Published online before print August 25, 2005, doi: 10.1161/01.RES.0000183880.49270.f9
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Submitted on May 10, 2005
Revised on August 11, 2005
Accepted on August 16, 2005
Enhanced Functional Gap Junction Neoformation by Protein Kinase A-Dependent and Epac-Dependent Signals Downstream of cAMP in Cardiac Myocytes
Satoshi Somekawa ;
From the Department of Structural Analysis (S.F., Y.N., H.F., N.M.), National Cardiovascular Center Research Institute, Suita, Osaka; the First Department of Internal Medicine (S.S., Y.S.), Nara Medical University, Kashihara, Nara, Japan.
* To whom correspondence should be addressed. E-mail: nmochizu{at}ri.ncvc.go.jp.
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 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 N-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.
Key words: gap junction • connexin43 • myocardial structure • cardiac gap junction connexins
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