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CONTROL OF GAP JUNCTION CONDUCTANCE BY CALCINEURIN-DEPENDENT CX-43 PHOSPHORYLATION: IMPLICATIONS OF ARRHYTHMOGENESIS
  1. A Waheed1,
  2. PD Lampe2,
  3. SC Salvage1,
  4. FS Hatch1,
  5. CH Fry1,
  6. RI Jabr1
  1. 1Department of Biochemistry and Physiology, University of Surrey, Guildford, Surrey, UK
  2. 2Fred Hutchinson Cancer Research Center, Seattle, WA, USA
  3. 3School of Physiology and Pharmacology, University of Bristol, Bristol, UK

Abstract

Slower action potential conduction, and consequent arrhythmias, follow decreased gap junction conductance, Gj. We investigated how the Ca2+-dependent phosphatase, calcineurin, regulates Gj through altering the phosphorylation of Cx43 protein, through a direct or indirect pathway.

Gj was estimated in guinea-pig ventricular myocardium from the frequency-dependent intracellular impedance1, in physiological (Na=147.4 mM) and low-Na solutions (Na=29.4 mM; used to raise intracellular [Ca2+]). Calcineurin inhibitors (cyclosporine-A, 5 µM; CAIP, 50 µM) and an inhibitor of the Ca2+-independent phosphatase PP1 (tautomycin, 5 nM) were used. Western blots of tissue lysates measured: 1) total-Cx43; 2) Cx43 phosphoserineS365 and phosphoserineS368, normalised to total-Cx43; 3) total and phosphothreonine 35-I1inhibitor-1 (I1; a PP1 regulator), normalised to GAPDH. Means (±SEM) were compared by ANOVA, the null hypothesis rejected at p<0.05.

Low-Na decreased Gj (59.8±3.6% control); reversed by CysA (85.5±5.5%; n=8) or CAIP (109.8±23.1%; n=3). Low-Na also decreased Cx43 phosphoserine365 vs control (0.57±0.02 vs 0.95±0.03; n=3), again reversed by cyclosporine-A (0.72±0.06, n=3; p<0.001) or CAIP (0.87±0.02, n=3). By contrast, Cx43 phosphoserine368 increased in low-Na (0.90±0.03 vs 0.08±0.02; n=6); also reversed by cyclosporine-A (0.15±0.9, n=6) and CAIP (0.13±0.03; n=6). Low-Na decreased phosphothreonine35-I1 (0.25±0.02 vs 0.83±0.00; n=6); reversed by CysA (0.37±0.02, n=6) and CAIP (0.71±0.08, n=6), suggesting calcineurin dephosphorylated phosphothreonine35-I1 and activated PP1. Tautomycin showed similar effects to calcineurin inhibitors in low-Na: reversing both the Gj decrease and alterations to phosphoserine365 and phosphoserine368.

Raised [Ca2+]i decreases Gj by a calcineurin-dependent pathway via dephosphorylating phosphoserine365-Cx43, and subsequently phosphorylating serine368. Phosphoserine365-Cx43 is targeted by PP1 through calcineurin-dependent pathway.

  • CARDIAC PROCEDURES AND THERAPY

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