Exchange protein directly activated by cAMP mediates slow delayed-rectifier current remodeling by sustained β-adrenergic activation in guinea pig hearts.

Rationale: β-Adrenoceptor activation contributes to sudden death risk in heart failure. Chronic β-adrenergic stimulation, as occurs in patients with heart failure, causes potentially arrhythmogenic reductions in slow delayed-rectifier K(+) current (IKs).

Objective: To assess the molecular mechanisms of IKs downregulation caused by chronic β-adrenergic activation, particularly the role of exchange protein directly activated by cAMP (Epac).

Methods And Results: Isolated guinea pig left ventricular cardiomyocytes were incubated in primary culture and exposed to isoproterenol (1 μmol/L) or vehicle for 30 hours. Sustained isoproterenol exposure decreased IKs density (whole cell patch clamp) by 58% (P<0.0001), with corresponding decreases in potassium voltage-gated channel subfamily E member 1 (KCNE1) mRNA and membrane protein expression (by 45% and 51%, respectively). Potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) mRNA expression was unchanged. The β1-adrenoceptor antagonist 1-[2-((3-Carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol dihydrochloride (CGP-20712A) prevented isoproterenol-induced IKs downregulation, whereas the β2-antagonist ICI-118551 had no effect. The selective Epac activator 8-pCPT-2'-O-Me-cAMP 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. 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate-AM acetoxymethyl ester (BAPTA-AM), cyclosporine, and inhibitor of nuclear factor of activated T cell (NFAT)-calcineurin association-6 (INCA6) prevented IKs reduction by isoproterenol and INCA6 suppressed isoproterenol-induced KCNE1 downregulation, consistent with signal-transduction via the Ca(2+)/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-2'-O-Me-cAMP 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 Ca(2+)/calcineurin/NFAT signaling. These results provide new insights into the molecular basis of K(+) channel remodeling under sustained adrenergic stimulation.