No abstract

Cytokines have significant roles in some cardiovascular disorders, but direct myocardial effects of cytokines remain to be elucidated. In the present study, we examined both the early and delayed effects of interleukin-6 (IL-6) on cultured chick embryo ventricular myocytes. Exposure of these cells to human recombinant IL-6 significantly decreased peak systolic [Ca2+]i (71.0 +/- 0.6% of the control value) and the amplitude of cell contraction (66.0 +/- 7.4% of the control value) within a few minutes. Pretreatment with NG-monomethyl-L-arginine (L-NMMA) or methylene blue completely inhibited the IL-6-induced early changes. Subsequent addition of L-arginine reversed the effects of L-NMMA. The levels of cGMP were significantly increased after 30 minutes of exposure to IL-6 (134.4 +/- 9.1% of the control value). Pretreatment with L-NMMA or EGTA significantly inhibited the IL-6-induced early elevation of cGMP. These results suggest that IL-6 acutely decreases intracellular Ca2+ transients and depresses cell contraction by nitric oxide (NO)-cGMP-mediated pathway. Therefore, IL-6 may enhance the Ca(2+)-dependent constitutive NO synthase activity in cardiac myocytes. On the other hand, 24-hour exposure to IL-6 also increased the levels of cGMP (159.0 +/- 22.8% of the control value) regardless of pretreatment with EGTA. These delayed increases in cGMP were also shown to be coupled with decreases in intracellular Ca2+ transients and the amplitude of cell contraction. Thus, IL-6 may induce Ca(2+)-independent NO synthase in cardiac myocytes. Together with the previous reports that have suggested the possible roles of IL-6 in myocardial stunning or endotoxic shock, this negative inotropic effect of IL-6 may contribute to these clinical settings.

A B S T R A C T To clarify conflicting reports concerning the effects of ischemia on left ventricular chamber stiffness, we compared the effects of hypoxia at constant coronary perfusion with those of global ischemia on left ventricular diastolic chamber stiffness using isolated, perfused rabbit hearts in which the left ventricle was contracting isovolumically. Since chamber volume was held constant, increases in left ventricular end diastolic pressure (LVEDP) reflected increases in chamber stiffness. At a control coronary flow rate (30 ml/min), 2 min of hypoxia and pacing tachycardia (4.0 Hz) produced major increases in postpacing LVEDP (10±+1 to 24+3 mm Hg, P < 0.01) and the relaxation time constant, T, (40+4 to 224±37 ms, P < 0.001), while percent lactate extraction ratio became negative (+18±2 to -48±15%, P < 0.001). Coronary perfusion pressure decreased (72+5 to 52±3 mm Hg, P < 0.01), and since coronary flow was held constant, the fall in coronary perfusion pressure reflected coronary dilation and a decrease in coronary vascular resistance. Following an average of 71±6 s reoxygenation and initial heart rate (2.0 Hz), LVEDP and relaxation time constant T returned to control. Hypoxia alone (without pacing tachycardia) produced similar although less marked changes (LVEDP, 10-+-1 to 20+3 mm Hg; and T, 32±3 to 119+22 ms; P < 0.01 for both) and there was a strong correlation between LVEDP and T (r = 0.82, P < 0.001).When a similar degree of coronary vasodilatation was induced with adenosine, no change in LVEDP Received for publication 7 July 1980 and in revised form 6 October 1980. occurred, indicating that the increase in end diastolic pressure observed during hypoxia was not secondary to vascular engorgement, but due to an acute effect of hypoxia on the diastolic behavior of the ventricular myocardium.In contrast, global ischemia produced by low coronary flow (12-15 ml/min) resulted in a decrease in LVEDP, as well as a marked fall in left ventricular systolic pressure. In 14 global ischemia experiments, pacing tachycardia led to a further decline in left ventricular systolic pressure, and no increase was noted in postpacing LVEDP. Changes in lactate extraction ratio were much smaller in magnitude than with hypoxia and constant coronary perfusion. In two experiments (one at normal coronary flow and one at 15 ml/min), left ventricular systolic pressure did not change markedly from control when tachycardia was superimposed, and postpacing LVEDP showed a marked rise (to >25 mm Hg), which gradually recovered over 1-2 min at the control heart rate.From these results, we conclude that left ventricular chamber stiffness increases when myocardial 02 demand exceeds supply. This change is usually masked in ischemic (reduced coronary flow) preparations, perhaps because of reduced turgor of the coronary vascular bed, marked reductions in systolic work (and therefore myocardial 02 requirements), and local accumulation of hydrogen ion and metabolites following acute severe reduction of coronary flow. The increased chamber...

Previous work has demonstrated that inducible NO synthase (iNOS) can be expressed in cardiac myocytes. In this study, we investigated transcriptional regulation of the iNOS gene in these cells. Lipopolysaccharide (LPS) induced iNOS mRNA and protein in cultured neonatal rat cardiac myocytes. H-89, dexamethasone, herbimycin, genistein, staurosporine, or pyrrolidine dithiocarbamate (PDTC) attenuated the iNOS induction by LPS. Forskolin, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma enhanced the LPS-induced iNOS expression. Combined stimulation of IL-6 and TNF-alpha also induced iNOS. The 5'-upstream sequence of the rat iNOS gene contains the nuclear factor-kappa B (NF-kappa B) site, CAAT box, IFN-gamma activation site (GAS), and IFN regulatory factor (IRF) site. DNase I footprinting assay revealed that the nuclear factors binding to these elements were increased by LPS exposure. Transient transfection assay suggested that these elements were indispensable for transcriptional regulation of the iNOS induction. Electrophoretic mobility shift assay revealed that LPS or TNF-alpha increased binding activity for the NF-kappa B site. A slower-migrating complex binding to the CAAT box gave rise after exposure to LPS or forskolin. Competition assay suggested that this slower-migrating complex consisted of a heterodimer between a member of CAAT box/enhancer binding (C/EBP) protein family and cAMP responsive element binding protein (CREB). LPS or IL-6 increased binding complexes for the IRF site, which was compatible with induction of IRF-1. LPS, IL-6, or IFN-gamma induced a novel binding complex for GAS, which also existed in the 5'-flanking region of the IRF-1 gene. These data suggest that (1) iNOS induction simultaneously requires both NF-kappa B activation and IRF-1 induction, and (2) the heterodimer between C/EBP and CREB has synergistic effects on the iNOS induction via the CAAT box.

We studied the effect of MgADP on the mechanical interaction of actomyosin in cardiac and skeletal muscles using an in vitro motility assay. The sliding velocities of fluorescently labeled actin filaments on rat cardiac and skeletal myosins were measured at various MgATP and MgADP concentrations. The filament velocity depended on MgATP concentration according to classic Michaelis-Menten kinetics with apparent Michaelis constants (Km) of 43 and 137 mumol/L and maximum velocity of 5.6 and 8.6 microns/s for cardiac and skeletal myosins, respectively. The presence of 2 mmol/L MgADP decreased the filament velocity and shifted the substrate concentration dependence of the velocity toward higher MgATP concentrations, yielding the inhibition constants of 194 and 478 mumol/L for cardiac and skeletal myosins, respectively. The activation energies determined by the temperature dependence of the velocity were 61 and 83 kJ/mol for rat V1 and rabbit cardiac myosins, which were similar to those of the dissociation rate constant of actomyosin-ADP complex reported in a solution study. The inhibition of the velocity by MgADP can be explained by the crossbridge scheme in which MgADP competes with MgATP for the substrate site on myosin molecules. In cardiac myosin, addition of a concentration of MgADP as low as 25 mumol/L significantly inhibited the velocity in the presence of 2 mmol/L MgATP, suggesting that increased intracellular MgADP may reduce the rate of crossbridge detachment, resulting in a decreased ATP consumption and an increased economy of force production under ischemic conditions. The present results support the idea that MgADP may be a physiologically important modulator of contraction in cardiac muscle.