Epac activation inhibits IL-6-induced cardiac myocyte dysfunction

Figures

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  Fig. 1. Effects of various cytokines on signal transducer and activator of transcription (STAT) phosphorylation and the time course of interleukin (IL)-6-mediated STAT phosphorylation. a Phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyrosine 705 (Tyr705) was examined at 30 min after treatment with IL-6 (30 ng/ml), IL-1β (10 ng/ml), IL-10 (10 ng/ml), IL-17 (10 ng/ml), interferon gamma (IFN-γ; 10 ng/ml), keratinocyte-derived chemokine (KC; 50 ng/ml), monocyte chemotactic protein-1 (MCP-1; 10 ng/ml), RANTES (regulated upon activation, normal T cell expressed and secreted; 10 ng/ml), and tumor necrosis factor alpha (TNF-α; 10 ng/ml). The phosphorylation was upregulated by 14-fold in response to IL-6 (n = 4, *significantly different at p < 0.05, Student’s t test). T-STAT Total STAT, P-STAT phosphorylated STAT. b Phosphorylation of signal transducer and activator of transcription 1 (STAT1) at Tyrosine 701 (Tyr701) was examined at 30 min after treatment with IL-6 (30 ng/ml), IL-1β (10 ng/ml), IL-10 (10 ng/ml), IL-17 (10 ng/ml), IFN-γ (10 ng/ml), KC (50 ng/ml), MCP-1 (10 ng/ml), RANTES (10 ng/ml) and TNF-α (10 ng/ml). The phosphorylation was upregulated by…

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  Fig. 2. Effects of exchange protein activated by cAMP (Epac) on the activation of the Janus kinase–signal transducers and activators of transcription (Jak-STAT) pathway in response to IL-6. a, b Pretreatment of neonatal rat cardiac myocytes with 8-(4-chlorophenylthio)-2′-O-Me-cAMP-AM (8-CPT-AM; 10 μmol/L) for 5 h significantly reduced both IL-6-mediated phosphorylation of STAT3 at Tyr705 by 57 ± 11.5% (a) (n = 5–6, **significantly different at p < 0.01, Student’s t test) and IL-6-mediated phosphorylation of STAT1 at Tyr701 by 65 ± 6.8% (b) (n = 6–8, ****significantly different at p < 0.0001, Student’s t test). c Pretreatment of neonatal rat cardiac myocytes with Epac1 selective inhibitor CE3F4 for 30 min significantly enhanced IL-6-mediated phosphorylation of STAT3 at Tyr705 by 35 ± 13.0% (n = 6, *significantly different at p < 0.05, one-way ANOVA). Data are presented as the mean ± SEM

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  Fig. 3. Time course of Epac-mediated expression of suppressor of cytokine signaling-3 and -1 (SOCS3, SOCS1, respectively) in cardiac myocytes. a, b mRNA expression of SOCS3 (a) and SOCS1 (b) was examined by quantitative real-time PCR in neonatal rat cardiac myocytes in the presence of 8-CPT-AM (10 μmol/L) for the indicated time. mRNA expression of SOCS3 and SOCS1 gradually increased with 8-CPT-AM treatment. a SOCS3 mRNA expression peaked at a significant increase of 1.7-fold at 1 h after 8-CPT-AM treatment (n = 4, *significantly different at p < 0.05 vs. baseline, one-way ANOVA). b SOCS1 mRNA expression peaked at a significant increase of 6.2-fold at 3 h after 8-CPT-AM treatment (n = 3–4, **significantly different at p < 0.01 vs. baseline, one-way ANOVA). NS Not significant. Data are presented as the mean ± SEM

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  Fig. 4. Effects of Epac activation on SOCS3 expression and IL-6 induced inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. a SOCS3 mRNA expression, examined by quantitative real-time PCR in neonatal rat cardiac myocytes in the presence of 8-CPT-AM (10 μmol/L) for 1 h, was significantly increased by 1.8-fold. However, Epac-mediated SOCS3 upregulation was blunted by the addition of a phospholipase C (PLC) inhibitor (U73122, 0.5 μmol/L) or protein kinase C (PKC) inhibitor (Ro-31-7549, 2 μmol/L) (n = 6–10, ***significantly different at p < 0.001 vs. baseline, one-way ANOVA). b SOCS3 protein expression, examined by western blotting in the presence of 8-CPT-AM (10 μmol/L) for 5 h, was significantly increased by 2.2-fold in the presence of 8-CPT-AM. This upregulation was also blunted by the addition of PLC inhibitor or PKC inhibitor (n = 6–11, ****significantly different at p < 0.0001 vs. baseline, one-way ANOVA). c Western blot analysis of iNOS expression in neonatal rat cardiac myocytes treated for the indicated times with IL-6 (30 ng/ml). iNOS expression increased gradually, peaking at 6 h. However, pretreatment with 8-CPT-AM (10 μmol/L) for 5 h inhibited the…

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  Fig. 5. Effects of Epac activation on Ca2+ concentration (a, b) and contractility of cardiac myocytes (c, d). a Intracellular Ca2+ concentration in response to increasing concentrations of isoproterenol (ISO; 0–10−5 mol/L) in adult rat cardiac myocytes with or without a 6-h IL-6 (30 ng/ml) pretreatment. b The effect of a 5-h 8-CPT-AM (10 μmol/L) pretreatment on the ISO-induced increase of Ca2+. Pretreatment with 8-CPT-AM restored the increase of Ca2+ transients suppressed by IL-6 (n = 29–64, **significantly different at p < 0.01, two-way ANOVA). c Cardiac cell shortening at baseline and in response to ISO (10−5 mol/L). d Pretreatment with 8-CPT-AM (10 μM) restored cell shortening suppressed by IL-6 (30 ng/ml) (n = 24–31, ****significantly different at p < 0.0001, two-way ANOVA). Data are presented as the mean ± SEM