The primers used in this study.

<p>The primers used in this study.</p

Tunicamycin suppresses the gp130-mediated activation of JAK1 and 2.

<p>Neonatal rat cardiac myocytes, pretreated with the indicated concentrations of Tm for 8 hours, were stimulated with LIF (300 U/ml) for 15 minutes. The activation of JAK1 and JAK2 was analyzed by immunoblotting with the phospho-JAK1 and phospho-JAK2 specific antibodies. Membranes were stripped and reprobed with anti-JAK1, anti-JAK2, or anti-GAPDH antibody, respectively. Representative images were shown (A). (B). For quantification, densitometric analyses for JAK1 and JAK2 phosphorylation were normalized with those of total JAK1 or total JAK2, respectively. Values were converted based on that of each group treated with LIF alone. Data were mean ± S.D. of three independent experiments. Dunnett test was performed for post-hoc multiple comparison test. *; <i>P</i><0.05 versus LIF alone.</p

Tunicamycin inhibits JAK/STAT3 pathway downstream of gp130 independently of ER stress, PTP1B and SOCSs.

<p>Neonatal rat cardiac myocytes were cultured with the indicated concentrations of Tm for 8 hours. Total RNA was prepared and applied for reverse transcription. Real time PCR system was used to detect the mRNA expression of CHOP (A), Grp78 (A), PTP1B (C), SOCS1 and 3 (E) as described under ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111097#s2" target="_blank">Material and Methods</a>’. The expression level of each gene was normalized with that of GAPDH, an internal control, and represented as value of fold induction relative to those of each non-treated group with Tm (control). Data were shown as mean ±S.D. (n = 3). **; <i>P</i><0.05 versus control at the multiple comparison test. After cardiac myocytes were pretreated with or without Tm (2 µg/mL) for 8 hours, cells were washed with medium and incubated for more 15 hours. Afterward, cells lysates were prepared for immunoblotting analyses with anti-CHOP, anti-Grp78 and GAPDH antibody. Experiments were repeated three times with similar results and representative data are shown in (B). Cardiac myocytes were pretreated with or without Tm (2 µg/mL) for 8 hours in the presence or absence of JTT551 (JTT), PTP1B inhibitor, and stimulated with LIF (300 U/I) for 15 minutes. Activations of STAT3 and ERK1/2 were analyzed by immunoblotting with anti-phospho-STAT3, anti-phopho-ERK1/2 and anti-GAPDH antibody. Representative images were shown in (D).</p

Tunicamycin does not influence the localization of gp130 in cardiomyocytes.

<p>Neonatal rat cardiomyocytes were treated with or without Tm (2 µg/mL) for 8 hours. Cultured cells were fixed and immunostained with anti-gp130 antibody or Hoechst for nuclei. Bar indicates 15 µm. Representative images were shown.</p

The combined treatment with IL-6 and sIL-6R fails to activate STAT3 in the presence of tunicamycin.

<p>Neonatal rat cardiac myocytes, pretreated with or without Tm (2 µg/mL) for 8 hours, were stimulated with IL-6 (20 ng/mL) plus sIL-6R (100 ng/mL) for 15 minutes. Activation of STAT3 and ERK1/2 were analyzed by immunoblotting with each phospho-specific antibody. Membranes were stripped and reprobed with anti-STAT3, anti-ERK1/2, or anti-GAPDH antibody, respectively. Representative images were shown (A). (B). For quantification, densitometric analyses for STAT3 or ERK1/2 phosphorylation were normalized with those of total STAT3 or total ERK, respectively. Values were converted based on that of each group treated with cytokine alone. Data were mean ± S.D. of three independent experiments. Dunnett test was performed for post-hoc multiple comparison test. *; <i>P</i><0.05 versus the combination of IL-6 and sIL-6R alone.</p

The Inhibition of N-Glycosylation of Glycoprotein 130 Molecule Abolishes STAT3 Activation by IL-6 Family Cytokines in Cultured Cardiac Myocytes

<div><p>Interleukin-6 (IL-6) family cytokines play important roles in cardioprotection against pathological stresses. IL-6 cytokines bind to their specific receptors and activate glycoprotein 130 (gp130), a common receptor, followed by further activation of STAT3 and extracellular signal-regulated kinase (ERK)1/2 through janus kinases (JAKs); however the importance of glycosylation of gp130 remains to be elucidated in cardiac myocytes. In this study, we examined the biological significance of gp130 glycosylation using tunicamycin (Tm), an inhibitor of enzyme involved in N-linked glycosylation. In cardiomyocytes, the treatment with Tm completely replaced the glycosylated form of gp130 with its unglycosylated one. Tm treatment inhibited leukemia inhibitory factor (LIF)-mediated activation of STAT3 and ERK1/2. Similarly, IL-11 failed to activate STAT3 and ERK1/2 in the presence of Tm. Interestingly, Tm inhibited the activation of JAKs 1 and 2, without influencing the expression of suppressor of cytokine signalings (SOCSs) and protein-tyrosine phosphatase 1B (PTP1B), which are endogenous inhibitors of JAKs. To exclude the possibility that Tm blocks LIF and IL-11 signals by inhibiting the glycosylation of their specific receptors, we investigated whether the stimulation with IL-6 plus soluble IL-6 receptor (sIL-6R) could transduce their signals in Tm-treated cardiomyocytes and found that this stimulation was unable to activate the downstream signals. Collectively, these findings indicate that glycosylation of gp130 is essential for signal transduction of IL-6 family cytokines in cardiomyocytes.</p></div

Tunicamycin inhibits the glycosylation of gp130 completely, but does those of LIFR and IL-11Rα partially.

<p>Neonatal rat cardiomyocytes were treated with the indicated concentrations of Tm for 8 hrs (A), or with 2 µg/mL of Tm for the indicated times (B), respectively. After each treatment, cell lysates were prepared and immunoblotted with anti-gp130, LIFR or IL-11Rα antibody, respectively. More than three independent experiments were performed with similar results and representative images were shown.</p

2-aminoethoxydiphenyl borate provides an anti-oxidative effect and mediates cardioprotection during ischemia reperfusion in mice

<div><p>Excessive levels of reactive oxygen species (ROS) and impaired Ca²⁺ homeostasis play central roles in the development of multiple cardiac pathologies, including cell death during ischemia-reperfusion (I/R) injury. In several organs, treatment with 2-aminoethoxydiphenyl borate (2-APB) was shown to have protective effects, generally believed to be due to Ca²⁺ channel inhibition. However, the mechanism of 2-APB-induced cardioprotection has not been fully investigated. Herein we investigated the protective effects of 2-APB treatment against cardiac pathogenesis and deciphered the underlying mechanisms. In neonatal rat cardiomyocytes, treatment with 2-APB was shown to prevent hydrogen peroxide (H₂O₂) -induced cell death by inhibiting the increase in intracellular Ca²⁺ levels. However, no 2-APB-sensitive channel blocker inhibited H₂O₂-induced cell death and a direct reaction between 2-APB and H₂O₂ was detected by ¹H-NMR, suggesting that 2-APB chemically scavenges extracellular ROS and provides cytoprotection. In a mouse I/R model, treatment with 2-APB led to a considerable reduction in the infarct size after I/R, which was accompanied by the reduction in ROS levels and neutrophil infiltration, indicating that the anti-oxidative properties of 2-APB plays an important role in the prevention of I/R injury <i>in vivo</i> as well. Taken together, present results indicate that 2-APB treatment induces cardioprotection and prevents ROS-induced cardiomyocyte death, at least partially, by the direct scavenging of extracellular ROS. Therefore, administration of 2-APB may represent a promising therapeutic strategy for the treatment of ROS-related cardiac pathology including I/R injury.</p></div

Hydrogen peroxide directly reacts with 2-APB.

<p>(A) Neonatal rat cardiomyocytes (NRCMs) were loaded with 5 μM DCF, and treated with 2-APB and H₂O₂. Average results of %Fluorescence increase were obtained from three independent experiments. (B) Intracellular ROS levels at 60 min after H₂O₂ stimulation were estimated from DCF fluorescence levels in NRCMs pretreated or not with 2-APB. Results of %Fluorescence increase are presented as mean values ± SEM obtained in three independent experiments. *<i>P</i><0.05 <i>vs</i>. untreated control, ^†<i>P</i><0.05 <i>vs</i>. H₂O₂ treated samples, obtained using one-way ANOVA. (C) NRCMs were loaded with 5 μM DCF and treated with 2-APB for 1 h, followed by stimulation with 2 mM PE. Average results of %Fluorescence increase were obtained from three independent experiments. (D) Intracellular ROS levels at 60 min after PE stimulation, estimated using DCF fluorescence intensity levels in NRCMs pretreated or not with 2-APB. Results of %Fluorescence increase are presented as mean values ± SEM obtained in three independent experiments. **<i>P</i><0.01 <i>vs</i>. untreated control, using one-way ANOVA. (E) Representative ¹H-NMR spectra obtained from 2-APB (spectrum A) or 2-APB in direct reaction with H₂O₂ for 5 min (spectrum B) or 60 min (spectrum C) are shown. (F) Representative thin-layer chromatography (TLC) images are presented. Reaction product indicates samples obtained from the reaction between 2-APB with H₂O₂ used for NMR measurement. Comparison between the reaction products and authentic H₂O₂ (left), 2-APB (middle), or phenol (right) is presented.</p

Treatment with 2-APB attenuated H₂O₂-induced cell death and Ca²⁺ influx in cardiomyocytes.

<p>(A) Analysis of the viability of neonatal rat cardiomyocytes (NRCMs) with or without 100 μM 2-APB pretreatment for 1 h, followed by subsequent stimulation with 100 μM H₂O₂ for 4 h. Results are presented as mean ± SEM obtained in five to seven independent experiments. (B) Representative fura-2 ratios from NRCMs pretreated with or without 2-APB for 1 h, followed by stimulation with H₂O₂. (C) NRCMs were pretreated or not with 5 μM BAPTA-AM, which was followed by H₂O₂ treatment and the degree of cell survival is presented as mean values ± SEM obtained in six or seven independent experiments. (D) Representative fura-2 ratios from NRCMs pretreated or not with 10 μM BAPTA-AM for 1 h, followed by H₂O₂ treatment. (E) Representative fura-2 ratios obtained using NRCMs treated with 100 μM H₂O₂ in HBSS buffer with or without Ca²⁺ prepared by the administration of EDTA. **<i>P</i><0.01, compared with H₂O₂-treated samples, using one-way ANOVA.</p