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Cardiovascular Research 2006 72(1):69-79; doi:10.1016/j.cardiores.2006.06.016
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Copyright © 2006, European Society of Cardiology

Chronic hemodynamic overload of the atria is an important factor for gap junction remodeling in human and rat hearts

Catherine Rucker-Martina,*, Paul Milliezb, Sisareuth Tana,1, Xavier Decrouyc,2, Michel Recouvreurd, Roger Vranckxe, Claude Delcayreb, Jean-François Renauda, Irene Duniad, Dominique Segretainc and Stéphane N. Hatemf

aCNRS-UMR 8162, Université Paris XI Sud, Hôpital Marie Lannelongue, 133 Avenue de la Résistance, 92350 Le Plessis Robinson, France
bCardiovascular Research Center Inserm Lariboisiere, Paris, France
cINSERM U570, Université Paris 5, France
dInstitut-Jacques-Monod-UMR7592 CNRS-Universités Paris 6/Paris 7, France
eINSERM U460, Paris, France
fINSERM, UMRS621; Université Pierre et Marie Curie-Paris6, Paris, France

* Corresponding author. Tel.: +33 1 40 94 25 20; fax: +33 1 40 94 25 22. Email address: catherine.rucker{at}ccml.u-psud.fr

Received 27 January 2006; revised 9 June 2006; accepted 13 June 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: The expression and distribution of connexins is abnormal in a number of cardiac diseases, including atrial fibrillation, and is believed to favor conduction slowing and arrhythmia. Here, we studied the role of atrial structural remodeling in the disorganization of gap junctions and whether redistributed connexins can form new functional junction channels.

Methods: Expression of connexin-43 (Cx43) was characterized by immunoblotting and immunohistochemistry in human right atrial specimens and in rat atria after myocardial infarction (MI). Gap junctions were studied by electron and 3-D microscopy, and myocyte–myocyte coupling was determined by Lucifer yellow dye transfer.

Results: In both chronically hemodynamically overloaded human atria in sinus rhythm and in dilated atria from MI-rats, Cx43 were dephosphorylated and redistributed from the intercalated disc to the lateral cell membranes as observed during atrial fibrillation. In MI-rats, the gap junctions at the intercalated disc were smaller (20% decrease) and contained very little Cx43 (0 or 1 gold particle vs. 42 to 98 in sham-operated rats). In the lateral membranes of myocytes, numerous connexon aggregates comprising non-phosphorylated Cx43 were observed. These connexon aggregates were in no case assembled into gap junction plaque-like structures. However, N-cadherin was well organized in the intercalated disc. There was very little myocyte–myocyte coupling in MI-rat atria and no myocyte–fibroblast coupling. Regression of the atrial remodeling was associated with the normalization of Cx43 localization.

Conclusion: Structural alteration of the atrial myocardium is an important factor in the disorganization of connexins and gap junction. Moreover, redistributed Cx43 do not form junction channels.

KEYWORDS Connexins; Gap junction; Atrial myocardium; Fibrosis; Atrial fibrillation

This article is referred to in the Editorial by R.J.P. Musters (pages 5–6) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
In the myocardium, gap junctions (GJ) are responsible for the rapid and coordinated propagation of excitation [1,2]. GJ channels are formed by the docking of two connexons (hemichannel) and each connexon consists of six connexin (Cx) subunits [1]. The role of GJ abnormalities in atrial fibrillation (AF) is actively studied [3–8]. In the atria, Cx43 and Cx40 are the principal connexins [9]. During AF, their levels of expression are altered and they both redistribute from the intercalated disk to the cell periphery. These changes in Cx expression are important arrhythmogenic factors contributing to inhomogeneities in electrical impulse propagation and to local conduction block [10]. However, mechanisms underlying atrial Cx disorganization are poorly understood. Beside rhythm disturbance [4,11], structural remodeling of the atrial myocardium could be an important pathogenic factor as reported in the myocardial zone bordering infarct scar [12] or in failing and hibernating ventricular myocardium [13,14]. Alterations in cell–cell and cell–extracellular interactions could be an important underlying mechanism of Cx disorganization [15]. Moreover, fibronectin and denatured collagen that accumulate in the interstitium of diseased myocardium could regulate Cx43 expression [16]. The functionality of the redistributed connexins are also open questions [17]. Increased transversal velocity of the electrical impulse and reduced anisotropy have been observed following Cx43 redistribution in a rat model of AF [4] but not in failing ventricle [18].

Cx43 demonstrated multiple electrophoretic isoforms including a non-phosphorylated (P0 or NP) Cx43 and two phosphorylated forms P1 and P2. Phosphorylation of Cx43 is an early event that occurs before Cx43 reaches the plasma membrane. In addition, phosphorylation regulates the assembly, degradation and gating of Cx43 [19]. Interestingly, during heart failure (HF), a large amount of Cx43 are hypophosphorylated, colocalized with the phosphatase 2A and could contribute to reduce gap junction functions [20]. However, very little is known on the regulation of Cx43 phosphorylation during atrial remodeling. Of note, profound alterations of myocyte phosphorylation which are partly responsible for changes in ionic channels properties have been described in dilated atria or during AF [21–23].

This study examined whether, beside AF, atrial remodeling contributes to GJ alterations. First, we studied the expression and distribution of connexins (i) in human right atrial biopsies from patients in sinus rhythm with dilated atria or in chronic AF and (ii) in rat atria after myocardial infarction (MI) with left ventricular (LV) dysfunction [24,25]. Second, we studied the GJ ultrastructural characters and dye coupling assays to examine whether the redistributed connexins form new junctional channels and how they contribute to cell–cell coupling. Third, we tested the hypothesis that alterations of GJ channel can reverse with treatment of atrial remodeling. We found that dilated atria in sinus rhythm with marked remodeling show a profound disorganization of GJ in human and rats. The redistributed connexins are mainly dephosphorylated and appear as not functional. Regression of atrial remodeling in rats is associated with the progressive normalization of GJ organization.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Human cardiac specimens
In accordance with the Helsinki Declaration and with approval of our institution Ethics Committee, biopsies of right atrial appendages (444±30 mg) were obtained from 15 patients undergoing cardiac surgery (Table 1). Three groups of patients were defined: group-A, patients in sinus rhythm with normal atria; group-B, patients in chronic AF (up to 6 months duration) often associated with atrial dilatation; and group-C, patients in stable sinus rhythm with atrial dilatation and structural myocardial alterations including myolysis and fibrosis [26,27]. In keeping with published studies [28,29], the following criteria were used for group C patients: (i) a clinical history known to be associated with an atrial remodeling such as mitral or tricupid valve diseases, HF and a pulmonary hypertension with right heart dilatation and (ii) a right atrial dilatation demonstrated by echocardiography and visual examination by the surgeon.


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  		Table 1 Clinical characteristics of patients

 
2.2. Experimental model and atria collection
The investigation conforms with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication 85-23, revised 1996). Myocardial infarction was induced in 50 male Wistar rats by ligating the left coronary artery [24,25]. After 3 months, surviving rats were untreated (MI-rats, n=20) or treated per os with a combination of lisinopril (1 mg/kg/day) and spironolactone (10 mg/kg/day) for 1 month (treated MI-rats, n=6). Sham-operated rats were used as controls (sham-rats, n=17). The cardiopathy was characterized between the third and the fourth month after surgery using transthoracic echocardiography as previously described [24,25].

All rats were sacrificed 4 months after surgery and left atrium (LA) was removed for processing as follows–fresh for dye coupling, frozen for immunoblotting, immunofluorescence and freeze fracture experiments, and chemically fixed for histology and conventional electron microscopy.

2.3. Western blot analysis
Western blot analysis was performed on lysates from frozen tissue as published [30]. Phosphorylated and dephosphorylated forms of Cx43 were detected using rabbit polyclonal anti-Cx43 antibody raised against the third cytoplasmic domain (1/100, Zymed-laboratories, CliniSciences, France). The non-phosphorylated Cx43 was detected using mouse monoclonal anti-Cx43 antibody which recognized Cx43 only when the serine-368 is unphosphorylated (clone CX-1B1, 1/20, Zymed-laboratories). Cx40 was detected using rabbit polyclonal anti-Cx40 antibody (1/20, Alpha-Diagnostic-International, Interchim, France). The bands were measured by densitometry using NIH image software; ratio of optical densities was calculated for each sample. Primary antibodies specificity was checked both by probing the membrane with the secondary antibody only and by pre-incubating the primary antibody with the immunizing peptide.

2.4. Histology and immunohistofluorescence
The interstitial collagen quantification was performed after Picrosirius Red F3BA coloration on five sections (8–10 fields/section) per animal [24,25]. Indirect immunofluorescence was performed on frozen sections as described [26]. Primary antibodies were rabbit polyclonal antibodies against Cx43 (1/50, Zymed-laboratories) or Cx40 (1/10, Alpha-Diagnostic-International) and mouse monoclonal antibodies against sarcomeric {alpha}-actinin (cloneEA-53, 1/200, Sigma) or Cx43 (clone CX-1B1, 1/10, Zymed-laboratories). Secondary antibodies were goat anti-rabbit IgG coupled to Alexa Fluor® 488 and goat anti-mouse IgG coupled to Alexa Fluor® 594 (1/100, MolecularProbes, Invitrogen). Images were collected with a multitracking confocal laser scanning microscope (CLM) (Zeiss LSM-510, Carl-Zeiss SAS). In control experiments primary antibodies were omitted.

2.5. Electronic microscopy
For thin section, small LA pieces (1 mm3) were fixed for 2 h in 2% glutaraldhehyde in 0.2 mol/L cacodylate buffer, pH7.4 and post-fixed for 1 h at 4 °C in 1% osmium tetroxide. After dehydration in gradual series of ethanol solutions, the samples were embedded in Epon-Araldite. Thin sections were stained with uranyl acetate and lead citrate before examination. We analyzed three samples from each group.

For freeze-fracture and immunogold-labeling, small pieces of non-fixed LA were placed on gold specimen holders (Balzers, Liechtenstein), rapidly frozen in liquid propane and stored in liquid nitrogen until replicated. Freeze-fractures and replication by platinum/carbon evaporation were carried out at –140 °C and 10–6 Torr vacuum in a freeze-fracture apparatus (model 301; Balzers). Replicas were processed for immunogold-labeling as previously described [31]. Replicas and thin sections were examined using a Philips CM12 electron microscope at 80 kV.

2.6. Dye coupling assay
For dye coupling assays, LA were cut into small pieces and immersed at 37 °C for 10 min in solution containing 0.5% Lucifer yellow (Sigma, St. Louis, MO) and 0.5% Rhodamine–dextran (Sigma) [32]. Tissues were incubated in 4% paraformaldehyde for 24 h and embedded in paraffin. Sections (7 µm) were observed using an upright fluorescent microscope (Nikon). As only few myocytes were damaged during the cutting procedure and, thus, were loaded with the dye containing solution, 30 randomly selected fields on three sections per heart were analyzed to quantify the myocyte–myocyte coupling. Values correspond to the percentage of coupled myocytes. We verified that the dye transfer was GJ mediated by pre-incubating heart pieces before dye coupling analysis with GJ coupling inhibitors: heptanol (3 mmol/L), oleamide (50 µmol/L), and glycyrrhetinic acid (100 µmol/L) [32].

2.7. Deconvolution microscopy
Deconvolution microscopy was carried out after indirect double immunofluorescence on 8-µm-thick frozen sections of LA as described [33]. Cx43 was detected with mouse monoclonal antibody directed against the 252–270 amino-acid sequence localized in the C-terminus (clone 2, 1/100, BD-Pharmingen, France). Pan-cadherin was stained with rabbit polyclonal antibody (1/100, Sigma). A Leica-DM-RXA microscope equipped with a piezoelectric translator (PIFOC, PI, Germany) placed at base of a 100x PlanApo objective (NA 1.4) and a 5 MHz Micromax 1300Y interline CCD camera (Roper Instruments, France) were used. Fluorescence images were collected automatically at 0.2 µm Z-intervals using Metamorph Software (Universal Imaging Corp., Downingtown, PA). Each Z-series was deconvoluted automatically using a measured Point Spread Function and an adapted constrained interactive deconvolution algorithm using custom-made software.

2.8. Statistical analysis
Data are expressed as means±S.E.M. and compared by one-way ANOVA followed by Fisher's test. Differences were considered significant at p<0.05. Frequencies were analyzed with the chi-squared test.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Connexins are redistributed in hemodynamic overloaded human atria in sinus rhythm
In atrial cryosections of group-A patients, most Cx43 and Cx40 appeared located in the intercalated disc (ID) and very little non-phosphorylated Cx43 was detected (Fig. 1A-upper panel). In group-B patients, Cx43 and Cx40 were present in the ID and along the lateral plasma membranes of myocytes where some of the redistributed Cx43 was non-phosphorylated (Fig. 1A–middle panel). We observed similar distribution patterns of Cx43 and Cx40 in group C-patients showing an intense staining of Cx43 and Cx40 along the plasma membranes of myocytes in areas where the interstitial space was enlarged. In this group, much of the redistributed Cx43 was non-phosphorylated (Fig. 1A–lower panel).


Figure 1
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  		Fig. 1 Connexins expression in human atria. (A) Double immunostaining of sarcomeric {alpha}-actinin (S{alpha}A, red) and Cx40 (Cx40, green) or Cx43, using the polyclonal antibody which recognizes the two forms of Cx43 (Cx43, green) or the monoclonal antibody directed against the non-phosphorylated form of Cx43 (Cx43nP, red) in atria of group-A, group-B and group-C patients, bar=20 µm. (B, C) Immunoblots showing recognition of phosphorylated (P1, 46-kDa) and non-phosphorylated (P0, 41-kDa) forms of Cx43 using the polyclonal anti-Cx43 antibody (B) and the selective detection of the non-phosphorylated form of Cx43 by the CX-1B1 antibody (C). (D) Ratio of the densitometric analysis of 46/41-kDa bands in atria of group-A, group-B and group-C patients (n=3/group). *p<0.01, ***p<0.001 vs. group-A. (E) Immunoblots of Cx40 in group-A, group-B and group-C patients.

 
Immunoblots using the polyclonal anti-Cx43 antibody (Fig. 1B) detected, in the three groups of patients, a 46-kDa and a 41-kDa protein which correspond to phosphorylated and non-phosphorylated Cx43, respectively. The CX-1B1 antibody detected only the 41-kDa protein (Fig. 1C). In group-B and group-C patients, the 46/41-kDa ratio was significantly lower than in group-A patients due to the increase amount of the non-phosphorylated Cx43 (Fig. 1B and D). In addition, a decrease of the expression of Cx40 (40 kDa) was observed in groups-B and -C as compared to group-A (Fig. 1E).

3.2. In dilated rat atria Connexin-43 is redistributed
To study the relation between myocardial remodeling of the atria and alterations of Cx43 expression we used the rat model of MI with LA dilation [24,25]. Four months after surgery the MI-rats had a LV dysfunction and an enlarged and fibrotic LA (Table 2). Whereas no episodes of AF were recorded on a 24-h electrocardiogram, P-wave duration was prolonged (30.2±1.3 ms vs. 17.6±0.26 ms, n=7; p<0.01) as well as the PR interval (55.0±2.5 ms vs. 38.5±1.1, n=7; p<0.01) (Fig. 2A). In MI-rats, Cx43 was redistributed to the myocyte plasmalemma where collagen accumulated and appeared almost completely dephosphorylated (Fig. 2B). Immunoblot analysis showed that the 46/41-kDa protein bands ratio was considerably lower in atria of MI-rats than sham-rats, due to the higher levels of non-phosphorylated Cx43 (Fig. 2C and D).


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  		Table 2 Echocardiographicand histological parameters of Sham- and MI-rats

 

Figure 2
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  		Fig. 2 Connexin-43 expression in MI-rat atria. (A) Electrocardiograms in sham-rats and MI-rats. (B) Picrosirius coloration (upper panel) showing extensive collagen in MI-rat atria, bar=50 µm; double immunostaining of S{alpha}A (red) and Cx43 using antibodies against the two forms of Cx43 (green) (middle panel) or the non-phophorylated form of Cx43 (red) and the two forms of Cx43 (green) (lower panel) in sham- and MI-rat atria, bar=20 µm. (C) Immunoblots using polyclonal anti-Cx43 antibody showing an increase of the non-phosphorylated form of Cx43 (P0, 41-kDa) in MI-rat atria. (D) Densitometric ratio of 46/41-kDa bands in sham- and MI-rat atria (n=3/group). ***p<0.001 vs. sham-rats.

 
3.3. Redistributed Cx43 does not form functional GJ in rat atria
Ultrastructure of GJ in diseased atria was further investigate by electron microscopy. In thin sections of sham-rat atria, ID was of normal appearance and desmosomes were clearly detected (Fig. 3A). GJ were identified as pentalaminar profiles along the longitudinal segment of ID (Fig. 3A). In MI-rat atria, GJ were also present at the ID but their size was reduced in comparison with those of sham-rat atria (Fig. 3B). In these MI-rat atria, a consistent accumulation of fibroblasts, pre-collagen and collagen fibers were present within the space between myocytes in comparison with sham-rat atria (Fig. 3C,D). It is noteworthy that in MI-rat atria, intercellular junctions were not detected between the increased amount of fibroblasts and myocytes (Fig. 3D).


Figure 3
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  		Fig. 3 Ultrastructure of myocytes and inter-myocyte space in sham- (A, C) and MI- (B, D) rat atria. (A) Thin section of sham-rat atria revealing regular and well-aligned intercalated disk. Arrows and rectangle point to GJ membrane profiles. Insert: higher magnification of the GJ profile outlined by the rectangle. (B) Thin section of MI-rat atria showing the irregular aspect of the intercalated disk. Arrows point to small GJ segments. Bars=500 nm. (C) Thin section of sham-rat atria inter-myocyte space occupied by a small cytoplasmic fragment of a fibroblast. (D) Thin section of inter-myocyte space from MI-rat atria showing that the interstitum is occupied by cytoplasmic fragments of fibroblasts (arrows), pre-collagen and collagen fiber bundles. Bars=250 nm.

 
To better identify GJ domains, we applied freeze-fracture (FF) and the fracture-labeling technique (FL). This method enables the high-resolution immunocytochemical analysis of the bi-dimensional distribution of membrane constituents on the freeze-fracture faces and allows to identify the membrane distribution of the phosphorylated and/or non-phosphorylated Cx43 isoforms on the myocytes plasmalemma. In sham-rat atria, phosphorylated Cx43 is mainly associated with the 9 nm GJ particles Cx43 (42 to 98 gold particles per GJ) (Fig. 4A); in contrast the non-phosphorylated Cx43 was absent (Fig. 4B).


Figure 4
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  		Fig. 4 Freeze-fracture and immunogold-labeling of sham- (A, B) and MI- (C, D) rat atrial myocytes junctional domains (10 nm gold particles). (A) FL of a large GJ in sham-rat atria. The fracture exposed both junctional profiles: junctional protoplasmic (jPF) and exoplasmic (jEF) fracture faces characterized by closely packed 9 nm intra-membranous particles on jPF and the corresponding arrays of pits on jEF. Both junctional fracture faces appear intensely gold-immunolabeled (arrows) with the antibody that recognizes the phosphorylated and non-phosphorylated forms of Cx43. Note the absence of gold-immunolabeling outside of the junctional plaque domain. (B) The gold-immunolabeling of the junctional plaque revealed on protoplasmic face (PF) is almost absent in sham-rat atria using the antibody directed against the non-phosphorylated form of Cx43. Bars=70 nm. (C) Extensive membrane domain of MI-rat atria exposed by the fracture showing the disassembly of the junctional domain resulting in irregular cluster of junctional particles. The disassembled junctional particles as well as isolated 9 nm intra-membranous particles (arrows) are poorly gold-immunolabeled using the antibody which recognizes the phosphorylated and non-phosphorylated forms of Cx-43, bar=100 nm. (D) MI-rat myocytes plasma membrane. The fracture exposed a large PF area characterized either by clusters or randomly distributed 9 nm particles. Note the intense gold-immunolabeling (arrows) using the monoclonal anti-Cx43 specific for the non-phosphorylated form, bar=50 nm. Shadowing direction is from bottom to top.

 
In MI-rat atria, fewer and smaller GJ profiles were detected at the ID compared to sham-rat atria (approximately –20% in MI-rats vs. sham-rats). They show a very poor gold immunolabeling of either phosphorylated or non-phosphorylated Cx43 (0 to 1 gold particle per GJ) (Fig. 4C). In these dilated atria, on the lateral plasma membrane of myocytes and on the longitudinal segment of the ID 9 nm intramembranous particles or small clusters gold-immunolabeled by non-phosphorylated anti-Cx43 were randomly distributed (Fig. 4D).

Functional coupling between myocytes was examined by incubating atrial tissues with a Lucifer Yellow (LY) and rhodamine–dextran containing solution. In sham-rat atria, 80±5% of myocytes loaded with the two dyes (yellow labeling) communicate through the GJ with adjacent myocyte (green labeling) while in MI-rat atria, only few myocytes stained green (6±15%) indicating a poor coupling (Fig. 5A and B). Around 20% of uncoupled myocytes in sham-rat atria may reflect less permeable GJ channel or some unspecific GJ alterations caused by the experimental procedure.


Figure 5
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  		Fig. 5 (A) Myocyte–myocyte coupling in sham- (a) and MI- (b) rat atria. Yellow fluorescence indicates that myocytes were loaded with both Lucifer Yellow (LY, green) and rhodamine–dextran (red). LY dye transfer through the GJ in sham-rat atria is indicated by the presence of a green myocyte adjacent to a yellow myocyte. In Mi-rat atria no diffusion of LY is observed (bar=20 µm). (B) Semi-quantitative analysis of myocyte–myocyte coupling (n=3/group, total cell number/rat=60), ***p<0.001. (C) Five distinct focal planes after deconvolution microscopy of N-cadherin (red) and Cx43 (green) organization in sham- (a) and MI- (b) rat atria (n=3/group). Nuclei are stained with DAPI.

 
All these findings indicate that redistributed Cx43 does not form new functional GJ in MI-rat atria.

3.4. Defective connexin targeting during atrial remodeling in rat
N-cadherin mediates cell–cell adhesion in the ID which is required for GJ plaque formation and normal Cx membrane targeting. Using 3D deconvolution microscopy with a high z-axis spatial resolution, we examined the localization of N-cadherin and Cx43. In contrast with sham-rat atria, in MI-rat atria only N-cadherin remained well organized in the ID, whereas Cx43 was scattered across the surface of the lateral membrane with very little overlay between the two proteins (Fig. 5C).

3.5. Reversion of GJ alteration in treated MI-rat
To further examine the relation between myocardial structural remodeling and Cx43 disorganization, some MI-rats were treated for 1 month with lisinopril and spironolactone (n=6). Before starting the treatment, we checked by echocardiography that rats had developed an ischemic cardiopathy complicated of HF with severe LV dysfunction (EF: 26.3±0.6%, n=6; p<0.001 vs. sham-rats) and marked LA dilation (LA diameter: 5.4±0.11 mm, n=6; p<0.001 vs. sham-rats). As previously shown [25], 1 month after treatment, there was a clear regression of the atrial remodeling. The LA weight/body weight ratio was decreased (0.14±0.01 mg/g vs. 0.32±0.05 mg/g in MI-rats; p<0.001), the P wave duration was reduced (27.8±0.5 ms, p<0.05 vs. MI-rats) while PR interval was not significantly decreased (50.6±1.3 ms) (Fig. 6A). There was also a decrease of the collagen area (17.7±2.7%; p<0.0001 vs. MI-rats) (Fig. 6B–upper panel). This was associated with the distribution of Cx43 in the ID (Fig. 6B–middle panel) and the decrease of non-phosphorylated Cx-43 (Fig. 6B–lower panel and C). Freeze-fracture of treated MI-rat atria show that large GJ plaques were assembled, characterized by the accumulation of 9 nm intra-membranous particles (Fig. 6D). Moreover, using 3D deconvolution microscopy, we found that most Cx43 localized near the N-cadherin at the level of the ID in contrast with non-treated MI-rat atria (Fig. 6E). These results indicate that alterations of GJ channel can reverse with the treatment of the atrial remodeling.


Figure 6
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  		Fig. 6 Reversion of atrial GJ alteration in MI-rat treated by lisinopril plus spironolactone. (A) Electrocardiograms and (B) Picrosirius coloration (upper panel), bar=20 µm; double immunostaining of S{alpha}A (red) and Cx43 using antibody against the two forms of Cx43 (green) (middle panel) or of the non-phophorylated form of Cx43 (red) and the two forms of Cx43 (green) (lower panel) in treated MI-rat atria, bars=20 µm. (C) Immunoblots of membrane proteins probed with polyclonal anti-Cx43 antibody showing a decrease of the non-phosphorylated form of Cx43 (P0, 41-kDa) in treated MI-rat atria. (D) FF of MI-rat atria after treatment showing an organized GJ plaque, bar=80 nm. (E) Five distinct focal planes after deconvolution microscopy of N-cadherin (red) and Cx43 (green) organization in treated MI-rat atria, bar=20 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study demonstrated that the structural myocardial remodeling of hemodynamic overloaded atria is associated with alterations of connexin expression and GJ remodeling as observed during AF. The redistribution of Cx43 visualized by fluorescent microscopy is consistent with the FF and FL observations showing the remodeling of the packing organization of the junctional plaque and the random distribution of non-phosphorylated Cx43 oligomers. Moreover, redistribution of Cx43 which does not form functional GJ channels results probably from the abnormal Cx43 targeting and could be associated with a reduced cell–cell coupling in diseased atria.

The spatial distribution of connexins is altered during AF in both human and experimental models [3–8]. In the rat, 24-h of pacing-induced AF is sufficient to cause the redistribution of Cx43 [4]. In the goat model of pacing-induced AF, the heterogeneity of connexin distribution correlates with AF duration [3]. These findings suggest that rapid arrhythmia contributes to the disorganization of GJ channels. Here, we show that connexins are also redistributed in patient in sinus rhythm with dilated atria. Moreover, similar alterations of Cx43 organization can be reproduced in an experimental model of atrial myocardial remodeling without AF [24,25]. In addition, regression of the atrial myopathy in treated MI-rats was associated with re-phosphorylation and assembly of organized GJ. All together these findings suggest that structural remodeling of the atrial myocardium notably fibrosis is another major factor responsible for GJ disorganization. A relationship between Cx43 remodeling and structural alterations of the myocardium in the ventricle has already been observed. In infarct scar tissue borders, it has been described by CLM that Cx43 is randomly distributed and scattered over the lateral surfaces of cells only in fibrotic area, pointing to the role of extracellular matrix remodeling [12]. Interstitial fibrosis may lead to alterations of the cytoskeleton network and adhesion proteins that contribute to normal GJ organization [34].

If redistributed (lateralized) Cx are functional, then they should form junctional channels with another hemichannel of an adjacent cell [1,35]. We showed in MI-rats that the presence of lateralized Cx43 in the absence of N-cadherin did not favor GJ formation. Lateralized Cx visualized by CLM and Cx oligomers identified by FF and FL were scattered in lateral plasma membrane without ultrastructural evidence of junctional plaque formation. Moreover, only few myocytes were stained with the LY dye, indicating reduced myocyte–myocyte coupling. No fibroblasts were stained with the LY dye, and there was evidence of GJ plaque between fibroblast and myocytes as observed in vitro [36,37]. Our findings suggest that lateralized connexins are not functional. The P wave prolongation in our model which suggests a depressed electrical conduction is also consistent with a poor myocyte–myocyte electrical coupling. A decreased longitudinal/transversal velocity ratio was observed in the rat model of atrial pacing [4]. The discrepancy between the two studies may be due to the severity of fibrosis in our model which is an important factor in impaired local electrical coupling. Interestingly, high optical action potential recording in dilated cardiomyopathy in dogs characterized by Cx43 redistribution and slow velocity of electrical propagation does not reveal abnormal anisotropy [18].

Another strong indication that lateralized connexins may not be functional is the observation that a large proportion of Cx43 was non-phosphorylated, like in failing ventricle [18,20,38]. Several phosphorylation sites regulate channel properties, assembly and targeting in junctional plaques [19,35]. For instance, dephosphorylated Cx43 from failing heart is responsible for depressed cell–cell coupling [20]. In addition, it is well known that Cx phosphorylation impairment might also contribute to a defect in its targeting and its assembly into GJ [19,39]. In dilated rat atria, our results showed the integrity of junctional membrane domains, a clear increase in Cx43 protein level due to a higher amount of non-phosphorylated Cx43 and numerous aggregates of Cx43 oligomers on myocytes plasma membrane. All together, our results suggest that redistribution of Cx43 does not result from the absence of junctional areas in the ID but rather from a defect in Cx43 targeting and the packing organization of the channel forming proteins [34,40,41].

4.1. Limitations of the study
First, the absence of spontaneous episodes of AF in this model, probably due to the small size of the rat atrium for the constitution of microreentry circuits, makes it difficult to study the relationship between GJ alterations and susceptibility to AF. Second, we focused our analysis on Cx43, rat atria do not express Cx40 [9]. However, Cx40 localization is also altered in diseased human atria as previously reported [4–7] and it will be of interest to examine whether redistributed Cx40 can form functional GJ channels.

Our observation that connexin are altered in hemodynamically overloaded atria suggests that GJ remodeling is an early event in the constitution of the AF substrate. Angiotensin II that affect connexin expression [42] or metalloproteinases that are involved in early changes of the extracellular matrix and alterations of myocyte–myocyte contacts [24,43] may be useful targets for the prevention of alterations of GJ and the occurrence of the arrhythmia.


   Acknowledgements
 
We thank Valérie Nicolas (IFR-141, Chatenay-Malabry, France) and Fabrice Cordelières (Institut Curie/CNRS-UMR-146, Orsay, France) for expert technical assistance. We are grateful to E. Deroubaix, F. Contard and E.L. Benedetti for constructive discussions.

This work was support by Fondation de France, Fondation pour la Recherche Médicale, Société Française de Cardiologie, ANR-05-PCOD-006-01.


   Notes
 
1 Current address: CNRS-UMR 5471, Talence, France. Back

2 Current address: Laboratoire de Chimie Physique, Université Paris-XI, Orsay, France. Back

Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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