiac reperfusion injury has been implicated in tissue damage caused when blood supply returns after the ischemia. During the reperfusion injury, transient receptor potential channel 1 (TRPV1) mediates cellular responses to stress such as heat, pain, and noxious stimuli. TRPV1 gates calcium and can regulate mitochondrial membrane potential. We hypothesize that the TRPV1 channel and its interaction with calcineurin have a specific role in mediating cardiac cellular injury. To test the hypothesis, adult cardiomyocytes from male Sprague-Dawley rats (225–300grams) were isolated and subjected to hypoxia-reoxygenation for one and two hours, respectively. Isolated adult cardiomyocytes were further treated with cell-permeable peptides and underwent cell death assays such as LDH measurement and Trypan Blue staining to measure cell viability. Moreover, flow cytometry assay was performed to identify the apoptotic rate of cell death upon peptide treatment. According to the results, AnnexinV-FITC/PI double staining demonstrates that cardiomyocytes undergoing hypoxia and reoxygenation (HR) enter the early apoptotic state more frequently than the necrotic state. There is also a significant two-fold difference between normoxia and HR groups in detecting the apoptotic rate. Synthesized peptide against the TRPV1-calcineurin interaction site, V1-cal, maintains a higher level of cell viability when cardiomyocytes were subjected to HR. V1-cal reduces the injury after hypoxia and reoxygenation in primary cardiomyocytes. These data reveal that TRPV1 has a significant role in cardioprotection. On a larger scale, controlling the interaction site between TRPV1 and calcineurin can possibly limit the reperfusion injury. Support or Funding Information The authors would like to thank American Physiological Society (APS) for providing financial support to Z. Zhanzak as a UGREF at Stanford University, School of Medicine, Department of Anesthesiology, Perioperative and Pain Medicine under the supervision of Professor Eric Gross. Annexin V-FITC/PI double staining flow cytometry of primary cardiomyocytes to detect apoptotic rate. LDH assay on primary adult cardiomyocytes Trypan Blue assay on H9C2 cells Immunofluorescence assay on transfected H9C2 cells. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

αB-crystallin regulates oxidative stress-induced apoptosis in cardiac H9c2 cells via the PI3K/AKT pathway

The present study was carried out to observe the protective effects of αB-crystallin protein on hydrogen peroxide (H2O2)-induced injury in rat myocardial cells (H9c2) and to investigate the mechanisms of these protective effects at the cellular level, which could provide the experimental basis for future applications of αB-crystallin in the treatment of cardiovascular disease. Western blotting was used to measure the expression of αB-crystallin in cultured H9c2 cells in vitro. A αB-crystallin recombinant expression vector, pcDNA3.1-Cryab, was constructed to transfect H9c2 cells for the establishment of cells that stably expressed αB-crystallin. A tetrazolium-based colorimetric assay (MTT test) was used to measure changes in the viability of the H9c2 cells at 1, 2, 3 and 4 h after induced by 150 μM H2O2 to establish a model of H2O2 injury to cells. H2O2 was applied to H9c2 cells that were stably transfected with αB-crystallin, and the effect of αB-crystallin overexpression on the viability of myocardial cells subjected to H2O2-induced injury was measured by the MTT assay. The effect of αB-crystallin overexpression on the H2O2-induced injury of H9c2 cells was also analyzed by flow cytometry. The mitochondrial components and cytoplasmic components of H9c2 cells were separated, and western blotting was used to measure the effect of αB-crystallin overexpression on the release of cytochrome c from the mitochondria. Western blotting was also used to measure the effect of αB-crystallin overexpression on the expression of the anti-apoptosis protein Bcl-2 and components of the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathway. The αB-crystallin recombinant expression vector pcDNA3.1-Cryab successfully transfected H9c2 cells, and H9c2 cells that were stably transfected with αB-crystallin were established after G418 selection. The measurements carried out by western blotting showed that αB-crystallin proteins are expressed in normal H9c2 cells, but the proteins' expression was much higher in pcDNA3.1-Cryab transfected cells (P < 0.01). The MTT assays showed that 4 h of H2O2 treatment induced significant injury in H9c2 cells (P < 0.01), but αB-crystallin overexpression can effectively antagonize the H2O2-induced injury to H9c2 cells (P < 0.05). The results of flow cytometry analysis showed that αB-crystallin overexpression can significantly reduce apoptosis in H2O2-injured H9c2 cells (P < 0.05). The results of western blotting showed that αB-crystallin overexpression in myocardial cells can reduce the H2O2-induced release of cytochrome c from the mitochondria (P < 0.05), antagonize the H2O2-induced downregulation of Bcl-2 (P < 0.05) and magnify the decrease in phosphorylated AKT levels induced by H2O2 injury (P < 0.05). The overexpression of αB-crystallin has a protective effect on H2O2-injured H9c2 cells, and αB-crystallin can play a protective role by reducing apoptosis, reducing the release of cytochrome c from the mitochondria and antagonizing the downregulation of Bcl-2 expression. The protective effects of αB-crystallin may be related to the PI3K/AKT pathway.

INSULIN AMELIORATES MIR-1-INDUCED INJURY IN H9C2 CELLS UNDER OXIDATIVE STRESS VIA AKT ACTIVATION

ObjectivesGrowing evidence indicates that aberrant up-regulation of microRNA-1 (miR-1) occurs in ischaemic myocardium. In addition, insulin elicits metabolism-independent cardioprotection against cardiovascular diseases. The aim of this study was to determine...

Heat shock protein 27 regulates oxidative stress-induced apoptosis in cardiomyocytes: mechanisms via reactive oxygen species generation and Akt activation

Increased reactive oxygen species (ROS) formation, which in turn promotes cardiomyocytes apoptosis, is associated with the pathogenesis and progression of various cardiac diseases such as ischemia and heart failure. Recent studies have shown that over expression of heat shock protein 27 (Hsp27) confers resistance to cardiac ischemia/reperfusion injury. However, not much is known about the regulation of myocyte survival by Hsp27. The rat cardiac cell line H9c2, with a stable overexpression of Hsp27, was established, with empty vector transfected H9c2 cells as controls. Following the cells challenged by Hydrogen Peroxide (H2O2), lactate dehydrogenase (LDH) release, apoptosis, intracellular ROS, cell morphology, mitochondrial transmembrane potential and the activation of serine/threonine kinase Akt were determined. Along with marked suppression of H2O2-induced injury by Hsp27 overexpression in H9c2 cells, ROS generation and the loss of mitochondrial membrane potential were also significantly depressed. Furthermore, augmented Akt activation was observed in Hsp27 overexpressed H9c2 cells following H2O2 exposure. Hsp27 inhibits oxidative stress-induced H9c2 damage and inhibition of ROS generation and the augmentation of Akt activation may be involved in the protective signaling.

Caveolin-1 Down-Regulation Inhibits Insulin-Like Growth Factor-I Receptor Signal Transduction in H9C2 Rat Cardiomyoblasts

Caveolin (Cav)-1, the major caveolar protein, directly interacts with IGF-I receptor (IGF-IR) and its intracellular substrates. To determine the role of Cav-1 in IGF-IR signaling, we transfected H9C2 cells with small interfering RNA specific for Cav-1-siRNA. The selective down-regulation of Cav-1 (90%) was associated with a smaller reduction of Cav-2, whereas Cav-3 expression was unaffected. A significant reduction of IGF-IR tyrosine phosphorylation in Cav-1-siRNA H9C2 cells was found compared with H9C2 control cells (Ctr-siRNA). The reduced IGF-IR autophosphorylation resulted in a decrease of insulin receptor substrate-1, Shc, and Akt activation. In addition, in Cav-1-siRNA H9C2 cells, IGF-I did not prevent apoptosis, suggesting that Cav-1 is required to mediate the antiapoptotic effect of IGF-I in cardiomyoblasts. The down-regulation of Cav-1 decreased IGF-IR activation and affected the ability of IGF-I to prevent apoptosis after serum withdrawal also in human umbilical vein endothelial cells. These results demonstrate that: 1) Cav-1 down-regulation negatively affects IGF-IR tyrosine phosphorylation; 2) this effect causes a reduced activation of insulin receptor substrate-1, Shc, and Akt; and 3) Cav-1 is involved in IGF-IR antiapoptotic signaling after serum deprivation.

Elucidating the Role of SNPs in AKT1 Gene Expression

AKT1 is part of the AKT family of intracellular kinases that regulates cell growth, proliferation, survival, and metabolism. In data pending publication, we identified a haplotype upstream of AKT1 that drives human variation in risk for metabolic syndrome and insulin resistance. Two SNPs in this haplotype, -C11898A and -C8166T, alter evolutionarily conserved nucleotides, and have been shown to alter enhancer and repressor activity in muscle, bone, and fat cells. Given the importance of AKT1 in the heart, we investigated the functional consequences of these upstream regulatory SNPs in heart cells. We transfected H9C2 cells with luciferase vectors, with (Pro) and without (Basic) the SV40 promoter, containing haplotype 1 (ancestral) or haplotype 2 (protects from metabolic syndrome) sequences derived from human subjects. In C8166T Pro vectors, haplotype 2 showed more relative light units (RLU) than haplotype 1, while C11898A Pro vectors did not show a difference in RLU between the two haplotypes. The C8116T region, consistent with haplotype 2 sequences that are protective against metabolic syndrome, shows greater enhancer activity in heart cells. This work is supported by funds from the Craig Summer Research Fellowship, UIC (JTY), by grant RO1 NS40606 and the Parsons Family Foundation (EPH), and by funds from the CCVR, UIC (PHG).

ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells.

Ischemia induces apoptosis as well as necrosis of cardiac myocytes. We recently reported the cloning of a cDNA that encodes an apoptotic inhibitor, ARC, that is expressed predominantly in cardiac and skeletal muscle. In the present study, we examined the ability of ARC to protect rat embryonic heart-derived H9c2 cells from apoptosis induced by hypoxia, a component of ischemia. We found that H9c2 cells express ARC and that exposure to hypoxia substantially reduces ARC expression while inducing apoptosis. Transfected H9c2 cells in which cytosolic ARC protein levels remain elevated during hypoxia were significantly more resistant to hypoxia-induced apoptosis than parental H9c2 cells or H9c2 cells transfected with a control vector. Loss of endogenous ARC in the cytosol of H9c2 cells was associated with translocation of ARC from the cytosol to intracellular membranes, release of cytochrome c from the mitochondria, activation of caspase-3, poly(ADP-ribose)polymerase (PARP) cleavage, and DNA fragmentation. All of these events were inhibited in H9c2 cells overexpressing ARC when compared with control cells. In contrast, caspase inhibitors prevented PARP cleavage but not cytochrome c release, suggesting that exogenously expressed ARC acts upstream of caspase activation in this model of apoptosis. These results demonstrate that ARC can protect heart myogenic H9c2 cells from hypoxia-induced apoptosis, and that ARC prevents cytochrome c release by acting upstream of caspase activation, perhaps at the mitochondrial level.

Expression of fibroblast growth factor receptor-1 in rat heart H9c2 myoblasts increases cell proliferation.

Basic fibroblast growth factor (FGF-2) plays an important role in myocardial growth and development and in particular cardiac myocyte proliferation. FGF-2 exerts its effects by binding to cell surface receptors (FGFR-1) of the tyrosine kinase family. We have detected the presence of both long and short isoforms of FGFR-1 in embryonic and adult mouse heart. In this report, we have examined the ability of long and short FGFR-1 isoforms to signal a mitogenic response. Assessment of RNA from rat myoblast H9c2 cells by reverse transcriptase-polymerase chain reaction and RNA blotting revealed that they were deficient in transcripts corresponding to long and short FGFR-1 species. Hybrid genes containing the cDNAs coding for long and short FGFR-1 isoforms directed by the myosin light chain-2 promoter and simian virus 40 enhancer sequences, were used to transiently transfect H9c2 cells. Total tyrosine phosphorylation was increased 2.0 and 2.6 fold in H9c2 cells transfected with the long and short FGFR-1 isoforms, respectively, compared to 'control' transfected H9c2 cells. This was accompanied by a 2.1 and 2.0 fold increase in DNA synthesis, as measured by tritiated thymidine incorporation, in H9c2 cells expressing the long and short FGFR-1 isoforms, respectively. To assess effects on proliferation, H9c2 cells were stably transfected with the myosin light chain-2/FGFR-1 cDNA genes. The rate of proliferation was increased 1.6 and 3.1 fold in H9c2 cells stably expressing the long and short FGFR-1 isoforms, respectively, compared to 'control' H9c2 cells. In contrast to non transfected H9c2 cells, treatment of H9c2 cells stably expressing long FGFR-1 with FGF-2 for 24 h resulted in a slight increase (1.3 fold, p < 0.02) in cell number. However, a greater response (1.5 fold, p < 0.0005) was observed with H9c2 cells stably expressing short FGFR-1 after treatment with FGF-2. These results suggest that both long and short FGFR-1 isoforms are capable of signalling a mitogenic response.