Exchange Protein Directly Activated by cAMP (Epac) Mediates Slow Delayed-Rectifier Current Remodeling by Sustained Beta Adrenergic Activation in Guinea Pig Hearts
Rationale: β-Adrenoceptor activation contributes to sudden-death risk in heart failure (HF). Chronic beta-adrenergic stimulation, as occurs in HF patients, causes potentially-arrhythmogenic reductions in slow delayed-rectifier K+-current (IKs).
Objective: To assess the molecular mechanisms of IKs-downregulation caused by chronic beta-adrenergic activation, particularly the role of Exchange protein directly activated by cyclic-AMP (Epac).
Methods and Results: Isolated guinea-pig left-ventricular (LV)-cardiomyocytes were incubated in primary culture and exposed to isoproterenol (1-µmol/L) or vehicle for 30 hrs. Sustained isoproterenol-exposure decreased IKs-density (whole-cell patch-clamp) by 58% (P<0.0001), with corresponding decreases in KCNE1 mRNA and membrane-protein expression (by 45%, 51% respectively). KCNQ1 mRNA-expression was unchanged. The β1-adrenoceptor antagonist CGP-20712A prevented isoproterenol-induced IKs-downregulation, whereas the β2-antagonist ICI-118551 had no effect. The selective Epac-activator 8-pCPT decreased IKs-density to an extent similar to isoproterenol-exposure, and adenoviral-mediated knockdown of Epac1 prevented isoproterenol-induced IKs/KCNE1-downregulation. In contrast, protein-kinase A inhibition with a cell-permeable highly-selective peptide blocker did not affect IKs-downregulation. BAPTA-AM, cyclosporine and INCA6 prevented IKs-reduction by isoproterenol and INCA6 suppressed isoproterenol-induced KCNE1-downregulation, consistent with signal-transduction via the Ca2+/calcineurin/NFAT pathway. Isoproterenol induced nuclear NFATc3/c4 translocation (immunofluorescence), which was suppressed by Epac1-knockdown. Chronic in-vivo administration of isoproterenol to guinea pigs reduced IKs-density and KCNE1 mRNA- and protein expression, while inducing cardiac dysfunction and action-potential prolongation. Selective in vivo activation of Epac via sp-8-pCPT-infusion decreased IKs-density and KCNE1 mRNA/protein-expression.
Conclusions: Prolonged β1-adrenoceptor stimulation suppresses IKs by downregulating KCNE1 mRNA and protein via Epac-mediated Ca2+/calcineurin/NFAT signaling. These results provide new insights into the molecular basis of K+-channel remodeling under sustained adrenergic stimulation.
- Received November 5, 2013.
- Revision received February 6, 2014.
- Accepted February 7, 2014.