Skip Navigation

Cardiovascular Research 2000 45(2):379-387; doi:10.1016/S0008-6363(99)00363-6
© 2000 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Litchenberg, W. H
Right arrow Articles by Gourdie, R. G
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Litchenberg, W. H
Right arrow Articles by Gourdie, R. G
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

The rate and anisotropy of impulse propagation in the postnatal terminal crest are correlated with remodeling of Cx43 gap junction pattern

Wanda H Litchenberg1, Lisa W Norman1, Adria K Holwell, Kylie L Martin, Kenneth W Hewett and Robert G Gourdie*

Department of Cell Biology, MUSC, 173 Ashley Avenue, Suite 601, Charleston, SC 29425, USA

* Corresponding author. Tel.: +1-843-792-8181; fax: +1-843-792-0664 gourdier{at}musc.edu

Received 22 July 1999; accepted 8 October 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Background: Disruptions to intermyocyte coupling have been implicated in arrhythmogenesis and development of conduction disturbances. At present, understanding of the relationship between the microscopic organization of intercellular coupling and the macroscopic spread of impulse in the normal and diseased heart is largely confined to theoretical analyses. Methods and results: The abundance and arrangement of gap junctions, as well as conduction properties, were assessed in terminal crest preparations isolated from the atria of neonate, weanling, and adult rabbits. We report that the connexin composition of terminal crest was uncomplicated, with Cx43 being the most prominent isoform detectable by Western blotting and immunostaining. Terminal crest myocytes showed little change in total Cx43-gap junction per cell during postnatal growth as assessed by stereology. However, marked non-uniformities emerged in the sarcolemmal distribution of Cx43-gap junctions. Cx43-gap junction area at myocyte termini increased 3.5-fold from birth to adulthood. Correlated with this change in Cx43, impulse propagation velocity parallel to the myofiber axis, as assessed by multi-site optical mapping using voltage-sensitive dye (di-4-ANEPPS), increased 2.4-fold. Conversely, the amount of Cx43-gap junctions on myocyte sides, and the conduction velocity transverse to the myofiber axis, remained relatively invariant during maturation. Hence, the increasing electrical anisotropy of maturing terminal crest was wholly accounted for by increases in conductance velocity along the bundle. This increase in longitudinal conduction velocity was correlated with changes in the sarcolemmal pattern, but not the overall density, of Cx43-gap junctions. Conclusions: This study provides the first correlative structure/function analysis of the relationship between the macroscopic conduction of impulse and the microscopic cellular organization of gap junctions in a differentiating cardiac bundle. Confirmation is provided for theoretical predictions which emphasize the importance of the cell-to-cell geometry of coupling in determining the spread and pattern of myocardial activation.

KEYWORDS Gap junctions; Atrial function


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Proper electromechanical function and rhythmic contraction of the heart depend in large part on the specialized conduction properties of distinct areas of myocardium. While activity of sarcolemmal voltage-dependent and time-dependent ion channels account for electrophysiological characteristics at the level of individual cardiomyocytes, it is becoming increasingly apparent that passive electrical properties, and tissue architecture, are as important in determining the overall electrophysiologic profile of the heart [1,2]. Intercellular coupling between myocytes is provided by gap junctions which allow the regulated flow of ions, metabolites, and electrical current from cell-to-cell. As such, gap junctional coupling provides the physical basis for action potential (AP) propagation throughout the developing and mature myocardium [3–5]. Spatial patterns of gap junction distribution, and region- or chamber-specific differences in gap junctional connexin expression, may account for the different conductile properties observed in specialized myocardium and atrial or ventricular working myocardium [6–8]. Consistent with a role in normal function, several studies have correlated alterations in gap junction distribution with cardiac pathophysiology and potential arrhythmogenic foci in ischemic heart disease and hypertrophic cardiomyopathy [9–13].

Although morphogenesis of the mammalian heart is near complete at birth, the physiology and differentiation state of myocytes remain immature [14]. During postnatal development, myocytes undergo profound changes in size, morphology and molecular phenotype. A striking reorganization of electrical connectivity also occurs in the ventricle during the period following birth [15,16]. In the early postnatal heart, gap junctions are distributed uniformly, being dispersed more or less randomly across the sarcolemma. During postnatal growth, this distribution changes with gap junctions becoming progressively more concentrated at intercalated discs. This progressive alteration in the geometry of connectivity between myocytes has been suggested as one of the main factors underlying changes in the rate and anisotropy of conduction during postnatal development [17,18]. Specifically, the preferential concentrations of electrical connections at myocyte termini have been speculated to account for the anisotropic or directional nature of impulse propagation in mature myocardial tissues, in which conduction velocity along the myocyte long axis exceeds that transverse to this axis. Uniform discontinuous anisotropic conduction of AP is an important component of stable electromechanical function. However, a decrease in transverse propagation velocity (and hence increased anisotropy), due to reductions in gap junctions at lateral contacts between aging myocytes, has been proposed to be a substrate for micro-reentry and arrhythmia [2]. It is therefore an important task to investigate the developmental mechanisms underpinning the generation of cellular non-uniformities in gap junction distribution, as such mechanisms may have a direct bearing on the genesis of pathological states in the more mature heart.

While a link between changing AP conduction properties and the distribution of gap junctions over the course of heart development has been implied, no direct study of the association has been done. The goal of this work was to carry out such a correlative structure/function studies using the rabbit terminal crest (tc). This uniformly anisotropic fascicle is a readily identifiable atrial bundle containing well-aligned strands of unbranched myocytes [19]. Quantitative assessment of conduction velocity is difficult in structurally more complex myocardial tissues, e.g. the developing ventricular myocardium, due to non-uniformities in fiber orientation, branching and the complexity of waveforms generated by such tissues. The utility of the tc as a model was readily apparent in data which indicated that Cx43 was the major gap junctional connexin found in this tissue. Our hypothesis that correlated increases in gap junction polarization and AP conduction anisotropy occur in the terminal crest over postnatal growth is supported by the data presented herein.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
All animals were treated and cared for in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 85-23, revised 1996, National Research Council, Washington, DC).

2.1 Isolation of living terminal crest
Adult (4–6 months) and weanling (6–9 weeks) New Zealand white rabbits received intravenous heparin (1000 U/kg) and sodium pentobarbital ear vein anesthesia (60 mg/kg). Neonates (3–12 days old) received the same via intraperitoneal injection. The heart was then removed and placed in cold HEPES-modified Krebs. Following removal of the sinoatrial node, its associated tissue, and the Eustachian valve, the terminal crest (tc) was dissected free from the right atrium at the atrioventricular (AV) ring.

2.2 Optical mapping of activation with fluorescent voltage sensitive dye
Isolated tc samples were placed endocardial-side down and superfused at 37°C with oxygenated HEPES-modified Krebs at 12 ml/min. A bipolar electrogram was recorded using platinum electrodes following pulse stimulation at 1 ms durations, voltage intensity of 2x threshold, and stimulus intervals of 300–400 ms. Prior to recording, isolated tc were superfused with 10.5 µM di-4-ANEPPS (Molecular Probes) at 37°C in HEPES-modified Krebs for 15 min. The preparation was then excited with monochromatic green light from a 250W Tungsten-Halogen source with a 500 nm filter. Emitted light was focused through a Nikon camera lens (F1.8) onto a 1.4x1.4 mm silicon photodiode array (144 elements, 375 µm center-to-center spacing Centronics, ND144-5T). Photodiode currents were conditioned with current-to-voltage amplifiers (50, 200 or 1000x) having AC coupling (0.2 Hz) and low pass filtering (1000 Hz). Optical signals were filtered by a second order, low pass analog filter with –3 db cutoff points of 22Hz, then digitized at 1000 samples per second per channel. All photodiode outputs (and the bipolar electrogram) were digitized to PC at 1 kHz by a 12-bit, AT-MIO-16E-2 A/D board (National Instruments). After digitization, cubic spline interpolation was used to produce an effective temporal resolution of 0.1 ms; first and second derivatives of the waveform were then estimated. Recordings were taken with the stimulus electrode placed at different locations while the location of the recording electrode remained fixed. The activation time for the area of tc monitored by each photodiode corresponded to the half amplitude on the upstroke of the waveform. Average conduction velocities, parallel and transverse to the long axis of each tc sample, and ratios of conduction anisotropy, were calculated from multiple maps of activation derived from each specimen.

2.3 Western blotting and immunoconfocal analysis of terminal crest samples and hela cell connexin transfectants
Western blotting of tc membrane preparations with antibodies against Cx43 (Chemicon), Cx40 (GP318) and Cx45 (GP42) were carried out as described previously [20]. Frozen sections of optically mapped and control tc samples were immunolabeled by anti-connexin antibodies using methods described in earlier publications [8,20,21]. Sections were also labeled with an anti-dystrophin antibody (1:20, Novocastra Laboratories) [22]. Samples were examined on a BioRad MRC-1024 SCLM using standard filter and collection modalities. Controls confirming antibody specificity included (i) immunolabeling and immunoblotting of Hela cell transfectants expressing either Cx43, Cx40, or Cx45 independently [23] (ii) immunolabeling of Hela cells and tc excluding incubation with primary antibody and (ii) immunolabeling with primary antibody in the presence of excess (25–100 µg/ml) immunogen.

2.4 Quantitative 3D stereology and statistics
Pairs of serial, or near serial, histological sections from each of three animals sampled per age group were labeled in sequence using anti-Cx43 or anti-dystrophin antibodies. For sampling, the sections were divided into quartiles of equal length. Two optical sections (40x1.4 NA objective at zoom=2) were taken, at random, within each quartile in the anti-Cx43 labeled section and in its anti-dystrophin-labeled sister section. Myocardial volume density (Vv-myocardium), sarcolemmal surface density (Sv-sarcolemma) and myocyte numerical density (Nm) per unit tissue volume were estimated from each image taken from dystrophin-labeled sections using the isotropic curvilinear test system of Merz (L100) according to the methods of Weibel [24].

Formula
where Vv=myocyte volume density, Pa=sum of test points on myocytes, and PT=sum of test points on cardiac tissue;

Formula
where Sv=myocyte sarcolemma surface density per unit volume (µm2/µm3), Ia=sum of intersections with myocyte boundaries, d=test line length;

Formula
where Pc=test points on cardiac tissue, k2=1, Na=number of discrete profiles, β=size distribution coefficient, K=shape coefficient. K and β were graphically derived according to Weibel [24] based on dystrophin-labeled profiles of longitudinally and transversely sectioned myocytes. β was derived from myocyte diameter coefficient of variation measured from transversely sectioned myocytes. To derive K, profiles of longitudinally sectioned myocytes were assessed. Based on the above, estimates of the volume (Vv -myocyte) and sarcolemmal surface area of individual myocytes (Sv-myocyte) were obtained. The overall surface density of Cx43 area per unit tissue volume (Sv-gj-tissue-Total) and Cx43 area per unit tissue volume at myocyte sides (Sv-gj-tissue-Sides) and ends (Sv-gj-tissue-Ends) were estimated from Cx43 immunolabeled sections using identical methodology to that described above for Sv. Cx43 indices were normalized by average Nm within tissue quartiles to give area of Cx43 immunolabeling per myocyte (Sv -gj-myocyte-Total), Cx43 at myocyte ends per myocyte (Sv-gj-myocyte-End) and Cx43 at sides per myocyte (Sv-gj-myocyte-Sides).

2.5 Data analysis
Data were processed with SAS 6.11 (SAS Institute). Normality was assessed by the univariate normality procedure. A log transformation was utilized if non-normality was found (P<0.05). Bartlett's test for homogeneity of variance was performed; the confidence interval for the Chi-square (P<0.05). The three age groups were compared by a one-way analysis of variance. For a significant difference, an F of P<0.05 was required. Pair-wise mean comparisons (given variances=S.E.M.) were performed using Student's t-test and corrected with Bonferroni bounds (P<0.05).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Cx43 is the main gap junctional protein expressed by the rabbit terminal crest (tc)
To determine which connexins were expressed in rabbit tc over postnatal development, immunohistochemistry and Western blotting were undertaken on tc isolated from neonates, weanlings and adults (Fig. 1). The anti-Cx40, anti-Cx43 and anti-Cx45 antibodies used were characterized as giving strong, specific signal by Western blotting and immunohistochemistry on Hela cells transfected with cDNAs overexpressing either Cx45 (Fig. 1a,b), Cx40 (Fig. 1e,f) or Cx43 (Fig. 1i,j). While Cx45 was detected in a margin of tissue corresponding to presumed SA node in some preparations, consistent with other reports [20], we found little evidence for Cx45 immunolabeling within the tc proper (Fig. 1c,d). Moderate levels of Cx40 immunolabeling were observed in coronary arterial endothelia (Fig. 1h). Punctate Cx40 immunolabeling was detected only at low levels in tc myocardia (Fig. 1g,h). At all stages, Cx43 was the most prominent connexin identifiable by immunohistochemistry (Figs. 1 and 2Go). In adults samples, Cx43 localized prominently to myocyte termini at intercalated disks (Figs. 1k,l and 2Go). Western blotting of neonatal and adult tc indicated bands consistent with Cx43 (Fig. 1i). Fig. 1e shows a blot of Cx40 in neonatal and adult tc and Hela cells overexpressing Cx40. At this exposure, signal is evident in the Hela-Cx40 cells, but no Cx40 is detected in tc at either stage. However, consistent with immunolabeling, prolonged exposures revealed a faint band corresponding to Cx40 in adult preparations (data not shown). In agreement with immunohistochemical data reported by Verheule et al. [25], Cx40 and Cx45 were detected in preparations from adult and neonatal atria (i.e. non-terminal crest atria). However, Cx45 was never detected, even after prolonged exposures in neonatal or adult tc (Fig. 1a).


Figure 1
View larger version (107K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 (a, e, i) Western blots of terminal crest (tc) membrane protein preparations from neonatal (N, 10 µg) and adult rabbit (A, 10 µg) tc were probed with antibodies to (a) Cx45, (e) Cx40, or (i) Cx43. Controls included transfected HeLa cell lines expressing Cx45 (H45), Cx40 (H40) or Cx43 (H43). (b, f, j) Confocal images of Hela cells transfected with (b) Cx45, (f) Cx40 and (j) Cx43 cDNAs and immunolabeled with either anti-Cx45, anti-Cx40 or anti-Cx43 antibodies. (c, g, k) Low magnification surveys of adult tc immunolabeled with the (c) anti-Cx45, (g) anti-Cx40 and (k) anti-Cx43 antibodies. (d, h, l) High magnifications of adult tc (with coronary artery) double-labeled using rhodamine-phalloidin (red) and (d) anti-Cx45, (h) anti-Cx40 and (l) anti-Cx43 antibodies (green). Scales=5 µm.

 

Figure 2
View larger version (45K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Isochronal maps of action potential (AP) propagation from (a) neonate, (c) weanling and (e) adult tc derived from multi-site optical recordings of AP. The stimulus electrode is indicated by an asterisk (*). Isochrones=1ms. Optically-mapped tc from (b) neonate, (d) weanling and (f) adult immunolabeled for Cx43. Intercalated discs are arrowed on b, d and f. Scale=5 µm.

 
3.2 Increases in conduction anisotropy with age are correlated with levels of Cx43 localized to myocyte ends
Action potential (AP) conduction velocities were calculated from optical recordings of activation obtained from preparations of neonate, weanling, and adult rabbit tc. Five representative AP recordings from photodiodes located near the center of the array are shown in Fig. 3. To validate the optical mapping, conduction velocities were measured on adult tc using conventional extracellular electrodes. Subsequently, the same samples were assessed using optical mapping. Extracellular recordings and conduction velocity measurements compared favorably with optical recordings (data not shown).


Figure 3
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 ms of action potential (AP) recordings from five photodiodes at the center of the photodiode array. The three vertical lines on the recordings (from left to right) indicate the beginning of the upstroke, mid-point, and peak.

 
Fig. 2 provides a summary of representative isochronal activation maps and Cx43 gap junction distribution within identical samples taken from each of the three age groups. The neonatal isochronal map shows uniform, but not highly anisotropic conduction of AP (Fig. 2a). Correspondingly, Cx43 immunolabeling was relatively uniformly distributed around myocytes (Fig. 2b). The weanling map indicated that conduction velocity along the myofiber long axis had increased relative to the transverse conduction velocity across the tissue strip (Fig. 2c). This change paralleled a relative increase in Cx43 immunolabeling at myocyte ends (Fig. 2d). In the adult terminal crest, longitudinal conduction velocity was more pronounced than that observed in the younger stages. As can be seen from the activation map at this stage, conduction is uniform and highly anisotropic (Fig. 2e). In the adult preparation, Cx43-positive puncta were prominently localized at myocyte ends with an apparent reduction of gap junctions located at lateral domains of sarcolemma (Fig. 2f).

3.3 Quantitation of the relationship between activation and Cx43 distribution
Activation times along the axis of myofiber orientation or perpendicular to this axis in the tc were used to calculate longitudinal and transverse conduction velocities (Fig. 4a). Longitudinal conduction velocity increased 2.3-fold from 22.8±1.9 cm/s in the neonate to 51.7±5.0 cm/s in the adult. Longitudinal velocity was significantly different (P<0.05) between adult and neonate samples and between adult and weanling samples. Transverse conduction velocity, 13.3±1.2 cm/s in the neonate, remained near constant with age, not significantly varying (P>0.05) between each of the three stages studied. In Fig. 4b, the data is replotted, as the ratio of longitudinal to transverse velocity, i.e., as conduction anisotropy. Conduction anisotropy increased 2.4-fold from 1.7 in the neonate to 4.1 in the adult. The conduction anisotropy ratios were significantly different between all groups: adult vs. weanling, adult vs. neonate, and weanling vs. adult. From this data, we concluded that increases in anisotropic conduction in the differentiating tc were explained entirely by increases in the longitudinal conduction velocity.


Figure 4
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 (a) Longitudinal (VL) and transverse (VT) conduction velocities (cm/ms; mean±S.E.M).) from neonatal, weanling, and adult preparations. (b) Conduction anisotropy (VL/VT) (mean±S.E.M) at each stage. *, Significant differences (P<0.05) in VL between neonate and adult groups (4a) and in VL/VT between neonate and weanling, neonate and adult, and weanling and adult groups (4b). (c) Gap junctional area ratios per myocyte (Ends/Sides (squares), Ends/Total (diamonds), and Sides/Total (circles)) plotted against postnatal age.

 
To examine the changes in myocyte dimensions and gap junctional distribution that accompany the increase in longitudinal conduction velocity, a stereological analysis was undertaken on the same tissues used for activation mapping (Table 1 and Fig. 4). Consistent with the well-established hypertrophic increases in myocyte size [14] myocyte surface area and volume increased 4.3-fold and 8.1-fold over postnatal maturation respectively, with highly significant differences (P<0.01) between each stage. The surface density of Cx43 area per unit myocardial tissue volume (Sv-gj-tissue-Total) fell dramatically (P<0.01) over postnatal development. The surface density of gap junctional area at myocyte termini (Sv-gj-tissue-Ends) and along the lateral surfaces (Sv-gj-tissue-Sides) per unit tissue volume indicated similar declines (Table 1). A significant 10-fold decrease was observed in myocyte numerical density (myocyte number/µm3 tissue–Nm) over postnatal development: neonate=0.299±0.021, weanling=0.091±0.012, and adult 0.029±0.003 (mean±S.E.M). Normalization of the gap junctional surface density data to myocyte numerical density indicated that, although myocyte surface area increased 4.3-fold between neonate and adult stages, total sarcolemma-localized Cx43 per myocyte (Sv-gj-myocyte-Total) only showed a marginally increasing trend with developmental age (Table 1). The main factor underlying variation in Cx43 pattern was revealed when the analysis of gap junctional area per myocyte was partitioned into lateral and terminal domains of sarcolemma. While there was no significant change in Cx43 along myocyte sides (Sv-gj-myocyte-Sides) between neonate and adult, there was a highly significant increase in the area of Cx43 immunolabeling polarized at the ends of myocytes (Sv-gj-myocyte-Ends). Gap junctional membrane at this domain increased 3.5-fold between neonate and adult. To investigate potential relationships between domain-specific and total Cx43 gap junctional area per myocyte values, ratios of Cx43 at ends to sides (ends/sides), ends to total (ends/total), and sides to total (sides/total) were calculated (Fig. 4c). As anticipated, the Cx43 ratios of ends/total and sides/total were inversely related with the former increasing 2.4-fold and the latter decreasing 2.0-fold over postnatal development. The ratio of Cx43 at myocyte ends to sides (ends/sides) increased steadily with increasing postnatal age, a 5-fold change between neonate and adult stages. As can be ascertained from comparing panels a, b and c of Fig. 4, variation in Cx43 ends/sides with postnatal age closely matched variation in conduction anisotropy ratio (correlation coefficient; r=0.99).


View this table:
[in this window]
[in a new window]

  		Table 1 Surface density (Sv) of Cx43 gap junction area per unit tissue volume (Sv-gj-tissue) and individual myocytes (Sv-gj-myocyte) within the whole sarcolemma (Total) and lateral (Sides) and terminal (Ends) sarcolemmal domainsa

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
It has been proposed that alterations in the sarcolemmal distribution of gap junctions may account for increases in the rate and anisotropy of impulse propagation during postnatal growth [16–18]. To date, there has been no experimental confirmation of this hypothesis. This represents a significant omission since it is now thought that breakdown or reversal of the differentiation process determining normal patterns of myocyte connectivity may be key to the formation of arrhythmogenic substrates in various cardiac pathologies [2,4,5,24–27]. In this study, the rabbit terminal crest (tc) was used to examine the relationship between gap junctional distribution and abundance and changes in electrical activation patterns. We find that while the total amount of Cx43 gap junction per myocyte is relatively invariant over postnatal growth, there is a significant reallocation of gap junction within the sarcolemmal pool to myocyte ends. This latter change shows a striking correlation with both increases in the velocity and directionality (i.e. anisotropy) of action potential (AP) propagation.

This study is the first to quantitatively examine developmental variation in Cx43 content at the level of the individual myocyte. Stereology indicates that Cx43 per myocyte rises slightly over postnatal growth. This latter observation was unexpected, as previous studies had reported that gap junctions and Cx43 mRNA and protein levels rose and fell dramatically over postnatal growth [28–30]. The difference between these earlier biochemical and ultrastructural studies, and the current study, is probably explained by the fact that our estimates of Cx43 are adjusted for variation in myocyte numerical density (Nm). Indeed, tissue surface densities measured here (i.e. gap junctional indices not normalized by Nm) correspond well to those reported previously for the surface density of gap junctions per unit myocardial volume in the developing rabbit ventricle [28]. A further factor may be that Cx43 quantitation in the present study was confined to gap junctions within the sarcolemma. Membrane levels of Cx43 may show only limited correlation with variation in cellular pools of Cx43. This proposition is supported by our studies of Cx43 expression in a mesodermal cell line [31], where it has been found that membrane localized protein, but not mRNA, varies in response to WNT-expression, suggesting a role for post-translational mechanisms in targeting Cx43 to the membrane.

Anisotropic conduction has been demonstrated in a variety of cardiac tissues [17,32–34]. This study provides the first direct analysis of the relationship between changes in coupling and myocardial activation over a developmental time course in which correlative structural and functional assessments were carried out on identical tissue preparations. The conduction velocities measured correspond favorably with those previously found for atrial tissue in mammalian postnates [6,17]. More pertinently, our optical measurements of longitudinal conduction velocity are in excellent agreement with those recently reported by Efimov and colleagues for tc in intact preparations from rabbit [35]. The present work also concurs with previous observations that transverse conduction velocity in atrial bundles (e.g. tc, Bachmann's bundle) undergoes little change relative to longitudinal velocity between birth and maturity [34]. In their classical paper, Spach and Dolber [17] observed an increased conduction anisotropy in atrial bundles from adults as compared to children. This change was suggested to be associated with a reduction in side-to-side gap junctions. In agreement with this, we and others demonstrated that gap junction on myocyte sides, measured as a fraction of that at myocyte ends and sides, decreased between birth and maturity [5]. Interestingly, our stereological data reveals an unforeseen aspect of this pattern. Although, decreasing as a fraction of the total (see Fig. 4c), the absolute amounts of gap junction positioned at myocyte sides show no significant variation between neonatal and adult stages (see Table 1). Put another way, it is not decreases in the size of the pool of gap junctions at lateral sarcolemma per se that correlates with the increasing anisotropy of the atrial bundle. Rather, these changes appear more related with progressive accretion of available Cx43 gap junction at the termini of differentiating myocytes. One interesting qualification is that 4–6 month old rabbits (i.e. relatively young adults) were the oldest studied. Given the suspected role of slowed transverse conduction in micro-reentry in elderly humans [17], it would be clinically relevant to ask whether conduction velocity across the bundle and gap junction levels in lateral sarcolemmal domains remain constant in older rabbits.

There are additional factors that may contribute to the developmental differences in myocardial conduction patterns. Structural considerations include decreased axial resistance as a function of increased cell cross-sectional area and increased fascicle formation [34]. Conduction velocity should, according to classical cable theory, vary in proportion to myocyte cross-sectional area if internal resistivity remains constant. Myocyte diameter increased from 5 µm to 15 µm between the neonatal and adult group, with cell length/width ratios remaining near constant over this same period. Based on this, myocyte diameter accounts for ~ 17 cm/sec of the 29 cm/sec increase in longitudinal conduction velocity between the neonate and the adult. This leaves just over 40% to be explained by other factors, including the lowered intercellular resistance that presumably results from a 3.5-fold increase in the density of end-to-end gap junctions. It also noted that while tc myocyte diameter increases, transverse conduction velocities and lateral gap junction levels remain comparably invariant over growth. Membrane current differences, particularly the sodium current and those determining the upstroke velocity could, in theory, also account for some of the conduction velocity changes. In mammalian ventricular myocardium [36], and specialized conduction tissue [37], upstroke velocity and amplitude of AP increases with development. However, atrial tissues may have very different developmental electrophysiologic profiles from that of ventricular myocardium [34,38]. Dolber and Spach found that in Bachmann's bundle, Vmax decreased but longitudinal conduction velocity increased with postnatal development [34]. This reversal of the usual relationship between Vmax and conduction velocity led the authors to propose that tissue structure rather than sarcolemmal electrical properties accounted for the postnatal increases in longitudinal conduction velocity. While the results of the present study supports this hypothesis, it would seem useful to further clarify the degree to which AP morphology contributes to the rate and directionality of impulse propagation in the tc. Ongoing studies are also needed to more precisely understand the relationship between developmental changes in cellular coupling pattern and impulse conduction characteristics. Such data will be of interest to clinicians as well as developmental biologists. There is mounting evidence that breakdown of normal gap junction organization in myocardial pathologies characterized by arrhythmia and other abnormalities of cardiac conduction [2,4,5,24–27]. Our study points to the existence of dynamic processes which may act to promote or maintain gap junction distribution in the normal myocardium. As we have proposed previously, this could include differences in gap junction turnover at specific sarcolemmal domains [16]. Sequestration of Cx43 via zo-1 linkage [39] may also have a role. Whatever the physical explanations, it is proposed that disruption of such processes explain the chaotic patterns of connectivity observed in cardiac disease.

Time for primary review 21 days.


   Acknowledgements
 
This work was supported by the NHLBI (HL56728) and NSF (9734406). The assistance of Ms Sandra Klatt is acknowledged with gratitude. Dr Klaus Willecke is thanked for providing Hela cells transfected with connexin cDNAs and Dr Nick Severs for antibody against Cx45 (GP42). The authors thank Drs David Sedmera and Madison Spach for their excellent critical input.


   Notes
 
1 Dr Litchenberg and Ms. Norman contributed equally to this work. Back


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

  1. Cranefield P.F. Channels, cables, networks, and the conduction of the cardiac impulse. Am J Physiol (1983) 245(6):H901–H910.[Web of Science][Medline]
  2. Spach M.S. Discontinuous conduction in the heart. Soonper P.M., Joyner R.W., Jalife J., eds. (1997) Armonk, N.Y: Futura Publishing. 5–51.
  3. Gros D.B., Jongsma H.J. Connexins in mammalian heart function. Bioessays (1996) 18(9):719–723.[CrossRef][Web of Science][Medline]
  4. Severs NJ, Dupont E, Kaprielian R, et al. Gap junctions and connexins in the cardiovascular system. Annual of Cardiac Surgery. London: Rapid Science Publishers, 1996:31–44.
  5. Gourdie R.G., Litchenberg W.H., Eisenberg L.M. Heart cell communication in health and disease. DeMello W.C., Janse M.J., eds. (1998) Boston/Dordrecht/London: Kluwer. 19–44.
  6. Thomas S.A., Schuessler R.B., Berul C.I., et al. 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]
  7. Simon A.M., Goodenough D.A., Paul D.L. Mice lacking connexin40 have cardiac conduction abnormalities characteristic of atrioventricular block and bundle branch block. Curr Biol (1998) 8(5):295–298.[CrossRef][Web of Science][Medline]
  8. Coppen S.R., Severs N.J., Gourdie R.G. Connexin45 delineates an extended conduction system in the embryonic and mature rodent heart. Dev Genet (1999) 24:82–91.[CrossRef][Web of Science][Medline]
  9. Smith J.H., Green C.R., Peters N.S., et al. Altered patterns of gap junction distribution in ischemic heart disease. An immunohistochemical study of human myocardium using laser scanning confocal microscopy. Am J Pathol (1991) 139(4):801–821.[Abstract]
  10. De Mello W.C. Renin-angiotensin system and cell communication in the failing heart. Hypertension (1996) 27(6):1267–1272.[Abstract/Free Full Text]
  11. Sepp R., Severs N.J., Gourdie R.G. Altered patterns of intercellular junction distribution in hypertrophic cardiomyopathy. Heart (1996) 76:412–417.[Abstract/Free Full Text]
  12. Peters N.S., Coromilas J., Severs N.J., Wit A.L. Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia. Circulation (1997) 95(4):988–996.[Abstract/Free Full Text]
  13. Spach M.S., Boineau J.P. Microfibrosis produces electrical load variations due to loss of side-to-side cell connections: a major mechanism of structural heart disease arrhythmias. Pacing Clin Electrophysiol (1997) 20(2 Pt 2):397–413.[CrossRef][Medline]
  14. Hirakow R., Gotoh T., Watanabe T. Quantitative studies on the ultrastructural differentiation and growth of mammalian cardiac muscle cells. I. The atria and ventricles of the rat. Acta Anat (1980) 108(2):144–152.[Web of Science][Medline]
  15. Gourdie R.G., Green C.R., Severs N.J., Thompson R.P. Immunolabelling patterns of gap junction connexins in the developing and mature rat heart. Anat Embryol (1992) 185(4):363–378.[Medline]
  16. Angst B.D., Khan L.U., Severs N.J., et al. Dissociated spatial patterning of gap junctions and cell adhesion junctions during postnatal differentiation of ventricular myocardium. Circ Res (1997) 80(1):88–94.[Abstract/Free Full Text]
  17. Spach M.S., Dolber P.C. Relating extracellular potentials and their derivatives to anisotropic propagation at a microscopic level in human cardiac muscle. Evidence for electrical uncoupling of side-to-side fiber connections with increasing age. Circ Res (1986) 58(3):356–371.[Abstract/Free Full Text]
  18. Peters N.S., Severs N.J., Rothery S.M., et al. Spatiotemporal relation between gap junctions and fascia adherens junctions during postnatal development of human ventricular myocardium. Circulation (1994) 90:713–725.[Abstract/Free Full Text]
  19. Spach M.S., Dolber P.C. Cardiac electrophysiology: a textbook. Rosen M.R., Janse M.J., Wit A.L., eds. (1990) Mt. Kisco, NY: Futura Publishing. 517–534.
  20. Coppen S.R., Kodama I., Boyett M.R., et al. Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal–crista terminalis border. J Histochem Cytochem (1999) 47:907–918.[Abstract/Free Full Text]
  21. Gourdie R.G., Wei Y., Klatt S., Mikawa T. Endothelin-induced conversion of heart muscle cells into impulse conducting Purkinje fibers. Proc Natl Acad Sci (1998) 95:6815–6818.[Abstract/Free Full Text]
  22. Byers T.J., Kunkel L.M., Watkins S.C. The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle. J Cell Biol (1991) 115(2):411–421.[Abstract/Free Full Text]
  23. Elfgang C., Eckert R., Lichtenberg-Frate H., et al. Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol (1995) 129(3):805–817.[Abstract/Free Full Text]
  24. Weibel E.R. Stereological Methods. (1979) vol. 1. London: Academic Press.
  25. Verheule S., van Kempen M.J.A., te Welscher P.H.J.A., et al. Characterization of gap junction channels in adult rabbit atrial and ventricular myocardium. Circ Res (1997) 80:673–681.[Abstract/Free Full Text]
  26. Gourdie RG, Lo CW. Cx43 gap junctions in development and disease. In: Perrachia C, editor. Gap Junctions. Academic Press, invited review, in press.
  27. Peters N.S., Wit A.L. Myocardial architecture and ventricular arrhythmogenesis. Circulation (1998) 97(17):1746–1754.[Free Full Text]
  28. Chen L., Goings G.E., Upshaw-Earley J., Page E. Cardiac gap junctions and gap junction-associated vesicles: ultrastructural comparison of in situ negative staining with conventional positive staining. Circ Res (1989) 64(3):501–514.[Abstract/Free Full Text]
  29. Fishman G.I., Hertzberg E.L., Spray D.C., Leinwand L.A. Expression of connexin43 in the developing rat heart. Circ Res (1991) 68(3):782–787.[Abstract/Free Full Text]
  30. Fromaget C., el Aoumari A., Dupont E., et al. Changes in the expression of connexin 43, a cardiac gap junctional protein, during mouse heart development. J Mol Cell Cardiol (1990) 22(11):1245–1258.[CrossRef][Web of Science][Medline]
  31. Eisenberg LM, Gourdie RG, Eisenberg CA. Wnt-11 is expressed in early avian mesoderm and required for the differentiation of the quail mesoderm cell line QCE-6. Development 1997;124(2):525-36.57.
  32. Bouman L.N., Duivenvoorden J.J., Bukauskas F.F., Jongsma H.J. Anisotropy of electrotonus in the sinoatrial node of the rabbit heart. J Mol Cell Cardiol (1989) 21(4):407–418.[CrossRef][Web of Science][Medline]
  33. Clerc L. Directional differences of impulse spread in trabecular muscle from mammalian heart. J Physiol (1976) 255(2):335–346.[Abstract/Free Full Text]
  34. Dolber P.C., Spach M.S. Structure of canine Bachmann's bundle related to propagation of excitation. Am J Physiol (1989) 257(5 Pt 2):H1446–H1457.[Web of Science][Medline]
  35. Efimov I.R., Fahy G.J., Cheng Y., et al. High-resolution fluorescent imaging does not reveal a distinct atrioventricular nodal anterior input channel (fast pathway) in the rabbit heart during sinus rhythm. J Cardiovasc Electrophysiol (1997) 8(3):295–306.[Web of Science][Medline]
  36. Pacioretty LM, Gilmour RF Jr. Developmental changes of action potential configuration and I(to) in canine epicardium. Am J Physiol 1995; 268(6 Pt 2).
  37. Rosen M.R., Reder R.F., Hordof A.J., et al. Age-related changes in Purkinje fiber action potentials of adult dogs. Circ Res (1978) 43:931–938.[Abstract/Free Full Text]
  38. Toda N. Age-related changes in the transmembrane potential of isolated rabbit sino-atrial nodes and atria. Cardiovasc Res (1980) 14(1):58–63.[Abstract/Free Full Text]
  39. Toyofuku T., Yabuki M., Otsu K., et al. Direct association of the gap junction protein connexin-43 with zo-1 in cardiac myocytes. J Biol Chem (1998) 273:12725–12731.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Physiol.Home page
C. M. Loughrey, T. Seidler, S. L. W. Miller, J. Prestle, K. E. MacEachern, D. F. Reynolds, G. Hasenfuss, and G. L. Smith
Over-expression of FK506-binding protein FKBP12.6 alters excitation-contraction coupling in adult rabbit cardiomyocytes
J. Physiol., May 1, 2004; 556(3): 919 - 934.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
F R Quinn, S Currie, A M Duncan, S Miller, R Sayeed, S M Cobbe, and G L Smith
Myocardial infarction causes increased expression but decreased activity of the myocardial Na+--Ca2+ exchanger in the rabbit
J. Physiol., November 15, 2003; 553(1): 229 - 242.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
R. J. Barker, R. L. Price, and R. G. Gourdie
Increased Association of ZO-1 With Connexin43 During Remodeling of Cardiac Gap Junctions
Circ. Res., February 22, 2002; 90(3): 317 - 324.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Litchenberg, W. H
Right arrow Articles by Gourdie, R. G
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Litchenberg, W. H
Right arrow Articles by Gourdie, R. G
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?