Cellular basis for the acute inhibitory effects of IL-6 and TNF-α on excitation-contraction coupling

There is controversy over whether nitric oxide (NO) mediates acute negative inotropic actions of cytokines including tumor necrosis factor-alpha (TNF-α). The reports from established laboratories have appeared inconsistent, which could be due to species differences. Thus, we tried to elucidate the mechanisms underlying negative inotropic action of interleukin-6 (IL-6) and TNF-α in the same model. We studied the effects of cytokines on [Ca²⁺](i) transients (using indo-1), cell shortening (CS) (using a video motion detector) and the L-type Ca²⁺ channel current (I(Ca)) (using the whole cell perforated patch clamp technique) in isolated guinea-pig ventricular myocytes. IL-6 (1000 U/ml) or TNF-α (500 U/ml) decreased both peak systolic [Ca²⁺](i) (IL-6: 0.43 ± 0.01 to 0.40 ± 0.01, n = 5, P < 0.05; TNF-α: 0.42 ± 0.02 to 0.39 ± 0.02, n = 5, P < 0.05) and the amplitude of CS (IL-6: 7.5 ± 0.9 to 6.2 ± 0.5 μm, n = 5, P < 0.05; TNF-α: 6.7 ± 0.7 to 5.8 ± 0.7 μm, n = 5, P < 0.05) without detectable reductions in I(Ca) (IL-6: 0.9 ± 0.1 to 0.9 ± 0.1 nA, n = 4, N.S.; TNF-α: 1.1 ± 0.3 to 1.1 ± 0.2 nA, n = 4, N.S.) within 5 min. The nitric oxide synthase (NOS) inhibitor, N(G)-monomethyl-L arginine (300 μmol/l), blocked the effects of IL-6 but not of TNF-α. When pretreated with 20 nmol/l isoproterenol, exposure to IL-6 decreased both I(Ca) (2.8 ± 0.5 to 2.0 ± 0.3 nA) and the amplitude of CS (10.4 ± 2.4 to 7.5 ± 1.9 μm) within 5 min. TNF-α also clearly depressed I(Ca) (2.9 ± 0.9 to 2.3 ± 0.7 nA) and the amplitude of CS (7.0 ± 1.4 to 5.5 ± 1.3 μm) in β-adrenergic stimulated cells. TNF-α significantly increased the content of sphingosine (product of sphingomyelin pathway) in isolated heart. The effects of low dose sphingosine (5 μmol/l) mimicked those of TNF-α on cardiac myocytes. IL-6 produced an acute negative inotropic effect through a NO-dependent pathway while TNF-α did so via a sphingomyelin-dependent pathway in isolated guinea-pig ventricular myocytes.