Novel Epac fluorescent ligand reveals distinct Epac1 vs. Epac2 distribution and function in cardiomyocytes

Laëtitia Pereira, Holger Rehmann, Dieu Hung Lao, Jeffrey R. Erickson, Julie B C Bossuyt, Ju Chen, Donald M Bers

Research output: Contribution to journalArticle

31 Scopus citations

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 (ℙ-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 ℙ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epacspecific antibodies tested, ℙ-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). ℙ-O-MecAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. ℙ-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.

Original languageEnglish (US)
Pages (from-to)3991-3996
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number13
DOIs
StatePublished - Mar 31 2015

Keywords

  • Cardiomyocytes
  • Epac1
  • Epac2
  • Fluorescence
  • Localization

ASJC Scopus subject areas

  • General

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