Supplementary Material for: Critical Role of Cx40 in Reduced Endothelial Electrical Coupling by Lipopolysaccharide and Hypoxia-Reoxygenation

Background: We discovered that lipopolysaccharide (LPS, an initiating factor in sepsis) and hypoxia-reoxygenation (H/R, a confounding factor) reduce electrical coupling between microvascular endothelial cells from wild-type (WT) but not Cx40-/-mice. Because Cx40 knockout could result in nonspecific effects, this discovery may not establish the causal relationship between Cx40 and reduced coupling. Using the same cell culture model, we aimed to address this uncertainty by using the rescue-of-function approach. Methods/Results: Electrical coupling between endothelial cells (hind-limb muscle origin) was determined by electrophysiology. LPS, H/R and concurrent LPS + H/R reduced coupling between WT but not Cx40-/- cells. The defect in Cx40-/- cells was rescued by ectopic expression of Cx40, after infecting the cells with adenovirus encoding Cx40. Cx40-/- cells were also engineered to express mutant Cx40 that lacked the carboxyl terminal domain beginning at residue 236 (Cx40Δ237-358) or 344 (Cx40Δ345-358). No response to inflammatory stimuli was observed in cells expressing either of these 2 mutants. Conclusion: Our data establish the causal relationship between Cx40 and reduced coupling and suggest that the 345-358 amino acid motif of the Cx40 carboxyl terminal is required for reduced coupling. Cx40 may participate in compromised conducted response in the microvasculature during sepsis.