Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction

Abstract

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 [Ca²⁺]_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.

Keywords: Calcineurin; Conduction velocity; Connexin 43; Gap junction conductance.

Fig. 1

Low-Na solution on gap junction conductance (G_j): effect of calcineurin inhibitors. a Values of G _j in Tyrode’s (control) before and after exposure to low-Na solution, data from 20 separate preparations. b Effect of cyclosporin-A (CysA, 5 μM) in Tyrode’s or low-Na solution on G _j, data expressed as a percentage of the value in Tyrode’s solution (control), n = 5. c Effect of calcineurin-inhibitory peptide (CAIP, 50 μM) in Tyrode’s or low-Na solution on G _j, n = 5 *p < 0.05 vs Tyrode’s; **p < 0.01 vs Tyrode’s; ***p < 0.001 vs Tyrode’s; #p < 0.05 vs low-Na; ###p < 0.0001 vs low-Na

Fig. 2

S368-Cx43 phosphorylation and gap junction conductance, G_j, in low-Na solution: effect of calcineurin and PKC inhibitors. a Western blots of phosphorylated pS368-Cx43 (pS368-Cx43) in control and low-Na solution, effect of CysA (left panels) and CAIP (right panels). Band densities normalised to total Cx43 (T-Cx43) levels in the lower panel. b Western blots of phosphorylated S368-Cx43 (pS368-Cx43) in control Tyrode’s and low-Na solution, effect of chelerythrin (CHE, 2 μM). Band densities normalised to total Cx43 (T-Cx43) levels in the lower panel. GAPDH levels are also shown as a housekeeping protein. c Effect of chelerythrine (CHE, 2 μM) in Tyrode’s or low-Na solution on G _j and normalised to levels in Tyrode’s solution. *p < 0.05 vs Tyrode’s; **p < 0.01 vs Tyrode’s; ***p < 0.001 vs Tyrode’s; #p < 0.05 vs low-Na solution; ##p < 0.01 vs low-Na; ###p < 0.001 vs low-Na (n = 4)

Fig. 3

S365-Cx43 phosphorylation in low-Na solution: effect of Cn inhibitors. a Western blots of T-Cx43 and pS365-Cx43 in control and in low-Na solution in the absence or presence of CysA or CAIP. b Band densities of pS365-Cx43 normalised to T-Cx43 in the different conditions. *p < 0.05 vs Tyrode’s; ***p < 0.001 vs Tyrode’s; ##p < 0.01 vs low-Na (n = 3)

Fig. 4

Thr35-I1 phosphorylation in low-Na solution: effect of Cn inhibitors. a Western blots of T-Cx43, GAPDH, T-I1 and pThr35-I1 in control and in low-Na solution in the absence or presence of CysA or CAIP. b Band densities of T-I1 normalised to GAPDH. c Band densities of pThr35-I1 normalised to T-I1 in different conditions. *p < 0.05 vs Tyrode’s; #p < 0.01 vs low-Na ; ##p < 0.01 vs low-Na (n = 3)

Fig. 5

Effect of tautomycin (TTM) on Cx43 and I1 phosphorylation and on gap junction conductance, G_j. a Western blots of T-Cx43, GAPDH and pS368-Cx43 in control, low-Na and low-Na with TTM. Band densities normalised to total Cx43 (T-Cx43) levels in the lower panel. b Western blots of T-Cx43 and pS365-Cx43 in control Tyrode’s, low-Na and low-Na with TTM. Band densities normalised to total Cx43 (T-Cx43) levels in the lower panel. c Values of G _j in Tyrode’s and low-Na solutions with and without TTM normalised to Tyrode’s (control). d Western blots of T-Cx43, GAPDH and pThr35-I1 in control Tyrode’s, low-Na, low-Na with FST. Band densities of pThr35-I1 normalised to GAPDH in lower panel. *p < 0.05 vs Tyrode’s; ***p < 0.001 vs Tyrode’s; ##p < 0.01 vs low-Na (n = 3)

Fig. 6

Effect of low-Na solution and increased stimulation rate on action potential (AP) morphology and conduction: effect of cyclosporin-A (CysA, 5 μM). a Upper panel: conducted APs in Tyrode’s and low-Na solution, effect of CsA. Lower panel: upstroke phases of the AP and (above) differential of these phases in Tyrode’s and low-Na solution, effect of CsA. b Upper panel: conducted APs in Tyrode’s at 1 and 5 Hz stimulation, effect of CsA. Lower panel: upstroke phases of the AP and (above) differential of these phases at 1 and 5 Hz stimulation, effect of CysA. Inset shows Fura-2 Ca²⁺ transients at 1 and 5 Hz

Fig. 7

Schema of proposed Cn-dependent intracellular pathways mediating changes to Cx43 phosphorylation and gap junction conductance, G_j. Under control conditions, Cx43 is highly phosphorylated at S365 which prevents phosphorylation of Cx43 at S368 by PKC—under this condition, G _j is high. Raised intracellular [Ca²⁺] activates Cn to dephosphorylate inhibitor 1 (I1) at Thr35 (pThr35) and the dissociation of PP1 from I1. This results in activation of PP1 to dephosphorylate pS365 enhance phosphorylation of S368 by PKC and thus decrease G_j