Abstract 13395: Exchange Protein Directly Activated by cAMP (Epac) and Combined Transcriptional/Translational Pathways Underlie Slow Delayed Rectifier Current Remodeling by Sustained Beta-Adrenergic Activation
Background: β-adrenoceptor (βAR) activation contributes to sudden death risk in heart failure (HF). K+-channel downregulation is important in HF arrhythmogenesis. This study assessed the molecular basis of changes in slow delayed rectifier current (IKs) with chronic βAR activation.
Methods: Isolated LV guinea pig cardiomyocytes were cultured and exposed to Isoproterenol (Iso, 1 μ M) or vehicle (CTL) for 30 hrs. Parallel sets of CTL and Iso, Iso + intervention or intervention cells were studied in each set of expts. IKswas recorded by 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 decreased KCNQ1 (-51%*, *P<0.01) and KCNE1 (-51%*) protein (immunoblots). KCNQ1 mRNA (qPCR) was unchanged but KCNE1 mRNA was reduced by 45% (**P<0.01).To understand underlying mechanisms, LV cells were incubated with blockers and/or activators targeting signaling pathways. Iso effects were blocked by β1AR but not β2AR blockers. 8-Br-cAMP (1 mM) and 8-CPT (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, suggesting Epac1 mediation. A cell-permeable protein kinase-A peptide inhibitor failed to alter Iso effects. Adenoviral-mediated knockdown (KD) of Epac1 in cultured cardiomyocytes prevented the effects of Iso, with mean IKs reduced by 82.5%** by Iso+GFP-virus vs 31% (p=NS vs.CTL) for Iso+KD. Calcineurin (0.8 µM) and INCA6 (NFAT blocker, 1 µM) prevented Iso-induced downregulation of IKs and KCNE1 mRNA. Immunostaining revealed increased nuclear translocation of NFATc3 (by 61%, P<0.01) and c4 (42%, P<0.05) with Iso. Iso upregulated miR-214, which is predicted to target KCNQ1. Luciferase reporter assays confirmed the inhibitory interaction between miR-214 and the 3’-UTR of KCNQ1.
Conclusions: βAR stimulation suppresses IKs by downregulating KCNE1 mRNA/protein and KCNQ1 protein via β1AR stimulation of EPAC-related Ca2+/calcineurin/NFAT signaling. NFAT downregulates KCNE1 mRNA and chronic βAR stimulation upregulates miR-214, translationally suppressing KCNQ1 protein. These results provide new insights into the fundamental molecular mechanisms underlying arrhythmogenic ion channel remodeling by chronic βAR-activation.
- © 2012 by American Heart Association, Inc.