(The FASEB Journal. 1999;13:S83-S89.)
© 1999 FASEB
In vitro reestablishment of cell–cell contacts in adult rat cardiomyocytes. Functional role of transmembrane components in the formation of new intercalated disk-like cell contacts
HANS M. EPPENBERGER1 and
CHRISTIAN ZUPPINGER
Department of Biology, Institute of Cell Biology, Swiss Federal Institute of Technology ETH, CH-8093 Zurich, Switzerland
1Correspondence: Institute of Cell Biology, ETH-Hoenggerberg, CH-8093 Zurich, Switzerland. E-mail: hme{at}cell.biol.ethz.ch
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ABSTRACT
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Primary adult rat cardiomyocytes (ARC)in culture are shown to
be a model system for cardiac cell hypertrophy in vitro.
ARC undergo a process of morphological transformation and grow only by
increase in cell size, however, without loss of the cardiac
phenotype. The isolated cells spread and establish new cell-cell
contacts, eventually forming a two-dimensional heart tissue-like
synchronously beating cell sheet. The reformation of specific
cell contacts (intercalated disks) is shown also between ventricular
and atrial cardiomyocytes by using antibodies against the gap junction
protein connexin-43 and after microinjection into ARC of N-cadherin
cDNA fused to reporter green fluorescent protein (GFP) cDNA. The
expressed fusion protein allowed the study of live cell cultures and of
the dynamics of the adherens junction protein N-cadherin during the
formation of new cell-cell contacts. The possible use of the formed ARC
cell-sheet cells under microgravity conditions as a test system for the
reformation of the cytoskeleton of heart muscle cells is
proposed.—Eppenberger, H. M., Zuppinger, C. In vitro
reestablishment of cell–cell contacts in adult rat cardiomyocytes.
Functional role of transmembrane components in the formation of new
intercalated disk-like cell contacts.
Key Words: cardiomyocytes • N-cadherin • gap junction • GFP
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INTRODUCTION
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HEART FAILURE still represents the major cause
of death (45%) in the industrial world and basic as well as clinical
research in molecular and cellular cardiology is a major task needed to
overcome this situation. Cardiac hypertrophy is known to be one of the
first adaptive responses to pathological conditions of the heart but
further deterioration leads to heart failure, representing the end
stage of cardiac hypertrophy and resulting almost unavoidably in death.
The need for an adequate rate of systemic circulation is met by
adaptation of the cardiac system to acute or chronically altered
demands. If an increased demand becomes long-lasting, e.g., through
training or through pathological changes like mechanical overload,
myocardial infarction caused by stress, the heart muscle reacts by
increasing its size (in fact by increasing the size of cardiomyocytes),
whereby genetic activities involved in cell structure formation and
building of the contractile apparatus may also be changed.
Although during embryonic, fetal, and early neonatal growth cardiac
enlargement involves an increase in cell size (hypertrophy) as well as
cell proliferation (hyperplasia), adult cardiomyocytes grow by
hypertrophy only. The loss of mitotic activity, however, depends on the
species. It occurs in small animals much earlier than, e.g., in humans;
DNA replication leading to multinucleation without cytokinesis is
nevertheless observed at an age when no more mitotic activity is seen
(1)
.
Taken together, heart muscle is able to compensate for increased demand
to a certain degree, through hypertrophy, and also for cell loss caused
by necrosis and/or apoptosis but is unable to replace dead cells
through new ones, i.e. no repair potential is available. This lead to
the idea of using cardiomyocyte cell cultures to eventually replace
dead cardiomyocytes, and possibly to repair, e.g., the site of an
infarction by implanting heart muscle cells derived from an ex
vivo-in vitro-in vivo system, thus possibly
avoiding the need for transplanting a whole heart.
We have shown that in vitro adult rat cardiomyocytes can
form new cell–cell contacts, a precondition for implantation, by
reconstructing intercalated disk-like structures and eventually forming
a synchronously beating heart tissue-like cellular layer
(2)
. This possibly could be used for implantation if
provided on a biocompatible matrix.
We will give a brief review of the techniques developed and used to
produce primary cardiomyocyte cultures
(ARC)2
in our laboratory. In addition we will present some recent results
obtained by using a fusion protein consisting of the coding sequence of
green fluorescent protein (GFP) (3)
and N-cadherin
(4)
. N-cadherin represents a major component of the
intercalated disk where it participates in forming the so-called
adherens junctions. It has been shown to be a necessary factor for
reestablishing new cell-cell contacts in primary cell cultures
(5)
.
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MATERIALS AND METHODS
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Cell culture
Ventricular and atrial cardiac muscle cells of adult female rats
(2 months old, from Sprague-Dawley-Ivanovas) were isolated by
retrograde perfusion of the hearts with type II collagenase
(Worthington Biochemical Corp., Freehold, NJ) according to a method
that has been previously described (6
, 7
).
Culture dishes were coated with 0.1% gelatin. Culture medium contained
M-199 (Animed AG, Basel, Switzerland), 20 mM creatine (Sigma Chemical
Co., St. Louis, MO), 1% penicillin/streptomycin (GIBCO, Grand
Island, NY), and 20% preselected, heat-inactivated (56°C) fetal calf
serum (FCS, GIBCO). To inhibit fibroblast outgrowth, cytosine
arabinoside (10 mM) was added throughout the culture period. Medium was
changed after 2, 7, 11, or 14 days. For live observations, cells were
plated on culture dishes with glass coverslips glued onto a hole in the
bottom of the dish. The coverslips were coated with laminin at a
concentration of 2 µg/cm2 (GIBCO). This coating procedure
resulted in a similar cellular phenotype as the standard gelatin-coated
cell culture dishes. Culture medium was based on M-199 (Amimed AG) and
contained 20 mM creatine (Sigma), 1% penicillin/streptomycin (GIBCO),
and 20% preselected, heat-inactivated FCS (GIBCO). To inhibit
fibroblast proliferation, cytosine arabinoside (10 µM) was added
throughout the culture period. Atrial ARC were isolated from the
auricles of already perfused hearts. The tissue was digested three
times for 15 min at 37°C in a solution containing collagenase (200
units/ml) and pancreatin (0.6 mg/ml; GIBCO-BRL, Life Technologies,
Basel). Culture medium was changed on days 3, 7, and 12. In cultures
repeatedly used for GFP-imaging medium was changed after each recording
session.
Microscopy and image processing
Immunocytochemical preparations and fluorescence in living cells
were analyzed using a confocal Leica TCS NT scanner on the Leica
DMIRB-E inverted microscope. Objectives used were x40 and x63 (1.4 NA
PL APO). Cross-talk of simultaneously recorded channels was corrected
by selective AOTF-tuning of laser intensity. Pinhole size for all
channels was set to the optimal value recommended by the manufacturer.
For single-channel image recording of EGFP-fluorescence, an appropriate
filterset was used (TK500 and wide band-pass filter 525/50). When used
for recording living cells, laser intensity was reduced to less than
20% of the maximal output. Further image processing was done on
Silicon Graphics workstations using Imaris (Bitplane, Zurich), a 3-D
multi-channel image processing software. Video time-lapse
phase-contrast images were recorded using an AVT-Horn camera and a
video digitizer board. A climate box on a heated microscope stage
(Zeiss) provided an incubator environment during the observation
period. Images were further processed in Adobe Photoshop (gamma
correction and layout) and printed on a Fuijtsu Pictography color
printer.
Electron microscopy
Cardiomyocytes cultured on laminin-coated glass coverslips were
fixed for 3 h with 2% glutaraldehyde (Fluka) in 0.1 M sodium
cacodylate buffer at 4°C. Samples were then postfixed for 1 h in
1% osmium tetroxide, dehydrated stepwise to 70% ethanol, and stained
en bloc for 1 h in 2% uranyl acetate in 70% ethanol.
After dehydration was completed, the cells were infiltrated with an
epoxy resin based on Epon 812 (Fluka) in a series of ascending mixtures
of epoxy/propylenoxide. Gelatin capsules filled with 100% resin were
placed on the coverslips and the samples were heat polymerized for
48 h at 60°C. Ultrathin sections were cut on a Diatom
ultramicrotome (Reichert), placed on carbon- and formvar-coated
copper grids (Provac, Balzers, FL), and poststained with uranyl acetate
and lead citrate. Preparations were examined in a JEM100C transmission
electron microscope at 80 kV.
Immunocytochemistry
Monoclonal anti-myomesin antibodies were prepared as previously
described (8)
. Polyclonal anti-connexin-43 antibodies were
a generous gift of D. Gros (Faculté des Sciences de Luminy,
Marseille). Secondary antibodies were from Jackson Immunochemicals
(fluorescein isothiocyanate, Texas Red). For immunocytochemistry,
culture dishes were rinsed with phosphate-buffered saline (PBS), fixed
with 3% paraformaldehyde in PBS for 15 min, and permeabilized for 5
min in 0.2% Triton X-100 in PBS. Incubation with the primary
antibodies diluted in blocking buffer containing bovine serum albumin
ranged from 2 h to overnight at 4°C.
Microinjection of plasmid constructs
ARC were grown 5 days before microinjection. Microinjection of
cDNA constructs was done using an Eppendorf Microinjector model 5242.
Desalted plasmid DNA was diluted to 0.1 µg/µl with phosphate
buffer. Before injection, ARC cultures were changed to L-15 medium
(GIBCO) supplemented as described above for the cardiomyocyte culture
medium and containing 60 mM BDM (2,3-butanedione monoxime) to inhibit
the beating activity of the cardiomyocytes and to reduce the risk of
hypercontraction during injection. ARC were analyzed when GFP
fluorescence became detectable, usually 24–48 h after injection.
Molecular genetic techniques
Chicken N-cadherin cDNA cloned as a 3.2-kb fragment in the pECE
eukaryotic expression vector (9)
was a generous gift from
B. Geiger (Weizmann Institute, Rehovot, Israel). A Sal I/Apa I fragment
was isolated and ligated in-frame into the multiple cloning site of the
expression vector pEGFP-N3 (Clonetech) upstream of the coding sequences
of the GFPmut1 variant (red-shifted) of GFP, subsequently named
enhanced GFP (EGFP). The expression of the fusion construct NcadEGFP
was driven by the CMV immediate early gene promotor. Linker size
between N-cadherin and the EGFP coding sequence was 18 bp of the
multiple cloning site. The vector was dephosphorylated with arctic
shrimp alkaline phosphatase (Promega) before ligation.
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RESULTS AND DISCUSSION
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Remodeling and adaptive flexibility of heart muscle is observed
in vivo under hypertrophy conditions and the capacity of
differentiated cardiac muscle to adapt to changing demands has been
demonstrated accordingly (10)
. Isolated ARC in primary
culture represent an excellent system to monitor changes in the
molecular and cellular behavior of the cells, which equally appear
within the cardiomyocytes of heart muscle in vivo. After
isolation and during cultivation ARC pass through several gradual
morphological transitions that can be followed by video time-lapse
microscopy (VTL). A sequence of four VTL stages is shown in
Figure 1
,a–e. Starting with the elongated, rod-shaped
cardiomyocytes present in adult cardiac muscle tissue, cells
progress to a rounded shape whereby many of the myotypic structures and
organelles like myofibrils and intercalated disks are degraded. The
general heart phenotype, however, is not lost; after some time, the ARC
attach to the substrate, followed by flattening and spreading into a
polymorphic cell morphology. The enlarged cells form new contacts and
electron microscopy of similar cells confirmed the formation and
existence of new adherens junctions containing intercalated disks (Fig. 1f
). The cells also resume contraction, a sure sign
that myofibrils have been reformed. The appearance, in a perinuclear
way, of newly built myofibrils gradually extending toward the periphery
of the cell has been described earlier (2)
. Figure 2
demonstrates that dissociated and isolated cultured cardiomyocytes
(Fig. 2b
), after some time, rebuild a cardiac tissue-like
cell sheet (Fig. 2c
) that is again electrically coupled,
beats rhythmically, and strongly resembles the tissue sections in
Figure 2a
. Other culture systems have been established,
especially those that simulate the mechanical environment of the
neonatal heart (or skeletal muscle) by progressive unidirectional
stretch (11)
. In such cultures, a confluent cardiomyocyte
population organizes into parallel and multilayered arrays of
rod-shaped cells. The strength of static cultures, as demonstrated in
this report, lies in the possibility to monitor single cells during the
culture period. In the stretched culture marked by organogenic growth,
single cells have to again be isolated enzymatically to determine
parameters such as cell size.
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Figure 1. Morphological changes, loss of cell polarity, and reestablishment of
new cell-cell contacts during long-term culture. The same field of a
culture dish is shown as a series of video time-lapse images starting
on day 1 after isolation (a) and going on by one image on
every following day (b–e). The sequence
illustrates the different spreading speed of individual cells (numbered
1–4) and the resulting change in morphology of hypercontracted
(a, number 1) or rod-shaped (a, number 2–4) ARC,
which frequently undergo rounding-up during the first days of culture
(b–d, number 4). First de novo
cell-cell contacts occur by side-to-side alignment during attachment
(number 2 and 3) or by the extension of pseudopods (for example number
1 in panel d). In panel e, all cells have
established contact, finally forming a monolayer of contracting cells.
(f) Transmission electron micrograph showing
ultrastructural details of a cell-cell contact site. Different junction
types are present: a gap junction plaque (GJ, arrowhead) flanked by
adhesive junctions. Short filaments terminating from both sides in the
desmosomal junction plaque can be recognized (arrows). Original
magnification x20,000.
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Figure 2. Comparison of gap junctions and myofibrillar array in heart tissue
(a), in freshly isolated cells (b), and in
long-term-cultured ARC (c). Cryosections of adult rat
ventricle, freshly isolated cells, and cell cultures were immunostained
for connexin-43 (green) and myomesin (red) and recorded by confocal
microscopy. Entire stacks of optical sections were then used to obtain
shadow projection images using the image processing software Imaris.
Arrows in panel c point to intercalated disk-like structures marked by
gap junctions.
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In the rat, as in other mammals, mitotic activity of cardiomyocytes
stops shortly after birth. This is one of the reasons that no repair
mechanism is effective after cell damage within the myocardium. A
compensatory mechanism to meet increased demand in the myocardium is
enlargement of the cells accompanied by formation of additional
myofibrils (hypertrophy). ARC in culture, as in in vivo,
cannot divide. However, as can be seen in Figure 1
, after some time in
culture ARC tend very strongly to reestablish contact with neighboring
cells. Because no increase in cell number takes place and because cells
are not able to move on the substrate under the given culture
condition, new cell-cell contacts can only be made by increasing the
size of the individual cell. Thus, an in vitro model for
cellular hypertrophy is available that shows changes in a number of
parameters that also occur upon remodeling in vivo under
hypertrophic conditions (10)
. An increase of surface area
of about five times compared to isolated rod-shaped cardiomyocytes has
been shown by Messerli et al. (12)
. This enlargement
necessitates changes in the cellular cytoskeleton that presumably also
occur in hypertrophic cells in vivo during remodeling of
ventricular cell mass (13)
. We think that influence of
microgravity on the reconstitution of heart tissue in cell cultures
could definitely be of particular interest for tissue engineering
purposes. A direct influence on the cytoskeletal and myofibrillar
behavior is to be expected, which is itself linked to the establishment
of cell-matrix and cell-cell contacts. The feasibility of heart
cultures under conditions of continuous free-fall was demonstrated for
cardiac tissue constructs that contracted spontaneously and
synchronously (14)
and for neonatal rat heart cells in
three-dimensional cultures kept in simulated microgravity
(15)
. The main argument for using adult cardiomyocytes
lies in the fact that they are derived from a terminally
differentiated heart tissue and that it is the type of cell that also
in vivo is confronted with hypertrophic stimuli, such as
overload or other pathological events (16)
. Further
detailed characterization of the cardiomyocyte phenotype cultured
under microgravity conditions may allow conclusions relevant to human
cardiovascular behavior.
The establishment of new cell-cell contacts in ARC is accompanied by a
renewal of adherens-type junctions and of gap junctions allowing
electrical coupling of the contact sites. The degradation of
intercalated disk junctions in the early stages of ARC culture
preparations has been described on a solely structural basis by
Claycomb et al. (17)
. Although the fate of gap junctions
during cultivation has attracted much interest (18
,
19
), adherens junction degradation and regulation has been
little studied so far. From our laboratory, some data have been
reported on the spatial and temporal appearance of N-cadherin,
plakoglobin, and catenin complex (20)
. Recently we
attempted to use co-cultures of ventricular and atrial cardiomyocytes
to demonstrate the formation of contact sites also between cell types
from independent locations within the heart. As seen in Figure 3
,the contact interface shows equal properties in both cell types. The
small rounded atrial heart muscle cell established contact with the
further-developed ventricular cardiomyocytes and new gap junctions were
formed by connexin-43 (Fig. 3d
). Metabolic and electrical
coupling were shown to be functional by transfer of lucifer yellow, a
low-molecular-weight dye, from atrial to ventricular cells. This result
is of particular interest when one considers the above-mentioned
possibility of grafting cardiac muscle cells into infarcted regions of
the heart muscle. It allows the assumption that, e.g., atrial
appendix-derived cardiomyocytes could be used after surgery as a source
for the preparation of transplantable cardio-sheets. Successful
preparations of primary cultures from human atrial cells have been
achieved in our laboratory (J. Feucht, unpublished
observations). So far, several authors have proposed the use of rat
embryo cardiomyocytes (21
, 22
) or skeletal
muscle cells (23)
for substituting lost cardiomyocytes in
diseased heart.
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Figure 3. Development of cell-cell contacts in a co-culture of ventricular and
atrial cardiomyocytes (a–c). Ventricular
cardiomyocytes were cultured for 7 days before freshly isolated atrial
cells (a, arrows) were added. After 3 days, the atrial ARC
had established contacts with ventricular cardiomyocytes while
the spreading process of both cell types went on (b). The
atrial cell on the left made contact by spike-like pseudopods (white
arrowheads), whereas the atrial cell to the right sent out long
filamentous pseudopods along the whole contact site (black arrowheads).
Three days later the spreading process of both cell types was completed
(c). The left atrial cell filled up the contact site (white
arrowhead), whereas the cell to the right partially retracted.
Co-cultures were then immunostained for myomesin (red) and connexin-43
(green). An example of such a heterotypic contact is shown in panel
d.
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The one protein thought to be the first component for establishing new
cell–cell contacts is N-cadherin, which is a transmembrane
glycoprotein that belongs to the Ca2+-dependent cell
adhesion protein family. The extracellular domain of cadherins mediates
the homophilic interaction among neighboring cells, whereas the
cytoplasmic carboxy-terminal tail contains binding domains for proteins
mediating the interaction with the cytoskeleton (24
,
25
). It has been demonstrated by Hertig et al.
(20
, 26
) that N-cadherin in ARC is the first
component of the disrupted intercalated disk to be degraded but also
the first one to reappear when new contacts were to be made. However,
by common immunohistochemical techniques no conclusive results could be
obtained as to what way new N-cadherin was expressed, transported to
the cell membrane, and introduced into the membrane in order to make
contact with neighboring cells. The usually employed technique required
fixation and hence, killing of the cell before analysis
(27)
. In addition, cross-linking of cytoplasmic proteins
through fixation and permeabilization of cells in order to allow
antibodies to enter the cell could lead to a loss of newly synthesized
protein pools. To answer our question on the dynamic distribution of
N-cadherin in isolated and newly interacting ARC we constructed a
fusion protein of N-cadherin and GFP as a reporter (Fig. 4
). Figure 5
demonstrates the ectopic expression under a CMV-promotor of the fusion
protein after microinjection in living cells. It became possible to
follow over several days the dynamics of N-cadherin on its way to the
cell membrane thanks to the green fluorescent reporter protein. The
fusion protein sorted to identical sites where endogenous N-cadherin
was also located.
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Figure 4. Schematic representation of a fusion protein consisting of N-cadherin
and the EGFP reporter (NcadEGFP). Depicted are, in dimensions of the
respective coding sequence, the five extracellular cadherin domains
(conventionally denoted as EC1–5), one transmembrane domain (TM), and
the cytoplasmic domain (CP) linked to EGFP.
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Figure 5. Establishment of intercalated disk-like structures and strengthening of
cell contacts visualized by the fluorescence of an N-cadherin-GFP
fusion protein in living cells. The expression vector was injected into
one nucleus of the cardiomyocyte in the middle that had been cultured
for 5 days and had not yet completed the spreading process. Living
cells were recorded 1 day later for the first time (a) and
again after 24 h (b). A phase-contrast image
(a, b) and the corresponding confocal
GFP-fluorescence image (average of the whole stack of optical sections)
is shown from each time point (a', b'). In panel
a, the contact site between the cell in the upper right
corner and the NcadEGFP-expressing cell (arrowheads) was still
incomplete and gaps devoid of NcadEGFP-fluorescence were observed
(arrows). Twenty-four hours later, the contact site was closed and a
connected line of NcadEGFP-fluorescence was observed (arrow).
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Adult cardiomyocytes in culture represent a valuable model for
studying the flexibility and the potential adaptability of cardiac
muscle cells to various conditions influencing heart development,
differentiation, and pathology. Gravity changes may directly influence
intracellular cytoskeletal structures in several ways, as has been
shown, e.g., for osteoblasts (28)
. It can be expected that
the cardiomyocyte morphology will be affected by changes in gravity
level and it would be of particular interest to study under
microgravity conditions the influence of growth factors like IGF-1 or
bFGF because a direct effect of the growth factors on cell size and
myofibril formation has been demonstrated (29)
. They may
be used to simulate a number of molecular and cellular conditions and
to study how cells cope with stress situations that in vivo
may result in irreversible cardiac damage. It is only under in
vitro conditions that one can elucidate the factors for the
formation of new cell contacts necessary for the creation of a cardiac
cell sheet and later on for a successful reconstruction of an
intercalated disk between heart muscle tissue and a possible
graft.
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ACKNOWLEDGMENTS
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This study was supported by Swiss National Science Foundation Grant
No. 31-40485.94. The authors would like to thank Lyudmilla Polonchuk
and Sigrid Aigner from our laboratory for providing parts of Figure 2
,
and the European Space Agency for the invitation to publish data from
our contribution to the Workshop Cell and Molecular Biology
Research in Space in Leuven 1998.
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FOOTNOTES
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2 Abbreviations: ARC, adult rat cardiomyocytes;
GFP, green fluorescent protein; FCS, fetal calf serum; PBS,
phosphate-buffered saline.
Received for publication September 28, 1998.
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S. Kostin and J. Schaper
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Circ. Res.,
May 11, 2001;
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[Abstract]
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ABSTRACT
INTRODUCTION
