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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.
Abstract
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)eCdependent 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)eCdependent 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 celleCcell 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 celleCcell contacts. Collectively, PKA and Epac cooperatively enhance functional GJ neoformation in cardiomyocytes.
Key Words: gap junction connexin43 myocardial structure cardiac gap junction connexins
Introduction
Gap junctions (GJs) are channels formed by two docking connexons; one connexon is provided by each of the two contiguous cells and is constituted of six connexin (Cx) molecules.1 Among the 20 Cx members, Cx40, Cx43, and Cx45 are expressed in the heart.2 Of the three, Cx43 is predominantly expressed in working heart muscle cells.3,4 GJs in the heart are characterized by their localization at the intercalated disk between each myocyte and also by their role in electrical conductance required for coordinated electrical excitation.5 Myocytes electrically coupled by GJs show synchronized contraction. The importance of Cx43 in electrical excitation in vivo is evident by cardiac-specific depletion of Cx43 leading to cardiac arrhythmia.6
The overall function of GJs depends on the number of GJs and the gating function of assembled GJs. GJs are upregulated by increased transcription of Cx, increased distribution of Cx at celleCcell contacts, and decreased degradation of Cx from the cell membrane. cAMP increases Cx43 mRNA.7 cAMP also enhances the trafficking of Cx43 from the endoplasmic reticulum/Golgi apparatus to the plasma membrane.8 Cx43 turnover is regulated by proteosomal and lysosomal degradation, and the half-life of Cx43 is less than two hours, suggesting that a rapid synthesis and trafficking system operates in cardiac myocytes.9
GJ is modulated by the phosphorylation of Cx43 on Ser and Tyr residues. The intercellular communication through Cx43 is decelerated and accelerated by its phosphorylation on Ser368 by protein kinase C and on Ser364 by protein kinase A (PKA), respectively.10,11 In addition to Ser phosphorylation, phosphorylated Cx43 on Tyr247 and Tyr265 is repressed from junctional communication.12 In addition to phosphorylation, GJ formation is regulated by Cx43-binding molecules. Cx43 binds to the junctional adhesion molecule-associating proteins zonula occludens-1 (ZO-1) and -catenin.13,14 Dominant-negative ZO-1, which dissociates the endogenous ZO-1 from Cx43, disturbs the localization of Cx43 at the celleCcell contacts, resulting in the reduced conductance of GJs.13 Wnt-1 signal prevents -catenin degradation, thereby increasing -catenin, which not only drives Cx43 expression but also associates with the Cx43 at the celleCcell contacts, where -catenin localizes with cadherin.14
cAMP-induced Cx43 assembly has been extensively characterized in terms of Cx43 synthesis, delivery to the plasma membrane, and phosphorylation, which is believed to depend exclusively on PKA.15 However, other downstream molecules of cAMP have not been elucidated in the neoformation of GJs. We and others have demonstrated that exchange protein directly activated by cAMP (Epac)/cAMP-GEF, a guanine nucleotide exchange factor (GEF) for Rap1, is activated by cAMP,16,17 and that cAMPeCEpac-Rap1 signal enhances the barrier function of vascular endothelial cells by stabilizing cadherin-mediated cell adhesion.18,19 Analogous to this Epac-induced cadherin-based cell adhesion, we hypothesized that Epac may be involved in GJ neoformation as a cAMP-triggered signaling molecule in cardiac myocytes.
In this study, we investigated the molecular mechanism by which GJ neoformation is regulated by cAMP using a PKA-specific activator and an Epac-specific activator. We analyzed the GJ accumulation at celleCcell contacts by immunostaining of Cx43 and the gating function of GJs by dye spreading in neonatal rat cardiomyocytes (NRCMs) stimulated with these activators. We demonstrate that the Cx43 accumulation at celleCcell contacts depends on Epac and that dye spreading depends on PKA. Therefore, PKA and Epac downstream of cAMP cooperatively enhance functional GJ neoformation in cardiac myocytes.
Materials and Methods
Reagents and cAMP Analogs
Dibutyryl-cAMP (dbcAMP) was purchased from Sigma-Aldrich, Epac-specific activator 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT) from Calbiochem; and PKA-specific activator N6-benzoyladenosine-3',5'-cyclic monophosphate (6Bnz) was from BIOLOG Life Science Institute. Other chemical compounds, antibodies, and adenoviruses are listed in the supplemental information (available online at http://circres.ahajournals.org).
Cell Culture
NRCMs were isolated from Wistar rats (1 to 2 days old; Kiwa Jikken Dobutsu, Japan) on a Percoll gradient as described previously.20 The details of cardiac myocyte preparation are described in the supplemental information. The NRCMs spread onto the glass-base dishes for 24 hours after isolation were subjected to immunostaining or dye transfer assay after drug treatment for another 12 hours. We observed that the adherens junctions (AJs) were not maturated, although NRCMs contacted each other before the drug treatment, indicating that we used the reassembling NRCMs for the experiments. Experiments using animals were approved by our institutional animal use and care committee. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals (NIH, revision 1996).
Immunocytochemistry
NRCMs stimulated with cAMP analogs were immunostained as described previously.21 Briefly, cells cultured on glass-base dish were blocked with PBS containing 4% BSA for 1 hour at room temperature (RT), then stained with anti-Cx43, antieCsarcomeric -actinin (S-A), and antieCneural (N)-cadherin at RT. Protein reacting with primary antibodies was visualized with Alexa 488eClabeled goat anti-rabbit IgG and Alexa 546eClabeled goat anti-mouse IgG. Images were recorded with a confocal microscope (BX50WI; Olympus). For quantitative immunofluorescence analysis, images were also recorded using an epifluorescence microscope (IX-71; Olympus) controlled by MetaMorph version 6.2 software (Molecular Devices). The number of Cx43-positive dots at the celleCcell contacts on the fluorescence images were counted as Cx43 puncta.
Gating Function of GJs Analyzed by Microinjected Dye Transfer
Microinjected dye transfer was performed as described by Doble et al, with minor modifications.22 The details of dye transfer method are described in the supplemental information.
RT-PCR Analysis
Total RNAs extracted from NRCMs and human cervical carcinoma cell line (HeLa) cells using Trizol (Invitrogen) were reverse-transcribed using SuperScript II and random primers (Invitrogen). The resultant DNAs were PCR-amplified using Epac-specific primers described in the supplemental information.
Western Blot Analysis and N-Cadherin Translocation Assay
NRCMs were lysed in buffer described in the supplemental information. Lysates precleared by centrifugation at 15 000g for 10 minutes were subjected to SDS-PAGE and immunoblotting with antibodies as indicated in Figures 3, 4, 5, and 6. Proteins reacting with primary antibodies were visualized by an enhanced chemiluminescence system (Amersham Biosciences) with peroxidase-conjugated and species-matched secondary antibodies and analyzed with an LAS-1000 system (Fuji Film). N-cadherin translocation assay was performed as described previously.18
Detection of GTP-Bound Form of Rap1
Rap1 activity was assessed by a modified Bos method as described previously.23 Briefly, NRCMs starved in DMEM for 3 hours were treated with the stimulants as indicated in Figures 3 and 6 and lysed at 4°C in a pull-down lysis buffer described in the supplemental information. GTP-bound Rap1 was collected on glutathione S-transferase fused with Rap1 binding domain of Ral guanine nucleotide dissociation stimulator precoupled to glutathione-Sepharose beads and subjected to SDS-PAGE followed by immunoblotting using anti-Rap1.
Statistical Analysis
The results were expressed as the mean±SD. Student t test was used to analyze differences between two groups. Group differences were assessed with one-way ANOVA or two-way ANOVA, followed by post hoc comparisons tested with Scheffe’s method. At least 3 fields randomly selected from each culture for analysis of Cx43 staining or at least 4 cells for dye transfer assay from each culture were used to yield a single value for each culture. The number of the cultures for analysis was indicated in the figure legends as n. Significant differences were indicated as P value <0.05 ().
Results
cAMP Enhances Functional GJ Neoformation in Cultured NRCMs
Because cAMP has been reported previously to enhance GJ formation,7 we confirmed the dbcAMPeCregulated functional GJ neoformation by quantitatively analyzing Cx43 accumulation at the celleCcell contacts by immunostaining and gating function of GJs by microinjected dye transfer assay. dbcAMP enhanced the Cx43 accumulation at the celleCcell contacts (Figure 1A and 1B). To neglect the possibility of cardiac fibroblast contamination in the NRCMs in the following experiments, and to show the confluence of the NRCMs, cells were immunostained for sarcomeric -actinin (Figure 1A, bottom). The Cx43 puncta in the cells treated with dbcAMP for 12 hours were clearly observed at the celleCcell contacts, where N-cadherin localized (Figure 1C), indicating that dbcAMP induces the accumulation of Cx43 at the celleCcell contacts. We investigated the effect of dbcAMP on gating function of GJs by microinjected dye transfer assays (Figure 1D and 1E). Microinjected dye was more widely transferred to the neighboring cells in dbcAMP-treated NRCMs than vehicle-treated cells (Figure 1D). The quantitative data are shown in Figure 1E. These results are in agreement with previous reports7,8 and validated the assays we used in this study.
PKA Is Required But Not Sufficient Alone for cAMP-Enhanced GJ Neoformation
Because PKA is involved in the enhancement of GJ formation,15 we first tested the effect of H89, a specific PKA inhibitor, on cAMP-enhanced accumulation of Cx43. Unexpectedly, H89 did not block the dbcAMP-induced accumulation of Cx43 (Figure 2A and 2B), although H89 did block cAMP-enhanced intercellular communication assessed by microinjected dye transfer assays (Figure 2C).
We next examined the effect of 6Bnz, a specific activator for PKA,24 on intercellular communication and Cx43 accumulation at celleCcell contacts to directly assess the involvement of PKA in cAMP-enhanced GJ formation. 6Bnz induced Cx43 accumulation slightly but to a much lesser extent than dbcAMP (Figure 2D and 2E). Notably, 6Bnz enhanced dye transfer to a greater extent than vehicle but to a lesser extent than dbcAMP (Figure 2F). These results indicate that PKA signaling is required but not sufficient alone for cAMP-enhanced GJ neoformation and suggest that there is a novel signaling downstream of cAMP in addition to PKA involved in Cx43 accumulation at celleCcell contacts for functional GJ neoformation.
cAMP Activates PKA and Epac-Rap1 Signaling in NRCMs
Epac has been identified as a novel cAMP target and a Rap1-specific GEF. We therefore hypothesized that Epac-Rap1 signaling may be involved in cAMP-enhanced GJ neoformation. RT-PCR analysis revealed the expression of Epac in NRCM but not in HeLa cells used as a negative control (Figure 3A). To test the hypothesis, we first examined whether dbcAMP induces the activation of Rap1 and the phosphorylation of cAMP response element binding protein (CREB) in NRCMs. As shown in Figure 3B, dbcAMP induced Rap1 and CREB activation in NRCMs. Rap1 activation by dbcAMP is dependent on time and concentration (supplemental Figure IA and IB, available online at http://circres.ahajournals.org). H89 inhibited dbcAMP-induced CREB phosphorylation but not dbcAMP-induced Rap1 activation (Figure 3B and 3C), indicating that Rap1 activation does not depend on PKA, whereas CREB phosphorylation depends exclusively on PKA. We next tested whether Rap1 activation and CREB phosphorylation are induced by 8CPT, which has been developed recently as a specific activator for Epac.25 8CPT only activated Rap1, not CREB. In striking contrast, 6Bnz induced CREB activation but did not affect Rap1 activity (Figure 3D and 3E). Together, these findings demonstrate that cAMP activates Epac-Rap1 and PKA signaling pathways in NRCMs.
Activation of Epac Signaling Leads to Cx43 Accumulation at CelleCCell Contacts
Because we observed Rap1 activation in response to dbcAMP, we proceeded to investigate the involvement of Epac-Rap1 signaling in cAMP-induced Cx43 accumulation at celleCcell contacts. Like dbcAMP, 8CPT significantly enhanced the accumulation of Cx43 at the celleCcell contacts (Figure 4A and 4B). 8CPT induced Cx43 accumulation at the celleCcell contacts to a similar extent to dbcAMP and to a greater extent than 6Bnz. 6Bnz only slightly increased the number of Cx43 puncta (Figure 4B) compared with vehicle and did not further increase the accumulation of Cx43 at celleCcell contacts caused by 8CPT alone. These results indicate that Epac-mediated signaling is mainly responsible for cAMP-induced Cx43 accumulation at the celleCcell contacts.
We excluded the possibility that increased synthesis of Cx43 on cAMP stimulation resulted in the accumulation of Cx43 at the celleCcell contacts. No discernible increase was observed in the cells stimulated with vehicle, dbcAMP, 8CPT, 6Bnz, and a combination of 8CPT and 6Bnz for 12 hours (Figure 4C and 4D), suggesting that distribution or functional augmentation of GJs is essential for cAMP-induced functional GJ neoformation. In addition, phosphorylation of Cx43 was not affected by dbcAMP, 8CPT, or 6Bnz, nor a combination of 8CPT and 6Bnz (Figure 4C and 4E).
Epac Enhances AJ Formation
Several lines of evidence suggest that AJ formation organized by N-cadherin is a prerequisite for GJ assembly in cardiomyocytes when reassembling and recoupling.26eC28 We used reassembling NRCMs before drug treatment. Recently, we and others revealed that Rap1 is involved in the celleCcell contacts mediated by epithelial (E)-cadherins and vascular endothelialeCcadherins (VE-cadherins).18,29 Thus, it is possible that cAMP enhances GJ neoformation by enhancing N-cadherineCmediated AJ formation preceding the GJ formation in NRCMs. To address this possibility, we investigated whether cAMP induces N-cadherineCmediated AJ formation in NRCMs. N-cadherin distribution at celleCcell contacts was enhanced by dbcAMP and 8CPT, whereas 6Bnz neither affected the distribution of N-cadherin nor enhanced the effect of 8CPT (Figure 5A).
To quantitatively analyze the localization of N-cadherin after drug treatment, we performed a biochemical N-cadherin translocation assay. Because N-cadherin is connected to actin cytoskeleton in maturated AJs, cadherin anchored to actin cytoskeleton can be detected in detergent-insoluble fractions of cell lysates. We found an increase in N-cadherin in Triton X-100eCinsoluble fraction when stimulated by dbcAMP and 8CPT (Figure 5B). However, 6Bnz did not change either basal- or 8CPT-increased levels of N-cadherin in the Triton X-100eCinsoluble fraction (Figure 5B and 5C). Collectively, these findings indicate that cAMP enhances AJ formation through Epac in NRCMs. We found no difference in N-cadherin expression in NRCMs stimulated with dbcAMP, 8CPT, or 6Bnz, or a combination of 8CPT and 6Bnz by immunoblotting (data not shown).
Rap1 Activation Is Essential for cAMP-Mediated Cx43 Redistribution and AJ Formation
We investigated the role of Rap1 in cAMP-induced Cx43 accumulation and AJ formation in NRCMs. To examine the effect of Rap1 on AJ and GJ formation, we inactivated Rap1 by adenovirus-expressing Rap1GAP1b, which specifically catalyzes the hydrolysis of GTP to GDP on Rap1.30 Endogenous Rap1 activity was almost completely suppressed by the expression of increasing amount of Rap1GAP1b in NRCMs (Figure 6A). Moreover, overexpression of Rap1GAP1b inhibited cAMP-induced Rap1 activity without affecting cAMP-stimulated CREB phosphorylation (Figure 6B), confirming that Rap1GAP1b specifically blocks Epac-Rap1 pathway but not PKA-mediated signaling.
Inactivation of Rap1 blocked the cAMP-induced accumulation of Cx43 and N-cadherin at the celleCcell contacts (Figure 6C and 6D). dbcAMP-induced translocation of N-cadherin to cytoskeleton-anchored fraction was inhibited by inactivation of Rap1 but not by LacZ overexpression (Figure 6E and 6F). These results suggest that cAMP induces N-cadherineCbased AJ assembly through an Epac-Rap1 signaling pathway, which may precede the accumulation of Cx43-based GJs.
PKA and Epac-Rap1 Signaling Cooperatively Enhances GJ Neoformation in NRCMs
Because we found that PKA alone is not sufficient for cAMP-enhanced GJ neoformation and that Epac-Rap1 signaling is involved in cAMP-induced accumulation of Cx43, we assessed the effect of PKA activation and Epac-Rap1 activation on gating function of GJs. 8CPT merely showed the weak enhancement of the intercellular connection, as revealed by microinjected dye transfer assay (Figure 7A). However, 8CPT significantly enhanced 6Bnz-mediated intercellular communication (Figure 7B). The effect of the combination of 8CPT and 6Bnz was comparable to that of dbcAMP. Given that 8CPT induces the Cx43 accumulation at the celleCcell contacts, cAMP potentiates functional GJ neoformation via a PKA-mediated enhanced gating function and Epac-Rap1 signal-mediated accumulation of Cx43 to celleCcell contacts.
Discussion
The function of GJs in the heart depends on the number of GJs between neighboring cells and the gating function of individual GJ at the celleCcell contacts. We investigated how cAMP induces Cx43 accumulation at celleCcell contacts and enhances gating function in NRCMs that were about to develop the mature celleCcell contacts. For the first time, we demonstrated the involvement of Epac-Rap1 signaling downstream of cAMP in GJ neoformation of cardiomyocytes. Although Cx43 accumulated at the celleCcell contacts on cAMP stimulation has been ascribed to PKA,7 this study demonstrated that Epac-Rap1 signaling activated by cAMP is mainly responsible for the redistribution of Cx43 to celleCcell contacts.
The number of GJs was increased by Epac-Rap1 downstream of cAMP as indicated by the increase in Cx43-positive puncta at celleCcell contacts. However, there was no increase in the amount of Cx43 after cAMP treatment, indicating the importance of the redistribution of Cx43 rather than increase of Cx43 transcription on cAMP. How does Epac signaling induce the accumulation of Cx43 at celleCcell contacts Epac-Rap1 activation resulted in enhancement of AJ formation accompanied by GJ formation, as evidenced by increases in N-cadherin and Cx43 at the celleCcell contacts after dbcAMP stimulation (Figure 5). AJ formation constituted by N-cadherin is a prerequisite for GJ neoformation.28,31 When adult myocytes are cultured, Cx43 is transported and accumulated at the plasma membrane, where N-cadherin accumulates on celleCcell contact.26 Therefore, GJ formation depends on N-cadherineCbased AJ maturation. We have shown previously that the Epac-Rap1 signal enhances the VE-cadherineCbased celleCcell contacts in vascular endothelial cells.18 In this study, we found that Epac activation resulted in the increased accumulation of N-cadherin at the intercellular junction of NRCMs. Thus, N-cadherin accumulation at the celleCcell contacts induced by the Epac-Rap1 signal may account for Cx43 accumulation in NRCMs by analogy to Epac-Rap1eCtriggered VE-cadherin accumulation in vascular endothelial cells.
The target of activated Rap1 for enhancement of cadherin-based AJ is still unclear. Rac belonging to Rho family GTPase and regulating actin cytoskeleton is suggested to function downstream of Rap1.32 Therefore, Rac may increase the chances of cell contacts and induce cadherin engagement by extending membrane downstream of Rap1. Maturated N-cadherin on Epac activation, which is detected in the cytoskeleton-anchored fraction, may be accompanied by translocation of Cx43 through cadherin-associating -catenin because Cx43 is capable of binding to -catenin.14 Because ZO-1 is recruited to AJs by binding to -catenin and is also capable of binding to Cx43,33 ZO-1 may participate in the accumulation of Cx43 during maturation of AJs.
Another factor affecting functional GJ neoformation in addition to the number of GJs is the gating function of individual GJs. PKA activation facilitates intercellular communication without accumulation of Cx43 at celleCcell contacts, concurring with previous reports underpinning that PKA and cAMP increases single channel conductance of the GJ,34 although the characteristics of single GJ channel conductance evoked by PKA activation still remains elusive.15 We found a marked increase in dye transfer on PKA activation with a slightly increased accumulation of Cx43 at the celleCcell contacts (Figures 4 and 7). These results indicate that PKA mainly contributes to the functional neoformation of GJs by enhancing gating function of GJs. Phosphorylation of Cx43 on Ser residues is required for intercellular communication of GJs.35 Because we found no significant increase in either total Cx43 or phosphorylated Cx43, PKA may indirectly modulate GJ conductance in addition to direct phosphorylation of Cx43 or may phosphorylate a critical Ser/Thr that was indistinguishable in the phosphorylated Cx43 band in our immunoblot for Cx43 (Figure 4C).
The enhanced gating function of GJs is mainly ascribed to PKA, whereas the accumulation of Cx43 to celleCcell contacts is mainly attributable to Epac-Rap1 signal. Hence, Epac-Rap1 signal may accelerate the trafficking of Cx43 to the plasma membrane or inhibit the endocytosis of Cx43 from the plasma membrane. We did not quantify the translocation of Golgi fraction to celleCcell contacts on cAMP stimulation. Previously, GJ trafficking was dynamically monitored by green fluorescence proteineCtagged Cx43.36 Therefore, it will be of great interest to observe the Cx43 dynamics on 8CPT stimulation to directly elucidate Epac-Rap1 signaling.
In conclusion, we demonstrated that cAMP potentiates functional GJ neoformation by a PKA-dependent increase in intercellular communication and by an Epac-Rap1eCdependent accumulation of Cx43 in NRCMs.
Acknowledgments
This work was supported in part by grants from the Ministry of Health, Labor, and Welfare Foundation of Japan; the Ministry of Education, Science, Sports, and Culture of Japan; the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan; the Japan Health Science Foundation; and Astellas Foundation for Research on Metabolic Disorders. We thank Michiyuki Matsuda and Akihiro Umezawa for their helpful input; Nobuo Shirahashi for statistical analysis; James T. Pearson and Michael E. Mendelsohn for critical reading; and Yuko Matsuura and Manami Sone for their technical assistance.
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