Published online before print April 27, 2001, doi: 10.1161/hh0901.089986
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© 2001 American Heart Association, Inc.
Integrative Physiology |
Tissue-Specific Patterns of Gap Junctions in Adult Rat Atrial and Ventricular Cardiomyocytes In Vivo and In Vitro
Presented in part at the 71st Scientific Sessions of the American Heart Association, Dallas, Tex, November 8–11, 1998, and published in abstract form (Circulation. 1998;98[suppl I]:I-816).
From the Department of Experimental Cardiology, Max-Planck-Institute, Bad Nauheim, Germany.
Correspondence to Sawa Kostin, MD, Max-Planck-Institute, Benekestrasse 2, D-61231 Bad Nauheim, Germany. E-mail skostin{at}kerckhoff.mpg.de
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Abstract |
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Abstract—To verify the hypothesis that tissue-specific patterns of gap junctions (GJs) are determined by intrinsic factors within myocytes forming different cardiac tissues, we have compared by quantitative transmission electron microscopy (TEM) the structural features of GJs in adult rat atrial myocytes (AMs) and ventricular myocytes (VMs) in vivo with those in developing GJs in cultured AMs and VMs in vitro. Quantitative TEM data revealed a 3-fold increase in the number of developing GJs per intercalated disk in both AMs and VMs from 6 to 15 days in culture. However, at days 12 and 15, the percentage of GJ length per intercalated disk and mean GJ length were 2-fold higher in VMs than in AMs. Measurements of connexin43 GJs by confocal microscopy confirmed TEM data and demonstrated respectively 2- and 4.5-fold greater mean values of GJ length and area in VMs than in AMs. These differences are attributable to the development of large GJs (>3 µm) in VMs, closely resembling those observed in VMs in vivo. Although large GJs in cultured VMs comprised
14% of the total number of GJs,
their contribution to total GJ length and area constituted >60% and
85%, respectively. In marked contrast, the number of large GJs in AMs
both in vitro and in vivo was <1% from the total number of GJs. These
data confirm our hypothesis and provide the first evidence that
tissue-specific patterns of GJs in AMs and VMs are determined primarily
by intrinsic factors within cardiac myocytes and are developmentally
regulated.
Key Words: gap junction • ventricle • atrium • morphometry • development
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Conduction of the cardiac impulse is dependent on both active sarcolemmal ionic currents and passive electrical properties determined by the myocardial tissue architecture and specialized junctions responsible for electrical coupling, the gap junctions (GJs). The influence of myocardial architecture on conduction is determined by the size and shape of individual cardiomyocytes1 and their packing geometry in the myocardium.2 The role of GJs in cardiac conduction is dependent on their constituent connexin isoforms, as well as on the size, number, and spatial distribution of these junctions. Connexin isoforms of GJs have been shown to vary in different cardiac tissues with the following specific conduction properties: the nodes,3 4 5 a specialized conduction system,3 6 7 8 9 10 11 and atrial and ventricular myocardium.12 13 14 15 The size, number, and spatial distribution of GJs vary also distinctly across the different types of myocytes with disparate conduction properties and therefore are considered an important structural determinant of conduction.2 16 17 18
Examples of tissues with disparate structural patterns of GJ interconnections are the atrial and ventricular myocardium.2 19 20 However, virtually nothing is known about the mechanisms of establishment and regulation of specific patterns of GJs in atrial myocytes (AMs) and ventricular myocytes (VMs) in vivo. Although different loading conditions imposed on these cardiac chambers may influence the structural patterns of cellular interconnections and their GJs, we hypothesized that intrinsic factors within cardiac myocytes may also play a role in determining specific patterns of GJ organization in AMs and VMs. To verify this hypothesis, we used a model of disaggregated and externally unloaded adult rat AMs and VMs in long-term culture. In this "dedifferentiation-redifferentiation" model, initial disassembly of GJs is followed by a subsequent reestablishment of GJs.21 22 23 Using transmission electron microscopy (TEM) and confocal scanning laser microscopy (CSLM), we have quantitatively compared the structural features of GJs in AMs and VMs in adult rat hearts in vivo with those of developing GJs in cultured AMs and VMs in vitro. In the present study, we show that structural differences in GJ organization between adult AMs and VMs in vivo are reproducible in vitro, such that cultured VMs, as compared with AMs, consistently develop a specific subpopulation of large GJs closely resembling those observed in adult VMs in vivo. These data provide the first evidence that tissue-specific patterns of GJs in AMs and VMs are determined primarily by intrinsic factors within cardiac myocytes and are developmentally regulated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments were performed according to protocols approved by Regierungpräsidium, Darmstadt, Germany. Adult hearts were obtained from 9 Wistar rats (90 days old). The animals were deeply anesthetized, and their hearts were excised and perfused with 2% glutaraldehyde for TEM (6 hearts) or with 1% paraformaldehyde for immunohistochemistry (3 hearts) as described.24 25 26 AMs and VMs were isolated from adult Wistar rats (90 days old) and maintained under identical conditions in culture according to methods previously described.21 27 TEM morphometry of GJs in AMs and VMs in vivo and in vitro was performed as previously described.2 21 28 29 30 To compare our data with those published, we define a large GJ of which the greatest length is >3 µm as reported by Luke and Saffitz.31 As described previously,2 28 31 we have separately analyzed TEM data of GJs in myocytes cut in a longitudinal plane (LP) and transverse plane (TP) to the long myocyte axis. Quantification of GJ size by CSLM was performed according to protocols described by Gourdie et al.19 32
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GJs in Adult Atrial and Ventricular Myocardium
We used TEM to study GJs in adult rat AMs and VMs in vivo. Representative micrographs of AMs in LP of section denote relatively simple intercalated disks (IDs) and GJs with a profile length <1 µm (Figure 1A
).
Figures 1B and 1C show representative GJs in
AMs in TP of section.
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VMs sectioned in LP showed highly tortuous IDs and numerous
GJs
(Figure 1D
). Large GJs (profile length >3 µm) in LP of
section were infrequent, but when observed (online Figures 1A and 1B,
available in an online data supplement at http://www.circresaha.org),
they were more common between VMs connected in a side-to-side
orientation (following the definition of Luke and
Saffitz).31 Large GJs were
observed more frequently in VMs sectioned in TP than in LP. This type
of GJ appeared in TEM as long (up to 10 µm) ribbonlike junctions
(following the original description of Hoyt et
al),28 exhibiting highly
folded and tortuous contours
(Figure 1E). Further examples of VMs in TP of section
displaying large GJs at the IDs are shown in online Figure 2. Large GJs
were found in VMs to be mainly confined to the periphery of the IDs
(Figure 1F) in close proximity to or being continuous with
the external sarcolemma. Further examples of peripherally
located large GJs are provided in online Figure 3. It is worthy to note
that large GJs were observed even in small-sized IDs (online Figure 4)
at the lateral cellular branches that are numerous in
VMs.2 33
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Quantitative Analysis of GJs In
Vivo
We used TEM to quantify GJ size in adult rat AMs and
VMs in vivo. The advantage of TEM resides in the high-resolution
identification of GJs as a structure that can be directly measured with
precision. Quantitative TEM analysis revealed that AMs are
interconnected by GJs having a profile length of 0.53±0.03 µm in TP
and 0.51±0.03 µm in LP, whereas in VMs the mean GJ length was
significantly greater in TP than in LP (1.04±0.04 µm versus
0.65±0.06 µm, respectively).
Histograms comparing the frequency distribution of different
GJ size classes in AMs versus VMs in both LP and TP (online Figure 5)
show no significant differences in this parameter between
AMs and VMs in LP. However, VMs sectioned in TP showed significantly
higher frequency values of large GJs than those in VMs in LP or in AMs
in both LP and TP. Thus, from the total 1346 GJs measured in VMs in TP
of section, 138 (10.2%) had a profile length >3 µm, whereas such
GJs identified in VMs in LP and AMs in LP and TP comprised,
respectively, only 1.5%, 0.8%, and 0.9% from the total number of
GJs. This difference is reflected in the relative contribution of large
GJs to the total GJ length (online Figure 6). Thus, large GJs
encompassed 49.3% of the total GJ length in VMs in TP and <15% in
VMs in LP. The contribution of large GJs to the total GJ length in AMs
averaged 5.5% in LP and 6.2% in TP.
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Taken together, quantitative data of adult rat hearts indicate that myocytes forming atrial and ventricular tissue are interconnected by GJs that differ in a tissue-specific manner in terms of their size and spatial distribution. In particular, these data show that large ribbonlike GJs are a structural peculiarity of VMs but not of AMs in vivo.
To investigate whether GJs are reestablished in tissue-specific patterns in vitro, we conducted experiments using isolated AMs and VMs from adult rat hearts and maintained in long-term culture.
Immunoconfocal Characterization of Cell
Cultures
Figures 2A through 2D are 3-dimensional views showing the
difference in the architecture of IDs and in the cell size between AMs
and VMs isolated from respective adult tissues. By immunolabeling for
N-cadherin, randomly selected AMs showed 5.5±1.5 IDs per cell, whereas
VMs possessed 9.8±2.1 IDs per cell
(P<0.01, n=100 cells). These
data are consistent with previously published
values.2 28 31 34 35
During subsequent maintenance in culture, isolated
AMs and VMs disassemble ID structures, spread on the substratum, and
tend to rapidly reestablish new
IDs,21 23 27
confirmed by immunolabeling for N-cadherin and 
-catenin
(Figures 2E and 2F).
Evaluation of GJ Formation by CSLM
CSLM revealed that in AMs cultured for 6 days,
connexin43 (Cx43) reappeared along the newly redeveloped IDs as
clusters of distinct small dots
(Figures 3A and 3B). From day 9 to day 15, AMs showed a
gradual increase in the size and number of GJs per ID
(Figures 3C through 3E). The majority of redeveloped GJs were
oval or discoid in shape.
Figures 3F through 3I show Cx43 GJs in VMs at different
developmental stages ranging from 6 to 15 days in culture. At day 6,
GJs were small and sparsely distributed along the IDs
(Figure 3F), whereas at day 9, GJs appeared larger and more
numerous
(Figure 3G). With increasing time in culture (12 to 15 days),
VMs showed a further and dramatic increase in length and area of GJ
plaques
(Figures 3H and 3I).
Ultrastructural Assessment of GJ
Formation
We used TEM to examine in further detail the sequences
of GJ formation in relation to phenotypic changes of AMs and VMs in
culture. As in tissue sections, we have analyzed GJs in
cultured myocytes in LP and TP to the substratum.
TEM of AMs at 6 days in culture revealed dedifferentiating
cells almost lacking myofibrillar structures while abundantly
containing secretory granules and Golgi complex
(Figure 4A). Reassembled IDs between AMs at 6 days showed the
presence of zipperlike adhesion junctions
(Figure 4B) and very close cell-to-cell appositions
(Figure 4C), representing typical GJs, as
observed at high magnifications in LP
(Figures 4D and 4E). Similar patterns of GJs were found in TP
(Figures 4F and 4G).
At day 9, as development further proceeded, AMs showed
nascent myofibrils with irregular Z-densities
(Figure 4H). As compared with 6 days, GJs at 9 days appeared
larger and more numerous as shown in LP at low and high magnifications
(Figures 4I through 4L). Similar patterns of GJs were found in
TP
(Figures 4M and 4N).
AMs at 12 days exhibited myofibrils with distinct and
regular Z-bands
(Figure 5A). GJs connecting these cells appeared as linear or
curvilinear junctions as shown in LP
(Figures 5B through 5D) and TP
(Figures 5E and 5F).
AMs at 15 days contained compactly arranged myofibrils and
secretory granules
(Figure 5G). Although the overall pattern of GJs at 15 days
(Figure 5H) did not differ substantially from that at 12
days, occasional GJs exceeding 1 µm in length tended to have folded
contours
(Figure 5I). Typical patterns of GJs observed in TP of
section in AMs at day 15 are shown in
Figure 5J.
TEM data of cultured VMs at early stages of GJ formation (6
to 9 days in culture) were described
previously.21 Here, we
report more detailed data of GJs in VMs at 12 to 15 days.
Redifferentiating VMs at these intervals showed mature myofibrils
(Figure 6A) and reorganized T-tubules
(Figure 6B), comparable with those found in vivo or in
freshly dissociated adult
VMs.36 VMs at day 12
developed large GJs with a ribbonlike appearance observable in LP
(Figures 6C and 6D) and TP (data not shown). VMs at day 15
showed long ribbonlike GJ profiles discernible in LP at low
(Figures 6E and 6F) and high
(Figures 6G through 6I) magnifications. Similar configurations
of GJs were found in TP
(Figures 6J and 6K). Some ribbonlike GJs had exceptionally
long profiles (>10 µm) as shown in LP
(Figures 6F and 6H) and TP
(Figure 6J).
Quantitative Analysis of GJ
Formation
We determined the size of individual GJs by TEM and by
CSLM using immunolabeling for Cx43. The results given in the
Table
show that a progressive increase in the number and size of GJs occurred
in both AMs and VMs with increasing time in culture. However, at 12 and
15 days, when comparable total lengths of IDs were examined by TEM, the
percentage of the total GJ length per ID was 2-fold greater in VMs
than in AMs. This difference is mainly attributable to the 2-fold
higher GJ profile length in VMs than in AMs, whereas the total number
of GJs per unit ID length was comparable between these
groups.
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Regardless of the time in culture in both AMs or VMs, no significant changes were observed in TEM parameters of GJs measured in LP versus TP of section. For example, in VMs at day 15, the average size of the total 81 GJs selected at random from sections cut in LP was 1.15 µm and did not differ from that of the total 75 GJs observed in TP (1.05 µm). In the corresponding AM cultures, the size of an average GJ profile was 0.57 µm in LP and 0.52 µm in TP.
Similar to TEM data, GJ morphometric parameters
obtained by CSLM in 12- to 15-day cultures and expressed as GJ profile
length and area were respectively 2- and 4.5-fold greater in VMs than
those in AMs
(Table).
Figures 7A and 7B compare the frequency distribution of GJ
size classes as determined by CSLM and TEM in AMs versus VMs in 12- to
15-day cultures. Although the histograms of GJ size distribution showed
a skew toward the smaller junctions in both types of myocytes,
statistical analysis revealed significant differences between
these cultures due mainly to the higher proportion of larger GJs in VMs
than in AMs. Thus, in VMs, from the total 2238 GJs measured by CSLM,
332 GJs (14.8%) had a profile length >3 µm. By contrast, AMs showed
a marked paucity of larger GJs, such that only 20 GJs of 2076 (0.96%)
exceeded 3 µm
(Figure 7A). The results obtained using CSLM showed a close
resemblance with those obtained by TEM in that 14.1% of GJs in VMs (42
of 298) had a profile length >3 µm
(Figure 7B). In AMs, such GJs comprised only 1% (3 of
286).
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The difference between AMs and VMs in the frequency
distribution of GJ sizes is reflected in the contribution of different
size classes to the total GJ length
(Figures 7C and 7D) and area
(Figure 7E). Thus, in marked contrast with the symmetrical
configuration of the histograms of the relative contribution of
different GJ sizes to total GJ length and area as observed in AMs, the
configuration of the corresponding histograms in VMs was markedly
skewed toward the higher values of the contribution of larger GJs to
the total GJ length and area. Compared with VM cultures, in which the
contribution of GJs with a length >3 µm to the total GJ length
averaged 64.7% and 60.4% (as determined respectively by CSLM and
TEM), the contribution of such GJs to the total GJ length in AMs was
respectively 9.5- and 7.2-fold lower. Furthermore, when the
contribution of the different GJ size classes was related to the total
GJ area
(Figure 7E), GJs with a length >3 µm encompassed 87.4%
and 25% of total GJ area in VMs and AMs, respectively. Finally, a
marked difference between AMs and VMs was revealed when the total GJ
length from each size class was added to plot the cumulative
contribution of different size classes to the total GJ length
(Figure 7F). Thus, in AMs, GJs with a profile length <3 µm
constituted 93.8% of the cumulative GJ length, whereas in the
corresponding VM cultures, these GJs constituted only
40.6%.
In summary, quantitative TEM and CSLM data substantiate and extend the conclusion that AMs and VMs in vitro differ markedly in the size of their GJs and in the proportion of large ribbonlike GJs. These data are comparable with the results obtained in vivo. Another obvious difference was a marked contribution of large GJs (>3 µm) to the total GJ length in VMs both in vitro and in vivo as compared with a very modest contribution of large GJs to the total GJ length in AMs both in vitro and in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous quantitative data of adult canine hearts have clearly demonstrated marked structural differences between atrial and ventricular tissues in the patterns of intercellular connections and in the size, number, and spatial distribution of their GJs.2 20 The present quantitative study performed in adult rat hearts confirms these findings and demonstrates that myocytes forming atrial and ventricular myocardium are interconnected by GJs that differ in a tissue-specific manner in terms of their size and 3-dimensional distribution. In particular, we show that a specific subclass of extremely large ribbonlike GJs represents a structural peculiarity of adult rat ventricular tissue, previously characterized in detail in adult canine ventricle.28 31
The specific aim of the present study was to verify the hypothesis that atrial and ventricular tissue–specific patterns of GJs in vivo are determined and regulated by intrinsic factors within the myocytes forming these cardiac tissues, rather than extrinsic factors, such as load or wall tension. The following findings support our hypothesis: (1) the observation that isolated and externally unloaded AMs and VMs in long-term culture in vitro reestablish patterns of GJ interconnections that are similar with disparate atrial and ventricular tissue–specific patterns of GJ interconnections as we documented quantitatively in adult hearts in vivo and (2) the demonstration that VMs in vitro consistently redevelop a specific subpopulation of large ribbonlike GJs, closely resembling those observed in adult VMs in vivo. These conclusions are based on direct measurements of GJs by TEM in adult AMs and VMs in vivo and in AMs and VMs in long-term culture in vitro. In addition, quantitative TEM data of GJ formation in vitro were confirmed by CSLM measurements of Cx43 GJs.
It should be emphasized that the data on the spatial
organization of GJs in vitro cannot be entirely extrapolated to the
3-dimensional situation in vivo, which is undoubtedly much more
complex. Such examples include (1) the presence in adult VMs in vivo of
an anisotropic pattern of GJ distribution at the IDs with higher GJ
length in TP than in LP of section, as reported in canine
hearts28 31 or
rat hearts (present study) and the lack of this pattern, with
almost no differences in GJ size in LP and TP, as documented in our in
vitro experiments, and (2) higher frequencies of large GJs in VMs in
vitro than in vivo. A plausible explanation for the observed
differences is a continuous process of remodeling of the intercellular
connections and GJs that occurs in the course of postnatal heart
development. This process is not species-specific but has conclusively
been described in a number of mammalian
species,37 38
including
rats.1 39 40
Moreover, this process continues over a relatively extended time after
birth41 ; in the human heart,
the adult pattern of GJ distribution is achieved at 6 years of
age.42 Certainly, these
distal steps in the establishment of adult patterns of GJ distribution
cannot be achieved in vitro. Nonetheless, our data showing that the
major structural differences in GJs between AMs and VMs in vivo are
regained in vitro strongly indicate intrinsic myocyte-specific
mechanisms in control of GJ size.
Currently, the precise function of the large ventricular GJs, both in vivo and in vitro, is poorly understood. However, as established in the present study, as well as in others,7 15 19 35 43 44 45 46 47 the position of large GJs at the periphery of the IDs, ie, directly in the path of the action potential, would predict an important role of these junctions in intercellular current transfer between VMs, probably contributing to the efficient anisotropic pattern of impulse conduction. New insights into the function of large GJs have recently emerged from studies carried out in Cx43-deficient mice. These showed that when Cx43 is diminished, it is functionally more advantageous for VMs to maintain GJ size rather than GJ number per ID.30 These data, together with the results of computer simulation studies of conduction under conditions in which GJ plaque size is varied,48 suggest an important role of large GJs in supporting safe conduction that is critical for normal ventricular conduction.
Apart from the function of large GJs, many experimental31 and clinical34 46 47 studies documented a striking and selective disruption of large GJs in diseased ventricular tissue. The recognition that these junctions are extremely vulnerable structures under different pathophysiological settings that are prone to arrhythmias emphasizes the clinical relevance of understanding the mechanisms of formation and stabilization of large ventricular GJs.30 We propose that isolated adult rat AMs and VMs in long-term culture differing markedly in the size of their GJs can serve as reliable models for studying the role of GJ size in myocardial tissue–specific intercellular communication and conduction, and merit further investigations.
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Acknowledgments |
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This work was supported by a Max-Planck-Gesellschaft grant (to S.K. and J.S.). We are grateful to Gunther Schuster and Gerhard Stämmler for computer assistance and Annemarie Möbs for photographic work.
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Footnotes |
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Original received May 22, 2000; resubmission received January 22, 2001; revised resubmission received March 9, 2001; accepted March 9, 2001.
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References |
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