Abstract 12705: Exchange Protein Directly Activated by cAMP (Epac): A Novel Signaling Pathway Underlying Ionic Remodeling by Beta-Adrenergic Activation in Guinea Pig Hearts
Background: Heart failure (HF) produces chronic β-adrenoceptor (βAR) activation, which contributes to sudden death risk. K+ channel downregulation is important in HF arrhythmogenesis. This study assessed the role of Epac1 protein, an alternative to the PKA pathway, in slow delayed rectifier current (IKs) downregulation with chronic βAR activation.
Methods: Isolated guinea pig LV cardiomyocytes were cultured and exposed to Isoproterenol (Iso, 1 μ M) or vehicle (CTL) for 30 hrs. Parallel sets of CTL, Iso, Iso + intervention or intervention-only cells were studied in each set of expts to have comparable conditions. IKs was recorded with whole cell patch clamp.
Results: Iso decreased IKs from 2.4±0.2 pA/pF to 1.0±0.2 pA/pF at +60 mV (*** P<0.001). Iso reduced KCNQ1 (-62%*, *p<0.05) and KCNE1 (-51%*) protein (immunoblots) and membrane immunofluorescence by 73%*** and 70%*** respectively. KCNQ1 mRNA (qPCR) was unchanged but KCNE1 mRNA was reduced by 50% (**P<0.01). To study underlying mechanisms, LV cells were incubated with blockers or activators targeting signaling pathways. 8-Br-cAMP (1 µM) and 8-CPT (Epac1 activator, 6 µM) decreased IKs from 4.2±0.2 (CTL) pA/pF to 1.9±0.4** and 2.1±0.2 pA/pF,** similar to Iso-exposed cardiomyocytes. Adenoviral-mediated knockdown (KD) of Epac1 in cultured cardiomyocytes prevented Iso effects, with IKs averaging 0.80±0.10 pA/pF for CTL, 0.14±0.01 pA/pF** (Iso+GFP-virus), and 0.55±0.08 pA/pF (Iso+KD, p<0.01 vs. Iso+GFP-virus, p=NS vs CTL). Immunostaining revealed nuclear NFATc3 translocation with Iso. BAPTA-AM (Ca2+ chelator, 10 μ M), cyclosporine (calcineurin inhibitor, 0.8 µM), and INCA6 (NFAT inhibitor, 1 µM) prevented IKs downregulation. Iso stimulation upregulated microRNAs 1 (targeting KCNE1) and 214 (KCNQ1) by 24% and 54% respectively.
Conclusions: βAR stimulation suppresses IKs by downregulating KCNE1 and KCNQ1 protein via β1AR stimulation of Epac-coupled Ca2+/calcineurin/NFAT signaling pathways. The mechanisms of expression changes are different for KCNE1 (primary decrease in message) vs. KCNQ1 (message unchanged, protein downregulated). MicroRNAs likely act as effectors. These results provide insights into arrhythmogenic ion channel remodeling in HF and potential therapeutic strategies.
- © 2011 by American Heart Association, Inc.