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Am J Physiol Heart Circ Physiol 293: H1231-H1241, 2007. First published April 13, 2007; doi:10.1152/ajpheart.00607.2006
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Effects of sterile pericarditis on connexins 40 and 43 in the atria: correlation with abnormal conduction and atrial arrhythmias

Kyungmoo Ryu,1,2 Li Li,1 Celeen M. Khrestian,2 Naomichi Matsumoto,2 Jayakumar Sahadevan,2 Mary L. Ruehr,3 David R. Van Wagoner,3 Igor R. Efimov,1 and Albert L. Waldo1,2

Departments of 1Biomedical Engineering and 2Medicine, Case Western Reserve University, and 3Department of Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio

Submitted 9 June 2006 ; accepted in final form 13 April 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The canine sterile pericarditis model is characterized by impaired conduction and atrial arrhythmia vulnerability. Electrical and structural remodeling processes caused by the inflammatory response likely promote these abnormalities. In the present study, we tested the hypothesis that altered distribution of atrial connexins is associated with markedly abnormal atrial conduction, thereby contributing to vulnerability to atrial flutter (AFL) and atrial fibrillation (AF) induction and maintenance. During rapid pacing and induced, sustained AFL or AF in five sterile pericarditis (SP) and five normal (NL) dogs, epicardial atrial electrograms were recorded simultaneously from both atria (380 electrodes) or from the right atrium (RA) and Bachmann's bundle (212 electrodes). Tissues from RA sites were subjected to immunostaining and immunoblotting to assess connexin (Cx) 40 and Cx43 distribution and expression. Transmural myocyte ({alpha}-actinin) and fibroblast (vimentin) volume were also assessed by immunostaining. RA pacing maps showed markedly abnormal conduction in SP, with uniform conduction in NL. Total RA activation time was significantly prolonged in SP vs. NL at 300-ms and 200-ms pacing-cycle lengths. Sustained arrhythmias were only inducible in SP [total: 4/5 (AFL: 3/5; AF: 1/5)]. In NL, Cx40, Cx43, {alpha}-actinin, and vimentin were homogeneously distributed transmurally. In SP, Cx40, Cx43, and {alpha}-actinin were absent epicardially, decreased midmyocardially, and normal endocardially. SP increased epicardial vimentin expression, suggesting fibroblast proliferation. Immunoblot analysis confirmed reduced expression of Cx40 and Cx43 in SP. The transmural gradient in the volume fraction of Cx40 and Cx43 in SP is associated with markedly abnormal atrial conduction and is likely an important factor in the vulnerability to induction and maintenance of AFL/AF in SP.

gap junctions

STERILE PERICARDITIS (SP) occurs as a consequence of open-heart surgery and provides an important substrate for the initiation and maintenance of atrial fibrillation (AF) and atrial flutter (AFL), arrhythmias commonly occurring in the first few days following open-heart surgery (15). The canine SP model was developed as an experimental counterpart to the postoperative patient and has been extensively studied in our laboratory for more than 20 years (12, 13, 15, 23, 28). The SP model is characterized by inhomogeneous atrial conduction, including conduction delay and formation of functional lines of block, especially at rapid rates (18, 23). There is also a high incidence of inducibility (>90%) of sustained AF/AFL (12, 15, 23). Moreover, irregular atrial activation (i.e., fibrillatory conduction) in the right atrium (RA) is often seen during AF due to a single, rapid, reentrant circuit in the left atrium (LA) (13, 19). However, the mechanisms underlying SP-induced conduction abnormalities and atrial arrhythmias have not been fully elucidated.

Gap junctions underlie the electrical coupling of cardiac myocytes (17, 26), with the predominant connexins expressed in the atria being connexin40 (Cx40) and connexin43 (Cx43) (26). Altered distribution of Cx40 and Cx43 in the atria may contribute to abnormal conduction and, thereby, to substrate changes required for initiation and maintenance of reentrant atrial arrhythmias (1, 8, 11, 16, 26). We tested the hypothesis that changes in the atrial distribution of Cx40 and Cx43 contribute to the markedly abnormal atrial conduction in the SP model, thereby contributing to increased vulnerability to AF and AFL induction and maintenance.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We studied five SP and five normal (control) mongrel dogs (17–23 kg). The SP model was created as previously described, 4 days before an epicardial-mapping study in the open-chest state (12, 15, 24, 28). All studies were performed in accordance with guidelines specified by the Institutional Animal Care and Use Committee of Case Western Reserve University and "Public Health Service Policy on Humane Care and Use of Laboratory Animals" of the National Institutes of Health, Office of Laboratory Animal Welfare.

Studies in the open-chest state. The heart was exposed under general anesthesia, and the dog was mechanically ventilated as previously described (12, 15, 24, 28). For SP dogs, pairs of stainless-steel wire electrodes previously sutured on the high RA (HRA), Bachmann's Bundle (BB), and the posterior-inferior LA (PLA) were used for pacing, along with body-surface leads I, II, and III for monitoring or recording (CardioLab; Prucka Engineering, Houston, TX). For normal dogs, these electrode pairs were sutured in place at the time of the open-chest study. Rapid pacing (for ~6 s) at twice the stimulus threshold (Bloom Stimulator, model DTU; Fischer Imaging, Denver, CO) was performed from the HRA electrode site at 300- and 200-ms cycle lengths (CLs) to assess the resulting atrial activation pattern in four normal dogs and four SP dogs. One normal dog was paced from BB. Pacing was not performed in one SP dog. To assess the consistency of pacing effects on atrial activation, three consecutive paced beats during a steady-state response to the pacing (i.e., 3–4 s after the initiation of pacing) were analyzed in all paced dogs. Unless AF or AFL had already been induced by prior pacing, programmed or burst pacing from the HRA, BB, or the PLA was used to induce AFL or AF.

Data acquisition. During both rapid atrial pacing and induced, sustained (lasting >5 min) AFL or AF, atrial activation maps were determined by recording from epicardial electrode arrays containing 380 electrodes placed on both atria, including BB (3 dogs), or from 212 electrodes placed on the RA and BB (7 dogs), as previously described (Fig. 1) (12, 24).


Figure 1
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  		Fig. 1. Location of epicardial electrode arrays on heart. Numbers in parentheses represent number of electrodes in each region. Thick black line indicates location of a transmural tissue section. At right is enlarged histological section of representative section from transmural right atrium (RA) free wall (RAFW). EPI, epicardium; ENDO, endocardium; BB, Bachmann's Bundle; PV, pulmonary vein; LAA, left-atrial appendage; IVC, inferior vena cava; SVC, superior vena cava; RAA, right-atrial appendage.

 
Immunohistochemistry: immunostaining and immunoblotting. Tissues from selected sites in the RA free wall (RAFW) were subjected to immunostaining and confocal imaging of transmural sections to measure volumes of Cx40 and Cx43 expression. Figure 1 demonstrates a representative, low-power, transmural, Masson's trichrome-stained section from the low RAFW of a SP dog. Transmural volumes of myocytes and fibroblasts were assessed by immunostaining of {alpha}-actinin and vimentin, respectively.

On completion of epicardial mapping studies, the heart was rapidly removed and submerged in cold Tyrode solution (composition, in mmol/l: 128 NaCl, 1.3 CaCl2, 4.7 KCl, 1.05 MgCl2, 1.19 NaH2PO4, 20 NaHCO3, 11.1 glucose). The heart was minimally exposed to air during cutting of selected RAFW sections. The RAFW sections were immediately embedded in tissue-freezing medium (Sakura) and were frozen in isopentane cooled by liquid nitrogen. All samples were stored in a –80°C freezer before being cryosectioned.

Frozen tissues were cryosectioned into 16-µm-thick preparations. Sections were fixed for 5 min in –20°C methanol, blocked in 10% normal horse serum (Sigma-Aldrich) for 1 h, and incubated with primary antibodies at 4°C overnight. Sections were then treated with secondary antibodies at room temperature for 2 h. For Cx43 staining, we used a combination of rabbit polyclonal anti-Cx43 IgG (1:1,000; Sigma-Aldrich), mouse monoclonal anti-{alpha}-actinin IgG (1:1,600; Sigma-Aldrich), and a guinea pig polyclonal anti-vimentin IgG (1:800; Progen Biotechnik) as the primary antibodies. For secondary antibodies, the combination of donkey anti-rabbit FITC-conjugated IgG (Chemicon, 1:100), donkey anti-mouse Cy3-conjugated IgG (Chemicon, 1:400), and donkey anti-guinea pig Cy5-conjugated IgG (Chemicon, 1:100) were used. For Cx40 staining, the primary antibodies included a rabbit polyclonal anti-Cx40 IgG (1:100; Chemicon) and the same mouse anti-{alpha}-actinin and guinea pig anti-vimentin antibodies as used in the Cx43 study. The same combination of secondary antibodies was used in the Cx40 and Cx43 studies. Omission of primary or secondary antibodies as negative controls was included in all immunostaining experiments.

Confocal immunofluorescence images of selected transmural RAFW sections cut transverse to the crista terminalis (Fig. 1) were obtained by using a Leica TCS SP AOBS spectral laser-scanning confocal microscope. Three-dimensional structures of each region were reconstructed from confocal immunofluorescence images recorded in z-series and analyzed by using digital image-processing software (Volocity; Improvision, Coventry, UK) to quantify the total volume of each signal (Cx43, Cx40, {alpha}-actinin, and vimentin). For normal dogs, confocal imaging of transmural RAFW sections focused on three distinct regions: 1) endocardium, 2) midmyocardium, and 3) epicardium. In contrast, transmural RAFW sections from SP dogs were analyzed as four regions: 1) endocardium, 2) endocardial/midmyocardial junction, 3) midmyocardial/epicardial junction, and 4) epicardium, to better characterize the transmural changes seen.

Western blot analysis was used to compare the level of Cx40 and Cx43 expression in SP and normal dogs. Tissues from the right-atrial appendage (RAA), RAFW, BB, and the left-atrial appendage (LAA) were homogenized for 20 s in ice-cold M-PER mammalian protein extraction buffer (Pierce) containing a protease-inhibitor mixture (Sigma). Proteins from the cell lysate were separated by using SDS-PAGE (10% acrylamide gel), transferred to a nitrocellulose membrane, and probed with rabbit anti-Cx40 (1:500, polyclonal; Zymed) or mouse anti-Cx43 (1:1,000, monoclonal; Sigma). Blots were incubated in the appropriate secondary antibody, washed, and exposed to X-ray film. Films were scanned by using NIH ImageJ software for densitometric analysis of immunoreactive bands.

To correct for variations in protein loading, blots were stained with Ponceau S before incubation with the primary antibody. Densitometry of the Ponceau S-stained blot was performed on an identically sized region of proteins for each lane running from 35 to 65 kDa. The cumulative density of the area within the box for each lane was used to normalize Cx40 and Cx43 values on the blot. The lane with the greatest protein density was set at unity. Loading variability was corrected by dividing the corresponding connexin value in each lane by the relative protein abundance (see Fig. 7 legend).


Figure 7
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  		Fig. 7. A and B: representative Western blots showing Cx40 (A) and Cx43 (B) expression in LAA, RAA, RAFW, and BB in normal and SP dogs. Ponceau S staining (used for normalization of protein loading) is shown below each immunoblot. C and D: column plots summarizing regional expression of Cx40 (C) and Cx43 (D), with immunoblot intensity of each lane normalized to protein loading.

 
Data analysis. Epicardial mapping data were analyzed as previously described (12, 24). The mapping system permitted selection of activation times and computation of isochrone maps with a maximum resolution of 1 ms. Raw atrial electrograms (AEGs) acquired in both unipolar and bipolar formats were used for the determination of activation times (12, 24). During steady-state responses to the pacing (i.e., 3–4 s after the initiation of pacing), the atrial activation sequence was analyzed. Activation-sequence maps were drawn with isochrones at 10-ms intervals during AFL and at 5-ms intervals during pacing for the resulting activation pattern due to pacing. Total RA activation time during pacing from the HRA site was calculated from the pacing stimulus to the latest site of epicardial atrial activation.

For the SP dog with sustained AF, frequency analysis using the Fast Fourier Transform (FFT) technique was performed from 4-s segments of all bipolar AEGs recorded from both atria as previously described (19–21). FFT results were displayed as power spectra, and the region between 1 and 15 Hz was retained for analysis. In some cases, harmonics of the fundamental frequency were present. During AF, a driver was defined as an area with stable, rapid, regular activation denoted by a single dominant frequency peak and with the highest power in the FFT analysis. Irregular atrial activation was defined as activation denoted by multiple frequency peaks with a broad band and without a dominant frequency in the FFT analysis. All signal processing and analyses were done using MATLAB software (MathWorks, Natick, MA).

Statistical analysis. Statistical comparisons between group means were obtained by Student's t-test. Differences with P < 0.05 were considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Transmural gradients of Cx40 and Cx43. Figures 2 and 3 demonstrate immunostaining results of Cx40, Cx43, {alpha}-actinin, and vimentin of the transmural RAFW tissue sections from normal and SP dogs. Figure 2 illustrates a representative example from a normal dog of confocal images from the endocardium, midmyocardium, and epicardium. Immunofluorescence signals of Cx40, Cx43, {alpha}-actinin, and vimentin demonstrate a relatively homogeneous distribution of myocytes, fibroblasts, and connexins in all transmural regions. Figure 3 illustrates representative examples of confocal images from a SP dog. In contrast to normal dogs, Cx40, Cx43, and {alpha}-actinin (myocytes) staining revealed a gradient, with these proteins undetectable in the epicardium, significantly decreased in the midmyocardium, and normally distributed in the endocardium. Endocardial staining of Cx40, Cx43, {alpha}-actinin, and vimentin in the SP dogs demonstrated a distribution pattern very similar to that of normal dogs. An increase of Cx40 in blood vessels and endothelial cells was observed in the midmyocardial/epicardial junction (Fig. 3B), perhaps due to pericarditis-induced neoangiogenesis (4, 22).


Figure 2
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  		Fig. 2. Representative examples of immunostaining of connexin (Cx) 43 (top) and Cx40 (bottom), {alpha}-actinin (myocytes), and vimentin (fibroblasts) from a normal dog from 3 different regions (region 1, endocardium; region 2, midmyocardium; region 3, epicardium) of the transmural RAFW.

 

Figure 3
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  		Fig. 3. Representative examples of immunostaining of Cx43 (top) and Cx40 (bottom), {alpha}-actinin (myocytes), and vimentin (fibroblasts) from a sterile pericarditis (SP) dog from 4 different regions of transmural RAFW. A: region 1, endocardium; region 2, endocardial/midmyocardial junction. B: region 3, midmyocardial/epicardial junction; region 4, epicardium. CSP, canine SP model.

 
Figure 4 graphically illustrates the calculated volumes of Cx40, Cx43, {alpha}-actinin, and vimentin in each region from normal and SP dogs. In all SP dogs, the volume fractions of Cx40 and Cx43 significantly decreased rapidly across the myocardium and were negligible in the epicardium. The volume fraction occupied by myocytes ({alpha}-actinin) was also significantly decreased from the endocardial/midmyocardial junction (region 2) to the midmyocardial/epicardial junction (region 3) and then to the epicardium (region 4). In contrast, the volume of fibroblasts increased significantly from region 2 toward the epicardium. In all normal dogs, there was no statistically significant difference in the distribution of any of these proteins between transmural regions. The relatively wide range in the volume of each region when comparing dogs within each group was likely due to physiological variability, but within each group (normal or SP) the data were always in the same direction.


Figure 4
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  		Fig. 4. A: transmural volumes of Cx43 (top) and Cx40 (bottom) in 5 SP dogs (left) and 5 normal dogs (right). B: transmural volumes of myocytes (top) and fibroblasts (bottom) in 5 SP dogs (left) and 5 normal dogs (right). P values between each region are shown under each chart.

 
The volumes of Cx40 and Cx43 in endocardial and epicardial regions in normal and SP dogs were compared. The volumes of Cx40 and Cx43 from the endocardium (region 1) did not show a significant difference between the two groups (normal, 26,235 ± 6,571 µm3 vs. SP, 18,649 ± 7,682 µm3, P = 0.132 for Cx40; normal, 28,843 ± 13,378 µm3 vs. SP, 26,723 ± 13,741 µm3, P = 0.811 for Cx43). In contrast, the volumes of Cx40 and Cx43 from the midmyocardium (region 2 in normal and regions 2and 3 in SP dogs) were significantly different (Cx40, P = 0.002 for control region 2 vs. SP region 2 and P = 0.001 for control region 2 vs. SP region 3; Cx43, P = 0.009 for control region 2 vs. SP region 2 and P = 0.003 for control region 2 vs. SP region 3). The epicardium (normal, region 3; SP, region 4) also showed a significant difference between the two groups (normal, 20,345 ± 6,752 µm3 vs. SP, 4 ± 2 µm3, P < 0.001 for Cx40; normal, 20,081 ± 9,579 µm3 vs. SP, 291 ± 231 µm3, P = 0.002 for Cx43), reflecting the virtual absence of myocytes in this region in SP dogs.

The loss of connexins visualized in confocal images of epicardium and midmyocardium of SP dogs might reflect either a loss of myocytes or changes in the distribution of connexins before changes in myocyte numbers. To compare the extent of connexin degradation relative to myocyte loss, the ratios of Cx40 and Cx43 expression to {alpha}-actinin (myocyte marker) expression were determined in all regions from the endocardium to the epicardium in normal and SP dogs (Fig. 5). Because no myocytes were present in the epicardial region of SP dogs, this region was excluded from the analysis. Figure 5A shows that the ratios of both Cx40 and Cx43 to {alpha}-actinin were significantly (P < 0.05) reduced from the endocardial region (Cx40, 0.22 ± 0.06; Cx43, 0.34 ± 0.19) to the midmyocardial region (Cx40, 0.10 ± 0.05; Cx43, 0.15 ± 0.05) and the epicardial region (Cx40, 0.05 ± 0.03; Cx43, 0.08 ± 0.06) in SP dogs. However, in normal dogs (Fig. 5B), the ratios of both Cx40 and Cx43 to myocytes were similar in all regions.


Figure 5
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  		Fig. 5. Ratio of connexin expression to {alpha}-actinin expression in different regions from SP (A) and normal (B) dogs.

 
Figure 6 demonstrates representative examples of global images of Cx43, {alpha}-actinin, and vimentin distribution in a low-power view of transmural RAFW sections from both normal (Fig. 6A) and SP (Fig. 6B) dogs. Transmural gradients of Cx43 and myocytes ({alpha}-actinin) were clearly present in SP but not in normal dogs.


Figure 6
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  		Fig. 6. A: representative examples of immunostaining of Cx43, {alpha}-actinin (myocytes), and vimentin (fibroblasts) from a control dog of transmural RAFW; a and b demonstrate enlargement of square sections at top. B: representative examples of immunostaining of Cx43, {alpha}-actinin (myocytes), and vimentin (fibroblasts) from a SP dog; a, b, and c are enlargements of square sections at top.

 
Connexin expression. Regional expression of Cx40 and Cx43 was determined from different areas throughout the atria in representative normal and SP dogs. Immunoblot analysis revealed that expression of both Cx40 and Cx43 was reduced in SP dogs. Figure 7 shows a representative Western blot (Fig. 7A) and bar plots summarizing the expression of Cx40 (Fig. 7C) and Cx43 (Fig. 7, B and D) from four different areas of the atria: the LAA, the RAA, the RAFW, and BB. The SP model was associated with reduced Cx43 expression in all sites, and Cx40 expression was significantly reduced in all regions except the RAA.

RA epicardial activation during rapid pacing. Atrial activation patterns analyzed during three consecutive cycles demonstrated consistency of atrial activation in during pacing. RA epicardial activation maps during pacing showed markedly abnormal conduction in SP dogs compared with uniform conduction in normal dogs. Figure 8A demonstrates representative examples of RA activation during HRA pacing at 300- and 200-ms CLs from a normal dog (Fig. 8A, left) and a SP dog on postoperative day 4 (Fig. 8A, right). Activation-sequence maps (atrial pacing at 300- and 200-ms CL) from a normal dog demonstrate relatively uniform impulse propagation in a craniocaudal direction in the RAFW. In contrast to atrial activation patterns in normal dogs, maps from SP dogs were characterized by a nonuniform slowing of conduction, denoted by relative crowding of isochrones and a marked increase in total RA activation time. In fact, the mean total RA activation time was prolonged in SP vs. normal dogs at both pacing CLs (300-ms pacing CL, 58 ± 5 vs. 46 ± 7 ms, P < 0.05; 200-ms pacing CL, 74 ± 12 vs. 47 ± 7 ms, P < 0.02) and was increased at the shorter pacing CL (200 ms) compared with the longer pacing CL (300 ms) in SP dogs (83 vs. 62 ms) but was unchanged in normal dogs.


Figure 8
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  		Fig. 8. A: representative examples of RA activation sequence maps during high RA (HRA) pacing at 300- and 200-ms cycle lengths (CLs) from normal dog (left) and a SP dog (right). Activation-sequence maps from a normal dog demonstrate homogeneous impulse propagation. Activation-sequence maps from a SP dog demonstrate abnormal conduction with areas of slow conduction and conduction block. Note marked increase in total RA activation time during pacing at a 200-ms CL vs. a 300-ms CL. Isochrones are at 5-ms intervals. Dashed line indicates pericardial reflection. B, left: representative RA activation-sequence map during induced, sustained atrial flutter (AFL; CL = 186 ms) in same dog as in A. Isochrones are at 10-ms intervals. Isochrone map demonstrates a single-loop reentry. Thick, black dashed lines indicate functional line of block. Wave front breaks through at BB and travels along right side of BB. It then travels down the RAFW, breaks through to the endocardium, and travels up the interatrial septum (gray dashed line with arrow). Gray line with arrows indicates direction of reentrant activation wave front in RAFW. Asterisk indicates epicardial breakthrough site, and diamond indicates entrance to endocardium of AFL reentrant wave front; a–h indicate recording sites of atrial electrograms (AEGs) shown at right. Right: bipolar AEGs recorded from selected epicardial sites (a–h) are illustrated. S, ventricular pacing stimulus. C: fast Fourier transform (FFT) analyses of an atrial fibrillation (AF) episode due to a stable driver in the left atrium (LA) with a short CL (117 ms). Lower tracingsare simultaneously recorded bipolar AEGs from a LA site (a) showing a regular atrial rhythm and a right atrium (RA) site (b) showing fibrillatory conduction during this episode of AF. A dominant peak at 8.54 Hz was found in LA that corresponded to 1:1 atrial activation pattern at the CL (117 ms). Broad bands with multiple peaks from 5.74 to 7.81 Hz were found in RA and part of LA that corresponded to fibrillatory conduction caused by wave fronts from LA driver.

 
AFL and AF. Sustained atrial arrhythmias were only inducible in SP dogs (total, 4/5; AFL, 3/5; AF, 1/5). AFL occurred after a line of functional block developed between the venae cavae (23, 28). Figure 8B illustrates a representative RA activation sequence map during induced, sustained AFL (186 ms CL) in the same SP dog as shown in Fig. 8A, right. The circulating reentrant wave front breaks through from the septum at BB and then travels down the RAFW, entering endocardially to travel up the interatrial septum. A line of block was present in the region between the venae cavae. In all three AFL episodes (mean AFL CL, 167 ± 30 ms), an area of relatively slow conduction and a functional line of block were present in the RAFW. The only sustained AF episode induced in a SP dog demonstrated stable, rapid (CL 117 ms), regular atrial activation in parts of the LA with irregular atrial activation in the rest of the atria (Fig. 8C) (13, 19, 28). A dominant frequency peak of 8.54 Hz present near the pulmonary veins and BB corresponded to 1:1 atrial activation at a mean CL of 117 ms (the driver). Broad bands with multiple frequency peaks from 5.74 to 8.54 Hz were present throughout the RA and part of the LA, were associated with an LA-to-RA frequency gradient, and corresponded to fibrillatory conduction produced by the LA driver.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings in this study were that in response to SP, the atria demonstrated a proliferation of epicardial fibroblasts, a loss of epicardial myocytes, and an altered distribution of Cx40 and Cx43. Both connexins were homogenously and similarly expressed in the endocardium. Although no transmural gradient in connexin expression was apparent in normal dogs, Cx40 and Cx43 expression was gradually attenuated beyond the endocardial layer to the epicardium in the SP dogs. Loss of connexins was greater that that of myocytes (detected with {alpha}-actinin) in the midmyocardium. These findings are consistent with the trauma (necrosis and/or apoptosis) caused by the severe inflammatory response accompanying the pericarditis (6). Previous studies have shown that Cx40 and Cx43 are critical elements underlying the function of atrial gap junctions and that they contribute to propagation of atrial activation (1, 8, 11, 16, 26). Thus changes in the transmural distribution of Cx40 and Cx43 and a loss of epicardial myocytes would be expected to contribute to abnormal atrial conduction and would likely provide the substrate for the markedly abnormal atrial conduction and the remarkable vulnerability to atrial tachyarrhythmias found in the SP model.

Relationship of these findings to other studies. The findings of a transmural gradient of the volume of Cx40 and Cx43, the loss of epicardial myocytes, and the proliferation of epicardial fibroblasts in the SP model may explain the unanticipated normal electrophysiological finding from Boyden and Hoffman's (3) study in dogs with chronic RA enlargement due to pulmonary artery banding and surgically created tricuspid insufficiency (TI). In their study, atrial arrhythmias were inducible in all (n = 8) TI dogs. Transmembrane action potentials could not be recorded from the epicardium because the glass microelectrodes broke when inserted in to the epicardial tissue. This presumably occurred secondary to changes due to the SP that necessarily resulted from the surgical procedure necessary to produce the model. Amplitude and duration of transmembrane action potentials recorded from the RA endocardium were similar to that of normal dogs (3). This suggests that the electrophysiological characteristics of the endocardium of TI dogs were not altered by surgically creating the model but that pathophysiological changes in other regions of the atrial wall were likely responsible for vulnerability to atrial arrhythmia induction and maintenance in TI dogs.

Distribution of Cx40 and Cx43 in other studies of AF. Distribution of Cx40 and Cx43 in the atria has been extensively studied in several animal models and humans, although the impact of pericarditis on connexin distribution has not been reported. In the RAA and the LAA from goats with persistent AF for at least 2 mo, the distribution of Cx43 seemed unchanged and homogeneous throughout the atrial preparations (25). However, the distribution of Cx40 became increasingly heterogeneous and the volume of Cx40 expression was decreased. Increased heterogeneity of Cx40 distribution in both appendages correlated with increased persistence of AF, whereas the expression of Cx43 remained unchanged. In contrast, a recent study (10) in 31 patients with AF undergoing a Maze procedure demonstrated that, compared with expression in sinus-rhythm patients, Cx43 volume was decreased by 56% in the RAFW and by 57% in the RAA; Cx40 volume was reduced 54% in appendages but showed a tendency to be increased in the RAFW. Polontchouk et al. (16) showed increased expression of Cx40 in the RAA of AF patients, and another study by Dupont et al. (5) also showed increased Cx40 in the RAA of patients who developed postoperative AF. In contrast, Nao et al. (14) reported a downregulation of Cx40 in the RAA patients with AF compared with normals. They also reported that Cx43 expression was unchanged in patients with AF. Moreover, Kanagaratnam et al. (8) showed no difference in expression or distribution of Cx40 and Cx43 detected with confocal microscopy between patients in chronic AF or sinus rhythm. However, the results from van der Velden et al. (25) in goats and Kostin et al. (10), Dupont et al. (5), and Nao et al. (14) in AF patients all demonstrated a heterogeneous distribution of Cx40.

In our study of the SP model, Cx40 expression was unchanged in the RAA but was significantly reduced in all other regions (Fig. 7, A and C). Expression of Cx43 was significantly reduced in all regions (Fig. 7, B and D). Our study reveals not only a decrease in the volume of connexins but also a transmural gradient of connexin volumes from the endocardium to the epicardium in the RAFW. The changes in volume and the distribution of Cx40 expression in the RAFW were very similar to the changes in Cx43 expression in the RAFW. The variable changes reported by other investigators in AF studies suggests that multiple connexin-remodeling processes may be associated with AF maintenance.

Abnormal conduction and Cx40 and Cx43. Complex interactions between Cx40 and Cx43 are likely to contribute to electrical propagation in atrial myocardium (2). Kleber's group (2) has shown that conduction velocity was lower in strands of atrial myocytes from mice with a deletion of Cx43 compared with wild type. Paradoxically, in strands of atrial myocytes from mice with a deletion of Cx40, conduction velocity was faster than in wild-type normals. Other studies have demonstrated that mice with a targeted deletion of the Cx40 gene showed a diminished atrial conduction velocity (9). In support of the notion that Cx40 is important for atrial conduction, atrial arrhythmias occurred spontaneously (9) or could be induced easily (7, 27). Our study suggests that the transmural gradient of Cx40 and Cx43 and reduced expression of Cx40 and Cx43 in the epicardial region contribute to abnormal conduction by producing transmural differences in atrial conduction in different regions of the RA. We have demonstrated that an increased pacing rate produces increased abnormalities in atrial conduction with markedly slow conduction areas in SP dogs, whereas atrial conduction remains uniform in normal dogs.

Limitations. Simultaneous endocardial activation mapping was not performed in this study. Also, the time course of Cx40 and Cx43 changes in SP dogs was not studied, because the study was limited to terminal studies on postoperative day 4. In addition, other cellular-remodeling processes during tissue injury caused by SP and their relationships to Cx40 and Cx43 remodeling or abnormal atrial conduction were not studied.

It is difficult to clearly differentiate distinct transmural layers in the atrium. In this study, epicardium, midmyocardium, and endocardium were identified by approximation of the distance from the epicardial layer and based on transmural changes in Cx40, Cx43, and {alpha}-actinin. The extent of fibrous tissues and inflammatory cells in the epicardium of the SP dogs was not examined in this study.

Immunostaining data were only compared between the epicardial and endocardial regions between control and SP dogs in this study. Immunostaining data from the midmyocardium of control dogs were not compared directly with the data from midmyocardium in the SP dogs due to the challenge of clearly identifying the location and extent of the midmyocardium in the atria. However, transmural gradients of Cx40, Cx43, and {alpha}-actinin were present even within the midmyocardial region in the SP dogs. Therefore, immunostaining data were taken from the junctions of the endocardium/midmyocardium and midmyocardium/epicardium to effectively demonstrate the changes in different regions in the SP model.

Myocytes were detected by using {alpha}-actinin immunostaining. Thus the mechanisms underlying myocyte death (apoptosis vs. necrosis) were not studied, and we cannot delineate the causes of myocyte injury and loss due to the SP.

Conclusion. The transmural distributions of Cx40 and Cx43 are markedly altered in the SP model in response to epicardial inflammation. The altered transmural gradient of Cx40 and Cx43 was associated with abnormal atrial conduction and, along with loss of epicardial myocytes and epicardial fibroblast proliferation, is likely critical in the vulnerability of the SP model to the induction and maintenance of AFL and AF.


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This work was supported in part by grants R01-HL-38408 and R01-HL-74189 from the National Heart, Lung, and Blood Institute.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. L. Waldo, Division of Cardiology, MS LKS 5038, Univ. Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106 (e-mail: albert.waldo{at}case.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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