Ventricular arrhythmias are initiated by abnormal action potential (AP) conduction partially caused by an increased gap junction (GJ) resistivity (Rj). Alterations in ventricular GJ electrical properties may result from either raised [Ca2+]i and/or changes in the phosphorylation state of GJ proteins, connexin 43 (Cx43). In particular, the phosphorylation of the serine residue at 368 site (Cx43-pS368) by protein kinase C (PKC). We have reported that the slowed AP conduction velocity induced by raised [Ca2+]i was reversed by cyclosporin A (CsA), an inhibitor of the Ca2+/calmodulin serine-threonine dependent protein phosphatase, calcineurin (Cn). In this study we tested the hypothesis that raised [Ca2+ ]i increases Rj as a result of increased Cx43-pS368 levels caused by a possible interaction between Cn and PKC. Guinea-pig left ventricular papillary muscles were dissected in Tyrode’s solution (total [Na]=147.4mM; 24mM NaHCO3, 5%CO2, 37°C). Tissue impedance (Z) was measured over a range of frequencies (0.02-100kHz) using the oil gap technique. Rj was calculated from Z values in control and low Na Tyrode’s solutions (29.4 mM Na, used to increase [Ca2+ ]i) in the absence/presence of Cn inhibitors (CsA, 5μM and Cn-autoinhibitory peptide,CAIP, 50μM) or PKC inhibitor (chelerythrine, CHE, 2μM). The protein expression of total Cx43 (T-Cx43) as well as Cx43-pS368 was determined from whole tissue lysates using western blotting. Cx43- pS368 bands were in turn normalised to their corresponding T-Cx43 (Cx43-pS368/T-Cx43). Data are means ± SEM, compared by ANOVA, the null hypothesis rejected at p<0.05. Rj was significantly increased by low Na (608 ± 104Ω.cm) when compared to control (403 ± 62Ω.cm; n=7; p<0.001). Such an effect was reversed by CsA (475±100 Ω.cm; n=7;p<0.001) which alone had no effect. Similar effects were observed with CAIP. Combination of low Na and CHE partially inhibited the rise in Rj induced by low Na alone (control:232.5Ω.cm; low Na:397.5Ω.cm; low Na and CHE: 316.6 Ω.cm; n=2). Moreover, T-Cx43 protein expression was unchanged during all interventions when compared with control. However, there was a significant increase of the Cx43-pS368/T-Cx43 in low Na (0.90±0.64; n=6; p< 0.05) when compared to control (0.05±0.02;n=6). Such an increase was reversed by CsA (0.15±0.04; n=6; p<0.001) or CHE (0.03±0.01; n=3; p<0.001) in the presence of low Na solution. Raised [Ca2+]i in ventricular myocardium is associated with CsA and CHE-sensitive increase in Rj caused by increased Cx43-pS368 protein levels. This site is known to be targeted by PKC and lowering Cx43 gap junction conductance. In conclusion, we propose a possible synergistic interaction between Cn and PKC evoked by raised [Ca2+ ]i slowing AP conduction a mechanism known to be proarrhythmogenic.