A characteristic pattern of systolic and diastolic cardiac dysfunction occurs during the clinical course of septic shock. We have previously demonstrated that endotoxin-induced cardiac dysfunction in guinea pigs is mediated by TNF-α. In addition, the concentration of intramyocyte calcium is significantly elevated and critically involved in the pathogenesis of myocardial dysfunction in vivo. The purpose of this study was to determine whether in vitro exposure of adult guinea pig myocytes to a cytokine-enriched medium would reproduce the abnormalities in intracellular calcium.

Cardiac myocytes were isolated using a Langendorff apparatus and collagenase, and yielded individual rod-shaped myocytes with a viability of 70-80%. Cytokine-enriched medium, used for myocyte stimulation, was produced by co-incubation of guinea pig peritoneal macrophages with E. coli endotoxin for 24 hours. Isolated myocytes were divided, and incubated with either cytokine-enriched medium or normal medium for indicated time periods. Intracytoplasmic calcium was measured with the fluorescent indicator FURA-2AM. Compared to control myocytes, cytokine-stimulated myocytes demonstrated no difference in viability, FURA-2AM compartmentalization, FURA-2AM or calcium leakage, or efficiency of FURA-2AM loading. Intracytoplasmic calcium concentration was no different between stimulated and control myocytes (125 nM versus 116nM) after 6 hours of incubation. However, after 18 hours of incubation, there was a significant increase in intracytoplasmic calcium in cytokine-exposed myocytes (131 nM versus 103 nM, p = 0.0051). By 24 hours, the intracytoplasmic calcium was indistinguishable (99nM versus 98 nM) between groups.

These data demonstrate that the abnormalities in intracellular calcium witnessed in vivo, as well as the time course for its development, can be reproduced in isolated myocytes following cytokine exposure. These data suggest that cytokines are sufficient to produce calcium abnormalities in the absence of cellular immunity and/or derangements in tissue perfusion.