Nitric oxide donors alter cardiomyocyte cytokine secretion and cardiac function

Objects: The mechanisms by which nitric oxide produces beneficial/detrimental effects on physiologic function are unclear. In this study, we hypothesized that nitric oxide promotes cyclic guanosine monophosphate (cGMP) formation, which, in turn, promotes cardiomyocyte secretion of inflammatory cytokines as well as accumulation of intracellular Na+/Ca2+; these factors contribute to altered cardiac contractile function. Design: Laboratory study. Setting: Medical Center. Subjects: Adult Sprague Dawley rats weighing 325-350 g. Interventions: Cardiomyocytes were prepared by collagenase perfusion of rat hearts; cells were plated (5 x 10(4) cells/microtiter well) and challenged with either vehicle or nitric oxide donor (S-nitroso-N-acetyl-penicillamine [SNAP] or PAPA NONOATE, 3-[2-Hydroxy-2-nitroso-1-propythdrazinol]-1-propanamine], NOC-15 [PAPA-NO], 0.3 or 1.0 mM of each nitric oxide donor) in the presence/absence of methylene blue (10 mu M/L to inhibit cGMP). After 3 hrs, supernatants were collected to measure nitrite/nitrate (nitric oxide), cytokines (tumor necrosis factor-alpha, interleukin-1 beta, interieukin-6), and cGMP levels; cells were then loaded with a fluorescent indicator (Fura-2AM or sodium-binding benzofurzan isophthalate) to measure myocyte Ca2+ or Na+, respectively. Parallel experiments included the addition of nitric oxide donor (0.3 or 1.0 mM SNAP or PAPA-NO) to perfused hearts in presence or absence of the methylene blue to examine cGMP-mediated effects on myocardial contraction -relaxation, while other experiments determined a) potential lipopolysaccharide contamination of myocyte preparations; and b) whether a cGMP analogue recapitulated the effects of nitric oxide donors on cytokine secretion. Measurements and Main Results: Nitric oxide donors produced a dose-dependent increase in cGMP levels in myocyte supernatants as well as an increase in myocyte cytokine secretion, increased myocyte loading of Na+/Ca2+, and produced myocardial contractile dysfunction. Addition of the cGMP analog, 8-bromo-cGMP, recapitulated the effects of nitric oxide donors on myocyte cytokine secretion. Nitric oxide donor-related effects were ablated by pretreatment of myocytes or isolated hearts with methylene blue. Treatment of myocytes with recombinant bactericidal/permeability-increasing protein to scavenge lipopolysaccharide confirmed that cytokine responses to nitric oxide donors were not related to lipopolysaccharide contamination of myocyte preparations. Conclusions: We suggest that nitric oxide synthesis in injury and disease promotes cGMP formation, which, in turn, modulates cardiac contraction/relaxation by a) altering cardiomyocyte secretion of inflammatory cytokines and b) altering myocyte handling of Na+/Ca2+.