Background: Tumor Necrosis Factor alpha (TNF alpha) upregulation during acute inflammatory response has been associated with numerous cardiac effects including modulating Connexin43 and vascular permeability. This may in turn alter cardiac gap junctional (GJ) coupling and extracellular volume (ephaptic coupling) respectively. We hypothesized that acute exposure to pathophysiological TNF alpha levels can modulate conduction velocity (CV) in the heart by altering electrical coupling: GJ and ephaptic. Methods and Results: Hearts were optically mapped to determine CV from control, TNF alpha and TNF alpha + high calcium(2.5 vs. 1.25 mM) treated guinea pig hearts over 90 mins. Transmission electron microscopy was performed to measure changes in intercellular separation in the gap junction-adjacent extracellular nanodomain-perinexus (W-P). Cx43 expression and phosphorylation were determined by Western blotting and Cx43 distribution by confocal immunofluorescence. At 90 mins, longitudinal and transverse CV (CVL and CVT, respectively) increased with control Tyrode perfusion but TNF alpha slowed CVT alone relative to control and anisotropy of conduction increased, but not significantly. TNF alpha increased W-P relative to control at 90 mins, without significantly changing GJ coupling. Increasing extracellular calcium after 30 mins of just TNF alpha exposure increased CVT within 15 mins. TNF alpha + high calcium also restored CVT at 90 mins and reduced W-P to control values. Interestingly, TNF alpha + high calcium also improved GJ coupling at 90 mins, which along with reduced W-P may have contributed to increasing CV. Conclusions: Elevating extracellular calcium during acute TNF alpha exposure reduces perinexal expansion, increases ephaptic, and GJ coupling, improves CV and may be a novel method for preventing inflammation induced CV slowing.