Cardioprotective Effects of Glucagon-like Peptide-1 (9-36) Against Oxidative Injury in H9c2 Cardiomyoblasts: Potential Role of the PI3K/Akt/NOS Pathway.

ABSTRACT: Glucagon-like peptide (GLP)-1(7-36), a major active form of GLP-1 hormone, is rapidly cleaved by dipeptidyl peptidase-4 to generate a truncated metabolite, GLP-1(9-36) which has a low affinity for GLP-1 receptor (GLP-1R). GLP-1(7-36) has been shown to have protective effects on cardiovascular system through GLP-1R-dependent pathway. Nevertheless, the cardioprotective effects of GLP-1(9-36) have not fully understood. The present study investigated the effects of GLP-1(9-36), including its underlying mechanisms against oxidative stress and apoptosis in H9c2 cells. Here, we reported that GLP-1(9-36) protects H9c2 cardiomyoblasts from hydrogen peroxide (H2O2)-induced oxidative stress by promoting the synthesis of antioxidant enzymes, glutathione peroxidase-1, catalase, and heme oxygenase-1. In addition, treatment with GLP-1(9-36) suppressed H2O2-induced apoptosis by attenuating caspase-3 activity and upregulating antiapoptotic proteins, Bcl-2 and Bcl-xL. These protective effects of GLP-1(9-36) are attenuated by blockade of PI3K-mediated Akt phosphorylation and prevention of nitric oxide synthase-induced nitric oxide production. Thus, GLP-1(9-36) represents the potential therapeutic target for prevention of oxidative stress and apoptosis in the heart via PI3K/Akt/nitric oxide synthase signaling pathway.

Phloretin Alleviates Arsenic Trioxide-Induced Apoptosis of H9c2 Cardiomyoblasts via Downregulation in Ca2+/Calcineurin/NFATc Pathway and Inflammatory Cytokine Release.

Arsenic trioxide (ATO) is among the first-line chemotherapeutic drugs for treating acute promyelocytic leukemia patients, but its clinical use is hampered due to cardiotoxicity. The present investigation unveils the mechanism underlying ATO-induced oxidative stress that promotes calcineurin (a ubiquitous Ca2+/calmodulin-dependent serine/threonine phosphatase expressed only during sustained Ca2+ elevation) expression, inflammatory cytokine release and apoptosis in H9c2 cardiomyoblasts, and its possible modulation with phloretin (PHL, an antioxidant polyphenol present in apple peel). ATO caused Ca2+ overload resulting in elevated expression of calcineurin and its downstream transcriptional effector NFATc causing the release of cytokines such as IL-2, IL-6, MCP-1, IFN-γ, and TNF-α in H9c2 cardiomyoblast. There was a visible increase in the nuclear fraction of NF-κB and ROS-mediated apoptotic cell death. The expression levels of cardiac-specific genes (troponin, desmin, and caveolin-3) and genes of the apoptotic signaling pathway (BCL-2, BAX, IGF1, AKT, ERK1, -2, RAF1, and JNK) in response to ATO and PHL were studied. The putative binding mode and the potential ligand-target interactions of PHL with calcineurin using docking software (Autodock and iGEMDOCKv2) showed the high binding affinity of PHL to calcineurin. PHL co-treatment significantly reduced Ca2+ influx and normalized the expression of calcineurin, NFATc, NF-κB, and other cytokines. PHL co-treatment resulted in activation of BCL-2, IGF1, AKT, RAF1, ERK1, and ERK2 and inhibition of BAX and JNK. Overall, these results revealed that PHL has a protective effect against ATO-induced apoptosis and we propose calcineurin as a druggable target for the interaction of PHL in ATO cardiotoxicity in H9c2 cells.

Overexpression and pre-treatment of recombinant human Secretory Leukocyte Protease Inhibitor (rhSLPI) reduces an in vitro ischemia/reperfusion injury in rat cardiac myoblast (H9c2) cell.

One of the major causes of cardiac cell death during myocardial ischemia is the oversecretion of protease enzymes surrounding the ischemic tissue. Therefore, inhibition of the protease activity could be an alternative strategy for preventing the expansion of the injured area. In the present study, we investigated the effects of Secretory Leukocyte Protease Inhibitor (SLPI), by means of overexpression and treatment of recombinant human SLPI (rhSLPI) in an in vitro model. Rat cardiac myoblast (H9c2) cells overexpressing rhSLPI were generated by gene delivery using pCMV2-SLPI-HA plasmid. The rhSLPI-H9c2 cells, mock transfected cells, and wild-type (WT) control were subjected to simulated ischemia/reperfusion (sI/R). Moreover, the treatment of rhSLPI in H9c2 cells was also performed under sI/R conditions. The results showed that overexpression of rhSLPI in H9c2 cells significantly reduced sI/R-induced cell death and injury, intracellular ROS level, and increased Akt phosphorylation, when compared to WT and mock transfection (p <0.05). Treatment of rhSLPI prior to sI/R reduced cardiac cell death and injury, and intra-cellular ROS level. In addition, 400 ng/ml rhSLPI treatment, prior to sI, significantly inhibited p38 MAPK phosphorylation and rhSLPI at 400-1000 ng/ml could increase Akt phosphorylation.

Maduramicin-activated protein phosphatase 2A results in extracellular signal-regulated kinase 1/2 inhibition, leading to cytotoxicity in myocardial H9c2 cells.

Maduramicin, a polyether ionophore antibiotic used as an anticoccidial agent in poultry industry, has been reported to be toxic to animals and humans if improperly used or by accident, resulting in heart failure, skeletal muscle degeneration and even death. However, the molecular mechanism underlying its cardiotoxicity remains elusive. Mitogen activated protein kinases (MAPKs) and protein phosphatases signaling pathways have been documented to be involved in the cell survival regulation. The present study was set to investigate the role of above pathways in maduramicin-induced myocardial cytotoxicity. Here we observed that maduramicin inhibited cell proliferation and reduced cell viability in H9c2 cells. Furthermore, we found that maduramicin suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a concentration-dependent manner. Ectopic expression of constitutively active MKK1 partially prevented the cytotoxicity of maduramicin. Moreover, we showed that maduramicin concentration-dependently activated protein phosphatase 2A (PP2A) by decreasing its phosphorylation and increasing its methylation. Inhibition of PP2A with okadaic acid attenuated maduramicin's toxicity. Overexpression of dominant negative PP2A partially rescued cells from maduramicin-inhibited ERK1/2 contributing to its cytotoxicity. The results indicate that maduramicin activates PP2A and consequently inhibits ERK1/2, leading to cytotoxicity in H9c2 cells. Our data suggest that manipulation of PP2A-ERK1/2 pathway may be a potential approach to prevent maduramicin-induced cardiotoxicity.

Berberine-induced cardioprotection and Sirt3 modulation in doxorubicin-treated H9c2 cardiomyoblasts.

Doxorubicin (DOX) is one of the most widely used anti-neoplastic agents. However, treatment with DOX is associated with cumulative cardiotoxicity inducing progressive cardiomyocyte death. Sirtuin 3 (Sirt3), a mitochondrial deacetylase, regulates the activity of proteins involved in apoptosis, autophagy and metabolism. Our hypothesis is that pharmacological modulation by berberine (BER) pre-conditioning of Sirt3 protein levels decreases DOX-induced cardiotoxicity. Our results showed that DOX induces cell death in all experimental groups. Increase in Sirt3 content by transfection-mediated overexpression decreased DOX cytotoxicity, mostly by maintaining mitochondrial network integrity and reducing oxidative stress. p53 was upregulated by DOX, and appeared to be a direct target of Sirt3, suggesting that Sirt3-mediated protection against cell death could be related to this protein. BER pre-treatment increased Sirt3 and Sirt1 protein levels in the presence of DOX and inhibited DOX-induced caspase 9 and 3-like activation. Moreover, BER modulated autophagy in DOX-treated H9c2 cardiomyoblasts. Interestingly, mitochondrial biogenesis markers were upregulated in in BER/DOX-treated cells. Sirt3 over-expression contributes to decrease DOX cytotoxicity on H9c2 cardiomyoblasts, while BER can be used as a modulator of Sirtuin function and cell quality control pathways to decrease DOX toxicity.

Compartment-specific Control of Reactive Oxygen Species Scavenging by Antioxidant Pathway Enzymes.

Oxidative stress arises from an imbalance in the production and scavenging rates of reactive oxygen species (ROS) and is a key factor in the pathophysiology of cardiovascular disease and aging. The presence of parallel pathways and multiple intracellular compartments, each having its own ROS sources and antioxidant enzymes, complicates the determination of the most important regulatory nodes of the redox network. Here we quantified ROS dynamics within specific intracellular compartments in the cytosol and mitochondria and determined which scavenging enzymes exert the most control over antioxidant fluxes in H9c2 cardiac myoblasts. We used novel targeted viral gene transfer vectors expressing redox-sensitive GFP fused to sensor domains to measure H2O2 or oxidized glutathione. Using genetic manipulation in heart-derived H9c2 cells, we explored the contribution of specific antioxidant enzymes to ROS scavenging and glutathione redox potential within each intracellular compartment. Our findings reveal that antioxidant flux is strongly dependent on mitochondrial substrate catabolism, with availability of NADPH as a major rate-controlling step. Moreover, ROS scavenging by mitochondria significantly contributes to cytoplasmic ROS handling. The findings provide fundamental information about the control of ROS scavenging by the redox network and suggest novel interventions for circumventing oxidative stress in cardiac cells.

MicroRNA-208a Potentiates Angiotensin II-triggered Cardiac Myoblasts Apoptosis via Inhibiting Nemo-like Kinase (NLK).

INTRODUCTION: MicroRNA-208a (miR-208a) exacerbated cardiomyocyte apoptosis via inhibiting nemo-like kinase (NLK). miR-208a is a crucial molecule in the regulation of heart diseases, however, the biological function and underlying mechanism of miR-208a in the progression of cardiomyocyte apoptosis is not clearly elucidated. We hypothesized that miR-208a might potentiate cardiomyocyte apoptosis through inhibiting NLK. METHODS: Male Sprague-Dawley rats were underwent permanent coronary artery ligation to establish myocardial infarction (MI) model. The quantitative real-time RT-PCR (qRT-PCR) was used to evaluate the expression of miR-208a and NLK mRNA. Western blot was applied to detect NLK and Bcl-2 proteins expression. Luciferase reporter assay was performed to indentify NLK as a target of miR-208a. The apoptosis of H9C2 cells was assessed by flow cytometry (FCM). RESULTS: miR-208a was upregulated accompanying with a significant decrease of NLK in response to MI, and stronger miR-208a staining was detected by in situ hybridization in the cytoplasm of cardiomyocytes in MI group compared to the sham group. In vitro, overexpression of miR-208a greatly enhance Ang II-induced the apoptosis of H9C2 cells through downregulating of NLK and the anti-apoptosis protein Bcl-2 expression, whereas these effects were reversed when miR-208a was downregulated. Dual luciferase reporter assay and western blot results demonstrated that NLK was a direct target of miR-208a. Interestingly, upregulation of NLK obviously increased Bcl-2 expression and reduced the percentage of apoptotic cells, while attenuation of NLK reduced the level of Bcl-2 and cells apoptosis after treatment with Ang II. CONCLUSIONS: miR-208a can promote Ang II-induced cardiomyocyte apoptosis via negatively regulating NLK expression, and inhibition of miR-208a may provide a novel therapeutic target for cardiomyocyte apoptosis.

The Protective Effect of INH2BP, a Novel PARP Inhibitor 5-Iodo-6-Amino-1,2-Benzopyrone, Against Hydrogen Peroxide-Induced Apoptosis Through ERK and p38 MAPK in H9c2 Cells.

INH2BP (5-iodo-6-amino-1,2-benzopyrone), a poly-ADP ribose polymerase inhibitor, has been shown to possess anti-cancer, anti-viral, and anti-inflammation properties. The aim of this study was to investigate the protective effect of INH2BP against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells. While the treatment of H9c2 cardiomyoblasts cells with hydrogen peroxide (H2O2) caused a loss of cell viability and an increase in the number of apoptotic cells, INH2BP significantly protected the cells against H2O2-induced cell death without any cytotoxicity. Our data also shows that INH2BP significantly scavenged intracellular reactive oxygen species (ROS), and markedly enhanced the expression of antioxidant enzymes such as Mn-SOD (superoxide) and Cu/Zn-SOD, and heme oxygenase-1, which was accompanied by the concomitant activation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation in H9c2 cells. The effects of INH2BP on ERK1/2 and p38 MAPK phosphorylation were abrogated by PD98059, an ERK1/2 inhibitor, and SB203580, a p38 inhibitor. In addition, inhibition of ERK1/2 and p38 MAPK by these inhibitors significantly attenuated INH2BP-mediated H9c2 viability as well as cleaved caspases-3, Bax, and Bcl-2 activation. Taken together, these results demonstrate that INH2BP prevents H2O2-induced apoptosis in H9c2 cells by reducing the production of intracellular ROS, regulating apoptotic-related proteins, and the activation of the ERK1/2 and p38 MAPK.

MicroRNA-210 decreases heme levels by targeting ferrochelatase in cardiomyocytes.

BACKGROUND: MicroRNA-210 (miR-210) increases in hypoxia and regulates mitochondrial respiration through modulation of iron-sulfur cluster assembly proteins (ISCU1/2), a protein that is involved in Fe/S cluster synthesis. However, it is not known how miR-210 affects cellular iron levels or production of heme, another iron containing molecule that is also needed for cellular and mitochondrial function. METHODS AND RESULTS: To screen for micro-ribonucleic acids (miRNAs) regulated by iron, we performed a miRNA gene array in neonatal rat cardiomyocytes treated with iron chelators. Levels of miR-210 are significantly increased with iron chelation, however, this response was mediated entirely through the hypoxia-inducible factor (HIF) pathway. Furthermore, miR-210 reduced cellular heme levels and the activity of mitochondrial and cytosolic heme-containing proteins by modulating ferrochelatase (FECH), the last enzyme in heme biosynthesis. Mutation of the 2 miR-210 binding sites in the 3' untranslated region (UTR) of FECH reversed the miR-210 response, while mutation of either binding site in isolation did not exert any effects. Changes mediated by miR-210 in heme and FECH were independent of ISCU, as overexpression of an ISCU construct lacking the 3' UTR does not alter miR-210 regulation of heme and FECH. Finally, FECH levels increased in hypoxia, and this effect was not reversed by miR-210 knockdown, suggesting that the effects of miR-210 on heme are restricted to normoxic conditions, and that the pathway is overriden in hypoxia. CONCLUSIONS: Our results identify a role for miR-210 in the regulation of heme production by targeting and inhibiting FECH under normoxic conditions.

ULK1 plays a critical role in AMPK-mediated myocardial autophagy and contractile dysfunction following acute alcohol challenge.

This study was designed to evaluate the role of ULK1 in AMPK-mediated myocardial autophagy and contractile dysfunction following acute alcohol challenge. Wild-type and AMPK knockout mice were challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial function was evaluated using echocardiography and edge-detection. Western blot analysis was employed to evaluate the levels of AMPK, Raptor, mTOR, the AMPK downstream signal ULK1 and autophagy markers Beclin-1 and LC3-II. siRNA was used to knockdown ULK1 in H9C2 myoblasts. GFP-LC3 puncta was used to evaluate autophagosome formation. Alcohol challenge compromised cardiac function as evidenced by decreased fractional shortening, peak shortening and intracellular Ca²âº rise, prolonged relengthening and intracellular Ca²âº decay in WT mice, the effects of which were mitigated by AMPK knockout. Ethanol exposure facilitated myocardial autophagy as evidenced by enhanced LC3-II level, as well as phosphorylation of AMPK, Raptor, and dephosphorylation of mTOR and ULKI in WT hearts, which were alleviated by AMPK knockout. Pharmacological inhibition of AMPK using compound C attenuated ethanol-induced autophagosome formation, AMPK phosphorylation, ULK1 dephosphorylation and apoptosis. Ethanol exposure-induced cardiomyocyte contractile defects and autophagosome accumulation were reversed by the autophagy inhibitor 3-MA. Similarly, knockdown of ULK1 using siRNA in H9C2 cells ablated ethanol-induced autophagosome accumulation, LC3-II expression and cell death. Lysosomal inhibition using bafilomycin, E64-D and pepstatin A potentiated ethanol-induced increase in autophagosome formation. Taken together, our results suggest that ULK1 may play a critical role in AMPK-mediated myocardial autophagy, apoptosis and contractile dysfunction following acute alcohol challenge.

Evidence that AMP-activated protein kinase can negatively modulate ornithine decarboxylase activity in cardiac myoblasts.

The responses of AMP-activated protein kinase (AMPK) and Ornithine decarboxylase (ODC) to isoproterenol have been examined in H9c2 cardiomyoblasts, AMPK represents the link between cell growth and energy availability whereas ODC, the key enzyme in polyamine biosynthesis, is essential for all growth processes and it is thought to have a role in the development of cardiac hypertrophy. Isoproterenol rapidly induced ODC activity in H9c2 cardiomyoblasts by promoting the synthesis of the enzyme protein and this effect was counteracted by inhibitors of the PI3K/Akt pathway. The increase in enzyme activity became significant between 15 and 30min after the treatment. At the same time, isoproterenol stimulated the phosphorylation of AMPKα catalytic subunits (Thr172), that was associated to an increase in acetyl coenzyme A carboxylase (Ser72) phosphorylation. Downregulation of both α1 and α2 isoforms of the AMPK catalytic subunit by siRNA to knockdown AMPK enzymatic activity, led to superinduction of ODC in isoproterenol-treated cardiomyoblasts. Downregulation of AMPKα increased ODC activity even in cells treated with other adrenergic agonists and in control cells. Analogue results were obtained in SH-SY5Y neuroblastoma cells transfected with a shRNA construct against AMPKα. In conclusion, isoproterenol quickly activates in H9c2 cardiomyoblasts two events that seem to contrast one another. The first one, an increase in ODC activity, is linked to cell growth, whereas the second, AMPK activation, is a homeostatic mechanism that negatively modulates the first. The modulation of ODC activity by AMPK represents a mechanism that may contribute to control cell growth processes.

Trichloroethylene induces methylation of the Serca2 promoter in H9c2 cells and embryonic heart.

Trichloroethylene (TCE) is a halogenated hydrocarbon used as a solvent in industrial settings and in house-cleaning products. Exposure to TCE has been linked to increased risk for congenital heart malformations in both human and animal models. Previous studies showed TCE exposure reduced the expression and function of the ATP-dependent calcium pump, Serca2a, which is important for regulating calcium flux in myocytes and maintaining physiological cardiac function. In this study, we investigated whether TCE reduced Serca2a expression by altering the methylation status of its proximal promoter region. Low doses of TCE exposure (10 ppb) induced DNA hyper methylation in the Serca2 promoter region in cardiac myoblast cells and rat embryonic cardiac tissue. TCE exposure induced DNA methylation in a region of the Serca2 promoter which is the target for SP1 binding site essential for regulation of Serca2a transcriptional activity. Chromatin immunoprecipitation data confirmed that TCE exposure reduced the binding of SP1 to the Serca2 promoter region adjacent to the methylated CpG dimer. Finally, low-dose TCE exposure reduced the concentration of S-adenosyl-methionine in exposed cells and embryos. These cumulative data indicate that epigenetic mechanisms, including DNA methylation, may be important in mediating the teratogenic effects of TCE in embryonic heart.

Upregulation of SIRT1 deacetylase in phenylephrine-treated cardiomyoblasts.

The sirtuin SIRT1 is an ubiquitous NAD(+) dependent deacetylase that plays a role in biological processes such as longevity and stress response. In cardiac models, SIRT1 is associated to protection against many stresses. However, the link between SIRT1 and heart hypertrophy is complex and not fully understood. This study focuses specifically on the response of SIRT1 to the α-adrenergic agonist phenylephrine in H9c2 cardiac myoblasts, a cell model of cardiac hypertrophy. After 24 and 48h of phenylephrine treatment, SIRT1 expression and deacetylase activity were significantly increased. SIRT1 upregulation by phenylephrine was not associated to changes in NAD(+) levels, but was blocked by inhibitors of AMP-activated Protein Kinase (AMPK) or by AMPK knockdown by siRNA. When SIRT1 was inhibited with sirtinol or downregulated by siRNA, H9c2 cell viability was significantly decreased following phenylephrine treatment, showing that SIRT1 improves cell survival under hypertrophic stress. We so then propose that the increase in SIRT1 activity and expression in H9c2 cells treated with phenylephrine is an adaptive response to the hypertrophic stress, suggesting that adrenergic stimulation of heart cells activates hypertrophic programming and at the same time also promotes a self-protecting and self-regulating mechanism.

MAPK signaling in H9c2 cardiomyoblasts exposed to cholesterol secoaldehyde--role of hydrogen peroxide.

3ß-Hydroxy-5,6-secocholestan-6-al (cholesterol secoaldehyde or ChSeco), an oxysterol known to be formed in ozone- and singlet oxygen-mediated oxidations of cholesterol, has been detected in the atherosclerotic plaque and in the brain of patients suffering from Alzheimer's disease and Lewy body dementia. Previously, we have shown that, in H9c2 cardiomyoblasts, ChSeco induces oxidative stress followed by apoptosis involving both intrinsic and extrinsic signaling pathways. In the present study, we investigated the nature of reactive oxygen species (ROS) and its associated redox signaling in H9c2 cells upon treatment with ChSeco. Both catalase and deferoxamine, which lowered intracellular ROS, were found to alleviate the ChSeco-induced cytotoxicity. ChSeco-treated H9c2 cells showed a significant decrease in the intracellular catalase activity, suggesting the involvement of H(2)O(2) in the associated cytotoxicity. Additionally, in ChSeco-exposed cells, there was a marked increase in lipid peroxidation and pre-treatment with SB 203580 (p38 MAPK inhibitor) and MEK1/2 inhibitor (ERK1/2 and JNK inhibitor) rendered protection against the cytotoxicity. An early increase in the expression of p-SAPK/JNK or delayed p38 MAPK did not alter ATF-2 but decreased c-Jun expression in these cells. Overall, these findings are consistent with MAPK signaling resulting from increased cellular H(2)O(2) in ChSeco-induced cytotoxicity in cardiomyoblasts.

Matrix metalloproteinases are less essential for the in-situ gelatinolytic activity in heart muscle than in skeletal muscle.

In order to determine whether the contribution of matrix metalloproteinases (MMPs) to the tissue gelatinolytic activity is similar between myocardium and skeletal muscle tissue, in-situ zymography was applied to myoblasts originated from myocardium or skeletal muscle of rodents as well as tissue sections of heart and soleus muscles of rats. Gelatinolyic activity was observed in cytoplasm and nucleus of both heart and skeletal myoblasts. The chelating agent EDTA blocked much of the gelatinolytic activity and the organomercurial activator of MMPs increased the activity in cells of both muscle origins. However, the inhibition of gelatinolytic activity by a broad spectrum MMP inhibitor was less profound in heart myoblasts than that in skeletal myoblasts. Gelatinolytic activity was also expressed in the endomysium and perimysium of tissue sections of heart and soleus muscles. Similar with findings in the cell studies, the gelatinase activity was increased by the MMP activator, mostly blocked by EDTA and partially inhibited by the MMP inhibitor. In the presence of the MMP inhibitor, the remaining gelatinolytic activity in the tissue sections was again higher in myocardium than that in soleus muscle. This observation was further supported by the gelatinolytic activity examined in tissue homogenates. Our findings suggest that other proteinases, in addition to MMPs, are more responsive for the tissue gelatinolytic activity in heart muscle as compared with that in skeletal muscle.

PKB/Akt activation inhibits p53-mediated HIF1A degradation that is independent of MDM2.

Cross-talk between the two transcription factors, p53 and hypoxia inducible factor 1alpha (HIF1A), is important in different pathophysiological conditions (Hammond and Giaccia, 2006, Clin Cancer Res 12:5007-5009) such as in the transition from myocardial hypertrophy to cardiac dilatation and heart failure. In that context, p53 induces HIF1A degradation which in turn provokes the transition from compensatory hypertrophy to myocardial thinning and chamber dilatation (Sano et al., 2007, Nature 446:444-448). In order to investigate the mechanism of p53-induced HIF1A degradation, we used the established in vitro model of deferroxamine (DFX)-induced HIF1A accumulation in H9c2 cardiac cells (Sano et al., 2007, Nature 446:444-448). Here, we report that opposite to HIF1A accumulation following exposure to DFX, prolonged DFX-induced p53 activation and HIF1A protein decrease, without any change in Hif1a mRNA. HIF1A protein decrease accompanied upregulated HIF1A ubiquitination. MDM2, an ubiquitin E3 ligase target gene of p53, was upregulated following prolonged DFX, but using p53/Mdm2 double-null mouse embryonic fibroblasts, we found that p53 upregulated HIF1A ubiquitination and degradation independently of MDM2. Moreover, with prolonged DFX treatment, an enhanced interaction between MDM2 and HIF1A was lacking. Instead, phospho-Akt(ser473) was decreased during the phase coinciding with HIF1A degradation, and inhibition of PKB/Akt phosphorylation using PI3K inhibitor (LY294002) upregulated HIF1A ubiquitination. In summary, we propose that p53-induced HIF1A degradation is not exclusively MDM2-mediated, but reversible by PKB/Akt phosphorylation.

NADPH oxidases participate to doxorubicin-induced cardiac myocyte apoptosis.

Cumulative doses of doxorubicin, a potent anticancer drug, lead to serious myocardial dysfunction. Numerous mechanisms including apoptosis have been proposed to account for its cardiotoxicity. Cardiac apoptosis induced by doxorubicin has been related to excessive reactive oxygen species production by the mitochondrial NADH dehydrogenase. Here, we explored whether doxorubicin treatment activates other superoxide anion generating systems such as the NADPH oxidases, membrane-embedded flavin-containing enzymes, and whether the subsequent oxidative stress contributes to apoptosis. We showed that doxorubicin treatment of rat cardiomyoblasts H9c2 triggers increases in caspase-3 like activity and hypoploid cells, both common features of apoptosis. Doxorubicin exposure also leads to a rapid superoxide production through NADPH oxidase activation. Inhibition of these enzymes using diphenyliodonium and apocynin reduces doxorubicin-induced reactive oxygen species production, caspase-3 like activity and sub-G1 cell population. In conclusion, NADPH oxidases participate to doxorubicin-induced cardiac apoptosis.

Glycogen synthase kinase-3beta is activated by matrix metalloproteinase-2 mediated proteolysis in cardiomyoblasts.

AIMS: Matrix metalloproteinase (MMP)-2 contributes to myocardial oxidative stress injury by degrading sarcomeric and cytoskeletal proteins in cardiomyocytes. Glycogen synthase kinase (GSK)-3beta is dysregulated during oxidative stress and is susceptible to proteolytic cleavage. Here we determined whether GSK-3beta is a MMP-2 substrate as a result of oxidative stress. METHODS AND RESULTS: MMP-2 and GSK-3beta were incubated and the cleavage fragments were identified by immunoblotting and silver stain. The intact protein and its primary cleavage fragment were subjected to trypsin digestion and the resultant peptides were analysed by LC-MS/MS. GSK-3beta kinase activity was measured using a peptide substrate and [gamma-(32)P]-ATP. Oxidative stress in H9c2 cardiomyoblasts was induced by H(2)O(2) and the levels and activities of MMP-2 and GSK-3beta were measured. Incubation of 47 kDa GSK-3beta with MMP-2 resulted in the time- and concentration-dependant cleavage of GSK-3beta as seen by appearance of an approximately 30 kDa fragment. MS analysis and Mascot database search yielded a peptide with an amino acid sequence of GSK-3beta lacking the N-terminal region. GSK-3beta kinase activity was significantly increased upon incubation with MMP-2 which was abrogated by the MMP inhibitor GM-6001. H(2)O(2) challenge of H9c2 cardiomyoblasts significantly increased the activity and level of MMP-2, reduced the level of GSK-3beta, and significantly increased GSK-3beta kinase activity. Both the loss of intact GSK-3beta and increase in its kinase activity were reduced with MMP inhibitors. MMP-2 pull-down assays in H9c2 cell lysates showed the association of MMP-2 with GSK-3beta. CONCLUSION: GSK-3beta may be a target of MMP-2 and its cleavage by MMP-2 enhances its kinase activity. MMP-2 may cleave off the N-terminal of GSK-3beta where the inhibitory phosphorylation of serine-9 occurs. MMP-2-mediated augmentation of GSK-3beta kinase activity may contribute to cardiac injury resulting from enhanced oxidative stress.

Mitochondrial monolysocardiolipin acyltransferase is elevated in the surviving population of H9c2 cardiac myoblast cells exposed to 2-deoxyglucose-induced apoptosis.

Cardiolipin (CL) is a major mitochondrial membrane phospholipid in the mammalian heart and the remodeling of CL is essential to maintain its unique unsaturated fatty acyl composition. We examined CL de novo biosynthesis and remodeling in the surviving population of H9c2 cardiac myoblast cells exposed to 2-deoxyglucose (2-DG). H9c2 cells were incubated in the absence or presence of 2-DG for 16 h with [1,3-3H]glycerol or [1-14C]linoleic acid (bound to albumin in a 1:1 molar ratio). Dead cells were removed and radioactivity was incorporated into CL. Its pool size, fatty acid composition, and the activities of the CL biosynthesis and remodeling enzymes were determined. The CL pool size, its fatty acid composition, and [1,3-3H]glycerol or [1-14C]linoleic acid incorporated into CL were unaltered in the surviving population of 2-DG-treated cells compared with controls. In addition, the activities of the CL de novo biosynthetic enzymes were unaltered. Cleaved caspase-3 and poly(ADP-ribose) polymerase were slightly elevated in the surviving population of 2-DG-treated cells compared with controls, indicating that apoptosis induction was occurring in these cells. Mitochondrial phospholipase A2 and monolysocardiolipin acyltransferase (MLCL AT) activities increased 33% (p < 0.05) and 63% (p < 0.05), respectively, in 2-deoxyglucose-treated cells compared with controls. In contrast, the activity of ALCAT1, an endoplasmic reticulum MLCL AT, decreased 77% (p < 0.05), but this was not due to a reduction in ALCAT1 mRNA expression. The mRNA expression of the Barth syndrome gene TAZ, encoding a mitochondrial CL transacylase, was unaltered in 2-DG treated cells. The increase in mitochondrial MLCL AT activity was due to an elevated expression in MLCL AT protein. Thus, an increase in MLCL AT activity and expression occurs to maintain the CL pool in the surviving population of H9c2 cells as a compensatory mechanism for the elevated phospholipase A2 activity seen in 2-DG-induced apoptosis. We hypothesize that increased mitochondrial MLCL AT activity and its expression, and hence, elevated CL resynthesis, may be a protective mechanism against monolysocardiolipin-mediated apoptosis.

Molecular cloning of a Trypanosoma cruzi cell surface casein kinase II substrate, Tc-1, involved in cellular infection.

In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of Trypanosoma cruzi, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of T. cruzi. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the T. cruzi genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked T. cruzi infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.