A new role for extracellular Ca2+ in gap-junction remodeling: studies in humans and rats

We wanted to elucidate whether extracellular calcium may regulate the expression of the cardiac gap-junction proteins connexin 40 and connexin43. In the free wall of the left atria of 126 cardiac surgery patients with either sinus rhythm (SR) or chronic atrial fibrillation (AF), we determined the expression of the cardiac gap-junction proteins Cx43 and Cx40 by Western blot and immunohistology. For deeper investigation, we incubated cultured neonatal rat cardiomyocytes at 2 or 4 mM Ca⁺⁺ for 24 h and determined intercellular coupling, Cx40, Cx43 protein and mRNA expression, protein trafficking and sensitivity to verapamil (10–100 nM), cyclosporin A (1 μM),and BMS605401 (100 nM), a specific inhibitor of Ca²⁺-sensing receptor (CaSR). We found in patients that both Cx are up-regulated in AF in the left atrium (by 100–200%). Interestingly, Cx40 was mainly up-regulated, if total serum calcium was ≥2.2 mM, while Cx43 was independent from extracellular [Ca⁺⁺]. In cultured cells, 4 mM Ca⁺⁺-exposure lead to up-regulation of Cx40, but not Cx43. We found enhanced Cx40 in the plasma membrane and reduced Cx40 in the Golgi apparatus. The membrane Cx40 up-regulation resulted in enhanced gap-junction intercellular coupling with a shift in the Boltzmann fit of voltage-dependent inactivation indicating a higher contribution of Cx40 as revealed by dual whole cell voltage clamp experiments. BMS605401 could prevent all Ca²⁺-induced changes. Moreover, cyclosporin A completely abolished the Ca²⁺-induced changes, while verapamil was ineffective. We conclude that extracellular calcium (24 h exposure) seems to up-regulate Cx40 but not Cx43.