Apoptosis In Cardiomyoblast Cells Research Articles

Benomyl induced oxidative stress related DNA damage and apoptosis in H9c2 cardiomyoblast cells

Benomyl, benzimidazole group pesticide, has been prohibited in Europe and USA since 2003 due to its toxic effects and it has been still determined as food and environmental contaminant. In the present study, the toxic effect mechanisms of benomyl were evaluated in rat cardiomyoblast (H9c2) cells. Cytotoxicity was determined by MTT and NRU assay and, oxidative stress potential was evaluated by reactive oxygen species (ROS) production and glutathione levels. DNA damage was assessed by alkaline comet assay. Relative expressions of apoptosis related genes were evaluated; furthermore, NF-κB and JNK protein levels were determined. At 4 μM concentration (at which cell viability was >70%), benomyl increased 2-fold of ROS production level and 2-fold of apoptosis as well as DNA damage. Benomyl down-regulated miR21, TNF-α and Akt1 ≥ 48.75 and ≥ 97.90; respectively. PTEN, JNK and NF-κB expressions were upregulated. The dramatic changes in JNK and NF-κB expression levels were not observed in protein levels. These findings showed the oxidative stress related DNA damage and apoptosis in cardiomyoblast cells exposed to benomyl. However, further mechanistic and in vivo studies are needed to understand the cardiotoxic effects of benomyl and benzimidazol fungucides.

Tid1-S attenuates LPS-induced cardiac hypertrophy and apoptosis through ER-a mediated modulation of p-PI3K/p-Akt signaling cascade.

Myocardial dysfunction is clinically relevant? repercussion that follows sepsis. Tid 1 protein has been implicated in many biological process. However, the role of Tid 1 in lipopolysaccharide (LPS)-induced cardiomyocyte hypertrophy and apoptosis remains elusive. In the current research endeavor, we have elucidated the role of Tid1-S on LPS-induced cardiac hypertrophy and apoptosis. Interestingly, we found that overexpression of Tid1-S suppressed TLR-4, NFATc3, and BNP protein expression which eventually led to inhibition of LPS-induced cardiac hypertrophy. Moreover, Tid1-S overexpression attenuated cellular apoptosis and activated survival proteins p-PI3K and pser473 Akt. Besides this, Tid1-S overexpression enhanced ER-a protein expression. Collectively, our data suggest that Tid1-S plausibly enhance ER-a protein and further activate p-PI3K and p ser473 Akt survival protein expression; which thereby led to attenuation of LPS-induced apoptosis in cardiomyoblast cells. Interestingly, our data suggest that Tid1-S is involved in attenuation of cardiomyoblast cells damages induced by LPS.

Erratum to: Hypoxia Augments Increased HIF-1a and Reduced Survival Protein p-Akt in Gelsolin (GSN)-Dependent Cardiomyoblast Cell Apoptosis.

Cytoskeleton filaments play an important role in cellular functions such as maintaining cell shape, cell motility, intracellular transport, and cell division. Actin-binding proteins (ABPs) have numerous functions including regulation of actin filament nucleation, elongation, severing, capping, cross linking, and actin monomer sequestration. Gelsolin (GSN) is one of the actin-binding proteins. Gelsolin (GSN) is one of the actin-binding proteins that regulate cell morphology, differentiation, movement, and apoptosis. GSN also regulates cell morphology, differentiation, movement, and apoptosis. In this study, we have used H9c2 cardiomyoblast cell and H9c2-GSN stable clones to understand the roles and mechanisms of GSN overexpression in hypoxia-induced cardiomyoblast cell death. The data show that hypoxia or GSN overexpression induces HIF-1α expression and reduces the expression of survival markers p-Akt and Bcl-2 in H9c2 cardiomyoblast cells. Under hypoxic conditions, GSN overexpression further reduces p-Akt expression and elevates total as well as cleaved GSN levels and HIF-1α levels. In addition, GSN overexpression enhances apoptosis in cardiomyoblasts under hypoxia. Hypoxic challenge further induced activated caspase-3 and cell death that was attenuated after GSN knock down, which implies that GSN is a critical therapeutic target against hypoxia-induced cardiomyoblast cell death.

Hypoxia Augments Increased HIF-1α and Reduced Survival Protein p-Akt in Gelsolin (GSN)-Dependent Cardiomyoblast Cell Apoptosis.

Cytoskeleton filaments play an important role in cellular functions such as maintaining cell shape, cell motility, intracellular transport, and cell division. Actin-binding proteins (ABPs) have numerous functions including regulation of actin filament nucleation, elongation, severing, capping, cross linking, and actin monomer sequestration. Gelsolin (GSN) is one of the actin-binding proteins. Gelsolin (GSN) is one of the actin-binding proteins that regulate cell morphology, differentiation, movement, and apoptosis. GSN also regulates cell morphology, differentiation, movement, and apoptosis. In this study, we have used H9c2 cardiomyoblast cell and H9c2-GSN stable clones to understand the roles and mechanisms of GSN overexpression in hypoxia-induced cardiomyoblast cell death. The data show that hypoxia or GSN overexpression induces HIF-1α expression and reduces the expression of survival markers p-Akt and Bcl-2 in H9c2 cardiomyoblast cells. Under hypoxic conditions, GSN overexpression further reduces p-Akt expression and elevates total as well as cleaved GSN levels and HIF-1α levels. In addition, GSN overexpression enhances apoptosis in cardiomyoblasts under hypoxia. Hypoxic challenge further induced activated caspase-3 and cell death that was attenuated after GSN knock down, which implies that GSN is a critical therapeutic target against hypoxia-induced cardiomyoblast cell death.

CREB Negatively Regulates IGF2R Gene Expression and Downstream Pathways to Inhibit Hypoxia-Induced H9c2 Cardiomyoblast Cell Death.

During hypoxia, gene expression is altered by various transcription factors. Insulin-like growth factor-II (IGF2) is known to be induced by hypoxia, which binds to IGF2 receptor IGF2R that acts like a G protein-coupled receptor, might cause pathological hypertrophy or activation of the mitochondria-mediated apoptosis pathway. Cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB) is central to second messenger-regulated transcription and plays a critical role in the cardiomyocyte survival pathway. In this study, we found that IGF2R level was enhanced in H9c2 cardiomyoblasts exposed to hypoxia in a time-dependent manner but was down-regulated by CREB expression. The over-expression of CREB in H9c2 cardiomyoblasts suppressed the induction of hypoxia-induced IGF2R expression levels and reduced cell apoptosis. Gel shift assay results further indicated that CREB binds to the promoter sequence of IGF2R. With a luciferase assay method, we further observed that CREB represses IGF2R promoter activity. These results suggest that CREB plays an important role in the inhibition of IGF2R expression by binding to the IGF2R promoter and further suppresses H9c2 cardiomyoblast cell apoptosis induced by IGF2R signaling under hypoxic conditions.

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

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

Impact of LPS-induced cardiomyoblast cell apoptosis inhibited by earthworm extracts.

Dilong is an earthworm extract with a dense nutritional content, widely used in Chinese herbal medicine to remove stasis and stimulate wound healing. Earthworm extracts are traditionally used by indigenous people throughout the world. How this Dilong inhibits Lipopolysaccharide (LPS)-induced cardiomyoblast cell apoptosis is still unclear. This study investigates the Dilong extract effect on LPS-induced apoptosis in H9c2 cardiomyoblast cells. LPS (1 μg/ml) administration for 24 h induced apoptosis in H9c2 cells. Cell apoptosis was detected using MTT, LDH, TUNEL assay and JC-1 staining. Western blot analysis was used to detect pro-apoptotic and anti-apoptotic proteins. Dilong extract totally blocked the LPS impact, leading to the activation of anti-apoptotic proteins, Bcl-2 and Bcl-xL, stabilized the mitochondria membrane and down-regulated the extrinsic and intrinsic pro-apoptotic proteins, TNF-α, active caspase-8, t-Bid, Bax, active caspase-9 and active caspase-3. Dilong could potentially serve as a cardio protective agent against LPS-induced H9c2 cardiomyoblast cell apoptosis.

Cyclosporin A induces apoptosis in H9c2 cardiomyoblast cells through calcium-sensing receptor-mediated activation of the ERK MAPK and p38 MAPK pathways

The cardiotoxicity of cyclosporine A (CsA) limits its clinical application in extensive and long-term therapies. Our group has shown that CsA induces myocardium cell apoptosis in vivo and increases calcium-sensing receptor (CaSR) expression. However, its molecular mechanism remains unknown. The purpose of this study was to determine whether CaSR plays an essential role in CsA-induced apoptosis in H9c2 cells and to investigate the role of the mitogen-activated protein kinase (MAPK) signaling cascade in this process. H9c2 cells were treated with CsA in a dose-dependent manner, and decreased Bcl-2 expression, increased Bax expression, and caspase-3 activation were observed. In a time-dependent manner, CsA increased CaSR expression, activated the extracellularly regulated kinase (ERK) and p38 MAPK pathways, and inactivated the c-Jun N-terminal kinase (JNK) MAPK signaling pathway. When H9c2 cardiomyoblast cells pretreated with gadolinium chloride (GdCl(3)), a CaSR activator, were treated with CsA, decreased phosphorylation of ERK1/2, increased phosphorylation of p38, decreased Bcl-2 expression, increased Bax expression, and activated caspase-3 were observed. Cells pretreated with the CaSR inhibitor NPS2390 inhibited this process. Furthermore, the MEK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580 markedly blocked the effect of CsA on cell apoptosis, apoptotic-related protein expression, and caspase-3 activation. These findings showed that CsA induced apoptosis in H9c2 cells in vitro, and CaSR mediated the degradation of ERK MAPK and the upregulation of the p38 MAPK pathway involved in CsA-induced H9c2 cardiomyoblast cell apoptosis.

Leu27IGF2 plays an opposite role to IGF1 to induce H9c2 cardiomyoblast cell apoptosis via Gαq signaling

This study examines the role of IGF2/mannose 6-phosphate receptor (IGF2R) signaling in the signaling transduction regulation and cell apoptosis in H9c2 cardiomyoblast cells. However, it is difficult to recognize the distinct activation of IGF2 signaling without interfacing with IGFI receptor (IGF1R) after exposure to IGF2. Leu27IGF2, an analog of IGF2 that interacts selectively with the IGF2R, was used to specifically activate IGF2R signaling in this study. DNA fragmentation and TUNEL assay revealed that in contrast to IGF1 treatment preventing angiotensin II and AG1024-induced cell apoptosis, Leu27IGF2 appears to synergistically increase apoptosis in those cells. We further found cell apoptosis induction and an increase in the active form of caspase 3 in the treatment of cells with Leu27IGF2, but not IGF1. To detect the interaction between IGF2R and Galphaq using the immunoprecipitation assay, we found that IGF2R could directly interact with Galphaq and after treatment with Leu27IGF2 the binding ability of Galphaq to IGF2R had increased. This sequentially resulted in the phosphorylation of phospholipase C-beta, a key downstream modulator of Galphaq, on serine 537. Moreover, disruption of the Galphaq protein by small interferon RNA reduced the cell apoptosis induced by Leu27IGF2. Our findings demonstrate that IGF2R activation appears to induce cell apoptosis via Galphaq-deriving signaling cascades and its effect is completely different from IGF1R survival signaling.

Enhancement of AG1024-Induced H9c2 Cardiomyoblast Cell Apoptosis via the Interaction of IGF2R with Gα Proteins and Its Downstream PKA and PLC-β Modulators by IGF-Ⅱ

Our previous studies found that insulin-like growth factor-I receptor (IGF1R) signaling blockade caused cardiac hypertrophy, and that apoptosis is required for upregulating the IGF-II and the IGF-II/ mannose 6-phosphate receptor (IGF2R) gene. However, the role of IGF-II in the regulation of cell apoptosis through IGF2R is little known. In this study, we hypothesized that IGF-II may induce cell apoptosis through IGF2R but is dependent on IGF1R activity. Western blots and TUNEL assay revealed that in the presence of IGF1R, exogenous IGF-II acts, like IGF-I, would increase phospho-Akt through IGF1R, but does not affect the caspase 3 activation and apoptotic induction in H9c2 cardiomyoblast cells. Conversely, AG1024, an inhibitor of IGF1R activity, causes cell apoptosis, and the treatment with IGF-II further enhances this process, implying that it occurs through IGF2R. Moreover, immunoprecipitation assay revealed that treatment with IGF-II could enhance the interaction of IGF2R with Galphai and Galphaq but reduce its binding with Galphas, resulting in the reduction of phospho-PKA and the activation of PLC-beta. Taken together, these data provide new insight into the dual role of IGF-II in the control of IGF1R dependent cell apoptosis and involved activation of IGF2R signaling. Improving IGF1R activity and suppressing IGF2R may be a good strategy to prevent the progression of heart disease with cardiomyocyte apoptosis.

Cardiomyoblast apoptosis induced by insulin-like growth factor (IGF)-I resistance is IGF-II dependent and synergistically enhanced by angiotensin II

This study explores the synergistic effect of cardiomyoblast apoptosis induced by angiotensin II (Ang II) and Insulin-like growth factor (IGF)-I resistance, and elucidates the role of IGF-II via IGF-II receptor (R) and calcineurin pathways in apoptosis induced by Ang II and IGF-I resistance. Apoptosis of cultured cardiomyoblast H9c2 cells was assessed by DNA fragmentation on agarose gel electrophoresis, nuclear condensation stained with DAPI, and Western blot analysis of pro-apoptotic Bad and cytochrome c in various combinations of control, Ang II, antisense IGF (I or II), IGF (I or II) antibody, IGF (I or II) receptor (R) antibody, or calcineurin inhibitor (Cyclosporine A, (CsA)). We found the following: (1) The combination of Ang II and IGF-I deficiencies had a synergistic effect on apoptosis, confirmed by DNA fragmentation, nuclei condensation, and increases in such pro-apoptotic proteins as Bad, cytochrome c, caspase 9, and caspase 3 in H9c2 cells. (2) IGF-II and IGF-IIR protein products were increased by antisense IGF-I and IGF-I resistance, but these IGF-II protein products were not affected by sense IGF-I and non-specific antibody IgG in H9c2 cells. (3) The alteration of Bad protein level and the release of cytochrome c, both induced by treatments containing combinations of Ang II and antisense IGF-I, IGF-I antibody or IGF-IR antibody, were inhibited by IGF-II antibody. (4) DNA fragmentation, Bad, and cytochrome c which was induced by treatments combining IGF-IR antibody with Ang II or combining IGF-IR antibody with IGF-II were remarkably attenuated by CsA. IGF-I deficiency and/or IGF-IR resistance induced apoptosis in cardiomyoblast cells. The apoptosis, which might have been caused by the upregulation of IGF-II and IGF-IIR genes possibly activated the downstream calcineurin pathway, was synergistically augmented by Ang II.