SummaryPrevious studies have demonstrated the presence of myocardial depression in clinical and experimental septic shock. This depression is associated with the presence of a circulating myocardial depressant substance with physical characteristics consistent with cytokines. The present study utilized an in vitro myocardial cell assay to examine the role of various human recombinant cytokines, including tumor necrosis factor (TNF)ot and interleukin (IL)113, in depression of cardiac myocyte contractile function induced by serum from humans with septic shock. The extent and velocity of electrically paced rat cardiac myocytes in tissue culture was quantified by a closed loop video tracking system. Individually, TNF-0c and IL-113 each caused significant concentration-dependent depression of maximum extent and peak velocity of myocyte shortening in vitro. In combination, TNF-cx and IL-1[3 induced depression of myocardial cell contractility at substantially lower concentrations consistent with a synergistic effect. Using immunoabsorption, removal of both TNF-cx and IL-113 (but not either alone) from the serum of five patients with acute septic shock and marked reversible myocardial depression resulted in elimination of serum myocardial depressant activity. IL-2, -4, -6, -8, -10, and interferon ~/failed to cause significant cardiac myocyte depression over a wide range of concentrations. These data demonstrate that TNF-0t and IL-113 cause depression of myocardial cell contraction in vitro and suggest that these two cytokines act synergistically to cause sepsis-associated myocardial depression in humans. Despite therapy with appropriate antibiotics and intensive supportive care, septic shock remains a serious disorder with significant morbidity and mortality. The typical human cardiovascular response to septic shock is characterized by hypotension, decreased systemic vascular resistance, and elevated cardiac index. In addition, myocardial depression manifested by reversible biventricular dilation and reduction of ejection fraction has been shown to be common in human septic shock (1-3).A previously described in vitro model of myocardial cell performance utilizes spontaneously beating rat cardiac myocytes in culture (4-6). This system allows assessment of myocardial cell performance (i.e., contraction) independent of changes in preload, afterload, and heart rate. In this system, serum from patients with acute septic shock produced in vitro depression of cardiac myocyte contractile function (decreased maximum extent and peak velocity of shortening) that correlated quantitatively and temporally with the

Previous studies have demonstrated the existence of a circulating myocardial depressant substance during human septic shock. We have recently identified this substance as a synergistic combination of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). This study utilized an in vitro cardiac myocyte assay to evaluate the potential mechanistic role of nitric oxide (NO) and cGMP in depression of myocyte contractility induced by TNF-α, IL-1β, TNF-α + IL-1β (at low concentrations), and human septic shock serum (HSS). TNF-α, IL-1β, TNF-α + IL-1β, and each of 5 sera from patients with acute septic shock caused depression of both maximum extent and peak velocity of cardiac myocyte shortening and an increase in intracellular cGMP concentration during 30 min of exposure (minimum P < 0.01). NO synthetase (NOS) and guanylate cyclase inhibitors such as N-methyl-l-arginine (l-NMA) and methylene blue prevented these effects; an excess ofl-arginine withl-NMA restored them (minimum P < 0.01). In contrast,d-arginine failed to reestablish cytokine-induced myocyte depression and cGMP accumulation prevented byl-NMA. Exposure of myocytes to TNF-α, IL-1β, or TNF-α + IL-1β produced a concentration-dependent increase in intracellular cGMP that paralleled the depression of cardiac myocyte contractility (minimum P < 0.001). In addition, TNF-α, IL-1β, TNF-α + IL-1β, or HSS application to cardiac myocytes resulted in increased NO gas generation, which was inhibited byl-NMA (minimum P < 0.01). Furthermore, unstimulated cardiac myocytes were shown to harbor constitutive but not inducible NOS activity. These data suggest that the sequential generation of NO by a constitutive NOS and cGMP by guanylate cyclase represents an important mechanism of cardiac myocyte depression by TNF-α, IL-1β, TNF-α + IL-1β, and the myocardial depressant substance(s) of septic shock.

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