Cardiac arrhythmias are associated with raised intracellular [Ca²⁺] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca²⁺-dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca²⁺-dependent phosphatase, calcineurin. Intracellular [Ca²⁺] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2. Raised [Ca^{2 +}]_i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca²⁺]_i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca²⁺-independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca²⁺]_i. PP2A had no role. Conduction velocity was reduced by raised [Ca²⁺]_i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca²⁺] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.

Atherosclerosis is a chronic inflammatory disease characterised by accumulation of monocytic cells and lipids within the sub-endothelial space by direct MO-to-EC contact through GJ. Both cell types express connexin (Cx)43 isoforms that permit formation of GJ. This is enhanced by adhesion molecules in the presence of pro-inflammatory stimuli such as TNF-α.TNF-α suggested to have a role on Cx43 expression through MAPK mainly over other intercellular pathways, however, so far the mechanism is still unclear which our hypothesis is to investigate it. Experiments were carried out in the absence and presence of 25ng/ml TNF-α.Human umbilical vein endothelial cells (HUVECs) monolayer functional integrity was assessed by measuring trans-endothelial electrical resistance (TEER). TEM assay used as a model for the transmigration of MOs to the sub-endothelial space. Upper and lower contents were collected, marked with CD14+ and measured by flow cytometry. MAPk inhibitors, Chelerythrine Chloride(ChC) and SB 203580. TEER measurements shown a reduction in the resistance in the presence of TNF-α compared to the control respectively (13.58±2.82 Ω.cm², 43.94±3.32 Ω.cm²; n=6), however, in the presence of PKC inhibitor; ChC, the resistant was the same as control (48.64±3.62 Ω.cm², 45.76±4.83 Ω.cm²; n=6). In TEM,TNF-α enhanced the migration of MOs (66.3±2.1%; n=3) compared to the control (9.9±4.5%; n=3; P<0.001). Such an effect was prevented in monolayer treated with ChC (13.70±2.45%; n=3; P<0.001). Also, this fraction was attenuated by GAP26 and GAP27 and was comparable to the fraction of HUVECs Cx43-siRNA (8.33±4.66%; n=3). These results show that GJs is formed between ECs and MOs. TNF-α increased TEM, reduced the TEER and had no effects in HUVECs with Cx43-siRNA.The effect of TNF-α is mediated through PKC as it is effect attenuated by PKC inhibitor; ChC.