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

作者：Pereira Laetitia; Rehmann Holger; Lao Dieu Hung; Erickson Jeffrey R; Bossuyt Julie; Chen Ju; Bers Donald M^*

来源：Proceedings of the National Academy of Sciences, 2015, 112(13): 3991-3996.

DOI：10.1073/pnas.1416163112

• cardiomyocytes
• Epac1
• Epac2
• localization
• fluorescence

Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in beta-adrenergicdependent 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 (Phi-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 Phi-O-Me- cAMP was enhanced by binding to Epac. Unlike several Epacspecific antibodies tested, F-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (K-d = 10.2 +/- 0.8 mu M) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine- 3'-, 5'-cyclicmonophosphate (OMe-CPT). F-O-MecAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. F-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.

• 出版日期2015-3-31