On the mechanism of the phospholipase C-mediated attenuation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells

The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected. Pulse labeling experiments with [1,3-³H]glycerol and pulse-chase labeling experiments with [1,3-³H]glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C. In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids. Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from [1,3-³H]glycerol indicating the involvement of 1,2-diacyl-sn-glycerol. The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacyl-sn-glycerol levels. Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from [methyl-³H]choline was markedly stimulated. These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells.     

Doxorubicin-induced cardiotoxicity: direct correlation of cardiac fibroblast and H9c2 cell survival and aconitase activity with heat shock protein 27

The use of doxorubicin (Dox) and its derivatives as chemotherapeutic drugs to treat patients with cancer causes dilated cardiomyopathy and congestive heart failure due to Dox-induced cardiotoxicity. In this work, using heat shock factor-1 wild-type (HSF-1(+/+)) and HSF-1 knockout (HSF-1(-/-)) mouse fibroblasts and embryonic rat heart-derived cardiac H9c2 cells, we show that the magnitude of protection from Dox-induced toxicity directly correlates with the level of the heat shock protein 27 (HSP27). Western blot analysis of normal and heat-shocked cells showed the maximum expression of HSP27 in heat-shocked cardiac H9c2 cells and no HSP27 in HSF-1(-/-) cells (normal or heat-shocked). Correspondingly, the cell viability, measured [with (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] after treatment with various concentrations of Dox, was the highest in heat-shocked H9c2 cells and the lowest in HSF-1(-/-) cells. Depleting HSP27 in cardiac H9c2 cells by small interfering (si)RNA also reduced the viability against Dox, confirming that HSP27 does protect cardiac cells against the Dox-induced toxicity. The cells that have lower HSP27 levels such as HSF-1(-/-), were found to be more susceptible for aconitase inactivation. Based on these results we propose a novel mechanism that HSP27 plays an important role in protecting aconitase from Dox-generated O(2)*(-), by increasing SOD activity. Such a protection of aconitase by HSP27 eliminates the catalytic recycling of aconitase released Fe(II) and its deleterious effects in cardiac cells.     

Azoramide improves mitochondrial dysfunction in palmitate-induced insulin resistant H9c2 cells

Molecular and Cellular Biochemistry - White adipose tissue (WAT) is the bulk of fatty tissues in humans. Enhancing the potential of WAT-derived stem cells (WATDCs) to generate cardiomyocytes may...     

Heat shock regulates the respiration of cardiac H9c2 cells through upregulation of nitric oxide synthase

Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium and cardiomyocytes against ischemic injury. In the present study, we have shown that heat shock regulates the respiration of cultured neonatal cardiomyocytes (cardiac H9c2 cells) through activation of nitric oxide synthase (NOS). The respiration of cultured cardiac H9c2 cells subjected to mild heat shock at 42 degrees C for 1 h was decreased compared with that of control. The O2 concentration at which the rate of O2 consumption is reduced to 50% was increased in heat-shocked cells, indicating a lowering of O2 affinity in the mitochondria. Western blot analyses showed a fourfold increase in the expression of heat shock protein (HSP) 90 and a twofold increase in endothelial NOS (eNOS) expression in the heat-shocked cells. Immunoblots of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) in the immunoprecipitate of HSP90 of heat-shocked cells showed that there was a sevenfold increase in eNOS and no changes in iNOS and nNOS. Confocal microscopic analysis of cells stained with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate showed higher levels of NO production in the heat-shocked cells than in control cells. The results indicate that heat shock-induced HSP90 forms a complex with eNOS and activates it to increase NO concentration in the cardiac H9c2 cells. The generated NO competitively binds to the complexes of the respiratory chain of the mitochondria to downregulate O2 consumption in heat-shocked cells. On the basis of these results, we conclude that myocardial protection by hyperthermia occurs at least partly by the pathway of HSP90-mediated NO production, leading to subsequent attenuation of cellular respiration.     

Plantainoside D protects adriamycin-induced apoptosis in H9c2 cardiac muscle cells via the inhibition of ROS generation and NF-kappaB activation

Plantainoside D (PD), was isolated from the leaves of Picrorhiza scrophulariiflora (Scrophulariaceae). The anti-oxidative activity of PD was evaluated based on scavenging effects on hydroxyl radicals and superoxide anion radicals. Adriamycin (ADR) is a potent anti-tumor drug known to cause severe cardiotoxicity. Although ADR generates free radicals, the role of free radicals in the development of cardiac toxicity has not been understood. This study was undertaken to investigate the protective effect of PD against ADR-induced apoptosis. In vitro, ADR caused dose-dependent toxicity in H9c2 cardiac muscle cells. Pre-treatment of the cardiac muscle cells with PD significantly reduced ADR-induced apoptosis of cardiac muscle cells. PD inhibited the ROS produced by ADR in the cardiac muscle cells. As well, PD increased GSH(glutathione), compared with ADR. In response to ADR, NF-kappaB was activated in H9c2 cells. However the treatment of PD reduced the activation of NF-kappaB. We also observed that the NF-kappaB inhibitor, PDTC, inhibited the cytotoxic effect on ADR-induced apoptosis in cardiac muscle cells. In parallel, IkappaBalpha-dominant negative plasmid-overexpression abrogated ADR-induced apoptosis in H9c2 cardiac muscle cells. In conclusion, these results suggest that Plantaionoside D can inhibit ADR-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS generation and NF-kappaB activation. The pure compound PD can be a potential candidate agent which protects cardiotoxicity in ADR-exposed patients.     

Copper inhibition of hydrogen peroxide-induced hypertrophy in embryonic rat cardiac H9c2 cells

Previous studies have shown that dietary copper deficiency causes cardiac hypertrophy and depression of vascular epithelial growth factor (VEGF) expression in mouse model. Copper replenishment in the diet reverses cardiac hypertrophy and restores VEGF expression. The present study was undertaken to specifically determine the role of VEGF in copper effect on cell hypertrophy. Embryonic rat cardiac H9c2 cells were exposed to hydrogen peroxide to develop hypertrophy, determined by increases in cell size and total protein content. Copper addition at 5 microM in cultures suppressed cell hypertrophy. In the presence of anti-VEGF antibody, copper inhibitory effect on cell hypertrophy was blunted, and VEGF alone mimicked the inhibitory effect of copper. The results thus demonstrated that VEGF is critically involved in copper inhibition of cell hypertrophy induced by hydrogen peroxide in the H9c2 cells.     

Differential effects of chloroquine on cardiolipin biosynthesis in hepatocytes and H9c2 cardiac cells

Chloroquine is a potent lysomotropic therapeutic agent used in the treatment of malaria. The mechanism of the chloroquine-mediated modulation of new cardiolipin biosynthesis in isolated rat liver hepatocytes and H9c2 cardiac myoblast cells was addressed in this study. Hepatocytes or H9c2 cells were incubated with [1,3-³H]glycerol in the absence or presence of chloroquine and cardiolipin biosynthesis was examined. The presence of chloroquine in the incubation medium of hepatocytes resulted in a rapid accumulation of radioactivity in cardiolipin indicating an elevated de novo biosynthesis. In contrast, chloroquine caused a reduction in radioactivity incorporated into cardiolipin in H9c2 cells. The presence of brefeldin A, colchicine or 3-methyladenine did not effect radioactivity incorporated into cardiolipin nor the chloroquine-mediated stimulation of cardiolipin biosynthesis in hepatocytes indicating that vesicular transport, cytoskeletal elements or increased autophagy were not involved in de novo cardiolipin biosynthesis induced by chloroquine. The addition of chloroquine to isolated rat liver membrane fractions did not affect the activity of the enzymes of de novo cardiolipin biosynthesis but resulted in an inhibition of mitochondrial cytidine-5-diphosphate-1,2-diacyl-sn-glycerol hydrolase activity. The mechanism for the reduction in cardiolipin biosynthesis in H9c2 cells was a chloroquine-mediated inhibition of glycerol uptake and this did not involve impairment of lysosomal function. The kinetics of the chloroquine-mediated inhibition of glycerol uptake indicated the presence of a glycerol transporter in H9c2 cells. The results of this study clearly indicate that chloroquine has markedly different effects on glycerol uptake and cardiolipin biosynthesis in hepatocytes and H9c2 cardiac cells     

Assessment of hydroxyl radical generation and radical scavenging activity of Chinese medicinal herbs using GC-MS

Aqueous extracts of ten Chinese herbs were evaluated for their radical scavenging activity by a GC-MS method based on the Fenton reaction system. Hydroxylation of salicylate and phenylalanine is widely used as an index of hydroxyl radical formation in vivo and in vitro. A problem associated with quantifying product from such reactions is the generation of complex reaction products that increase background 'noise' and reduce sensitivity for the target product. The aim of this investigation was to develop a GC-MS methodology to assess in vitro hydroxyl radical production. In this method, hydroxyl radical was trapped by p-hydroxyphenylacetic acid to form 3,4-dihydroxyphenylacetic acid (DOPAC) which was then selectively extracted from the reaction mixture using aluminium oxide and assayed by GC-MS. Selective adsorption and desorption of the catechol nucleus from aluminium oxide was shown to eliminate interference from non-catechol reaction products effectively. This system was applied to examine the hydroxyl radical scavenging activity of different herbal extracts. The results showed that the herb Dimocaepus Longan Lour exhibited the highest radical scavenging activity of all the herbs examined. With the use of a stable isotope-labelled internal standard, this system could be readily applied to in vitro methods which use 4-hydroxybenzoic acid as a substrate for the hydroxyl radical.     

Lysophosphatidylcholine induces arachidonic acid release and calcium overload in cardiac myoblastic H9c2 cells.

Lysophosphatidylcholine (lyso-PC) and arachidonate are products of phosphatidylcholine hydrolysis by phospholipase A(2). In this study, the modulation of arachidonate release by exogenous lyso-PC in rat heart myoblastic H9c2 cells was examined. Incubation of H9c2 cells with lyso-PC resulted in an enhanced release of arachidonate in both a time- and dose-dependent fashion. Lyso-PC species containing palmitoyl (C(16:0)) or stearoyl (C(18:0)) groups evoked the highest amount of arachidonate release, while other lysophospholipid species were relatively ineffective. Cells treated with phospholipase A(2) inhibitors resulted in the attenuation of the enhanced arachidonate release in the presence of lyso-PC. Lyso-PC caused the translocation of phospholipase A(2) from the cytosol to the membrane fraction and induced an increase in Ca2+ flux from the medium into the cells. Nimodipine, a specific Ca(2+)-channel blocker, partially attenuated the lyso-PC-induced rise in intracellular Ca2+. Concurrent with Ca2+ influx, lyso-PC caused an enhancement of protein kinase C activity. The lyso-PC-induced arachidonate release was attenuated when cells were pre-incubated with specific protein kinase C and mitogen activated protein kinase kinase inhibitors. Taken together, these results strongly indicate that the lyso-PC-induced increases in levels of intracellular calcium and stimulation of protein kinase C lead to the activation of cytosolic phospholipase A(2) which results in the enhancement of arachidonate release in H9c2 cells.     

Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.     

Zinc inhibits doxorubicin-activated calcineurin signal transduction pathway in H9c2 embryonic rat cardiac cells

Elevation of the zinc-binding protein metallothionein (MT) in the heart inhibits doxorubicin (DOX)-induced myocardial apoptosis and heart hypertrophy. Zinc release from MT in response to oxidative stress has been suggested as a mechanism of action of MT protection from DOX toxicity, and calcineurin is involved in the signaling pathways leading to myocardial apoptosis and heart hypertrophy. The present study was undertaken to determine if zinc can modulate the DOX-activated calcineurin signaling pathway. H9c2 cells were treated with 1 muM DOX, and zinc release was monitored by a zinc ion-specific fluorophore, zinquin ethyl ester. Additionally, DOX-activated calcineurin signaling was detected by a calcineurin-dependent nuclear factor of activated T-cell reporter. DOX treatment induced an increase in intracellular labile zinc and activated calcineurin signaling. Pretreatment of H9c2 cells with a zinc-specific, membrane-permeable chelating agent, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), inhibited the increase in intracellular labile zinc and increased the DOX-activated calcineurin signaling. Pretreatment of H9c2 cells with exogenously added zinc attenuated the DOX-activated calcineurin signaling in a dose-dependent manner. However, neither TPEN nor addition of exogenous zinc affected DOX-induced cellular hypertrophy or DOX-induced decrease in cell viability. Additionally, inhibition of DOX-induced calcineurin signaling with the calcineurin inhibitors cyclosporine A or tacrolimus (FK506) failed to restrict the DOX-induced decrease in cell viability. These results indicate that zinc suppresses DOX-induced calcineurin signaling in H9c2 cells; however, calcineurin signaling is not involved in the DOX-induced decrease in cell viability in H9c2 cells. (It had been shown previously that calcineurin is also not necessary for DOX-induced H9c2 cell hypertrophy.).     

Tetrodotoxin attenuates isoproterenol-induced hypertrophy in H9c2 rat cardiac myocytes

Thyroid stimulating hormone (TSH) is shown to have definite anabolic effects on skeletal metabolism. Previous studies have demonstrated that Insulin-like growth factors (IGF-I and IGF-II) and their six high affinity binding proteins (IGFBPs 1-6) regulate proliferation and differentiation of bone-forming osteoblasts. The current study was intended to determine whether the anabolic effects of TSH on human osteoblastic (SaOS2) cells are mediated through insulin-like growth factor system components. TSH given at 0.01 ng to 10 ng/ml dose levels for 24 and 48 h significantly increased human osteoblastic (SaOS2) cell proliferation and alkaline phosphatase activity, the differentiation marker. TSH significantly increased IGFs (IGF-I and IGF-II) mRNA expression after 6 and 24 h and their protein levels after 24 and 48 h of treatment, respectively. Unlike the IGFs, the IGFBPs responded differently to TSH treatment. Though there were some inconsistencies in the regulation of stimulatory IGF binding protein-3 and -5 by TSH treatment, there was an overall increase at the mRNA abundance and protein levels. Again, the inconsistency persisted at the regulation of the inhibitory IGFBPs 2, 4, and 6 especially at the level of mRNA expression due to TSH treatment, there is an overall decrease in the levels of IGFBP-2, 4, and 6 in the conditioned media (CM) of SaOS2 cell cultures. The IGFBP proteases which control the availability of IGFs are also regulated by hormones. Pregnancy-Associated Plasma Protein-A (PAPP-A) is responsible for the proteolysis of IGFBP-4. TSH treatment significantly unregulated the expression of PAPP-A both at mRNA and protein levels. In conclusion, TSH promotes human osteoblastic (SaOS2) cell proliferation and differentiation by upregulating IGFs and their stimulatory IGF binding proteins and down regulating the inhibitory IGF binding proteins.     

N-acetylcysteine prevents glucose/glucose oxidase-induced oxidative stress,mitochondrial damage and apoptosis in H9c2 cells

AimsHigh blood glucose may auto-oxidize and generate free radicals, which are proposed to induce apoptosis in cardiac cells. The aim of the present study was to investigate the cell damage induced by glucose/glucose oxidase-dependent oxidative stress and the protective effect of N-acetylcysteine (NAC) on H9c2 cardiac muscle cells.Main methodsH9c2 cells were exposed to 33 mM glucose (G) + 1.6 milliunits (mU) of glucose oxidase (GO) and termed G/GO. Cell apoptosis, generation of reactive oxygen species (ROS-super oxide anion and hydrogen peroxide) and reactive nitrogen species (RNS-peroxinitrite), and the change in mitochondrial membrane potential (ΔΨm) was studied using flow cytometry and confocal microscopy, and cytochrome c release was measured using confocal microscopy. The expression of Bcl-2, Bax and the activation of procaspase-9 was studied by western blot.Key findingsExposure of H9c2 cells to G/GO resulted in a significant increase in cellular apoptosis (P < 0.05) and the generation of ROS and RNS (P < 0.001). Further, G/GO treatment led to a decrease in ΔΨm, release of cytochrome c, decrease in Bcl-2, increase in Bax expression and the activation of procaspase-9. Treatment with NAC significantly decreased apoptosis (P < 0.05) and reduced the levels of ROS and RNS (P < 0.001). NAC was also able to normalize ΔΨm, inhibit cytochrome c release, increase Bcl-2 and decrease Bax expression and procaspase-9 activation.SignificanceOur studies suggest that NAC has antioxidative and antiapoptotic activity against G/GO-induced oxidative stress through the inhibition of mitochondrial damage in H9c2 cells.     

Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H₂O₂)-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H₂O₂-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H₂O₂-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H₂O₂-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.     

Silibinin protects H9c2 cardiac cells from oxidative stress and inhibits phenylephrine-induced hypertrophy: potential mechanisms

Cardiac hypertrophy is the main response of the heart to various extrinsic and intrinsic stimuli, and it is characterized by specific molecular and phenotypic changes. Recent in vitro and in vivo studies indicate the involvement of reactive oxygen species in the hypertrophic response. In this study, silibinin, a plant flavonolignan extracted from milk thistle with potent antioxidant activity, was evaluated for its effects in (a) preventing hydrogen peroxide (H₂O₂)-induced cellular damage and (b) blocking the phenylephrine-induced hypertrophic response. Using the in vitro model of embryonic rat heart-derived H9c2 cells, we showed that silibinin has a rather safe profile as concentrations up to 200 μM did not affect cell viability. Pretreatment of H9c2 cells with silibinin resulted in better protection of H9c2 cells under conditions of H₂O₂-induced cellular stress compared to untreated cells as indicated by cell viability and DNA fragmentation assays. Furthermore, silibinin attenuated the phenylephrine-induced hypertrophic response as evidenced by the measurement of cell surface, up-regulation of atrial natriuretic peptide and increase of cellular protein levels. Moreover, silibinin repressed the phenylephrine-induced phosphorylation of ERK1/2 kinases, while it appeared to inhibit the weakly activated by phenylephrine phosphorylation of Akt. Based on our results, silibinin may attenuate the phenylephrine-induced hypertrophic response of H9c2 cells via antioxidant mechanisms involving mainly the inhibition of the intracellular signaling pathways mediated by ERK1/2 MAPKs and Akt.     

H9c2细胞对B组柯萨奇病毒3型重组株的易感性

H9c2细胞是来源于大鼠胚胎心脏组织的成肌细胞系,B组柯萨奇病毒(group B Coxsackievirus,CVB)是心肌炎和扩张型心肌病的主要病原.本研究观察了CVB3在H9c2细胞中的感染性,探讨H9c2细胞是否可用于CVB致心肌疾病的实验研究.用整合了增强型绿色荧光蛋白(EGFP)或海肾荧光素酶(RLuc)的...     

Protein acylation in the cardiac muscle like cell line,H9c2

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with ³H-palmitate or ³H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 Ci palmitate (PA, 2.3 nmol) or 125 Ci myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 ± 0.25 to 1.25 ± 0.56 Ci/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 ± 0.12 to 0.77 ± 0.36 Ci/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with ³H-palmitate. Incorporation into phospholipids (PL) increased from 40–60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of ³H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.