Thus, the two Epac isoforms may present functionally distinct therapeutic targets to be explored in the treatment of cardiac hypertrophy and arrhythmia. Thus, for all four of these known downstream Epac functional effects in cardiac myocytes (5, 11, 21, 23), Φ-O-Me-cAMP behaves like an antagonist (i.e., partial agonist effects were not apparent). As in OMe-CPT, this fluorescent cAMP analog has specificity for Epac over PKA. A truncated version representing a pseudogene (RDXP2) was assigned to Xp21.3. In contrast, Epac1 (in Epac2-KO cells) did not show sarcomeric striations (Fig.
Differences between Epac1 and Epac2 may dictate distinct subcellular localization and function.
The permeability, specificity, and binding affinity of Φ-O-Me-cAMP make it a useful tool to define endogenous Epac1 and Epac2 distribution in cardiomyocytes. We did not detect Epac1 to be highly concentrated at either the sarcolemma or mitochondria. In Epac1-KO, Epac2 was not detected around the nucleus but was especially concentrated in the T tubules, the site of RyR and Ca-induced Ca-release during EC coupling (38, 39). Addgene is open for ordering and depositing; find up-to-date details here. 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. Isolated rat cardiomyocytes were lysed by sonication in 20 mM Hepes buffer (pH = 7.2) with a protease inhibitor mixture (Calbiochem). S3). Neither 10 µM Φ-O-Me-cAMP nor 10–100 µM OMe-CPT activated PKA activity (Fig. Indeed, both bands disappear indicating that they represent two forms of Epac1 (Fig. This is consistent with our findings here that Epac-dependent nuclear export of histone deacetylase 5 (HDAC5) is abolished in Epac1-KO mice and mediated by β1-AR. This propensity for arrhythmias could also be exacerbated by an Epac-mediated down-regulation of slow delayed rectifier potassium channels (40, 41) mediated by Epac translocation, which could prolong action potential duration and increase arrhythmogenic risk. Our characterization of the novel Epac ligand Φ-O-Me-cAMP, instrumental here, should allow this tool to become more broadly useful for understanding Epac localization and signaling in other cell types. Avec les informations extraites de ce document, on peut proposer une explication à l’augmentation du nombre d’hématies chez les personnes qui vivent habituellement à basse altitude lorsqu’elles séjournent à des altitudes élevées.
Epac is a GEF for the small GTPases Rap1 and Rap2 and is directly activated by cAMP. There is a problem with the plasmid I received. (D) PKA activity assessed with Ad-AKAR FRET-based sensor under isoproterenol (ISO, 100 nM; n = 40), OMe-CPT (10 μM, n = 20; 100 μM, n = 20), and Φ-O-Me-cAMP (n = 12) vs. WT (n = 50). (bp), COVID-19 and Coronavirus Plasmids & Resources page, Genome (B) Mean of peak interval and full width at half maximum of peak (FWHM) for data as in A. 3 A and B). Significance was evaluated by using unpaired or paired Student’s t test, or two-way ANOVA as appropriate. 2 A and B), which is between values reported for cAMP (Kd = 30–45 μM for Epac 1) and OMe-CPT (Kd = 2 μM) (26⇓–28, 30). These compartments had been reported as sites of Ad-Epac1 or Epac1 FRET sensors expression in cells (16⇓⇓⇓⇓⇓–22). Ancillary Agreement for Plasmids Containing FP Materials, Standard format: Plasmid sent in bacteria as agar stab. Epac2 was recently identified as the isoform responsible for Epac-mediated SR Ca leak and arrhythmia (14), suggesting isoform-specific signaling pathways. (A) Confocal images of Φ-O-Me-cAMP binding in WT (±100 μM OMe-CPT) and Epac1/2 double knockout mice (DKO). Finally, our work excludes participation of Epac2 in HDAC5 dependent hypertrophy, because HDAC5 nuclear export was abolished by Epac1 deletion.
This material is available to academics and nonprofits only. 2D). La concentration sanguine d’EPO augmente ce qui active la multiplication des cellules souches d’hématies et entraîne donc une polyglobulie. We could detect a residual and saturable signal when Φ-O-Me-cAMP was used in the DKO (Fig. 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. Fig.
Epac1-KO abolished OMe-CPT–induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5.
Comme tous les facteurs de transcription, il contrôle l’expression d’autres gènes parmi lesquels se trouvent des gènes impliqués dans le métabolisme énergétique des cellules et le gène EPO. Cardiomyocytes were loaded with Φ-O-Me-cAMP together with the voltage-sensitive dye di-8ANEPPS (to assess T tubules; Fig. Epac2 localized at T tubules and can mediate cardiac arrhythmias, whereas Epac1 at the nuclear envelope can regulate β1-AR–mediated gene transcription. To test for Φ-O-Me-cAMP binding to Epac in cells, intact cardiac myocytes were exposed to Φ-O-Me-cAMP in physiological solution (Fig. 6C) and was abolished by 2-APB [an inositol-1,4,5-trisphosphate (IP3) inhibitor].
6 E and F). In contrast, Φ-O-Me-cAMP signal was greatly reduced both in DKO myocytes and by competition with the Epac-selective agonist OMe-CPT, indicating the specificity that is key for a useful fluorescent probe (35). Cyclic nucleotide research—still expanding after half a century, Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP, A family of cAMP-binding proteins that directly activate Rap1, Epac activation, altered calcium homeostasis and ventricular arrhythmogenesis in the murine heart, Epac activation induces histone deacetylase nuclear export via a Ras-dependent signalling pathway, Epac mediates beta-adrenergic receptor-induced cardiomyocyte hypertrophy, cAMP-binding protein Epac induces cardiomyocyte hypertrophy, Epac and phospholipase Cepsilon regulate Ca, Epac-mediated activation of phospholipase C(epsilon) plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca, The cAMP binding protein Epac modulates Ca, Epac enhances excitation-transcription coupling in cardiac myocytes, Sustained Epac activation induces calmodulin dependent positive inotropic effect in adult cardiomyocytes, Developmental changes in gene expression of Epac and its upregulation in myocardial hypertrophy, Epac2 mediates cardiac β1-adrenergic-dependent sarcoplasmic reticulum Ca2+ leak and arrhythmia, Epac: Defining a new mechanism for cAMP action, Critical role of the N-terminal cyclic AMP-binding domain of Epac2 in its subcellular localization and function, Microtubule-associated protein 1B-light chain 1 enhances activation of Rap1 by exchange protein activated by cyclic AMP but not intracellular targeting, Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments, Epac, in synergy with cAMP-dependent protein kinase (PKA), is required for cAMP-mediated mitogenesis, Novel single chain cAMP sensors for receptor-induced signal propagation, Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator, Cell cycle-dependent subcellular localization of exchange factor directly activated by cAMP, Direct spatial control of Epac1 by cyclic AMP, The protein kinase A anchoring protein mAKAP coordinates two integrated cAMP effector pathways, mAKAP and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope, A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK, Ligand-mediated activation of the cAMP-responsive guanine nucleotide exchange factor Epac, cAMP analog mapping of Epac1 and cAMP kinase. Indeed, Epac activation by adenoviral Epac1 expression or the specific Epac agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3-,5-cyclic monophosphate (OMe-CPT), can activate hypertrophic transcription regulators MEF2 and NFAT, via phospholipase C (PLC)/IP3/Ca/Ca-calmodulin kinase II (CaMKII)/HDAC4-HDAC5 (5, 11) and H-Ras/Ca/calcineurin pathways (6, 7), respectively. What strain of bacteria does my stab contain? 4 A and B). Principal Investigator, cite the article in which the plasmids were described,
We recently showed that activation of Epac2, but not Epac1, induces β1-AR–induced arrhythmias through CaMKII-dependent diastolic SR Ca release (14).