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Circulation Research. 2006;99:1216-1224
Published online before print October 19, 2006, doi: 10.1161/01.RES.0000250607.34498.b4
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(Circulation Research. 2006;99:1216.)
© 2006 American Heart Association, Inc.

Cellular Biology

Relative Contributions of Connexins 40 and 43 to Atrial Impulse Propagation in Synthetic Strands of Neonatal and Fetal Murine Cardiomyocytes

Philippe Beauchamp, Kathryn A. Yamada, Alex J. Baertschi, Karen Green, Evelyn M. Kanter, Jeffrey E. Saffitz, André G. Kléber

From the Department of Physiology (P.B., A.G.K.), University of Bern, Switzerland; Department of Neuroscience (A.J.B.), University of Geneva, Switzerland; and the Department of Medicine and Center for Cardiovascular Research (K.A.Y., E.M.K., J.E.S.) and the Department of Pathology (K.G., J.E.S.), Washington University, St Louis, Mo. Present address for J.E.S.: Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass.

Correspondence to André G. Kléber, MD, Department of Physiology, University of Bern, Bühlplatz5, CH-3012 Bern, Switzerland. E-mail kleber{at}pyl.unibe.ch


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Atrial tissue expresses both connexin 40 (Cx40) and 43 (Cx43) proteins. To assess the relative roles of Cx40 and Cx43 in atrial electrical propagation, we synthesized cultured strands of atrial myocytes derived from mice with genetic deficiency in Cx40 or Cx43 expression and measured propagation velocity (PV) by high-resolution optical mapping of voltage-sensitive dye fluorescence. The amount of Cx40 and/or Cx43 in gap junctions was measured by immunohistochemistry and total or sarcolemmal Cx43 or Cx40 protein by immunoblotting. Progressive genetic reduction in Cx43 expression decreased PV from 34±6 cm/sec in Cx43+/+ to 30±8 cm/sec in Cx43+/– and 19±11 cm/sec in Cx43–/– cultures. Concomitantly, the cell area occupied by Cx40 immunosignal in gap junctions decreased from 2.0±1.6% in Cx43+/+ to 1.7±0.5% in Cx43+/– and 1.0±0.2% in Cx43–/– strands. In contrast, progressive genetic reduction in Cx40 expression increased PV from 30±2 cm/sec in Cx40+/+ to 40±7 cm/sec in Cx40+/– and 45±10 cm/sec in Cx40–/– cultures. Concomitantly, the cell area occupied by Cx43 immunosignal in gap junctions increased from 1.2±0.9% in Cx40+/+ to 2.8±1.4% in Cx40+/– and 3.1±0.6% in Cx40–/– cultures. In accordance with the immunostaining results, immunoblots of the Triton X-100–insoluble fraction revealed an increase of Cx43 in gap junctions in extracts from Cx40-ablated atria, whereas total cellular Cx43 remained unchanged. Our results suggest that the relative abundance of Cx43 and Cx40 is an important determinant of atrial impulse propagation in neonatal hearts, whereby dominance of Cx40 decreases and dominance of Cx43 increases local propagation velocity.


Key Words: atrial myocyte • basic science • cardiac gap junction connexins • cardiovascular genomics • cell culture • conduction velocity • connexin 40 • connexin 43 • mapping • neonatal mouse cardiomyocytes • optical mapping


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Connexin (Cx) proteins enable the intercellular exchange of ions and small regulatory molecules and are important determinants of cardiac electrical propagation.1 Three major connexins, Cx43, Cx40, and Cx45, are expressed in heart.2,3 Cx43 is abundant in ventricular and atrial myocardium; Cx40 is expressed in atrial tissue and in the Purkinje system; Cx45 is present in the sinoatrial (SA) and atrioventricular (AV) nodes and colocalizes with Cx43 in ventricular myocardium.4,5 Cx43 and Cx40 each form channels with relatively large pores, whereas Cx45 forms narrow channels (unitary channel conductances of 75, 130, and 30 pS, respectively).6 Ventricular myocytes express abundant Cx43 and small amounts of Cx45. In contrast, atrial myocytes express large amounts of Cx43 and Cx40. In ventricular myocytes and transfected cells, Cx43 and Cx45 colocalize in gap junctions and may form heteromeric/heterotypic channels.5,7–10 Heterotypic Cx43/Cx40 gap junction channels have been described in HeLa cell pairs, whereas the role of heteromeric Cx43/Cx40 channels has not been fully clarified.7,11

Changes in cell-to-cell coupling by remodeling of gap junctions is considered an important factor in arrhythmogenesis.12–14 In the setting of atrial fibrillation (AF), conflicting results concerning gap junction remodeling have been reported. One early report described an increase in Cx43 in pacing-induced AF in dogs15; most other reports observed no major changes in Cx43 gap junctional or cellular protein and mRNA.14,16,17 Controversial reports have also been published about Cx40 remodeling, expression being upregulated in chronic human AF16,18 or downregulated in a heterogeneous pattern in goats and humans.14,17,19,20 Kanagaratnam et al21 showed a strong correlation of electrical propagation velocity with the ratio of Cx43 (or Cx40) to total Cx immunosignal ([Cx43+Cx40]). Thus, Cx43/[Cx43+Cx40] was directly and Cx40/[Cx43+Cx40] inversely related to propagation velocity. Importantly, these results suggest interaction between Cx43 and Cx40 function and/or expression in the atria.

Over the past years, several genetically engineered mice with modified expression of Cx43, Cx40, and Cx45 have been produced and the effects on cardiac structure, function, and development have been described.5,22–26 Macroscopic atrial conduction has been assessed in Cx40-ablated mice using extracellular electrode arrays and optical mapping.27

Recently, we developed a model of synthetic strands of murine cardiac myocytes in culture and assessed the effect of Cx43 deletion on ventricular propagation.5,28,29 In the present study, our goal was to assess the roles of Cx43 and Cx40 in atrial propagation. To this aim, we applied the patterned growth technique to create strands from murine atrial cardiomyocytes lacking Cx43 or Cx40.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Synthesis of Cardiac Strands of Specific Genotypes
The technique used to produce patterned growth of neonatal mouse ventricular myocytes has previously been described.28 Hearts were obtained from mice maintained in an inbred colony (C57BL/6J background for Cx43 and Cx40 mice; Cx43 mice were obtained from The Jackson Laboratory, Bar Harbor, Me; Cx40 mice were a gift from Prof Klaus Willecke, University of Bonn, Germany). Embryos from mice heterozygous (Cx43+/–) or homozygous (Cx43–/–) for a Cx43-null allele and wild-type mice (Cx43+/+) were obtained at embryonic day 20 (E20), ie, 1 day before birth. Wild-type (Cx40+/+) and Cx40-deficient (Cx40+/– and Cx40–/–) animals were obtained the first day post partum (D1). The genotype of each embryo was determined by PCR using standard protocols.

After careful and selective excision of the 2 atria and enzymatic digestion, a cell suspension was obtained that contained approximately 10 000 cells. This suspension was preplated to eliminate fibroblasts and seeded on coverslips to produce patterned growth.5,30 One cell suspension and 1 patterned culture dish (see Figure 1A) was obtained from each heart. Myocytes were grown in 4.5-mm long growth channels either 40 or 60 µm in width. All measurements were performed on days 4 to 5 in culture. The study complied with the ethical principles of the Swiss Academy of Medical Science.


Figure 1
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  		Figure 1. Synthesized atrial strands from neonatal cardiomyocytes. A, Low-magnification micrograph showing ring-shaped growth pattern with emerging cardiomyocytes strands. B, Double-labeling of Cx43 (red fluorescence) and Cx40 (green fluorescence) in Cx40+/+ strands. C and D, Synthetic atrial strands (Cx43+/+) showing antibody fluorescence against Cx40 (C) and Cx43 (D). Note consistent staining of cell circumferences with both antibodies. E and F, Red fluorescence visualizes presence of PAM-1 in atrial (E) and ventricular (F) strands. G, Quantitative comparison (y-axes in relative units) of PAM-1 fluorescence between atrial (A) and ventricular (V) strands from Cx40+/+ (n=12), Cx40+/– (n=12), and Cx40–/– (n=10) mice (n denotes number of analyzed cells).

Immunohistochemistry and Confocal Microscopy
In strands synthesized from ventricular myocytes, Cx40 is observed in only 2% to 3% of cells, which most likely originate from the specific ventricular conduction system.5 Thus, the abundance of Cx40 staining can be used as a marker of the atrial origin of the cells in the strands, with the exception of cultures made from Cx40–/– animals. To obtain a connexin-independent marker for atrial cell origin, we stained the cells with antibodies against peptidyl-glycine {alpha}-amidating monooxygenase (PAM)-1, a vesicular protein highly expressed in atrial tissue.31

To identify and quantify the amounts of Cx40 and Cx43 at intercellular junctions, cultured cells were fixed in paraformaldehyde and immunostained with monospecific antibodies using previously described protocols.28 The amount of immunoreactive signal in discrete spots located at intercellular junctions was quantified using laser scanning confocal microscopy and digital image processing algorithms validated in previous studies.32

Immunoblotting, Triton X-100 Extraction, and Membrane Fractionation
To measure the total tissue content of Cx43 and Cx40 in Cx40 deficient atrial tissue, we performed immunoblot analyses in atrial tissues of all genotypes as described previously.4 Whole atria (right and left) were homogenized; 30 µg of protein was loaded on each gel. Cx band densities were divided by their respective GAPDH density values. Corrected values were normalized to wild-type values. Moreover, to assess junctional versus nonjunctional connexin expression we performed Cx43 analysis in Cx40+/+, Cx40+/–, and Cx40–/– neonatal atria based on Triton X-100 (TX100) solubility.33

Optical Action Potential Measurement and Analysis of Propagation in Synthetic Strands
Staining of cell cultures with the voltage-sensitive dye RH237, the technique of multiple site optical recording of transmembrane potential by means of a light-sensitive diode array (8x8 diodes), and determination of conduction velocity, {theta}, have been described in detail elsewhere.5,28,34 Cultured cell strands were stimulated >1 mm from the recording site (cycle length, 400 to 500 ms; rectangular pulse of 5-ms duration; 1.5-fold threshold strength). The spatial resolution of the system was 15 µm with a x40 objective and 6 µm with a x100 magnification objective. Time resolution was 80 µs between data points.

Statistics
All data are given in means±SD.

For methodological details, see the online data supplement, available at http://circres.ahajournals.org.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Synthetic Strands From Fetal and Neonatal Atrial Murine Myocytes
Because Cx43 germline knockout animals die immediately after birth, patterned cultures from these animals were made from atria obtained 1 day before birth (E20) and compared with atrial cultures from Cx40 germline knockout animals obtained on the first day post partum (D1). In contrast to ventricular cells,28 atrial cells had an elliptical shape and consistently large nuclei (Figure 1C and 1D).

Cx43 and Cx40 (Figure 1B through 1D) were abundantly present in strands of wild-type atrial cells. In general, Cx43 and Cx40 colocalized in the same gap junctions, but small junctions showing only Cx43 or Cx40 fluorescence were also observed in double-labeling experiments (online data supplement). PAM-131 was found to be abundantly present in atrial strands and far less in ventricle. In control ventricular strands from Cx40–/– mice, PAM-1 signal was <10% of the signal in atrial strands, thus confirming homogeneity of specific atrial cell types in the latter (Figure 1E through 1G).

Effects of Cx43 Deletion on Cx40 Expression and Atrial Electrical Propagation
Similar to ventricular myocardium,5 germline knock out of Cx43 produced the expected ablation of Cx43 immunosignal in gap junctions, as summarized in Table 1 and illustrated in Figure 2A and 2B. Knock out of Cx43 had a distinct effect on the area occupied by Cx40 in the gap junctions. The area occupied by Cx40 immunoreactive signal in gap junctions decreased by 15% in Cx43+/– strands and by 50% in Cx43–/– strands. The decrease of Cx40 in Cx43–/– strands was attributable to a decrease in the number of Cx40 gap junctions, whereas the mean gap junction size occupied by the Cx40 signal showed no major change (Figure 2B and Table 1). In contrast to the immunohistochemical results, there was a nonsignificant tendency of total atrial Cx40 protein to increase with Cx43 ablation (Figure 2C). Thus, Cx40 increased 1.1±0.8-fold in Cx43+/– and 1.5±0.5-fold in Cx43+/– atria (n=6).


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  		Table 1. Cx43 and Cx40 Immunohistochemistry and Conduction Velocity in Atrial Synthetic Strands


Figure 2
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  		Figure 2. Immunohistochemistry of Cx43 and Cx40 proteins in gap junctions in atrial strands with deletion of Cx43 and atrial Western blots. A, Changes of the areas occupied by the Cx43 and Cx40 signals in the 3 genotypes. B, Changes of the number of Cx43 and Cx40 gap junctions. C, Changes in total atrial Cx43 and Cx40 protein with Cx43 ablation relative to the respective wild type.

An action potential of a typical atrial cell, measured by optical mapping of the fluorescence change in RH237 is shown in Figure 3A. Action potentials from mouse atrial myocytes showed the typical morphology, with a relative slow upstroke and lack of a plateau (see Kléber et al35). The change in propagation velocity in atrial strands from mice with progressive deletion of Cx43 is shown in a representative experiment in Figure 3B. Group data are shown in Figure 3C. The regular shape of the isochrones suggests absence of major discontinuities in propagation. The isochrone maps in Figure 3B reveal a crowding of isochrones in the Cx43–/– strand versus the Cx43+/+ strand, corresponding to a decrease in propagation velocity from 33 cm/sec to 19 cm/sec. On average, propagation velocity decreased from 34 cm/sec in Cx43+/+ strands by 12% in Cx43+/– strands and by 44% to 19 cm/sec in Cx43–/– strands (Figure 3C and Table 1). The maximal upstroke velocity of the action potential, dV/dtmax, taken as an indirect estimate of the ionic current driving propagation, was not different among the genotypes (online data supplement).


Figure 3
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  		Figure 3. Multisite, high-resolution optical mapping of transmembrane in atrial stands with Cx43 deletion. A, Optical measurement of atrial transmembrane action potential with typical atrial shape. B, Isochrone maps of propagation in intervals of 40 µs from red to blue (resolution, 15x15 µm per diode). Propagation velocity decreases from 33 cm/sec in the Cx43+/+ strand (top) to 19 cm/sec in the Cx43–/– strand (bottom). C, Changes in average values of propagation velocity in synthetic atrial strands with Cx43 deletion.

Effects of Cx40 Deletion on Cx43 Expression and Atrial Electrical Propagation
Comparison of the immunohistochemistry and electrophysiology between Cx43+/+ and Cx40+/+ strands synthesized from cells harvested within a 24-hour gestational window (E20 versus D1) showed no statistically significant difference, with the exception of the number of Cx40-positive gap junctions that was larger in Cx40+/+ versus Cx43+/+ strands (Table 1). Moreover, there was no difference in propagation velocities between Cx40+/– atrial strands at D1 versus E20 (online data supplement). Progressive genetic deletion of Cx40 had the expected effect of reducing the Cx40 immunosignal (Figure 4A and Table 1). Ablation of Cx40 had an unexpected effect on Cx43 expression in gap junctions. In contrast to the effects of Cx43 ablation, which led to a decrease in the area occupied by Cx40 in gap junctions, Cx40 deletion was associated with a significant increase in the area occupied by Cx43 immunosignal in gap junctions. The Cx43 signal increased from 1.2% area in Cx40+/+ strands by 2.3-fold in Cx40+/– and by 2.6-fold in Cx40–/– strands.


Figure 4
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  		Figure 4. Cx43 and Cx40 proteins in gap junctions in atrial strands with ablation of Cx40. A, Changes in the areas occupied by the Cx43 and Cx40 signals in the three genotypes. B, Changes in the number of Cx43 and Cx40 gap junctions. C, Immunoblots from whole atrial neonatal tissue for Cx43 and Cx40 in mice with Cx40 ablation. D, Junctional Cx43 protein expression. Top, Immunoblot showing TX100-soluble (sol) (nonjunctional) and -insoluble (insol) (junctional) proteins separated by SDS-PAGE. The numbers below the immunoblot are relative Cx43 band densities normalized to the Cx43+/+ insoluble band. Bottom, Coomassie blue–stained gel of the blot shown above. Light staining is a result of the small amount of protein (2.5 µg) loaded in each lane.

Immunoblots of total protein homogenates of isolated neonatal atria (Figure 4C) showed the expected decrease in Cx40 content with progressive Cx40 ablation (Cx40+/+, 1.0±0.2, n=6; Cx40+/–, 0.5±0.4, n=9, P<0.01 versus Cx40+/+; Cx40–/–, not detectable, n=3). However, in contrast to results observed by immunostaining in which the amount of Cx43 signal in gap junctions was increased, no significant increase in total Cx43 protein content was observed among the Cx40+/+, Cx40+/–, and Cx40–/– genotypes (Cx40+/+, 1.0±0.3, n=6; Cx40+/–, 1.1±0.2, n=8; Cx40–/–, 0.8±0.4, n=3). These results suggest a shift in the proportion of total Cx43 present within junctional and nonjunctional compartments. Finally, to assess whether the increase in Cx43 immunoreactive signal observed in Cx40-deficient atria represented increased Cx43 protein in junctional membranes, we determined relative Cx43 protein in TX100-insoluble and -soluble fractions of atrial homogenates. Cx40 ablation produced a 2.4-fold increase in Cx43 in the junctional membrane (Figure 4D), consistent with the immunostaining data. As expected, a small amount of faster migrating Cx43 was observed in the TX100-soluble, or nonjunctional, compartments. This result was supported by additional experiments involving membrane fractionations of Cx43 protein (online data supplement).

In contrast to Cx43-deficient strands, in which conduction velocity decreased as Cx43 was ablated, electrical propagation velocity increased with progressive Cx40 deletion. Comparison of isochrone maps from strands composed of Cx40+/+ versus Cx40–/– atrial myocytes is shown in a representative experiment in Figure 5A. The propagation velocity of 34 cm/sec in the Cx40+/+ strand increased to 48 cm/sec in the Cx40–/– strand. As with Cx43 ablation, the isochrones showed no major discontinuities in the Cx40–/– strands. The summarized data (Figure 5B and Table 1) confirmed the increase in electrical propagation velocity by 33% (Cx40+/–) and by 50% (Cx40–/–). The maximal upstroke velocity of the action potential, dV/dtmax, taken as an indirect estimate of the ionic current driving propagation, was not different among the genotypes (online data supplement).


Figure 5
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  		Figure 5. Changes in propagation velocities in strands of atrial myocytes with Cx40 deletion. A, Isochrone maps of propagation in intervals of 40 µs from red to blue (resolution, 15x15 µm per diode). Propagation velocity increases from 34 cm/sec in the Cx40+/+ strand (top) to 48 cm/sec in the Cx40–/– strand (bottom). B, Average values for propagation velocity in synthetic atrial strands (filled columns) and ventricular strands (open columns). Note increase of atrial propagation velocity with Cx40 ablation.

For comparison, synthetic ventricular strands were fabricated from the same hearts.5 Figure 5B and Table 1 show that in the Cx40–/– genotype, atrial propagation velocity approached ventricular velocity.

Macroscopic atrial conduction abnormalities have been reported in adult Cx40–/– mice.36 To clarify this seemingly paradoxical difference, we used quantitative confocal microscopy to compare Cx40 and Cx43 expression in gap junctions in intact atrial tissue from neonatal and adult mice with Cx40 ablation. Moreover, a second antibody (AB-2) against Cx43 was used in these experiments, in addition to Cx43 AB-1 used in cell cultures, to exclude epitope mapping as a cause of the observed increase in Cx43 signal. As shown in Figure 6A and Table 2, whole neonatal atria showed the same pattern of Cx40 and Cx43 expression in gap junctions found in the synthetic atrial strands, characterized by significantly increased amounts of Cx43 signal in gap junctions with Cx40 ablation. No statistically significant difference was present between the data obtained with the 2 Cx43 antibodies. This contrasted with the measurements in adult (mice >8 weeks old) atrial tissue, in which Cx40 ablation did not produce any significant changes in Cx43 expression in gap junctions. The potential relevance of this finding for atrial remodeling in disease is discussed below.


Figure 6
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  		Figure 6. Cx43 immunohistochemical staining in gap junctions. Whole neonatal and adult atria from mice with Cx40 deletion. A, Cx43 and Cx40 immunohistochemical staining in gap junctions (neonatal atria). Cx43 staining was performed with 2 different antibodies (AB-1 and AB-2), each showing an increase in the area occupied by Cx43 signal with Cx40 deletion. B, Cx43 immunohistochemical staining in gap junctions (adult atria). No differences among Cx40+/+, Cx40+/–, and Cx40–/– genotypes are observed.


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  		Table 2. Cx43 and Cx40 Immunohistochemistry in Neonatal and Adult Murine Atria


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
As the major result, our experiments show that genetic deletion of Cx43 in synthetic strands of neonatal atrial myocytes produces a decrease in propagation velocity and a concomitant decrease in the Cx40 immunosignal and in the number of Cx40-containing gap junctions. Conversely, deletion of Cx40 in neonatal atrial myocytes leads to an increase in electrical propagation velocity and a concomitant increase in Cx43 in atrial gap junctions.

The purity of atrial strands in cell cultures was verified by immunostaining with antibodies against Cx40 and PAM-1. The latter protein is a specific atrial protein involved in shaping of the pro-ANP vesicles.31 The use of synthetic atrial strands may have advantages and disadvantages with respect to the analysis of electrical properties in vivo. Although our technique can provide accurate information regarding 2D cellular electrical activation, it cannot reveal potential differences in conduction properties in different atrial regions because the strands were made of a cell mixture of left and right atrial myocytes. On the other hand, optical measurement of electrical propagation in atria in vivo may be affected by difficulties in determination of local activation time because of interference between the relatively long optical action potential upstroke,37 the very short intraatrial conduction times and atrial malformations.38 Because we combined right and left atria, our results do not address the role of differences in content of Cx43 and Cx40 between the left and right atria, as described in human atrial tissue.39

Electrical impulse spread in whole murine Cx40–/– hearts has revealed a number of conduction abnormalities, including SA, intraatrial, AV nodal, and intraventricular (His–Purkinje) conduction defects.27,36,40–43 However, studies of conduction defects in intact atria have been controversial. Whereas studies in anesthetized mice have revealed a marked prolongation in the duration of the P wave,27,36,40,41 prolongation of the P wave has been reported to be minor or absent in conscious animals.42,43 Direct measurements with optical mapping revealed a highly abnormal pattern of electrical excitation and propagation in the right atria of adult Cx40–/– mice,43 attributable to circumscribed intraatrial conduction slowing in only a small fraction ({approx}10%) of atrial sites. This finding may be related to the high incidence of cardiac malformations found in these animals.38 Alternatively, conduction disturbances limited to specific sites in the right atrium could be related to phenotypic diversity among atrial myocytes. More than 40 years ago, James44 postulated the presence of a specific intraatrial conduction system with Purkinje-like morphology.44,45 Although this hypothesis was later discounted, it was evident that the atria contain a small number of so-called "clear cells" sharing morphological and electrical features with cells of the Purkinje system.45,46 Atrial cell groups with a Purkinje cell–like phenotype might produce localized propagation block in the case of Cx40–/– ablation, if the phenotype of such cells would include the same type of gap junction expression as that found in ventricular Purkinje fibers (presence of Cx40, absence of Cx43).

Our finding of increased propagation velocity in atrial strands from neonatal Cx40–/– myocytes stands in contrast to observations made in adult mice, as summarized above. An indication that this property may indeed become lost during growth and development is the fact that the pattern of Cx43 in gap junctions observed in neonatal animals (Figure 4) was not present in adult murine atria (Figure 6). The molecular mechanisms responsible for this developmental change are not yet evident. However, this process may be pertinent to atrial electrical remodeling in states of mechanical overload and hypertrophy characterized by emergence of a gene expression program that shares close similarities with gene expression in neonatal hearts. Importantly and in agreement with our experiments, 1 study of human atrial tissue from patients undergoing cardiac surgery showed a consistent relationship between the ratios of Cx43 or Cx40 to total connexin protein (Cx43/[Cx43+Cx40] and Cx40/[Cx43+Cx40]) in atrial gap junctions and local propagation velocity.21

The seemingly paradoxical observation of increased atrial propagation velocity with deletion of Cx40 described in the present report may have several causes. The findings of Valiunas and colleagues7,11 involving formation of mixed Cx40 and Cx43 gap junction channels in transfected HeLa cell pairs are in line with our experiments in Cx40–/– strands. Interestingly, coexpression of Cx40 and Cx43 in HeLa cell pairs has been reported to lead to a decrease in intercellular electrical conductance compared with that seen for pure homomeric/homotypic channels. In the situation of Cx40/Cx43 coexpression, heteromeric connexons were biochemically detectable, but functional gap junction channels seemed to consist of homotypic and/or heterotypic channels.7 In the study by Valiunas et al7 levels of Cx43 or Cx40 expression were similar, suggesting that upregulation of Cx43 with Cx40 deletion, as observed in our experiments, would not be a prerequisite for the explanation of the increase in propagation velocity. However, our observations correspond with the notion of a real increase of Cx43 in gap junctions with Cx40 deletion; we observed that (1) the same increase of the Cx43 immunosignal was observed with 2 different antibodies; (2) the number of Cx43-positive gap junctions increased; and (3) the fraction of Cx43 in gap junctions and the sarcolemma increased in presence of unchanged total Cx43. To what extent this shift contributed to the changes in propagation velocity cannot be answered quantitatively with our experiments. Moreover, changes in phosphorylation of atrial connexins with genetic ablation cannot be excluded as a further modulator of the electrical properties.

The mechanism of the decrease of microscopic electrical propagation velocity with Cx43 ablation and the concomitant decrease of the Cx40 immunosignal in gap junctions by 50% have not yet been explained. Nevertheless, our findings correspond closely to the aforementioned study of Kanagaratnam et al in humans,21 in which a decrease in atrial propagation velocity was reported at sites where the fraction Cx43/[Cx43+Cx40] was decreased.

Cx45, a connexin protein forming channels with a small unitary conductance, has been reported to be present in very small amounts in atrial tissue.2,3,39 In our study, as in the study by Vozzi et al,39 it was not possible to quantify the very low levels of Cx45 immunosignal. To what extent Cx45 may play a role as a modulator of atrial cell-to-cell communication, especially with Cx43 or Cx40 deletion, remains to be elucidated.

Comparison of measurements of propagation velocities in different atrial regions in a variety of species yielded fast average electrical propagation in the crista terminalis (1.1 m/sec) and Bachmann’s bundle (1.6 m/sec) and slower propagation in the atrial appendages (0.6 m/sec).35 This indicates that atrial conduction is faster at sites that connect major structures and ensure synchronized cardiac excitation, such as the crista terminalis (propagation form the SA to the AV node) and Bachmann’s bundle (propagation from the right to the left atrium). The findings of Kanagaratnam et al,21 Valiunas et al,7 and the present work suggest that the fractions of Cx40 and Cx43 in atrial cells could represent one of the physiologically important determinants of atrial propagation.


*    Acknowledgments
 
We thank G. Rigoli, K. de Peyer, A. Roatti, and E. M. Gribben for help with experiments.

Sources of Funding

Supported by the Swiss National Science Foundation (to A.G.K. and A.J.B), the Swiss Heart Foundation (to A.G.K. and A.J.B), Swiss University Conference, and grants HL58507 (to J.E.S.) and HL66350 (to K.A.Y.) from the NIH.

Disclosures

None.


*    Footnotes
 
Original received February 14, 2006; revision received September 18, 2006; accepted October 11, 2006.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Willecke K, Eiberger J, Degen J, Eckardt D, Romualdi A, Guldenagel M, Deutsch U, Sohl G. Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem. 2002; 383: 725–737.[CrossRef][Medline] [Order article via Infotrieve]
  2. Saffitz JE, Davis LM, Darrow BJ, Kanter HL, Laing JG, Beyer EC. The molecular basis of anisotropy: role of gap junctions. J Cardiovasc Electrophysiol. 1995; 6: 498–510.[Medline] [Order article via Infotrieve]
  3. Davis LM, Rodefeld ME, Green K, Beyer EC, Saffitz JE. Gap junction protein phenotypes of the human heart and conduction system. J Cardiovasc Electrophysiol. 1995; 6: 813–822.[Medline] [Order article via Infotrieve]
  4. Johnson CM, Kanter EM, Green KG, Laing JG, Betsuyaku T, Beyer EC, Steinberg TH, Saffitz JE, Yamada KA. Redistribution of connexin45 in gap junctions of connexin43-deficient hearts. Cardiovasc Res. 2002; 53: 921–935.[Abstract/Free Full Text]
  5. Beauchamp P, Choby C, Desplantez T, de Peyer K, Green K, Yamada KA, Weingart R, Saffitz JE, Kleber AG. Electrical propagation in synthetic ventricular myocyte strands from germline connexin43 knockout mice. Circ Res. 2004; 95: 170–178.[Abstract/Free Full Text]
  6. Harris AL. Emerging issues of connexin channels: biophysics fills the gap. Q Rev Biophys. 2001; 34: 325–472.[Medline] [Order article via Infotrieve]
  7. Valiunas V, Gemel J, Brink PR, Beyer EC. Gap junction channels formed by coexpressed connexin40 and connexin43. Am J Physiol Heart Circ Physiol. 2001; 281: H1675–H1689.[Abstract/Free Full Text]
  8. Polontchouk LO, Valiunas V, Haefliger JA, Eppenberger HM, Weingart R. Expression and regulation of connexins in cultured ventricular myocytes isolated from adult rat hearts. Pflugers Arch. 2002; 443: 676–689.[CrossRef][Medline] [Order article via Infotrieve]
  9. Desplantez T, Halliday D, Dupont E, Weingart R. Cardiac connexins Cx43 and Cx45: formation of diverse gap junction channels with diverse electrical properties. Pflugers Arch. 2004; 448: 363–375.[Medline] [Order article via Infotrieve]
  10. Elenes S, Martinez AD, Delmar M, Beyer EC, Moreno AP. Heterotypic docking of Cx43 and Cx45 connexons blocks fast voltage gating of Cx43. Biophys J. 2001; 81: 1406–1418.[Abstract/Free Full Text]
  11. Valiunas V, Weingart R, Brink PR. Formation of heterotypic gap junction channels by connexins 40 and 43. Circ Res. 2000; 86: e42–e49.[Medline] [Order article via Infotrieve]
  12. Peters NS, Green CR, Poole-Wilson PA, Severs NJ. Reduced content of connexin43 gap junctions in ventricular myocardium from hypertrophied and ischemic human hearts. Circulation. 1993; 88: 864–875.[Abstract/Free Full Text]
  13. Kostin S, Klein G, Szalay Z, Hein S, Bauer EP, Schaper J. Structural correlate of atrial fibrillation in human patients. Cardiovasc Res. 2002; 54: 361–379.[Abstract/Free Full Text]
  14. van der Velden HM, Ausma J, Rook MB, Hellemons AJ, van Veen TA, Allessie MA, Jongsma HJ. Gap junctional remodeling in relation to stabilization of atrial fibrillation in the goat. Cardiovasc Res. 2000; 46: 476–486.[Abstract/Free Full Text]
  15. Elvan A, Huang XD, Pressler ML, Zipes DP. Radiofrequency catheter ablation of the atria eliminates pacing-induced sustained atrial fibrillation and reduces connexin 43 in dogs. Circulation. 1997; 96: 1675–1685.[Abstract/Free Full Text]
  16. Dupont E, Ko Y, Rothery S, Coppen SR, Baghai M, Haw M, Severs NJ. The gap-junctional protein connexin40 is elevated in patients susceptible to postoperative atrial fibrillation. Circulation. 2001; 103: 842–849.[Abstract/Free Full Text]
  17. Nao T, Ohkusa T, Hisamatsu Y, Inoue N, Matsumoto T, Yamada J, Shimizu A, Yoshiga Y, Yamagata T, Kobayashi S, Yano M, Hamano K, Matsuzaki M. Comparison of expression of connexin in right atrial myocardium in patients with chronic atrial fibrillation versus those in sinus rhythm. Am J Cardiol. 2003; 91: 678–683.[CrossRef][Medline] [Order article via Infotrieve]
  18. Polontchouk L, Haefliger JA, Ebelt B, Schaefer T, Stuhlmann D, Mehlhorn U, Kuhn-Regnier F, De Vivie ER, Dhein S. Effects of chronic atrial fibrillation on gap junction distribution in human and rat atria. J Am Coll Cardiol. 2001; 38: 883–891.[Abstract/Free Full Text]
  19. Ausma J, van der Velden HM, Lenders MH, van Ankeren EP, Jongsma HJ, Ramaekers FC, Borgers M, Allessie MA. Reverse structural and gap-junctional remodeling after prolonged atrial fibrillation in the goat. Circulation. 2003; 107: 2051–2058.[Abstract/Free Full Text]
  20. van der Velden HM, van Kempen MJ, Wijffels MC, van Zijverden M, Groenewegen WA, Allessie MA, Jongsma HJ. Altered pattern of connexin40 distribution in persistent atrial fibrillation in the goat. J Cardiovasc Electrophysiol. 1998; 9: 596–607.[Medline] [Order article via Infotrieve]
  21. Kanagaratnam P, Rothery S, Patel P, Severs NJ, Peters NS. Relative expression of immunolocalized connexins 40 and 43 correlates with human atrial conduction properties. J Am Coll Cardiol. 2002; 39: 116–123.[Abstract/Free Full Text]
  22. Eloff BC, Lerner DL, Yamada KA, Schuessler RB, Saffitz JE, Rosenbaum DS. High resolution optical mapping reveals conduction slowing in connexin43 deficient mice. Cardiovasc Res. 2001; 51: 681–690.[Abstract/Free Full Text]
  23. Guerrero PA, Schuessler RB, Davis LM, Beyer EC, Johnson CM, Yamada KA, Saffitz JE. Slow ventricular conduction in mice heterozygous for a connexin43 null mutation. J Clin Invest. 1997; 99: 1991–1998.[Medline] [Order article via Infotrieve]
  24. Morley GE, Vaidya D, Samie FH, Lo C, Delmar M, Jalife J. Characterization of conduction in the ventricles of normal and heterozygous Cx43 knockout mice using optical mapping. J Cardiovasc Electrophysiol. 1999; 10: 1361–1375.[Medline] [Order article via Infotrieve]
  25. Thomas SA, Schuessler RB, Berul CI, Beardslee MA, Beyer EC, Mendelsohn ME, Saffitz JE. Disparate effects of deficient expression of connexin43 on atrial and ventricular conduction: evidence for chamber-specific molecular determinants of conduction. Circulation. 1998; 97: 686–691.[Abstract/Free Full Text]
  26. Vaidya D, Tamaddon HS, Lo CW, Taffet SM, Delmar M, Morley GE, Jalife J. Null mutation of connexin43 causes slow propagation of ventricular activation in the late stages of mouse embryonic development. Circ Res. 2001; 88: 1196–1202.[Abstract/Free Full Text]
  27. Verheule S, van Batenburg CA, Coenjaerts FE, Kirchhoff S, Willecke K, Jongsma HJ. Cardiac conduction abnormalities in mice lacking the gap junction protein connexin40. J Cardiovasc Electrophysiol. 1999; 10: 1380–1389.[Medline] [Order article via Infotrieve]
  28. Thomas SP, Bircher-Lehmann L, Thomas SA, Zhuang J, Saffitz JE, Kleber AG. Synthetic strands of neonatal mouse cardiac myocytes: structural and electrophysiological properties. Circ Res. 2000; 87: 467–473.[Abstract/Free Full Text]
  29. Thomas SP, Kucera JP, Bircher-Lehmann L, Rudy Y, Saffitz JE, Kleber AG. Impulse propagation in synthetic strands of neonatal cardiac myocytes with genetically reduced levels of connexin43. Circ Res. 2003; 92: 1209–1216.[Abstract/Free Full Text]
  30. Rohr S, Scholly DM, Kleber AG. Patterned growth of neonatal rat heart cells in culture. Morphological and electrophysiological characterization. Circ Res. 1991; 68: 114–130.[Abstract/Free Full Text]
  31. Labrador V, Brun C, Konig S, Roatti A, Baertschi AJ. Peptidyl-glycine alpha-amidating monooxygenase targeting and shaping of atrial secretory vesicles: inhibition by mutated N-terminal ProANP and PBA. Circ Res. 2004; 95: e98–e109.[Abstract/Free Full Text]
  32. Saffitz JE, Green KG, Kraft WJ, Schechtman KB, Yamada KA. Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium. Am J Physiol Heart Circ Physiol. 2000; 278: H1662–H1670.[Abstract/Free Full Text]
  33. VanSlyke J, Musil L. Biochemical analysis of connexon assembly. In: Bruzzone R, Giaume C, eds. Connexin Methods and Protocols. Totowa, NJ: Humana; 2001: 117–134.
  34. Fast VG, Darrow BJ, Saffitz JE, Kleber AG. Anisotropic activation spread in heart cell monolayers assessed by high-resolution optical mapping. Role of tissue discontinuities. Circ Res. 1996; 79: 115–127.[Abstract/Free Full Text]
  35. Kléber AG, Janse MJ, Fast VG. Normal and abnomal conduction in the heart. In: Page E, Fozzard H, Solaro R, eds. The Handbook of Physiology. Volume 1: The Heart. New York: Oxford University Press; 2002: 455–530.
  36. Hagendorff A, Schumacher B, Kirchhoff S, Luderitz B, Willecke K. Conduction disturbances and increased atrial vulnerability in Connexin40-deficient mice analyzed by transesophageal stimulation. Circulation. 1999; 99: 1508–1515.[Abstract/Free Full Text]
  37. Hyatt CJ, Mironov SF, Vetter FJ, Zemlin CW, Pertsov AM. Optical action potential upstroke morphology reveals near-surface transmural propagation direction. Circ Res. 2005; 97: 277–284.[Abstract/Free Full Text]
  38. Gu H, Smith FC, Taffet SM, Delmar M. High incidence of cardiac malformations in connexin40-deficient mice. Circ Res. 2003; 93: 201–206.[Abstract/Free Full Text]
  39. Vozzi C, Dupont E, Coppen SR, Yeh HI, Severs NJ. Chamber-related differences in connexin expression in the human heart. J Mol Cell Cardiol. 1999; 31: 991–1003.[CrossRef][Medline] [Order article via Infotrieve]
  40. Kirchhoff S, Kim JS, Hagendorff A, Thonnissen E, Kruger O, Lamers WH, Willecke K. Abnormal cardiac conduction and morphogenesis in connexin40 and connexin43 double-deficient mice. Circ Res. 2000; 87: 399–405.[Abstract/Free Full Text]
  41. Kirchhoff S, Nelles E, Hagendorff A, Kruger O, Traub O, Willecke K. Reduced cardiac conduction velocity and predisposition to arrhythmias in connexin40-deficient mice. Curr Biol. 1998; 8: 299–302.[CrossRef][Medline] [Order article via Infotrieve]
  42. Tamaddon HS, Vaidya D, Simon AM, Paul DL, Jalife J, Morley GE. High-resolution optical mapping of the right bundle branch in connexin40 knockout mice reveals slow conduction in the specialized conduction system. Circ Res. 2000; 87: 929–936.[Abstract/Free Full Text]
  43. Bagwe S, Berenfeld O, Vaidya D, Morley GE, Jalife J. Altered right atrial excitation and propagation in connexin40 knockout mice. Circulation. 2005; 112: 2245–2253.[Abstract/Free Full Text]
  44. James TN. The connecting pathways between the sinus node and A-V node and between the right and the left atrium in the human heart. Am Heart J. 1963; 66: 498–508.[CrossRef][Medline] [Order article via Infotrieve]
  45. Janse M, Anderson R. Specialized internodal atrial pathways— fact or fiction? Eur J Cardiol. 1974; 2: 117–136.
  46. Hogan PM, Davis LD. Electrophysiological characteristics of canine atrial plateau fibers. Circ Res. 1971; 28: 62–73.[Abstract/Free Full Text]

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