title: Novel Epac fluorescent ligand reveals distinct Epac1 vs. Epac2 distribution and function in cardiomyocytes.
abstract: Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in β-adrenergic-dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2'-O-methyladenosine-3',5'-cyclic monophosphate (Φ-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of Φ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, Φ-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3'-,5'-cyclicmonophosphate (OMe-CPT). Φ-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. Φ-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT-induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.
authors.affiliation: Department of Pharmacology, University of California, Davis, CA 95616;
authors.forename: Laëtitia
authors.initials: L
authors.lastname: Pereira
authors.affiliation: Molecular Cancer Research, University Medical Center Utrecht, NL-3584 Utrecht, The Netherlands;
authors.forename: Holger
authors.initials: H
authors.lastname: Rehmann
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0612; and.
authors.forename: Dieu Hung
authors.initials: DH
authors.lastname: Lao
authors.affiliation: Department of Pharmacology, University of California, Davis, CA 95616; Department of Physiology, University of Otago, Dunedin, New Zealand 9013.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Erickson
authors.affiliation: Department of Pharmacology, University of California, Davis, CA 95616;
authors.forename: Julie
authors.initials: J
authors.lastname: Bossuyt
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0612; and.
authors.forename: Ju
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, University of California, Davis, CA 95616; dmbers@ucdavis.edu.
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D009202:Cardiomyopathies / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057809:HEK293 Cells; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic
chemical_list: C555650:8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate acetoxymethyl ester; C482601:Epac protein, mouse; D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; C116122:RAPGEF3 protein, human; C442864:RAPGEF4 protein, human; C526973:Rapgef3 protein, rat; C486707:Rapgef4 protein, mouse; C531571:Rapgef4 protein, rat; D011943:Receptors, Adrenergic, beta; D000242:Cyclic AMP; C498618:Hdac5 protein, mouse; D006655:Histone Deacetylases; D002118:Calcium
keywords: Epac1; Epac2; cardiomyocytes; fluorescence; localization
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