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Interleukin (IL)-6 has been shown to decrease cardiac contractility via a nitric oxide synthase (NOS)-dependent pathway during acute exposure. We previously reported that IL-6 decreases contractility and increases inducible NOS (iNOS) in adult rat ventricular myocytes (ARVM) after 2 h exposure. The goal of this study was to investigate the cellular mechanism underlying this chronic IL-6-induced negative inotropy and the role of iNOS. Pretreatment for 2 h with 10 ng ml−1 IL-6 decreased the kinetics of cell shortening (CS) and contractile responsiveness to Ca2+o ([Ca2+]o from 0 to 2 mm) in ARVM. We first examined whether IL-6 reduced Ca2+ influx via L-type Ca2+-channel current (ICa,L). Whole-cell ICa,L in ARVM was measured under conditions similar to those used for CS measurements, and it was found to be unaltered by IL-6. The sarcoplasmic reticular (SR) function was then assessed by examining postrest potentiation (PRP) and caffeine responsiveness of CS. Results showed that treatment with IL-6 for 2 h significantly decreased PRP, which was concomitant with a decrease in the phosphorylation of phospholamban. Following removal of IL-6, PRP and responsiveness to 10 mm caffeine were also reduced. Meanwhile, the IL-6-induced increase in nitric oxide (NO) production after 2 h (but not 1 h) was abolished by NG-monomethyl-l-arginine (l-NMMA) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; a selective inhibitor of iNOS). Furthermore, IL-6-elicited suppressions of PRP and responsiveness to caffeine and Ca2+o were abolished by L-NMMA and AMT. Thus, these results suggest that activation of iNOS mediates IL-6-induced inhibition of SR function in ARVM during chronic exposure.

We previously showed that chronic exposure to interleukin (IL)-6 decreases contractile and sarcoplasmic reticular (SR) function assessed by postrest potentiation (PRP) via a nitric oxide (NO)-dependent mechanism in adult rat ventricular myocytes (ARVM). Cyclic GMP (cGMP) has been associated with NO-associated negative inotropic effects of IL-6 during acute exposure; however, its role in chronic cardiac effects of IL-6 remains unclear. The present study examined the roles of cGMP and peroxynitrite (ONOO−) in chronic IL-6-induced negative inotropy in ARVM. After ARVM were exposed to IL-6 for 2–24 h, intracellular cGMP contents were time dependently increased; this was mimicked by a NO donor and abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), or Rp-8-Br-cGMP, an inhibitor of cGMP-dependent protein kinase G (PKG). Meanwhile, the IL-6-induced decrease in PRP at 2 h was blocked by ODQ or Rp-8-Br-cGMP. By contrast, ODQ or Rp-8-Br-cGMP only attenuated the inhibition of PRP induced by IL-6 after 24 h exposure. Furthermore, IL-6 time dependently increased superoxide anion production and ONOO− formation; the latter was abolished by 5,10,15,20-tetrakis-(4-sulphonatophenyl)-porphyrinato iron (III) (FeTPPS), an ONOO− decomposition catalyst. Interestingly, FeTPPS had no effect on the IL-6-elicited decrease in PRP at 2 h, but attenuated it after 24 h exposure. Moreover, inhibition of sGC/cGMP/PKG, but not ONOO− formation, abolished the IL-6-induced inhibition of kinetics of myocyte contraction during 24 h exposure. We conclude that while the sGC/cGMP/PKG pathway was the primary mechanism for chronic IL-6-induced negative inotropy at 2 h, both sGC/cGMP/PKG and ONOO−, at least in part, mediate the IL-6-induced inhibition of SR function after 24 h exposure.

Interleukin (IL)-6 decreases cardiac contractility via a nitric oxide (NO)-dependent pathway. However, mechanisms underlying IL-6-induced NO production remain unclear. JAK2/STAT3 and ERK1/2 are two well known signaling pathways activated by IL-6 in non-cardiac cells. However, these IL-6-activated pathways have not been identified in adult cardiac myocytes. In this study, we identified activation of these two pathways during IL-6 stimulation and examined their roles in IL-6-induced NO production and decrease in contractility of adult ventricular myocytes. IL-6 increased phosphorylation of STAT3 (at Tyr(705)) and ERK1/2 (at Tyr(204)) within 5 min that peaked at 15-30 min and returned to basal levels at 2 h. Phosphorylation of STAT3 was blocked by genistein, a protein tyrosine kinase inhibitor, and AG490, a JAK2 inhibitor, but not PD98059, an ERK1/2 kinase inhibitor. The phosphorylation of ERK1/2 was blocked by PD98059 and genistein but not AG490. Furthermore, IL-6 enhanced de novo synthesis of iNOS protein, increased NO production, and decreased cardiac contractility after 2 h of incubation. These effects were blocked by genistein and AG490 but not PD98059. We conclude that IL-6 activated independently the JAK2/STAT3 and ERK1/2 pathways, but only JAK2/STAT3 signaling mediated the NO-associated decrease in contractility.