Enhanced functional gap junction neoformation by protein kinase A-dependent and Epac-dependent signals downstream of cAMP in cardiac myocytes.
Gap junctions (GJs) constituted by neighboring cardiac myocytes are essential for gating ions and small molecules to coordinate cardiac contractions. cAMP is suggested to be a potent stimulus for enhancement of GJ function. However, it remains elusive how cAMP potentiates the GJ of cardiomyocytes. Here we demonstrated that the gating function of GJ is enhanced by the protein kinase A (PKA)-dependent signal, and that the accumulation of connexin43 (Cx43), the most abundant Cx in myocytes, is enhanced by an exchange protein directly activated by cAMP (Epac) (Rap1 activator)-dependent signal. The gating function of GJs was analyzed by microinjected dye transfer method. The accumulation of Cx43 was analyzed by quantitative immunostaining. Using the PKA-specific activator N6-benzoyladenosine-3',5'-cyclic monophosphate (6Bnz) and Epac-specific activator 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT), we could delineate the two important downstream signals of cAMP for enhanced GJ neoformation. Whereas 6Bnz potentiated gating function of GJs with slight accumulation of Cx43 at cell-cell contacts, 8CPT remarkably enhanced the accumulation of Cx43 with a slight effect on gating. We further noticed that adherens junctions (AJs) were maturated by 8CPT, as marked by increased neural-cadherin immunostaining. Because AJ formation precedes the GJ formation, AJ formation accelerated by Epac-Rap1 signal may result in enhanced GJ formation. The involvement of Epac-Rap1 signal in GJ neoformation was further confirmed by evidence that inactivation of Rap1 by overexpression of Rap1GAP1b perturbed the accumulation of Cx43 at cell-cell contacts. Collectively, PKA and Epac cooperatively enhance functional GJ neoformation in cardiomyocytes.
Somekawa
Satoshi
S
Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Fukuhara
Shigetomo
S
Nakaoka
Yoshikazu
Y
Fujita
Hisakazu
H
Saito
Yoshihiko
Y
Mochizuki
Naoki
N
D000818:Animals; D015820:Cadherins / Q000032:analysis; D002450:Cell Communication; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D017629:Gap Junctions / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology*; D020827:rap1 GTP-Binding Proteins / Q000502:physiology
D015820:Cadherins; D018031:Connexin 43; C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D020827:rap1 GTP-Binding Proteins