Elevated ROS depress mitochondrial oxygen utilization efficiency in cardiomyocytes during acute hypoxia.

Mitochondria are important sites for the production of ATP and the generation of ROS in cells. However, whether acute hypoxia increases ROS generation in cells or affects ATP production remains unclear, and therefore, monitoring the changes in ATP and ROS in living cells in real time is important. In this study, cardiomyocytes were transfected with RoGFP for ROS detection and MitGO-Ateam2 for ATP detection, whereby ROS and ATP production in cardiomyocytes were respectively monitored in real time. Furthermore, the oxygen consumption rate (OCR) of cardiomyocytes was measured. Similar results were produced for adult and neonatal rat cardiomyocytes. Hypoxia (1% O2) reduced the basal OCR, ATP-linked OCR, and maximal OCR in cardiomyocytes compared with these OCR levels in the cardiomyocytes in the normoxic group (21% O2). However, ATP-linked OCR, normalized to maximal OCR, was increased during hypoxia, indicating that the electron leakage of complex III exacerbated the increase of ATP-linked oxygen consumption during hypoxia and vice versa. Combined with the result that cardiomyocytes expressing MitGO-Ateam2 showed a significant decrease in ATP production during hypoxia compared with that of normoxic group, acute hypoxia might depress the mitochondrial oxygen utilization efficiency of the cardiomyocytes. Moreover, cardiomyocytes expressing Cyto-RoGFP or IMS-RoGFP showed an increase in ROS generation in the cytosol and the mitochondrial intermembrane space (IMS) during hypoxia. All of these results indicate that acute hypoxia generated more ROS in complex III and increased mitochondrial oxygen consumption, leading to less ATP production. In conclusion, acute hypoxia depresses the mitochondrial oxygen utilization efficiency by decreasing ATP production and increasing oxygen consumption as a result of the enhanced ROS generation at mitochondrial complex III.

Thymosin alpha 1 suppresses proliferation and induces apoptosis in breast cancer cells through PTEN-mediated inhibition of PI3K/Akt/mTOR signaling pathway.

Thymosin alpha 1 (Tα1), an immunoactive peptide, has been shown to inhibit cell proliferation and induce apoptosis in human leukemia, non-small cell lung cancer, melanoma, and other human cancers. However, the response and molecular mechanism of breast cancer cells exposed to Tα1 remain unclear. PTEN, a tumor suppressor gene, is frequently mutated in a variety of human cancers. In the present study, we aimed to investigate the biological roles of PTEN in the growth inhibition of human breast cancer cells exposed to Tα1. Using wild-type and mutant PTEN-expressing cells, we found a strong correlation between PTEN status and Tα1-mediated growth inhibition of breast cancer cells. The growth inhibition effect was more pronounced in breast cancer cells in which Tα1 enhanced PTEN expression, whereas endogenous PTEN knockdown reversed the growth inhibition effect of Tα1 in breast cancer cells. Further investigation revealed that PTEN up-regulation, which was induced by Tα1, can inhibit the activation of the PI3K/Akt/mTOR signaling pathway, leading to the growth inhibition of breast cancer cells. The addition of the synergy between Tα1 and the inhibition of PI3K/Akt/mTOR activation could strongly block cell viability in PTEN down-regulated breast cancer cells. PTEN-overexpressing cells not only up-regulated Bax and cleaved caspase-3/9 and PARP expression but also down-regulated Bcl-2 compared to the treatment with Tα1 alone. Together these findings suggest that PTEN mediates Tα1-induced apoptosis through the mitochondrial death cascade and inhibition of the PI3K/Akt/mTOR signaling pathway in breast cancer cells.

ROS-Induced Nuclear Translocation of Calpain-2 Facilitates Cardiomyocyte Apoptosis in Tail-Suspended Rats.

Isoproterenol (ISO) induced nuclear translocation of calpain-2 which further increased susceptibility of cardiomyocyte apoptosis in tail-suspended rats. The underlying mechanisms remain elusive. In the present study, the results showed that ISO (10 nM) significantly elevated NADPH oxidases (NOXs) activity and NOXs-derived ROS productions which induced nuclear translocation of calpain-2 in cardiomyocytes of tail-suspended rats. In contrast, the inhibition of NADPH oxidase or cleavage of ROS not only reduced ROS productions, but also resisted nuclear translocation of calpain-2 and decreased ISO-induced apoptosis of cardiomyocyte in tail-suspended rats. ISO also increased the constitutive binding between calpain-2 and Ca(2+)/calmodulin-dependent protein kinase II δB (CaMK II δB) in nuclei, concomitant with the promotion of CaMK II δB degradation and subsequent down-regulation of Bcl-2 mRNA expression and the ratio of Bcl-2 to Bax protein in tail-suspended rat cardiomyocytes. These effects of ISO on cardiomyocytes were abolished by a calpain inhibitor PD150606. Inhibition of calpain significantly reduced ISO-induced loss of the mitochondrial membrane potential, cytochrome c release into the cytoplasm, as well as the activation of caspase-3 and caspase-9 in mitochondrial apoptotic pathway. In summary, the above results suggest that ISO increased NOXs-derived ROS which activated nuclear translocation of calpain-2, subsequently nuclear calpain-2 degraded CaMK II δB which reduced the ratio of Bcl-2 to Bax, and finally the mitochondria apoptosis pathway was triggered in tail-suspended rat cardiomyocytes. Therefore, calpain-2 may represent a potentially therapeutic target for prevention of oxidative stress-associated cardiomyocyte apoptosis.

Multisite phosphorylation of Bcl-2 via protein kinase Cδ facilitates apoptosis of hypertrophic cardiomyocytes.

Activated protein kinase Cδ (PKCδ) associated with cardiac hypertrophy moves from the cytoplasm to the mitochondria and subsequently triggers the apoptotic signalling pathway. The underlying mechanisms remain unknown. The aim of the present study was to investigate whether mitochondrial translocation of PKCδ phosphorylates multiple sites of Bcl-2, resulting in an imbalance between Bcl-2 and Bak or Bax, thus enhancing the susceptibility of hypertrophic cardiomyocytes to angiotensin II (AngII)-induced apoptosis. Chronic pressure overload was induced by transverse aortic constriction (TAC) in rats. The apoptotic rate increased in hypertrophied cardiomyocytes. In AngII-treated hearts (10 nmol/L, 60 min), there was an increase in the number of TERMINAL deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL)-positive cells; PKCδ inhibition with 500 nmol/L δV1-1 for 60 min prevented the AngII-induced increase in apoptosis. In the hypertrophied myocardium, PKCδ expression increased, whereas that of Bcl-2 decreased compared with the synchronous control. Treatment of hearts with 10 nmol/L AngII for 60 min activated PKCδ and induced translocation of PKCδ to the mitochondria, where activated PKCδ facilitated the phosphorylation of Bcl-2 at serine-87 and serine-70 sites. The multisite phosphorylated Bcl-2 was released from the mitochondria, and exhibited reduced affinity for Bak and Bax. The imbalance between Bcl-2 and Bak/Bax induced the release of mitochondrial cytochrome c and then activated the caspase 3 apoptotic pathway during AngII stimulation (10 nmol/L, 60 min) of hypertrophied cardiomyocytes. Inhibition of PKCδ reduced these effects of AngII. The results suggest that PKCδ can counteract the anti-apoptotic effect of Bcl-2 and may promote cardiomyocyte apoptosis through multisite phosphorylation of Bcl-2 in hypertrophied cardiomyocytes.

Porcine reproductive and respiratory syndrome virus nonstructural protein 4 antagonizes beta interferon expression by targeting the NF-κB essential modulator.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(I·C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I·C)-induced IFN-ß production by PRRSV was dependent on the blocking of NF-κB signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-ß expression. Moreover, we verified that nsp4 inhibited NF-κB activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKKß. nsp4 was shown to target the NF-κB essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-ß production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response. IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs. Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-κB signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-κB-mediated IFN-ß production. We found that nsp4 interfered with the NF-κB signaling pathway through the cleavage of NEMO (a key regulator of NF-κB signaling) at the E349-S350 site, leading to the downregulation of IFN-ß production induced by poly(I·C). The data presented here may help us to better understand PRRSV pathogenesis.

Enhanced N-terminal degradation of troponin I blunts cardiac function responsiveness to isoproterenol in 4-week tail-suspended rats.

The N-terminal extension of cardiac troponin I (cTnI) is important in regulating cardiac function. Although the normal rat myocardium shows some cTnI N-terminal degradation (cTnI-ND), exposure to 4 weeks of tail-suspension markedly increased cTnI-ND. We hypothesized that the increased cTnI-ND in tail-suspended rats may affect cardiac function, particularly during ß-adrenergic (ß-A) stimulation. The increase in cardiac output with isoproterenol (ISO) treatment was smaller in tail-suspended rats compared with controls. Left ventricular end-diastolic pressure was elevated and increases in maximal rates of left ventricular pressure development and relaxation were lower during ISO treatment in tail-suspended rats. Response to ISO, forskolin, DB-cAMP and IBMX was also lower in cardiomyocytes from tail-suspended rats. The increase in shortening and re-lengthening the rates of cardiomyocytes at a maximal dose of ISO, forskolin, DB-cAMP and IBMX treatment was limited in tail-suspended rats. There was no difference in Ca2+ sensitivity of the isometric force between tail-suspended and control rats, although Ca2+ sensitivity was decreased less in tail-suspended rats versus control rats during PKA phosphorylation. There was no difference in PKA protein expression and activation during ISO stimulation between the two groups. Due to the increase in cTnI-ND, ISO-induced phosphorylation of cTnI was reduced in tail-suspended rats. The total phospholamban expression and phosphorylation by ISO was unaltered in tail-suspended rat hearts. These data suggest that enhanced cTnI-ND following 4-week tail-suspension is a major component of the ß-A receptor signaling pathway, depressing cardiac function under ISO stimulation.

Activation of BK(Ca) channels in zoledronic acid-induced apoptosis of MDA-MB-231 breast cancer cells.

BACKGROUND: Zoledronic acid, one of the most potent nitrogen-containing biphosphonates, has been demonstrated to have direct anti-tumor and anti-metastatic properties in breast cancer in vitro and in vivo. In particular, tumor-cell apoptosis has been recognized to play an important role in the treatment of metastatic breast cancer with zoledronic acid. However, the precise mechanisms remain less clear. In the present study, we investigated the specific role of large conductance Ca(2+)-activated potassium (BK(Ca)) channel in zoledronic acid-induced apoptosis of estrogen receptor (ER)-negative MDA-MB-231 breast cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: The action of zoledronic acid on BK(Ca) channel was investigated by whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, and flow cytometry assays. Cell proliferation was investigated by MTT test and immunocytochemistry. In addition, such findings were further confirmed with human embryonic kidney 293 (HEK293) cells which were transfected with functional BK(Ca) α-subunit (hSloα). Our results clearly indicated that zoledronic acid directly increased the activities of BK(Ca) channels, and then activation of BK(Ca) channel by zoledronic acid contributed to induce apoptosis in MDA-MB-231 cells. The possible mechanisms were associated with the elevated level of intracellular Ca(2+) and a concomitant depolarization of mitochondrial membrane potential (Δψm) in MDA-MB-231 cells. CONCLUSIONS: Activation of BK(Ca) channel was here shown to be a novel molecular pathway involved in zoledronic acid-induced apoptosis of MDA-MB-231 cells in vitro.

High glucose alters apoptosis and proliferation in HEK293 cells by inhibition of cloned BK Ca channel.

It has been reported that diabetic vascular dysfunction is associated with impaired function of large conductance Ca(2+) -activated K(+) (BK(Ca) ) channels. However, it is unclear whether impaired BK(Ca) channel directly participates in regulating diabetic vascular remodeling by altering cell growth in response to hyperglycemia. In the present study, we investigated the specific role of BK(Ca) channel in controlling apoptosis and proliferation under high glucose concentration (25 mM). The cDNA encoding the α+ß1 subunit of BK(Ca) channel, hSloα+ß1, was transiently transfected into human embryonic kidney 293 (HEK293) cells. Cloned BK(Ca) currents were recorded by both whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry and analysis of fragmented DNA by agarose gel electrophoresis. Cell proliferation was investigated by flow cytometry assays, MTT test, and immunocytochemistry. In addition, the expression of anti-apoptotic protein Bcl-2, intracellular Ca(2+) , and mitochondrial membrane potential (Δψm) were also examined to investigate the possible mechanisms. Our results indicate that inhibition of cloned BK(Ca) channels might be responsible for hyperglycemia-altered apoptosis and proliferation in HEK-hSloα+ß1 cells. However, activation of BK(Ca) channel by NS1619 or Tamoxifen significantly induced apoptosis and suppressed proliferation in HEK-hSloα+ß1 cells under hyperglycemia condition. When rat cerebral smooth muscle cells were cultured in hyperglycemia, similar findings were observed. Moreover, the possible mechanisms underlying the activation of BK(Ca) channel were associated with decreased expression of Bcl-2, elevation of intracellular Ca(2+) , and a concomitant depolarization of Δψm in HEK-hSloα+ß1 cells. In conclusion, cloned BK(Ca) channel directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemia condition.

[The influence of contraction modes on the phosphorylation of p38/Akt].

OBJECTIVE: Muscle contraction may prompt glucose uptake through non-insulin-dependent ways, and it may be due to the enhanced activation of key proteins known to regulate glucose metabolism, like p38 and Akt. Our experiment focused on the impact of different contraction modes on the phosphorylation of the molecules, thus to explore effective ways to lower blood glucose. METHODS: Isolated muscle strips perfusion technique and Western blot analysis were employed to investigate the influence of different modes of contraction on the activation of the molecules. RESULTS: Muscle contraction led to an increase in p38 phosphorylation, with the greatest effect observed after 5 minutes of 10% DC (duty cycle) contraction and 5 minutes of 1% DC contraction. However, phosphorylation of Akt were not altered by the two contraction modes. CONCLUSION: The level of phosphorylation of p38 was higher at the optimal contraction modes, but these modes could not increase the level of phosphorlation of Akt.

[Activation of cloned BKCa channels in Ang II-induced proliferation of HEK293 cells].

AIM: To investigate the mediating and regulating role of BK(Ca) channels in Ang II-induced cell proliferation. METHODS: Using Lipofectamine 2000, the pIRES-hSloalpha plasmid was transfected into HEK293 cells. The concentration-dependent cuve of Ang II-induced cell proliferation was tested by MTT colorimetry. The effect of IbTX, Ang II, Ang II + IbTX on the proliferating cell nuclear antigen (PCNA) expression and cell cycle of transfected HEK293-hSloalpha cells were detected by immunocytochemistry and flow cytometry, respectively. RESULTS: Ang II induced proliferation of transfected HEK293-hSloalpha cells in a concentration-dependent manner. PCNA expression of transfected HEK293-hSloalpha cells was enhanced by Ang II, and the percentage of S phase HEK293-hSloalpha cells was increased after Ang II treatment. However this effect was inhibited by IbTX, a selective BK(Ca) channel blocker. CONCLUSION: BK(Ca) channels play an potential role in mediating Ang II-induced proliferation of HEK293-hSloalpha cells.

Coexpression and characterization of the human large-conductance Ca(2+)-activated K (+) channel alpha + beta1 subunits in HEK293 cells.

Large-conductance Ca(2+)-activated K(+) channel is formed by a tetramer of the pore-forming alpha-subunit and distinct accessory beta-subunits (beta1-beta4) which contribute to BK(Ca) channel molecular diversity. Accumulative evidences indicate that not only alpha-subunit alone but also the alpha + beta subunit complex and/or beta-subunit might play an important role in modulating various physiological functions in most mammalian cells. To evaluate the detailed pharmacological and biophysical properties of alpha + beta1 subunit complex or beta1-subunit in BK(Ca) channel, we established an expression system that reliably coexpress hSloalpha + beta1 subunit complex in HEK293 cells. The coexpression of hSloalpha + beta1 subunit complex was evaluated by western blotting and immunolocalization, and then the single-channel kinetics and pharmacological properties of expressed hSloalpha + beta1 subunit complex were investigated by cell-attached and outside-out patches, respectively. The results in this study showed that the expressed hSloalpha + beta1 subunit complex demonstrated to be fully functional for its typical single-channel traces, Ca(2+)-sensitivity, voltage-dependency, high conductance (151 +/- 7 pS), and its pharmacological activation and inhibition.

Effects of essential oil from Croton tiglium L. on intestinal transit in mice.

AIM OF THE STUDY: Croton tiglium (Croton tiglium L., Euphorbiaceae) is widely used as a herb for treatment of gastrointestinal disturbances. Previous studies established its purgative and inflammational properties. The present study aimed to investigate the effects of Croton tiglium oil (CO) on intestinal transit in mice. MATERIALS AND METHODS: Gastrointestinal transit in mice and contractile characteristics of isolated intestinal strips from mice were evaluated. Intestinal inflammation was confirmed by histological examination. RESULTS: Low dose of CO increased the gastrointestinal transit of charcoal and barium meal as well as the production of fecal pellets in mice. In contrast, high dose exerted inhibitory effects. For normal colonic circular strips, both high and low dose of CO inhibited the contractile frequency. Low doses (0-20 microg/ml) of CO enhanced the phasic contractions, while high doses (>40 microg/ml) reduced them. Colonic longitudinal strips in CO-treated mice were less sensitive to electrical field stimulation than those in control mice. The contraction of colonic longitudinal, colonic and jejunal circular strips in CO-treated mice was more sensitive to atropine than that in control mice. CONCLUSIONS: CO might modulate gastrointestinal motility and induce intestinal inflammation related to immunological milieu and motor activity. Our findings may highlight the ethno-medical uses of Croton tiglium on intestinal disorders.

[Non-specific effect of myosin inhibitor BDM on skeletal muscle contractile function].

AIM: In order to elucidate the underlying mechanism of depressed maximal isometric twitch tension normalized by cross sectional area of muscle strip in unloaded soleus. METHODS: The soleus and extensor digitorum longus (EDL) muscle strips were perfused in vitro and treated by 2,3-Butanedione monoxime (BDM). RESULTS: The BDM decreased Pt of soleus and EDL in a concentration-dependent manner. The Pt could restored completely to normal level after washing out BDM. The isometric twitch duration was not altered during 1 mmol/L BDM of perfusion, but was shortened at 10 mmol/L dose. The time from maximal to half Pt in EDL was shorter than that in soleus during 10 mmol/L BDM of perfusion. The inhibitory effects of BDM on myosin ATPase activity were higher in EDL than in soleus. The inhibitory extent of BDM on myosin ATPase activity of soleus and EDL was lower than that on Pt. CONCLUSION: These results suggest that reduction in cross-bridge function of skeletal muscle may be one of reasons induced a decrease in its Pt. BDM is not a specific inhibitor on myosin ATPase activity and can affect multiple parts of excitation-contraction coupling in skeletal muscle.

An R111C polymorphism in wild turkey cardiac troponin I accompanying the dilated cardiomyopathy-related abnormal splicing variant of cardiac troponin T with potentially compensatory effects.

Cardiac muscle contraction is regulated by Ca(2+) through the troponin complex consisting of three subunits: troponin C (TnC), troponin T (TnT), and troponin I (TnI). We reported previously that the abnormal splicing of cardiac TnT in turkeys with dilated cardiomyopathy resulted in a greater binding affinity to TnI. In the present study, we characterized a polymorphism of cardiac TnI in the heart of wild turkeys. cDNA cloning and sequencing of the novel turkey cardiac TnI revealed a single amino acid substitution, R111C. Arg(111) in avian cardiac TnI corresponds to a Lys in mammals. This residue is conserved in cardiac and skeletal muscle TnIs across the vertebrate phylum, implying a functional importance. In the partial crystal structure of cardiac troponin, this amino acid resides in an alpha-helix that directly contacts with TnT. Structural modeling indicates that the substitution of Cys for Arg or Lys at this position would not disrupt the global structure of troponin. To evaluate the functional significance of the different size and charge between the Arg and Cys side chains, protein-binding assays using purified turkey cardiac TnI expressed in Escherichia coli were performed. The results show that the R111C substitution lowered binding affinity to TnT, which is potentially compensatory to the increased TnI-binding affinity of the cardiomyopathy-related cardiac TnT splicing variant. Therefore, the fixation of the cardiac TnI Cys(111) allele in the wild turkey population and the corresponding functional effect reflect an increased fitness value, suggesting a novel target for the treatment of TnT myopathies.