Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes

We tested the hypothesis that bacterial lipopolysaccharide (LPS) must be in ternalized to facilitate endotoxin-dependent signal activation in cardiac m yocytes. Fluorescently labeled LPS was used to treat primary cardiomyocyte cultures, perfused heart preparations, and the RAW264.7 macrophage cell lin t. Using confocal microscopy and spectrofluorometry, we found that LPS was rapidly internalized in cardiomyocyte cultures and Langendorff-perfused hea rts. Although LPS uptake was also observed in macrophages, only a fraction of these cells were found to internalize endotoxin to the extent seen in ca rdiomyocytes. Colocalization experiments with organelle or structure-specif ic fluorophores showed that LPS was concentrated in the Golgi apparatus, ly sosomes, and sarcomeres. Similar intracellular localization was demonstrate d in cardiomyocytes by transmission electron microscopy using gold-labeled LPS. The internalization of LPS was dependent on endosomal trafficking, bec ause an inhibitor of microfilament reorganization prevented uptake in both cardiomyocytes and whole hearts. Inhibition of endocytosis specifically res tricted early activation of extracellular signal-regulated kinase proteins and nuclear factor-kappaB as well as later tumor necrosis factor-alpha prod uction and inducible nitric oxide synthase expression. In conclusion, we ha ve demonstrated that bacterial endotoxin is internalized and transported to specific intracellular sites in heart cells and that these events are obli gatory for activation of LPS-dependent signal transduction.