Exchange Protein Directly Activated by cAMP Mediates Slow Delayed-Rectifier Current Remodeling by Sustained β-Adrenergic Activation in Guinea Pig Hearts

Abstract

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 (I Ks ). Objective: To assess the molecular mechanisms of I Ks 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 I Ks 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 I Ks downregulation, whereas the β 2 -antagonist ICI-118551 had no effect. The selective Epac activator 8-pCPT-2′-O-Me-cAMP decreased I Ks density to an extent similar to isoproterenol exposure, and adenoviral-mediated knockdown of Epac1 prevented isoproterenol-induced I Ks /KCNE1 downregulation. In contrast, protein kinase A inhibition with a cell-permeable highly selective peptide blocker did not affect I Ks 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 I Ks 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 I Ks 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 I Ks density and KCNE1 mRNA/protein expression. Conclusions: Prolonged β 1 -adrenoceptor stimulation suppresses I Ks 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.