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 microM),and BMS605401 (100 nM), a specific inhibitor of Ca(2+)-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 >or=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 Ca(2+)-induced changes. Moreover, cyclosporin A completely abolished the Ca(2+)-induced changes, while verapamil was ineffective. We conclude that extracellular calcium (24 h exposure) seems to up-regulate Cx40 but not Cx43.

Effects of metoprolol therapy on cardiac gap junction remodelling and conduction in human chronic atrial fibrillation.

BACKGROUND AND PURPOSE: We investigated the influence of metoprolol on gap junction proteins connexin43 (Cx43) and connexin40 (Cx40) in atrial tissue from patients with/without atrial fibrillation (AF). EXPERIMENTAL APPROACH: Left atrial tissue samples from 160 patients with AF or sinus rhythm (SR) with or without metoprolol (mean daily dose: 65.2 ± 9.1 mg·day⁻¹) were analysed for Cx43 and Cx40 by Western blot and immunohistology. Transverse and longitudinal conduction velocities were determined by 64 multi-electrode mapping. KEY RESULTS: Both Cx43 and Cx40 expression were significantly increased in patients with AF versus SR. Cx43-expression in AF was significantly higher in patients receiving metoprolol, while Cx40 expression was unaffected by metoprolol treatment. In AF, the ratio of lateral/polar expression of Cx43 and Cx40 was enhanced due to increased expression at the sides of the cells (lateral) and a loss at the cell poles. This AF-induced increase in lateral/polar expression of Cx43, but not of Cx40, was significantly antagonized by metoprolol treatment. Functionally, in AF patients, transverse conduction velocity in atrial samples was significantly enhanced and this change was also significantly antagonized by metoprolol. CONCLUSIONS AND IMPLICATIONS: AF induced enhanced lateral expression of Cx43 and Cx40 together with enhanced transverse conduction velocity in left atrial tissue. Alterations in localization of Cx43 and conduction changes were both antagonized by metoprolol, showing that pharmacological modulation of gap junction remodelling seems, in principle, possible. This finding may open new approaches to the development of anti-arrythmic drugs.