Inhibition of sarcoplasmic reticular function by chronic interleukin‐6 exposure via iNOS in adult ventricular myocytes

Interleukin (IL)-6, a pro-inflammatory cytokine, is produced during the acute phase of the immune response by immune and nonimmune cells (for review, see Akira et al. 1990). After binding to its receptor, IL-6 elicits numerous biological effects, including antibody induction, haematopoiesis, thrombocytopoiesis and acute-phase protein synthesis (for reviews, see Akira et al. 1990; Kishimoto et al. 1995). Recently, significant increases in serum levels of IL-6 and its mRNA expression and protein production in cardiac tissues have been reported in patients with chronic cardiac failure (Satoh et al. 1996; Steele et al. 1996; Roig et al. 1998; Cesari et al. 2003), myocarditis (Satoh et al. 1996), myocardial infarction (Ikeda et al. 1992; Guillen et al. 1995; Neumann et al. 1995), endotoxaemia (Hack et al. 1989), and the injury associated with ischaemia/reperfusion (Sawa et al. 1998) and cardiopulmonary bypass (Wan et al. 1996). In patients undergoing cardiopulmonary bypass, the increase in plasma IL-6 levels in the coronary sinus begins 5 min after aortic declamping, peaks at 1 h, and is sustained for at least 2 h (Wan et al. 1996). Therefore, IL-6 has been associated with the severity of several cardiovascular pathophysiological states (Cesari et al. 2003). In vitro studies on cardiac contractile function have shown that exposure to IL-6 for 2–3 min decreased contractility in papillary muscle isolated from hamster heart (Finkel et al. 1992). Studies with single myocytes also showed that IL-6 suppressed peak systolic [Ca2+]i and cell shortening (CS) within 5 min in chick embryonic cardiac myocytes (Kinugawa et al. 1994) and in adult guinea-pig ventricular myocytes (Sugishita et al. 1999). This acute negative inotropic effect of IL-6, accompanied by an increase in cell cGMP production (Kinugawa et al. 1994), was blocked by NG-monomethyl-l-arginine (l-NMMA), an inhibitor of nitric oxide synthase (NOS) (Finkel et al. 1992; Kinugawa et al. 1994; Sugishita et al. 1999). Thus, acute IL-6-induced suppression of cardiac contractility and [Ca2+]i have been suggested to be mediated by a NO-dependent pathway via activation of NOS (Finkel et al. 1992; Kinugawa et al. 1994; Sugishita et al. 1999), probably a constitutive endothelial isoform (eNOS) (Kinugawa et al. 1994). In contrast, studies on chronic cardiac effects of IL-6 showed that after 24 h incubation, IL-6 also increased cell cGMP, which was blocked by l-NMMA but not by EGTA, suggesting the involvement of a Ca2+-independent NOS (Kinugawa et al. 1994). However, the subtype of NOS that is involved in chronic cardiac effect of IL-6 has not been defined. As mentioned above, an in vivo study showed that elevated IL-6 levels were observed 1–2 h after aortic declamping in cardiopulmonary bypass (Wan et al. 1996). The direct cardiac effect of IL-6 during this period of time remains undefined. We recently reported that de novo synthesis and activation of iNOS induced by IL-6 can be detected in adult rat ventricular myocytes (ARVM) as early as 2 h after exposure (Yu et al. 2003). This earlier study also demonstrated that the IL-6-elicited iNOS activation and decrease in postrest potentiation (PRP) of contraction in ARVM are mediated by activation of Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3, the upstream mediators of IL-6 signalling (Yu et al. 2003). However, whether iNOS is the downstream mediator of IL-6-induced negative inotropy remains undefined. In the present study, we examined the effect of chronic exposure (for 2–24 h) of ARVM to IL-6 on contractility, contractile responsiveness to Ca2+o, L-type Ca2+-channel current (ICa,L), and sarcoplasmic reticular (SR) function. SR function was assessed using two protocols: PRP and caffeine-induced contraction (Bassani et al. 1993, 1994; Bers et al. 1998). We also examined the role of iNOS in IL-6-induced changes in SR function. We found that IL-6 decreased contractility and Ca2+o responsiveness of ARVM, primarily resulting from suppression of SR function accompanied by reduction in phosphorylation of phospholamban (PLB), a SR Ca2+-pump regulatory protein. This chronic cardiac effect of IL-6 was sustained after removal of IL-6. We also showed that inhibition of iNOS and NO production blocked the IL-6 negative inotropic action. A preliminary report of some of these results has been presented in an abstract (Yu et al. 2000).