Ordered assembly of the adhesive and electrochemical connections within newly formed intercalated disks in primary cultures of adult rat cardiomyocytes.

Abstract

The intercalated disk (ID) is a complex structure that electromechanically couples adjoining cardiac myocytes into a functional syncitium. The integrity of the disk is essential for normal cardiac function, but how the diverse elements are assembled into a fully integrated structure is not well understood. In this study, we examined the assembly of new IDs in primary cultures of adult rat cardiac myocytes. From 2 to 5 days after dissociation, the cells flatten and spread, establishing new cell-cell contacts in a manner that recapitulates the in vivo processes that occur during heart development and myocardial remodeling. As cells make contact with their neighbors, transmembrane adhesion proteins localize along the line of apposition, concentrating at the sites of membrane attachment of the terminal sarcomeres. Cx43 gap junctions and ankyrin-G, an essential cytoskeletal component of voltage gated sodium channel complexes, were secondarily recruited to membrane domains involved in cell-cell contacts. The consistent order of the assembly process suggests that there are specific scaffolding requirements for integration of the mechanical and electrochemical elements of the disk. Defining the relationships that are the foundation of disk assembly has important implications for understanding the mechanical dysfunction and cardiac arrhythmias that accompany alterations of ID architecture.

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Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2010, Article ID 624719, 14 pages
doi:10.1155/2010/624719
Research Article
OrderedAssemblyof the AdhesiveandElectrochemical
ConnectionswithinNewlyFormedIntercalated Disks inPrimary
Culturesof Adult Rat Cardiomyocytes
Sarah B. Geisler,1Kathleen J. Green,2Lori L. Isom,3,4Sasha Meshinchi,5Jeffrey R. Martens,4
Mario Delmar,3,6andMark W.Russell1
1Division of Pediatric Cardiology, Department of Pediatrics and Communicable Diseases, University of Michigan,
1150 West Medical Center Dr., Ann Arbor, MI 48109, USA
2Department of Pathology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Chicago, IL 60611, USA
3Department of Molecular and Integrative Physiology, University of Michigan, 1150 West Medical Center Dr., Ann Arbor,
MI 48109, USA
4Department of Pharmacology, University of Michigan, 1150 West Medical Center Dr., Ann Arbor, MI 48109, USA
5Department of Cell and Developmental Biology, University of Michigan, 1150 West Medical Center Dr., Ann Arbor, MI 48109, USA
6Department of Internal Medicine, University of Michigan, 1150 West Medical Center Dr., Ann Arbor, MI 48109, USA
Correspondence should be addressed to Mark W. Russell, mruss@med.umich.edu
Received 21 November 2009; Accepted 17 February 2010
Academic Editor: Guy M. Benian
Copyright © 2010 Sarah B. Geisler et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
The intercalated disk (ID) is a complex structure that electromechanically couples adjoining cardiac myocytes into a functional
syncitium. The integrity of the disk is essential for normal cardiac function, but how the diverse elements are assembled into
a fully integrated structure is not well understood. In this study, we examined the assembly of new IDs in primary cultures of
adult rat cardiac myocytes. From 2 to 5 days after dissociation, the cells flatten and spread, establishing new cell-cell contacts in
a manner that recapitulates the in vivo processes that occur during heart development and myocardial remodeling. As cells make
contact with their neighbors, transmembrane adhesion proteins localize along the line of apposition, concentrating at the sites
of membrane attachment of the terminal sarcomeres. Cx43 gap junctions and ankyrin-G, an essential cytoskeletal component
of voltage gated sodium channel complexes, were secondarily recruited to membrane domains involved in cell-cell contacts. The
consistent order of the assembly process suggests that there are specific scaffolding requirements for integration of the mechanical
and electrochemical elements of the disk. Defining the relationships that are the foundation of disk assembly has important
implicationsforunderstandingthemechanicaldysfunctionandcardiacarrhythmiasthataccompanyalterationsofIDarchitecture.
1.Introduction
Cardiac myocytes are linked to each other along their axis of
contraction by complex connections called intercalated disks
(reviewed in [1]). The cardiac intercalated disk (ID) is com-
posed of three types of cell-cell contacts: adherens junctions,
gap junctions, and desmosomes. These connections serve as
anchorage sites for the actin cytoskeleton, the intermediate
filament network, microfilaments, microtubules, and the
terminal ends of the myofibrils. They stabilize the cellular
cytoskeleton and electromechanically couple adjoining car-
diac myocytes to form a functional syncitium. Alterations in
the structure of the disk, either due to mutation of one of
its components or aberrant remodeling during heart failure,
can lead to progressive contractile dysfunction and cardiac
arrhythmias [2].
ID remodeling may be an important ventricular adap-
tation to congestive heart failure. In response to dilated
cardiomyopathy (DCM) and congestive failure, there is
a marked upregulation of adherens junction constituents,

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2Journal of Biomedicine and Biotechnology
including N-cadherin, and β-catenin, and the plicate region
of the intercalated disk becomes much more undulated
[3]. Since the increased undulation will decrease the angle
of incidence between the working myofibrils and their
membrane anchorage sites, this will result in an improved
force transmission and diminished shear stress at the inter-
calated disk. Therefore, structural adaptations within the
intercalated disk, specifically within the adherens junctions,
may in part compensate for the increased myocardial strain
that accompanies congestive heart failure and DCM and may
enable optimization of force transmission in this setting.
In contrast to the adhesive complex proteins of the
intercalated disk, connexin43 (Cx43), a gap junction protein
is not upregulated and, according to some reports, even
downregulated in the setting of DCM [4]. Several studies
have also shown significant redistribution of Cx43 in the
setting of cardiomyopathies and heart failure [5]. This
suggeststhattherearedistinctregulatorypathwaysgoverning
the expression of the mechanical and electrical elements of
the ID, and that these pathways can be affected by disease.
Additional studies have shown a close interaction between
the mechanical and electrical components of the intercalated
disk since loss of N-cadherin disrupts connexin localization
andcardiacimpulsepropagation[6],andcorrecttargetingof
Cx43 to the ID depends on transport of the intact protein to
cadherin-containing adherens junctions of the disk [7]. This
suggests that the disruption of the mechanical components
of the disk may lead to destabilization of the gap junctions
and ventricular arrhythmias.
Perhaps the most direct evidence of the important rela-
tionship between the structural and electrophysiologic prop-
ertiesof the IDinvolves theclinical syndrome of Arrhythmo-
genic Right Ventricular Cardiomyopathy/Dysplasia (ARVC).
ARVC is a heterogenous disorder characterized by severe
ventricular arrhythmias and, commonly, fibro-fatty replace-
ment of regions of the right and/or left ventricle (reviewed in
[8]).Ithasbeenestimated that,althoughARVCis arelatively
rare condition, affecting approximately 1 in 5000 people in
the U.S., it is responsible for approximately 10% of the cases
of sudden death in individuals under the age of 35 [9–11]. To
date, ten different genes have been determined to be respon-
sible for causing ARVC, including desmocollin, desmoglein,
desmoplakin, plakophillin-2 (PKP2), plakoglobin, desmin,
ZASP,TransmembraneProtein43(TP43),andTransforming
Growth Factor β3 (TGF β3). For many of these genes,
mutations have only been identified in a few pedigrees
worldwide [8] or lead to “atypical” forms of ARVD.
The great majority of ARVC cases with identified
genetic mutations are linked to genes coding for proteins
of the desmosome. Since none of these proteins directly
participate in propagation of the cardiac impulse, alteration
in the properties of the desmosome must have important
secondary effects on the electrochemical connections within
the ID including the gap junctions and/or the voltage-gated
sodiumchannels(VGSC).Recentstudieshavesupportedthis
assertion, demonstrating that plakophilin-2 (PKP2), a part
of the desmosomal complex, is involved in stabilization of
gap junctions and VGSCs within the ID. Loss of PKP-2 was
associated with redistribution of Cx43 gap junctions in cell
culture models and in patients with ARVC [12–14] and with
decreased sodium current and lower conduction velocity
in cultured cardiac myocytes [15]. Yet the relationships
between the assembly and organization of the desmosomes
and the recruitment and stabilization of the gap junctions
andVGSCshavenotbeenwellcharacterized.Inthisstudy,we
examinedtheassemblyofIDsinremodelingadultratcardiac
myocytes in primary culture. The findings suggest pro-
gressive and coordinated maturation of the transmembrane
adhesioncomplexes,intracellularcytoskeletalnetworks,and,
ultimately, specialized sarcolemmal domains.
2.MaterialsandMethods
2.1. Cell Culture. Primary cultures of cardiac myocytes were
isolated from adult female rats using enzymatic dissociation
of myocardial tissue with trypsin and collagenase as previ-
ously described [16]. Cells were cultivated on coverslips in
199 medium as described [17].
2.2. Immunohistochemistry. Cells on coverslips were washed
with PBS, fixed in methanol for two minutes, and rinsed
with PBS. Coverslips were incubated in PBS+0.1%TX100+1
mg/ml BSA for 30 minutes, rinsed three times with PBS, and
incubated with primary antibody diluted in PBS+BSA for
1.5 hours at room tempretare. Following three PBS washes,
coverslips were incubated with secondary antibodies. Cells
were incubated with monoclonal and polyclonal antibodies
together, followed by combined corresponding secondary
antibodies. Controls for immunostaining were the omission
of both primary antibodies and omission of both secondary
antibodies. Sequential staining was performed when two
monoclonal antibodies were used on one sample. After
the first primary/secondary incubations, coverslips were
incubated at 4◦C overnight with goat antimouse IgG, then
washed with PBS and incubated with Fab fragment of goat
antimouse IgG. The second primary/secondary antibodies
were then applied. Additional controls included reversing
orderofsecondaryantibodiesandreversingorderofprimary
incubations.
The following primary antibodies were used: 4B2
against Dsg [18], PKP2 (1:20, Biodesign International,
Meridian Life Science, Saco, ME. K44262M), pan-cadherin
(1.4mg/ml, Sigma C3678), NW6 against desmoplakin
(1:100), ankyrinG (10μg/mL, SCBT, SC28561), connexin43
(750μg/mL, Sigma C8093), plakoglobin (1:100, Sigma
P8087), Nav1.5 (40μg/mL, Alamone ASC-005). FITC
conjugated goat-antimouse IgG, Texas Red conjugated
goat-antimouse IgG, FITC conjugated goat anti-rabbit IgG,
and Texas Red-conjugated goat antirabbit IgG were obtained
from Jackson ImmunoResearch Laboratories Inc. (West
Grove, PA). Coverslips were mounted on slides using the
Prolong Gold antifade reagent with DAPI (Molecular Probes
Inc., Eugene, OR). Immunostained samples were examined
with an Olympus FV-500 Meta confocal microscope using a
60x objective.
2.3. Electron Microscopy. Cells were plated on laminin-
coated coverslips as above. Five days post plating, the cells

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Journal of Biomedicine and Biotechnology3
20μm
1d
(a)
20μm
3d
(b)
20μm
5d
(c)
20μm
7d
(d)
20μm
14d
(e)
20μm
(a?)
20μm
(b?)
20μm
(c?)
20μm
(d?)
20μm
(e?)
Figure1:Reassemblyofcell-cellcontactsinremodelingadultratcardiacmyocytesinculture.(a,b)Ondays1–3postplating,thecellrounds
up,theIDsdisassemble,andcadherinsredistributealongtheperipheryofthecell(arrowinb).(c,d)Betweendays5–7,newcell-cellcontacts
form and cadherin localizes to specific domains along the line of apposition (arrows). (e) By 14 days post plating, the cell-cell contacts have
remodeled to form more localized and mature structures (arrow). Scale bars are 20μm.
were fixed overnight at 4◦C in 2.5% glutaraldehyde and 2.0%
paraformaldehyde and post fixed in 1% osmium tetroxide.
After washing, they were stained with saturated uranyl
acetate, and embedded in Epon 812. Ultra-thin sections were
prepared and stained with saturated uranyl acetate and lead
citrate. The sections were examined with a Philips CM100
transmission electron microscope, at an accelerating voltage
of 60kV.
3.Results
3.1. Time Course of ID Remodeling in Primary Culture.
Adult rat cardiac myocytes grown in primary culture in the
presence of serum disassemble their contractile structures
and remodel their cellular cytoskeleton [19]. Over the course
of 2–5 days, the cells extend processes, form new cell-matrix
and cell-cell contacts, assemble new myofibrils, and resume
spontaneous beating. During the remodeling process, the
components of the ID are dismantled and subsequently
reassembled at the new sites of cell-cell contact. To assess
the time course of intercalated disk reassembly, primary
cultures were immunolabeled with a pan-cadherin and
PKP2 antibodies at timepoints from 1 to 14 days post
plating (Figures 1 and 2). It was noted that, as a result
of cell spreading and redistribution of adhesive junction
components, new cell-cell contacts began to form as early as
3 days post plating along the boundaries between adjoining
cells(Figure 2).Overthenext1-2weeksinculture,thedegree
of myofibril alignment increased and the new IDs became
focused at the polar ends of the myocyte along the new axis
of contraction (see Figures 1(e) and 1(e?)).
Based on these studies, the 5 day post-plating timepoint
was selected for further analyses. This timepoint allowed
the evaluation of the order of incorporation of cytoskeletal
elements into more complex and organized assemblies
(Figure 3). At later timepoints, contractile activity and
ongoing remodeling made it more difficult to differentiate
assembling from disassembling structures.
3.2. Adhesive Junction and Armadillo Family Proteins Colocal-
ize during the Early Phase of ID Assembly. To determine the
scaffolding requirements for each element of the ID, their
inclusion into new cell-cell contacts of varying complexity
and maturity was examined at 5-days post plating. The order
of incorporation was determined by pairwise comparison of
double immunolabelings with an array of ID components.
If two elements precisely colocalized in all cells sampled
then they were scored as essentially concurrent in their
incorporation into the new cell-cell contact. For some pairs,
one of the two elements was never noted at cell contacts in
the absence of the other; that element was determined to be
recruited after the other to the new cell contacts. For some
pairs, the localization patterns did not precisely overlap and
either protein could localize to the new contact without the
other. In those cases, the localization was determined to be
simultaneous but not strictly interdependent.
Using this approach, the earliest components of the ID
to localize to new cell-cell contacts were the transmembrane
proteins, including N-cadherin and desmocollin, and the
armadillo family proteins, including β-catenin (data not
shown), plakoglobin (PG), and PKP2 (Figure 4). Of these,
N-cadherin seemed to be required for adhesive contact
formation as none of the other components were noted
along the line of cell apposition in its absence. There was
some variability in the ratio of PKP2 to desmocollin (Figures
4(d)–4(f)), indicating that there may be a nonstoichiometric
relationship between the two, at least early in the assembly
process or that PKP2 can localize to contacts that do not

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(a)
20μm
(b)
10μm
(c)
10μm
(d)
10μm
(e)
Figure 2: Early remodeling of the intercalated disks of isolated
adult rat ventricular cardiomyocytes in primary culture. (a) One
day post plating, PKP2 (green) and N-cadherin (red) relocalize
from the intercalated disks to the lateral sarcolemma. (b)–(e) By
day 3 post plating, very immature cell-cell contacts have begun
to form between the spreading cardiomyocytes. N-cadherin (red)
specifically localizes to these contact sites before PKP2 (green).
Note that these new cell contacts predominantly contain either N-
cadherin (arrowheads in (b)–(f)) or both N-cadherin and PKP2
((b)–(f): yellow) but not PKP2 alone. Scale bars are 20μm (b) and
10μm (c)–(f).
containdesmocollin.However,thelocalizationpatternswere
nearly identical, indicating simultaneous or virtually simul-
taneous incorporation of the cadherin-armadillo protein
complexes of both desmosomal and adherens junctions
subtypes into the new cell adhesive domains. The most
notable differences were in regions where N-cadherin was
noted along projections within the cell extending from the
site of cell-cell contact. These may represent filopodial-like
cellular projections that, once the contact with the adjoining
cellisestablished,recruitdesmosomalcomponentsincluding
PKP2 (Figures 4(a)–4(c)).
Localization of the desmosomal cadherins, PKP2 and
PG to overlapping sarcolemmal domains closely followed N-
cadherin incorporation. Variations in the relative abundance
of the two within the adhesive contacts (Figure 5) suggests
that, while PKP-2 and PG are localizing to the same
sarcolemmal regions concurrently, the presence of one may
notberequiredforthelocalizationoftheotherandbothmay
be dependent on the presence of other components, namely,
the cadherins, for proper incorporation into the adhesion
complex.
Insertion of the intermediate filaments into the desmo-
somal complex requires desmoplakin (DP), a plaque protein
that binds to intermediate filaments and to desmosomal
proteins such as PKP2, PG, desmocollin, and desmoglein.
DP appeared to be recruited to the newly-formed cell
contacts after the assembly of complexes of the cadherin and
armadillo family of proteins. In addition, it appeared to be
recruited to only a subset of the contact sites. In contrast
to PKP2 which distributed along the entire adhesive contact,
DP localization was more granular in appearance, indicating
the assembly of desmosomal plaques (Figure 6).
3.3. Connexin43 Concentrates at New IDs after the Assembly of
theAdhesiveJunction-ArmadilloProteinComplexes. Previous
studies have demonstrated that gap junction formation
is dependent on the prior assembly of adhesive contacts
in multiple cell types including cardiac myocytes [20–
22]. Disruption of either desmosomal or adherens junction
complexes results in the loss of gap junctions from the
intercalated disk and the development of malignant ventric-
ular arrhythmias [23, 24]. Prior work by Kostin et al. [25]
demonstrated that, in primary adult rat cardiac myocytes in
primary culture, both adherens junctions (as determined by
immunolocalization of N-cadherin and α- and β-catenin)
and desmosomal contacts (as determined by desmoplakin
and PG) were recruited to new cell-cell contacts prior to the
formation of gap junctions. We similarly noted the recruit-
ment of gap junctions selectively to membrane domains
involved in adhesive contacts (Figure 7). Subsarcolemmal
assemblyofconnexonsprecededtheirinsertionintoadhesive
domains (Figures 7(a), 7(b), and 7(d)).
3.4. Recruitment VGSCs to the Intercalated Disk. Previous
studies examining the localization of VGSC signaling com-
plexes to specific subcellular membrane domains have high-
lighted the role of ankyrins in the targeting and stabilization
process. It has been demonstrated that Nav1.5 physically
interacts with ankyrin-G [26]. A mutation in SCN5A, the
gene encoding Nav1.5, that impairs its ability to bind
ankyrin-Gresultsinlossofthechannelfromthesarcolemma
and causes Brugada Syndrome, an electrophysiologic defect
of the heart associated with ventricular fibrillation and

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Journal of Biomedicine and Biotechnology5
(a)
(d)
(e)
(f)(g)
(b)
(c)
Figure 3: Ultrastructural analysis of new cardiomyocyte cell-cell contacts at 5-days post plating. At this timepoint, cell contacts form
and develop rapidly, allowing the observation of both nascent (a, b, d) and more mature (c, e, f, g) contact sites. (a) Remodeling
cardiomyocytes extend fingerlike projections (arrowheads) that interact with neighboring cells to form adhesive contacts (arrows) thereby
electromechanically linking the two cells. (b, d) Higher magnifications of an adhesive domain indicated in (a) shows insertion of actin
filaments into nascent adherens junctions (AJs) (arrows in (b)). Note the nascent (white arrowheads in (d)) and more mature (black
arrowhead in (d)) desmosomal contacts in close proximity to the maturing AJ. (c) A mature adhesive domain again demonstrates
desmosomal contacts (arrowheads) near a newly formed adherens junction (arrow). (e)–(g) More mature adhesive contacts demonstrate
adherens junctions (arrows in (e)) at the insertion of the terminal actin filaments of newly formed myofibrils, along with closely associated
desmosomal contacts (arrowhead in (f)) and gap junctions (arrowhead in (g)). Scale bars are 500nm (a)–(f) and 100nm (g).
sudden death [27]. Recent studies have further demon-
strated an interaction between PKP2 and Nav1.5, suggesting
functional integration of the VGSC with other membrane
proteins such as ankyrin and with desmosomal adhesive
complexes [15].
Therefore, to determine the structural requirements
for the recruitment of the VGSC to newly formed IDs,
the pattern of localization of ankyrin-G, Nav1.5, and the
desmosomal protein PKP2 was examined at 5 and 13 days
post plating. By 5 days post plating, ankyrin-G was specifi-
cally recruited to sarcolemmal domains involved in cell-cell
contacts as determined by PKP2 colocalization (Figure 8).
It appeared to arrive at the new ID significantly prior to
the Nav1.5 α subunit, consistent with ankyrin-G recruiting
VGSCs to the sarcolemma. Furthermore, it suggests that
ankyrin-G is likewise dependent for its localization on
targetingmechanismsthatspecificallydirectittositesofcell-
cell contact.
The Nav1.5 α subunit itself arrives at the newly formed
ID much later than ankyrin-G. At the 5-day timepoint,
when new cell-cell contacts are forming and ankyrin-G is
beginningtoco-localizetocellcontactsites,immunostaining
for Nav1.5 demonstrates a more diffuse localization (Figures
9(a)–9(d)). The antibody detects epitopes closely associ-
ated with newly assembled myofibrils. Previous work has
demonstrated that membrane domains are closely associated
with these new myofibrils [28]. Whether or not this truly
represents localization of Nav1.5 to membrane structures