THE

JOURNAL • RESEARCH • www.fasebj.org

Cardiomyocyte–myofibroblast contact dynamism

is modulated by connexin-43
Francisca Schultz,*,1,2 Pamela Swiatlowska,*,1 Anita Alvarez-Laviada,*,1 Jose L. Sanchez-Alonso,*
Qianqian Song,* Antoine A. F. de Vries,† Daniël A. Pijnappels,† Emily Ongstad,‡ Vania M. M. Braga,§
Emilia Entcheva,{ Robert G. Gourdie,‡ Michele Miragoli,k,#,3 and Julia Gorelik*,4
*National Heart and Lung Institute, Imperial College London, London, United Kingdom; †Department of Cardiology, Leiden University
Medical Center, Leiden, The Netherlands; ‡Center for Heart and Regenerative Medicine, Virginia Tech Carilion Research Institute, Roanoke,
Virginia, USA; §Department of Respiratory Sciences, Faculty of Medicine, National Heart and Lung Institute, Imperial College London,
London, United Kingdom; {Department of Biomedical Engineering, George Washington University, Washington, DC, USA; kHumanitas
Clinical and Research Center, Milan, Italy; and #Department of Medicine and Surgery, University of Parma, Parma, Italy

ABSTRACT: Healthy cardiomyocytes are electrically coupled at the intercalated discs by gap junctions. In infarcted
hearts, adverse gap-junctional remodeling occurs in the border zone, where cardiomyocytes are chemically and
electrically influenced by myofibroblasts. The physical movement of these contacts remains unquantified. Using
scanning ion conductance microscopy, we show that intercellular contacts between cardiomyocytes and myofi-
broblasts are highly dynamic, mainly owing to the edge dynamics (lamellipodia) of the myofibroblasts. Decreasing
the amount of functional connexin-43 (Cx43) at the membrane through Cx43 silencing, suppression of Cx43 traf-
ficking, or hypoxia-induced Cx43 internalization attenuates heterocellular contact dynamism. However, we found
decreased dynamism and stabilized membrane contacts when cellular coupling was strengthened using 4-
phenylbutyrate (4PB). Fluorescent-dye transfer between cells showed that the extent of functional coupling be-
tween the 2 cell types correlated with contact dynamism. Intercellular calcein transfer from myofibroblasts to
cardiomyocytes is reduced after myofibroblast-specific Cx43 down-regulation. Conversely, 4PB-treated myofi-
broblasts increased their functional coupling to cardiomyocytes. Consistent with lamellipodia-mediated contacts,
latrunculin-B decreases dynamism, lowers physical communication between heterocellular pairs, and reduces Cx43
intensity in contact regions. Our data show that heterocellular cardiomyocyte–myofibroblast contacts exhibit high
dynamism. Therefore, Cx43 is a potential target for prevention of aberrant cardiomyocyte coupling and myofi-
broblast proliferation in the infarct border zone.—Schultz, F., Swiatlowska, P., Alvarez-Laviada, A., Sanchez-Alonso,
J. L., Song, Q., de Vries, A. A. F., Pijnappels, D. A., Ongstad, E., Braga, V. M. M., Entcheva, E., Gourdie, R. G., Miragoli,
M., Gorelik, J. Cardiomyocyte–myofibroblast contact dynamism is modulated by connexin-43. FASEB J.
33, 000–000 (2019). www.fasebj.org
KEY WORDS: heart failure • myocardial infarction • Cx43 • heterocellular coupling

ABBREVIATIONS: 4PB, 4-phenylbutyrate; CM–CM, cardiomyocyte– Communication between cells is necessary to maintain
cardiomyocyte; CM–MFB, cardiomyocyte–myofibroblast; Cx43, connexin- proper organ development and function. Coupling between
43; KD, knockdown; MFB–MFB, myofibroblast–myofibroblast; shRNA, both cells of the same cell type as well as between different cell
short hairpin RNA; siRNA, small interfering RNA; SICM, scanning ion
conductance microscopy; a-SMA, a-smooth muscle actin types (heterocellular coupling) occurs ubiquitously through-
1
These authors contributed equally to this work.
out numerous tissues (1–5). Formation of cell–cell contacts
2
Current affiliation: Springer Nature, London, United Kingdom. via stable junctions is the basis of cellular crosstalk. Twelve
3
Correspondence: Department of Medicine and Surgery, University of connexin-hemichannel proteins form a stable gap junction
Parma, Via Gramsci 14, 43124 Parma, Italy. E-mail: michele.miragoli@
unipr.it
between 2 cells. This coupling has been shown to be present in
4
Correspondence: National Heart and Lung Institute, 4th Floor, Imperial different cell types, such as neurons, vascular smooth muscle
Centre for Translational and Experimental Medicine, Imperial College cells, and cardiomyocytes (1–7). Independently of the tissue
London, Hammersmith Campus, Du Cane Rd., London W12 0NN,
United Kingdom. E-mail: j.gorelik@imperial.ac.uk and composition of different connexin subunits, these gap
junctions perform the same electrical and chemical functions.
doi: 10.1096/fj.201802740RR
This article includes supplemental data. Please visit http://www.fasebj.org to Intercellular communication controls critical physio-
obtain this information. logic processes, facilitates dynamic tissue responses, and

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 promotes tissue remodeling triggered by pathologic
events. In the healthy heart, operating at the highest level
of multicellular synchronicity of action, cellular packing
and strong electrical communication are essential for its
operation. Interdispersed among the highly coupled adult
ventricular cardiomyocytes are a large number of fibro-
blasts and myofibroblasts (8, 9). These mesenchymal cells
are 2 to 3 times more abundant than cardiomyocytes, but
because of their smaller size, they only comprise ;20% of
the cardiac muscle tissue (8). Interactions between car-
diomyocytes and fibroblasts occur in the heart at different
levels. The paracrine functions of fibroblasts have been
characterized, including secretion of TGF-b and other
soluble factors (10, 11). Of particular note, fibroblasts
synthesize extracellular matrix fibers, such as collagen and
fibronectin, which form the 3-dimensional scaffold of the
heart that supports the cardiomyocytes. It has also been
shown that cardiomyocytes and fibroblasts in the heart are
mechanically coupled either via the extracellular matrix or
via adherens junctions; however, little is known about the
importance of such interactions (12). Finally, electrical
coupling may occur between the 2 cell types (9), although
to what extent this occurs and what the role of hetero-
cellular coupling in the normal heart is remains poorly
understood. In contrast to cardiomyocytes, fibroblasts are
motile cells that constantly form new contacts with
neighboring cells and that remodel the extracellular ma-
trix; both of these processes depend on the actin cytoskel-
eton motility of cardiac fibroblasts. The potential impact of
the motility of fibroblasts on the coupling between car-
diomyocytes and fibroblasts has not yet been investigated.
After an ischemic event, such as myocardial infarction,
myofibroblasts proliferate, specifically in the region in
which the injury occurred, as well as in the surrounding
area. Fibroblasts are replaced by, or differentiate into,
myofibroblasts, which are characterized by a number of
distinct markers, including de novo expression of a-smooth
muscle actin (a-SMA) (13). Myofibroblasts have been im-
plicated in the formation of myocardial scars and scar
expansion, which in turn has been linked to an increase in
arrhythmogenesis (14–18). A narrow atypical zone of
myocardial tissue that surrounds the scar is where close
contacts between myofibroblasts and cardiomyocytes oc-
cur; this is in contrast to the scar proper, where few car-
diomyocytes survive because of necrosis (19).
Transformation or infiltration of myofibroblasts is
thought to be triggered by a number of signals, includ-
ing local inflammatory reactions, mechanical stress, and
various cytokines and biochemical factors (9, 17). Cul-
tured myofibroblasts have been shown to couple to car-
diomyocytes through gap junction-based interactions (14).
However, it is not clear whether gap-junctional coupling
occurs between myofibroblasts and cardiomyocytes in
the diseased myocardium in vivo, although a recent study
has shown that myofibroblasts can electrically couple to
cardiomyocytes in the infarct border zone of isolated,
Langendorff-perfused mouse hearts ex vivo (20). Camelliti
et al. (21) demonstrated that the main connexin sub-
types that are expressed by resident fibroblasts in infarct
scars are connexin-43(Cx43) and connexin-45 (Cx45) (14,
21, 22). It is thought that when an ischemic event occurs,
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cardiomyocytes undergo structural remodeling, with Cx43
moving from the intercalated discs at the polar ends of the
cell to the lateral surfaces. This lateralization of Cx43 in-
creases the likelihood of cardiomyocyte-to-myofibroblast
(CM–MFB) coupling and the associated risk of ar-
rhythmia (21).
Myofibroblasts appear to have complex roles, as,
depending on the setting, they have been implicated in
both pro- or antiarrhythmic modulation. Heterocellular
electrotonic communication has been shown to be det-
rimental to cardiac function in computer-modeling
and experimental studies because myofibroblasts
are thought to directly depolarize cardiomyocytes,
slowing action potential conduction and promoting
ectopic electrical activity (14, 23–26). On the other hand,
myofibroblast–cardiomyocyte coupling by gap junctions
can have a positive impact on conduction across discon-
tinuities in networks of cardiomyocytes (15). In a study
of recipient and donor atria, where a scar formed in the
region of the suture, evidence of successful restoration
of electrical activity was found (27). By acting as passive
electrical conduits for current flow, myofibroblasts were
thus shown to contribute to the electrical synchronization
between host and graft tissue following heart trans-
plantation. Similarly, expression of exogenous Cx43 in
infarct scar myofibroblasts has been demonstrated to
improve conduction and stabilize the electrical rhythm in
preclinical animal models of cardiac injury (28). In addi-
tion to the modulation of electrical properties, there is
growing evidence that heterocellular mechanical interac-
tions have a pivotal role in the structure and function of
the CM–MFB coupling zone and scar (29, 30).
Therefore, myofibroblasts appear to have complex
regulatory roles; depending on the setting, they are im-
plicated in either proarrhythmic or antiarrhythmic mod-
ulation of the heart after a pathologic event. Nevertheless,
the detailed cellular and molecular mechanisms by which
physical heterocellular contacts mediate effects in cardiac
disease remain poorly characterized. In this study, we
developed an in vitro model of heterocellular contacts that
are formed during heart failure and we investigated
whether electrical and mechanical coupling are indepen-
dent processes or whether they act in synergy during
CM–MFB zone expansion. We show that dynamism of
interactions with this model depends on gap-junctional
Cx43 levels.
MATERIALS AND METHODS
Neonatal rat cells used for contact motility studies
Isolation of neonatal rat ventricular cardiomyocytes and myofi-
broblasts from 1–2-d-old Sprague-Dawley rats was done in ac-
cordance with the guidelines of the Home Office Animal
(Scientific Procedures) Act of 1986 of the United Kingdom. Car-
diomyocytes and myofibroblasts were isolated and cultured as
previously described (31). Monocultures of cardiomyocytes or
myofibroblasts were seeded into MatTek dishes (13 cm2; MatTek,
Ashland, MA, USA) or plastic-bottom dishes (35 mm, CytoOne;
USA Scientific, Ocala, FL, USA). For the coculture experiments,
cardiomyocytes were also plated in dishes, with myofibroblasts
SCHULTZ ET AL.
 seeded on top on the subsequent day. The myofibroblasts were
detached as previously described (31), spun, and resuspended in
1 ml HBSS per 1 million cells. The myofibroblasts were then
labeled by incubating for 20 min at 37°C and 1% CO2 with either
Vybrant DiI (excitation/emission: 549/565 nm; Thermo Fisher
Scientific, Waltham, MA, USA) or WGA-488 (wheat germ
agglutinin-488, excitation/emission: 495/519 nm; Thermo Fisher
Scientific) for recognition afterward. The myofibroblasts were
then seeded in with the cardiomyocytes and were left to make
gap junctions for 24 h before imaging.
Adult rat fibroblasts used for contact
motility studies
Studies involving adult rats were performed in accordance with
the Guide for the Care and Use of Laboratory Animals [National
Institutes of Health (NIH), Bethesda, MD, USA]. For the myo-
cardial infarction model, male adult Sprague-Dawley rats
(250–300 g) were anesthetized and underwent proximal coro-
nary ligation to induce chronic myocardial infarction. Rats
were allowed to recover for 16 wk to induce heart failure.
Time-matched control animals that did not undergo surgery
were used as control. After 16 wk, in vivo pressure–volume
analysis was performed before the hearts were explanted,
weighed, and prepared for cell isolation as previously described
(32–34). Fibroblasts were collected from the supernatant of the
cardiomyocyte isolation. Cardiomyocytes were spun at 400 g for
1 min, after which the supernatant containing the fibroblasts was
collected. This supernatant was then spun at 1000 g for 10 min to
pellet the fibroblasts. The supernatant was removed, and the
pellet was resuspended in DMEM containing 10% fetal bovine
serum, 1% antibiotic/antimycotic solution, and 1% L-glutamine.
Myofibroblasts were plated in 25-cm2 flasks and grown until
confluency.
Modulation of gap junctions and Cx43
Various methods to modulate gap junctions were used. Over-
expression of Cx43 fused at its C terminus to enhanced green-
fluorescent protein (eGFP) was achieved using a first-generation
adenovirus vector (Cx43–eGFP) and knockdown (KD) was car-
ried out using small interfering RNA (siRNA) (199927; Thermo
Fisher Scientific, Waltham, MA, USA) or vesicular stomatitis vi-
rus G-protein-pseudotyped lentiviral vectors encoding GFP
and a Cx43-specific short hairpin RNA (shRNA) (35, 36). Cx43
modulation by zonula occludens-1 (ZO-1) was inhibited by a
membrane-permeable peptide inhibitor that contains the Cx43
C-terminal postsynaptic density-95/disks-large/ZO-1 (PDZ)-
binding domain (aCT1 peptide) (37). Cardiomyocytes and
myofibroblasts were incubated, separately or in cocultures, for
;4 h with the aCT1 peptide (100 mM). Increase in cellular
coupling was also achieved using a small molecule, 4PB
(Calbiochem, San Diego, CA, USA), which was previously
published to act as a gap-junction agonist, as well as shown to
increase Cx43 expression in cardiomyocytes (38). We therefore
added 1 mM 4PB to myofibroblasts alone or both cardiomyocytes
and myofibroblasts for 48 h at 37°C. 1-Heptanol was used to
completely uncouple gap junctions (39). Hypoxia leads to in-
ternalization of Cx43 (40). Hypoxia was induced by incubation
at 37°C, 1% CO2, and 8% O2 overnight (;15 h). Single cultures
were seeded at least 24 h before hypoxia, whereas in cocultures,
myofibroblasts were seeded into 1–2-d-old cardiomyocytes ;4 h
before hypoxia treatment. For the dynamism experiments with
hypoxia, cells were first treated with hypoxia, after which the
closed dish was taken out of the incubator and rapidly placed in
the small, self-contained chamber that is necessary for recording.
We used each dish for a maximum of 1 h and did not find any
CARDIOMYOCYTE-MYOFIBROBLAST CX43-DEPENDENT MOVEMENT
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differences in speed between the start and end. Oxygen levels
were measured at the end of the session and were found to not
have returned to normoxia before the end of the set of scans.
Cocultures were treated with latrunculin-B (100 mM), at 37°C for
24 h in order to disrupt actin stress fibers (41).
Scanning ion conductance microscopy
Scanning ion conductance microscopy (SICM) was used to
image the contact area between cells (Fig. 1A). In brief, SICM
provides continuous topographical images in hopping mode via
a feedback-controlled nanopipette scan with minimal spatial drift
(a few nm) (23). SICM is a noncontact method that allows the
imaging of the topography of intact cells (23, 42, 43). Surface
topographical images of cardiomyocytes and myofibroblasts
were acquired by SICM at 25°C in a low-calcium physiologic
solution (144 mM NaCl (MilliporeSigma, Burlington, MA, USA),
5 mM KCl (MilliporeSigma), 1 mM MgCl2 (MilliporeSigma),
10 mM HEPES, in 2 l dH2O, pH 7.4) or in HBSS, in order to avoid
contraction. A large scan (60–100 3 60–100 mm) was carried out
to identify the contact areas between cells. After that, a smaller,
higher resolution scan of ;15 3 15 mm of the contact area was
carried out and repeated for 1 h (loop) to visualize changes in the
contact area between 2 cells (Fig. 1A, C). Afterward, the images
(15 3 15 mm) were processed in chronological order using
the CellTrack software (44) (http://bio.cse.ohio-state.edu/CellTrack/;
Fig. 1A, C). The program returns the average of the speed (pixel
per frame) from the randomly selected points within the selected
contact area (Fig. 1C). By knowing the time of each acquired
image (hh:mm:ss) and the number of pixels for each image
(512 3 512 pixels), it was possible to evaluate the average
movement velocity (mm/min) of a selected area.
SICM in combination with confocal microscopy
Surface topographical images of cardiomyocytes and myofibro-
blasts were acquired by SICM at 25°C in low-calcium buffer. For
the visualization of Cx43 at the junctions, cells were incubated for
24 h with the first-generation adenovirus vector encoding
Cx43–GFP (Adeno-X Expression System; Takara, Kyoto, Japan)
at a multiplicity of infection of 9. Cx43–GFP was excited at 473 nm
with a Stradus 473 laser (Vortran, Sacramento, CA, USA) and
confocal images were made at 3100 magnification using a Pho-
tomultiplier Detection System (PTI). Using the SICM setup, the
tip of the pipette was aligned with the laser beam. The surface of
the cell was then scanned as a conventional SCIM image. The
same area was subsequently scanned with the laser to visualize
the Cx43. The resulting images could be overlapped with the
corresponding topography image of the surface (Fig. 1B).
Immunofluorescence microscopy
Immunofluorescence microscopy of vimentin (anti-chicken
PA1-16759, 1:3,000; Thermo Fisher Scientific), a-SMA (anti-
mouse MA511547, 1:500; Thermo Fisher Scientific), Cx43
(anti-rabbit C6219, 1:1000; MilliporeSigma), and DAPI (33342,
1:1000; Thermo Fisher Scientific) were used to assess the inter-
nalization of Cx43 after hypoxia and the effect of latrunculin-B
on the heterocellular contact.
Protein extraction and Western blots
Western blots for the gap-junctional protein, Cx43, were carried
out as previously described (45); b-actin was used as a loading
control. In brief, cells were plated in 35-mm dishes (0.5 million
3
 Figure 1. SICM and the analysis
of contact movement. A) Sche-
matic of the SICM setup (left),
cartoons representing the dif-
ferent cell configurations used
here (top right), and the
analysis using CellTrack (44)
(bottom right). CellTrack (44)
selects a set of random points
(yellow squares) in the contact
area that is selected (circled)
and follows these throughout
the scans to calculate the move-
ment of the cell contact. B)
Representative images of a
CM–MFB contact, first scanned
with SICM (left) and subse-
quently with the laser confocal
(middle); an overlay showing
the junction (SICM scan) and
GFP-Cx43 in the junction
(green) is shown on the right.
C ) Representative set of scans
of the looped scans at represen-
tative time points. Each scan
takes 4–6 min, generating be-
tween 10 and 20 images, al-
though some may not be used
as debris on the cell may in-
terfere with the SICM scan. t,
time; z, cell height. D) Repre-
sentative image of CM–CM and
CM–MFB Cx43 distribution at the
cell–cell contacts. Red, b-catenin;
green, Cx43; pink, vimentin; blue,
DAPI. Cx43 was expressed at
the CM–MFB interface. The white
arrows indicate the presence of
Cx43 at the cell–cell contacts.
Scale bars, 10 mm.

cells per well) and cultured for 72 h with or without treatment. incubated on a shaker for 15 min on ice, and centrifuged for 15
The cells were subsequently washed with cold PBS and the min at 4°C, 14,000 g. Protein concentrations were determined
protein was extracted using lysis buffer containing protein in- using a BCA Protein Assay according to the manufacturer’s
hibitors. Collected lysates were sonicated for 1 min at 0°C, protocol (Pierce, Rockford, IL, USA). For Western blotting, each

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 protein sample was fractioned in denaturating conditions by
SDS–PAGE electrophoresis. A Trans-Blot Turbo System (Bio-Rad,
Hercules, CA, USA) was used to transfer the proteins to the
membrane (Bio-Rad). Membranes were blocked for 1 h in 5% milk
(MilliporeSigma) and then incubated overnight at 4°C in primary
antibody (Cx43, C6219, anti-rabbit 1:1,000, MilliporeSigma;
b-actin, A3854, anti-mouse 1:50,000; MilliporeSigma) diluted in
5% milk. The next day membranes were washed 3 times in
Tris-buffered saline with Tween-20 (TBST; 1 M Tris pH 8.0, NaCl
diluted in distilled water) and incubated with horseradish per-
oxidase (HRP)-conjugated secondary antibodies (7074, donkey
anti-rabbit 1:1000, Cell Signaling Technology, Danvers, MA,
USA; 7076, donkey anti-mouse 1:1000; Cell Signaling Technology)
diluted in TBST for 1 h at room temperature. Blots were visualized
using Luminata Forte Western HRP substrate (MilliporeSigma).
ImageJ software (NIH) was used to quantify bands that were
normalized to b-actin.
Parachute assays
Myofibroblasts were plated on 35-mm dishes and, upon con-
fluency, treated with the indicated treatments. For 4PB experi-
ments, myofibroblasts were treated for 48 h to increase Cx43
expression. To achieve Cx43 KD, myofibroblasts were either
transfected with Cx43-specific siRNA or transduced with lentiviral
vector particles expressing a Cx43-specific shRNA to silence Cx43
and eGFP to detect the transfected cells, prior to the dye staining.
Cells were incubated with 5 mM Cell Tracker Orange/FarRed
(Thermo Fisher Scientific) and 1 mM Calcein/Calcein Orange
(Thermo Fisher Scientific) for 30 min at 37°C, trypsinized and
plated on cardiomyocytes in a 1:2 ratio to form heterocellular
contacts. Cells were cocultured for 24 h. Live-cell imaging was
performed on a Zeiss Laser Scanning Microscope 780 Confocal.
Only cells that were calcein-positive and that were directly ad-
jacent to dually labeled fibroblasts (transferring cells) were
counted.
Cell boundary measurement
Quantification of images was performed using a custom-made
computer vision program to identify cell–cell contact (courtesy of
V.B.). In brief, the cell boundary was selected based on the a-SMA
protein staining and manually calibrated. The threshold and di-
lation cycle measurements were set for the interface area to pick
up only Cx43 expressed at the cell boundary, and these were kept
constant throughout all image analyses. Cx43 labeling was
identified and calculated as the intensity in Cx43 area (arbitrary
units/pixel2). Data are presented as means 6 SEM.
Statistical analysis
A statistical analysis was carried out in Prism 7 (GraphPad, La
Jolla, CA, USA) for all the scanning data and Western blots. All
data are represented as means 6 SEM. All data were tested for
normality before a Kruskal–Wallis test with post hoc tests (scans,
Western blotting) or Student’s t test (parachute assays) was carried
out. For all scanning data, n represents the number of 1 h scan sets
that were used. More image sets were carried out but may not have
been used for various reasons, including noise in the scan due to
debris on the cell, which interferes with the scan of the membrane.
All data were carried out with at least 1 dish of cells; dishes were
changed after 1–2 scan sets, especially for the hypoxia work, as the
scans were carried out at normal oxygen tension, which meant that
changes induced by hypoxia may have reverted back after 1–2 h.
Most experiments were carried out with cells from at least 2 sep-
arate isolations (10–15 pups/isolation).
CARDIOMYOCYTE-MYOFIBROBLAST CX43-DEPENDENT MOVEMENT
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RESULTS
Dynamic measurement of cell–cell contacts
We used neonatal rat cardiomyocytes and myofibroblasts
to establish a baseline (control) for the dynamism. After an
area was identified to contain a cluster of both myofibro-
blasts (identified by Vibrant DiI or WGA-488) and car-
diomyocytes (in the heterocellular cultures), or clusters of
either cell type (in homocellular cultures), a large scan was
obtained using SICM, followed by recording a set of scans
for 60 min. We then followed the contact over time and
analyzed the dynamism (see Materials and Methods). The
presence of gap junctions at the cellular interfaces was
confirmed using cardiomyocytes expressing Cx43 with
GFP fused to its C terminus (Cx43–GFP; Fig. 1). First, a
topographical image was obtained and subsequently, us-
ing confocal microscopy, the same area was imaged and
the Cx43–GFP was visualized. Each scan took ;5 min,
representative SICM and confocal images of Cx43–GFP at
the junction of a cardiomyocyte and myofibroblast
(CM–MFB) are shown in Fig. 1B. An example of the dis-
tribution of Cx43 at the cardiomyocyte–cardiomyocyte
(CM–CM) and CM–MFB contacts is presented in Fig. 1D.
Cx43-mediated coupling has implications for
cell–cell border zone dynamism
We evaluated the dynamism of neonatal rat ventricular
CM–CM, CM–MFB, and myofibroblast–myofibroblast
(MFB–MFB) contacts (Fig. 2). The CM–CM pairs, known
to be coupled by the electrical gap-junctional protein Cx43,
displayed minimal movement [0.04 6 0.005 mm/min, n =
14 scan sets (data were obtained from $2 dishes per iso-
lation from n $ 3 isolations, unless otherwise indicated)],
whereas the border zone, modeled by the coculture of
cardiomyocytes and myofibroblasts, showed a significant
increase in dynamism (0.14 6 0.033 mm/min, n = 11 scan
sets, P # 0.001). Interestingly, MFB–MFB contacts dis-
played a considerable level of dynamism in culture (0.44 6
0.22 mm/min, n = 7 scan sets, P # 0.05), compared to
heterocellular cell pairs.
Membrane Cx43-deficient cells show
limited dynamism
The involvement of Cx43 in the CM–MFB and MFB–MFB
dynamism was elucidated by infecting myofibroblasts
(or cardiomyocytes in CM–CM control experiments)
with lentiviral vector particles encoding GFP and a Cx43-
specific shRNA (Cx43–GFP shRNA) or by using siRNA
against Cx43 (Fig. 3). Green fluorescence was used to
identify myofibroblasts in which Cx43 was successfully
silenced. The myofibroblast membrane was also labeled
with a lipophilic membrane dye (Vybrant DiI, Thermo
Fisher Scientific; see Supplemental Fig. S1). Cx43 KD sig-
nificantly slowed the cell–cell dynamism of contacts in-
volving myofibroblasts: CM–MFB (0.017 6 0.013 mm/
min, n = 6 scan sets, n = 2 isolations, P # 0.01) and
MFB–MFB (0.027 6 0.007 mm/min, n = 7 scan sets, n = 2
5
 Figure 2. Dynamism in control CM–CM, CM–MFB, and MFB–MFB contacts. A) Representative set of scans for each
configuration. Cartoons indicate the configuration, as shown in Fig. 1. The red line is included for the contact between
MFB–MFB for clarity. t, time; z, cell height. B) Movement speed of the contacts. All data are represented as means 6 SEM. *P #
0.05, ***P # 0.001.

isolations, P # 0.01) but did not significantly affect car-
diomyocytes alone [0.035 6 0.012, n = 5 scan sets (from a
single isolation)] (Fig. 3A, B). By contrast, 50 mM heptanol
(a gap-junctional uncoupler) did not slow the dynamism
in CM–MFB (0.047 6 0.010, n = 7 scan sets, n = 2 isolations)
and MFB–MFB (0.059 6 0.016, n = 6 scan sets, n = 2 iso-
lations), suggesting that the presence of gap junctions on
the myofibroblast membrane, and not their function, is
required to activate the dynamic coupling (Supplemental
Fig. S2).
To confirm that the gap junctions between car-
diomyocytes and myofibroblasts were functional and to
investigate the extent of direct physical coupling, para-
chute assays were carried out (Fig. 3C, D). Control acceptor

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 Figure 3. Cx43 KD leads to
decreased dynamism and re-
duced dye transfer. A) Repre-
sentative set of scans for each
configuration. Illustrations in-
dicate the configuration, as
shown in Fig. 1. t, time; z, cell
height. B) Movement speed of
the contacts after Cx43 KD
using siRNA in only the myofi-
broblasts (in CM–MFB cultures)
or in either cell type in the
monocultures. C–E ) Cx43 KD
using Cx43-specific siRNA or
Cx43–GFP shRNA in only the
myofibroblasts leads to de-
creased dye transfer and there-
fore a reduced number of
functional gap junctions. C )
Analysis of the parachute assay.
Cx43 siRNA, n = 52 images (for
all parachute assays: the total
number of images from 2 dishes
per isolation for n = 3 isolations
are indicated); control, n = 61
images. Cx43–GFP shRNA, n =
29 images; control, n = 29
images. D) Representative im-
ages of control and Cx43 siRNA.
Calcein (green) is used as a
cell-permeable dye, whereas
CellTracker Orange (red) is a
cell-impermeable dye. E ) Rep-
resentative images of control
and Cx43–GFP shRNA. GFP
(green) is used to identify
myofibroblasts with Cx43 KD (only
shown in the shRNA-treated im-
ages). Calcein Orange (orange) is
used as a cell-permeable dye,
whereas CellTracker FarRed
(magenta) is a cell-impermeable
dye. Scale bars, 100 mm. All data
are represented as means 6 SEM.
*P # 0.05, **P # 0.01, ***P #
0.001.

cells (cardiomyocytes) were plated and control or myofibroblasts had been dually labeled with permeable
Cx43-deficient myofibroblasts [Cx43 siRNA (Fig. 3D, bot- (Calcein or Calcein Orange) and impermeable (CellTracker
tom left) or Cx43–GFP shRNA (Fig. 3D, bottom right)] were Orange or CellTracker FarRed) dyes. Myofibroblasts with
added to cultures of cardiomyocytes. Prior to addition, reduced Cx43 expression due to treatment with either Cx43

CARDIOMYOCYTE-MYOFIBROBLAST CX43-DEPENDENT MOVEMENT 7

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 siRNA [50.50 6 6.457%, n = 52 images (for all parachute
assays: the total number of images from 2 dishes per iso-
lation for n = 3 isolations are indicated); control, 100 6
6.236%, n = 61 images; P # 0.001] or Cx43–GFP shRNA
(55.63 6 2.662%, n = 29 images; control, 100 6 7.333%, n = 29
images; P # 0.001) showed significantly attenuated calcein
transfer compared to the respective control conditions
(normalized to 100%).
Increased cellular coupling induced by 4PB
A small molecule (4PB) has previously been shown to act as
an epigenetic modulator that increases cellular functional
coupling between cardiomyocytes (38). We used this mol-
ecule as a method to strengthen Cx43 coupling between
cardiomyocytes and myofibroblasts (Fig. 4). 4PB induced a
significant reduction in dynamism in the CM–MFB con-
figuration (Fig. 4C), when both cell types were incubated
with the small molecule (0.063 6 0.012, n = 29, P # 0.05), but
not when only the myofibroblasts were incubated with 4PB
(0.183 6 0.086, n = 55). Using an in vitro assay of direct
cell–cell communication in the parachute assay, there is a
significant increase in dye transfer in both cases; 4PB
CM–MFB: 159.3 6 10.64%, n = 29 vs. control (100 6 8.479%,
n = 33) and myofibroblasts only treated with 4PB: 159.3 6
9.961%, n = 55 vs. control (100 6 7.425%, n = 53) (Fig. 4A, B).
To confirm KD of Cx43 and to investigate whether 4PB
has a similar effect on myofibroblasts as was previously
published in cardiomyocytes (38), Western blotting was
carried out (Fig. 4D, E). 4PB treatment did not lead to a
significant increase in Cx43 levels in cardiomyocytes or
myofibroblasts, in contrast to previous findings in car-
diomyocytes (38). However, this may be caused by dif-
ferences in incubation time or technical repeats (n = 3–4)
and a potentially lower baseline level of Cx43 expression
in myofibroblasts compared to cardiomyocytes. Fol-
lowing Cx43 KD, Cx43 protein levels were reduced in

Figure 4. Increased cellular

coupling induced by 4PB. A)
Representative images of con-
trol cocultures and cocultures
with 4PB-treated myofibroblasts.
Calcein (green) is used as
a cell-permeable dye, whereas
CellTracker Orange (red) is a
cell-impermeable dye. Scale
bars, 100 mm. B) Analysis of the
parachute assay. 4PB treatment
of cocultured cardiomyocytes
and myofibroblasts (n = 29
images, n = 3 isolations) or
myofibroblasts alone (n = 55
images, n = 3 isolations) leads
to increased calcein transfer. C )
Treatment of both cardiomyo-
cytes and myofibroblasts, but not
myofibroblasts alone, leads to
reduced movement speed in
the contact. D, E) Western blot
analysis of Cx43 (n = 3–4 in-
dependent isolations). Quantifi-
cation (D) and representative
blots (E) are shown. AU, arbi-
trary units. All data are repre-
sented as means 6 SEM. *P #
0.05, ***P # 0.001.

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 cardiomyocytes to 69% of control (P = 0.057), and although
not significant, a trend toward reduction was found in
myofibroblasts; Cx43 expression was reduced to 41% of
control values (n = 3–4, P = 0.094; Fig. 4D, E). As Cx43
expression is generally lower in myofibroblasts compared
to cardiomyocytes (35, 46), this reduction was found to be
sufficient to induce the observed changes in membrane
motility and calcein transfer.
Hypoxia-induced internalization of Cx43
induced a decrease in dynamism
The presence of Cx43 on the membranes of isolated car-
diomyocytes and myofibroblasts was assessed by cultur-
ing the cells in hypoxic conditions (8% O2) for 12–16 h
before measuring cell–cell dynamism (Fig. 5B). Hypoxia is
known to internalize the connexons in the plasmalemma of
cardiomyocytes (40) and myofibroblasts (Fig. 5B, E and
Supplemental Fig. S3C). As postulated, for both CM–MFB
[0.067 6 0.021, n = 7 scan sets (3 dishes each from 2 isola-
tions), P # 0.05] and MFB–MFB (0.100 6 0.025, n = 13 scan
sets, P # 0.05) models, limited dynamism was measured
when cells were cultured in hypoxic conditions compared
to normoxic (control) conditions. This indicates that the
presence of Cx43, and thus the capacity to establish cell–cell
electrical coupling, is necessary for cell–cell border zone
movement. In terms of functional coupling, chronic hyp-
oxia also caused a reduction in dye transfer from neonatal
myofibroblasts to neonatal cardiomyocytes (Supplemental
Fig. S3A, B).
Inducing recruitment of Cx43 to the cell
membrane using the mimetic aCT1 peptide
A second approach was used to examine the effects of
increased levels of Cx43-mediated contact. The mimetic
Cx43 peptide aCT1 has previously been shown to pro-
mote the extent of Cx43 gap junction-mediated contacts
between cells (37), as well as reducing inducible arrhyth-
mias at the CM–MFB zone following ventricular cry-
oinjury (19). We therefore investigated the effect of the
peptide in our cellular heterocellular model (Fig. 5). In both
cardiomyocyte and myofibroblast monocultures, as well
as in cocultures of these cells, the aCT1 peptide induced
the occurrence of Cx43 at the contact regions between cells.
The increased presence of Cx43-mediated contacts in-
creased CM–MFB dynamism [0.415 6 0.090 mm/min, n =
5 scan sets (1–2 dishes from 3 isolations)], indicating a
novel “mechanical” role for an “electrical” intercellular
connection (Fig. 5C, F). By contrast, hypoxic conditions, in
the presence of the aCT1 peptide [0.057 6 0.015 mm/min,
n = 6 scan sets (3 dishes each from 2 isolations)], or hypoxia
after a 4-h preincubation with the aCT1 peptide [0.050 6
0.018 mm/min, n = 3 (3 dishes from a single isolation)],
significantly reduced dynamism (Fig. 5) despite Cx43 lo-
calization at heterocellular junctions (data not shown).
Therefore, we investigated whether enhanced Cx43-
mediated coupling had a functional role in intercellular
communication between cardiomyocytes and myofibro-
blasts (Fig. 5G). Myofibroblasts were dually labeled with the
CARDIOMYOCYTE-MYOFIBROBLAST CX43-DEPENDENT MOVEMENT
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cell-permeable dye Calcein and cell-impermeable dye Cell-
Tracker Orange and treated with either the aCT1 or reverse
(control) peptide. The myofibroblasts were then parachuted
onto cardiomyocytes, which were also treated with either the
aCT1 or reverse peptide (Fig. 5G; n = 4 isolations/treatment).
Although not significant, a small decrease in gap junction
intercellular communication, after treatment with the aCT1
peptide, was observed (P = 0.12) with respect to control.
Actin filament-based modification of Cx43
Myofibroblasts are known to promote conduction slowing
and ectopic activity in cardiac tissue (25, 47). As previously
shown (41), these arrhythmogenic properties can be sup-
pressed by disruption of a-SMA stress fibers using phar-
macological manipulation. Moreover, trafficking of Cx43
to the membrane is mediated by a-actin filaments. To in-
vestigate the role of a-SMA in cell membrane mobility
and to prevent intercellular gap-junctional communication
and functional Cx43 connexon expression on the sarco-
lemma, neonatal CM–MFB cocultures were treated with
latrunculin-B, an inhibitor of actin polymerization. After
incubation for 24 h, the myofibroblast dynamism was sig-
nificantly decreased [0.03 6 0.006 mm/min, n = 9 scan sets
(2 and 4 dishes from 2 isolations), P # 0.05; Fig. 6A, C]. Cell
membrane mobility was then examined in myofibroblasts
isolated from a rat model of myocardial infarction (16 wk
postinfarction) (32). a-SMA ablation resulted in lower cell
dynamism (0.03 6 0.004 mm/min, n = 13 scan sets) com-
pared to untreated myofibroblasts from the same model
(0.12 6 0.041 mm/min, P # 0.01; Fig. 6E, G). We assessed the
functionality of CM–MFB contacts in both models using a
parachute assay and observed a significant reduction in
calcein transfer following treatment with latrunculin-B in
cocultures of neonatal cardiomyocytes and myofibroblasts
(latrunculin-B, 20.18 6 5.113%, n = 38 images, n = 3; control,
100 6 4.744%, n = 35 images, n = 3; P # 0.001; Fig. 6B, D) and
among adult rats with myocardial infarction (latrunculin-B,
25.8 6 3.8%, n = 14 images, n = 3; control, 100 6 10.36%, n =
14 images, n = 3; P # 0.001) (Fig. 6F, H). Similar to the
decrease in calcein transfer seen after latrunculin-B treat-
ment of myofibroblast cultures derived from adult rats with
myocardial infarction, control (sham) rat myofibroblast
cultures also showed a reduction (42%, P , 0.001) in dye
transfer after latrunculin-B treatment (Supplemental Fig.
S4C, D); this was accompanied by a decrease in cell dyna-
mism (Supplemental Fig. S4A, B). This indicates that
myofibroblasts derived from both adult rats with myo-
cardial infarction and sham-treated adult rats are sensitive
to the effects of latrunculin-B. Significant effects of both
condition [sham vs. myocardial infarction (P = 0.0006)] and
treatment [control vs. latrunculin-B (P = 0.0364)] on contact
dynamism was found (2-way ANOVA), but the interaction
between treatment and condition was not significant.
Changes in the level of Cx43 expression in the
different treatment groups
In order to further validate that the changes seen were due
to the functional effects of Cx43, protein levels were
9
 Figure 5. Removing Cx43 by hypoxia and forcing Cx43 to the membrane by aCT1 peptide. A–D) Representative scans of control
and treatments. z, cell height. E ) Hypoxia reduces the dynamism between cells. F ) aCT1 treatment leads to increased dynamism
in control, but not in hypoxic, conditions. G) Treatment with the aCT1, but not the reverse, peptide leads to decreased dye
transfer, showing that, although the plaque size increases, the number of functional gap junctions decreases. All data are
represented as means 6 SEM. *P # 0.05, ***P # 0.001. For representative Western blots and quantification of Cx43 protein
expression in cardiomyocytes and myofibroblasts treated with hypoxia or aCT1, see Fig. 7A, B.

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 Figure 6. Treatment with latrunculin-B abolishes contact dynamism and dye transfer. A, B) Representative SICM scans (A) and images of
parachute assays (B) of control CM–MFB cultures and CM–MFB cultures treated with latrunculin-B from neonatal rats. z, cell height. C,
D) Treatment with latrunculin-B leads to reduced dynamism (C) and a reduction in the number of functional contacts (D). B, D,
Control, n = 35 images, n = 3 isolations; Latrunculin-B, n = 39 images, n = 3 isolations. For representative Western blots and quantification
of Cx43 protein expression in cardiomyocytes and myofibroblasts treated with latrunculin-B, see Fig. 7A, B. E, F) Representative SICM
scans (E) and images of parachute assays (F) of myofibroblasts cultures derived from adult rats with myocardial infarction that were
treated with latrunculin-B or vehicle control (MFB–MFB). For experiments carried out using myofibroblasts from sham-treated adult
rats, see Supplemental Fig. S4. G, H) Treatment with latrunculin-B leads to reduced dynamism (G) and a reduction in the number of
functional contacts (H). F, H, Control, n = 24 images, n = 3 isolations; Latrunculin-B, n = 25 images, n = 3 isolations. CTO, CellTracker
Orange; LatB, latrunculin-B. All data are represented as means 6 SEM. Scale bars, 100 mm. *P # 0.05, **P # 0.01, ***P # 0.001.

CARDIOMYOCYTE-MYOFIBROBLAST CX43-DEPENDENT MOVEMENT 11

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 assessed in separate cultures of neonatal cardiomyocytes
and fibroblasts using Western blot. Hypoxic conditions
reduce the levels of Cx43 in both cell types. A marked drop
in Cx43 expression is also observed in the myofibroblast
cultures treated with latrunculin-B. By contrast, aCT1
treatment notably increased the level of the Cx43 expres-
sion in myofibroblast samples (Fig. 7A, B).
Immunofluorescence staining was performed to visu-
alize the contacts that had formed. Heterocellular connec-
tions were further quantified using a custom-made cell
boundary measurement tool (V.B. laboratory). Quantify-
ing the Cx43 intensity in the Cx43 area, we observed that
the disruption of a-SMA reduced the functionality of
CM–MFB Cx43-based coupling compared to the untreated
sample (n = 15 images, n = 3 isolations, P # 0.01; Fig. 7A, B),
which is consistent with the parachute assay data of cul-
tures treated with latrunculin-B.
DISCUSSION
Pathologic events, such as myocardial infarction, trigger
complex changes and dynamic remodeling of the cardiac
tissue, which occurs over time. Through coordinated reg-
ulation of the gap-junctional proteins and the extracellular
matrix in the affected region, the irreversibly damaged
myocardium becomes electrically quarantined from the
rest of the heart to restrict the spread of adverse effects; the
border zone area between the infarct and the healthy tissue
undergoes highly dynamic changes in cell–cell commu-
nication. It has been reported that myofibroblasts coupled
to cardiomyocytes show an increase in Cx43 expression
and an associated increase in gap junction formation (21).
Cx43-based contact between myofibroblasts and car-
diomyocytes is thought to contribute to ectopic activity and
arrhythmias (25, 47). In the present study, we show a novel

Figure 7. Quantification of Cx43

expression in control, hypoxia-,
latrunculin-B-, and aCT1-treated
samples. A) Cx43 and loading
control [glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH)]
intensity bands in neonatal
cardiomyocyte and myofibroblast
cultures (n 5 2–4 independent
isolations). B) Quantification of
Western blots shown in A. All
data are represented as means 6
SEM; n = 2–4 independent isola-
tions. C ) Latrunculin-B treat-
ment significantly decreases the
Cx43 intensity in the Cx43
clusters (n = 15 images, n = 3
isolations). All data are repre-
sented as means 6 SEM. **P #
0.01. D) Representative images
of immunostaining of CM–MFB
contacts. Red, a-SMA; green,
Cx43; pink, vimentin; blue, DAPI.
Cx43 was expressed at the
CM–MFB interface. Scale bars,
10 mm. AU, arbitrary units;
latrunculin-B, LatB.

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 Cx43-dependent aspect of myofibroblast behavior that may
contribute to heart failure pathology. Specifically, myofi-
broblasts demonstrate highly motile contacts with each
other and with cardiomyocytes. This unique dynamism is
characterized by the rapid formation of new areas of contact
and similarly efficient remodeling of existing interactions.
By contrast, cardiomyocytes in culture form stable gap
junctions with each other and have very low movement in
zones of mutual Cx43-based interactions—consistent with
the idea that myocardial cell-to-cell coupling is important
for the propagation of electrical impulse (48).
This dynamic ability to remodel cell–cell contacts appears
to be necessary for the supportive roles of myofibroblasts.
An interesting question arises as to what the relationship
between these physical contact dynamics and the drivers of
electrical coupling, Cx43 gap junctions, is. In this study, us-
ing a variety of methods to modulate assembly of Cx43 and
actin, we aimed to address this question. Our results indicate
that this relationship (contact dynamism and electrical
coupling) is not simple and monotonic. The levels of
gap-junctional interaction require tight control to ensure
that, while sufficient levels of intercellular coupling are in
place, gap junction-mediated adhesions are not so strong as
to restrict the dynamism of heterocellular contacts. Follow-
ing myocardial infarction, myofibroblasts showed an in-
crease in Cx43 expression and associated level of gap
junction formation (21).
In our experiments, CM–MFB cultures exhibited much
higher membrane contact dynamics than cardiomyocyte
monocultures. This difference in the vigor of cell–cell
membrane contact dynamics was associated with differ-
ences in Cx43 expression and the stability of intercellular
Cx43 connections. We show that, although myofibroblasts
had lower Cx43 expression (35) than cardiomyocytes, Cx43
functionally couples cardiomyocytes and myofibroblasts,
as indicated by intercellular calcein transfer. It appears that
Cx43 serves both as an affector and as a sensor of contact
dynamism between cells. Furthermore, we determined that
decreasing the level of Cx43 through Cx43 silencing, by
preventing Cx43 trafficking, or through Cx43 internaliza-
tion reduced contact dynamism.
This is correlated with a reduction in dye transfer and,
thus, a reduction in gap-junctional coupling between
myofibroblasts and cardiomyocytes. By contrast, increas-
ing membrane Cx43 levels did not necessarily lead to an
increase in coupling and dynamism. We found that 4PB
increased coupling between cardiomyocytes and myofi-
broblasts without changing Cx43 protein levels (Fig. 4);
however, interestingly, it decreased dynamism.
In human glioblastoma cells, it was shown that levels of
unphosphorylated Cx43 were increased after treatment
with 4PB, whereas the phosphorylated form remained sta-
ble (49). We assume that the stabilization at the membrane
that we have previously shown (38) occurs through a
combined increase of both forms. Notably, 4PB affects Cx43
epigenetically; therefore, there is not necessarily a change in
expression. 4PB is known to exert multiple effects. 4PB is
an epigenetic modulator that increases cellular functional
coupling between 2 cardiomyocytes (unpublished data),
2 myofibroblasts or between a cardiomyocyte and myofi-
broblast. Among the nonepigenetic effects of 4PB is its
CARDIOMYOCYTE-MYOFIBROBLAST CX43-DEPENDENT MOVEMENT
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recognized action as a chemical chaperone, which can re-
duce endoplasmic reticulum stress and can attenuate the
unfolded protein response (50, 51). Better folding and im-
proved trafficking to the membrane can give rise to better
and more stable (low dynamism) electrical coupling even if
Cx43 transcription is not significantly changed, which is
probably the case here.
This suggests that there may be an optimal level of
Cx43-mediated electrical coupling that allows for high dy-
namism of CM–MFB contacts. When Cx43 levels are too low
in the contact area, the edge motility of myofibroblasts and
heterocellular contact dynamics are reduced. When the
electrical coupling is too high in the contact area, this becomes
too restrictive and dynamism is also reduced. Myofibroblasts
are normally only lightly coupled to each other and to car-
diomyocytes, with contacts breaking and reforming contin-
uously; they are highly mobile and show a lot of plasticity.
For instance, in vitro studies have shown that they are capa-
ble of bridging gaps among cardiomyocytes of up to 300 mm
(15, 21). Increasing the number of Cx43 connexons on the
myofibroblast membrane that are effectively coupled to their
counterparts in the cardiomyocyte membrane increases the
strength of the CM–MFB contacts, and as the pair starts be-
having as one, the motility of the myofibroblast is reduced.
Treatment with the aCT1 peptide has been shown to lead
to an increase in Cx43 plaque size and a decrease in Cx43
hemichannels (37, 52). Although treatment with the aCT1
peptide led to an increase in dynamism, this was associated
with a small decrease in dye transfer. This can be explained
by the fact that, after treatment of the cells with the aCT1
peptide, the phosphorylation status of Cx43 is likely to
have changed, as phosphorylation is one of the main ways
in which Cx43 channels are regulated through post-
translational modification (53–55). We have previously re-
ported that aCT1 increased Cx43 phosphorylation at S368 at
the interface between cardiomyocyte and myofibroblast in
vivo (19), a post-translational modification known to be as-
sociated with decreased Cx43 channel activity (56). We have
observed that aCT1 similarly increased S368 phosphoryla-
tion in our in vitro CM–MFB model (57). The observed re-
duction in dye transfer between coupled cells is therefore
likely to be explained by this change in Cx43 phosphoryla-
tion status, despite an increase in Cx43 expression and plaque
size. In addition, with recruitment of Cx43 to the membrane,
myofibroblasts treated with the aCT1-peptide showed in-
creased lamellipodia formation, which may indicate their
search for stable connections with neighboring cells.
It may be hypothesized that other modifications, such
as ubiquitination (which would decrease the amount of
protein at the junctions) are reduced or changed (for ex-
ample, acetylation or methylation) after treatment with
our interventions; however, it is beyond the scope of this
study to analyze these modifications further.
It has been shown that Cx43 trafficking to the sarco-
lemma of cardiomyocytes is dependent on both the mi-
crotubular network (58) and actin cytoskeleton (59).
Previously, the role of a-SMA stress fibers in myofibro-
blasts has been investigated, and it has been shown that
pharmacological ablation of these cytoskeletal elements
eliminates the arrhythmogenic effects of myofibroblasts
on cardiomyocytes (12, 41, 59). We therefore expanded on
13
 this research by studying the effect of pharmacological
ablation of the actin cytoskeleton in cocultures of car-
diomyocytes and myofibroblasts to investigate the impact
on heterocellular contact dynamism. Disruption of the
actin cytoskeleton significantly reduced CM–MFB contact
movement in cocultures of neonatal rat cardiomyocytes
with either neonatal rat myofibroblasts or myofibroblasts
derived from adult rats with myocardial infarction. Sub-
sequently, we investigated the diminishing heterocellular
contact functionally. Using a custom-made cell–cell mea-
surement software (V.B. laboratory), we found a partial
drop in Cx43 intensity in the Cx43 area after a-SMA stress
fiber removal, which could be related to the actin-dependent
Cx43 pool that has previously been described (59). These
results indicate that Cx43 not only affects contact dyna-
mism, but also that the opposite is true—Cx43 coupling
senses and reacts to actin dynamics.
How the Cx43-dependent myofibroblast dynamism
characterized in our study affects the formation of proar-
rhythmic substrates at the heterocellular coupling area
should be investigated further. The irregularity of the in-
terface between surviving cardiac muscle and the scar tissue
of myocardial infarction is a well-characterized determinant
and originator of re-entrant arrhythmias (60, 61). Modeling
studies have shown that microstructural variations in car-
diac tissue facilitate the formation of isolated sites of wave-
front breakthrough that may enable abnormal electrical
activity; these small regions of heterogeneous diseased tissue
can develop more widespread re-entrant activity (62, 63).
The relationship between Cx43 expression and level of
heterocellular contact dynamism that we report here likely
has implications for the disposition of mechanical forces
imparted by myofibroblasts on cardiomyocytes. How
Cx43-based mechano-interactions at the microscale shape

Figure 8. Schematic of our current view on the role of Cx43 in CM–MFB dynamism. Max., maximum; min., minimum.

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 the macrostructure of the extended heterocellular interface
thus may be of key importance to understanding the for-
mation and pathogenesis of arrhythmic re-entrant circuits.
In summary, here we reveal the complex relationship
between Cx43-mediated heterocellular electrical coupling
and physical contact dynamism (Fig. 8). We show that
Cx43 is not only involved in electrical coupling between
cardiomyocytes and myofibroblasts, but is also important
for the mechanical properties of the contacts. We provide
new insights into Cx43-mediated coupling between car-
diomyocytes and nanoscale evidence of the formation and
dynamism of heterocellular contacts. Mechanistically, we
demonstrate that Cx43 is necessary for both the dynamism
of structural membrane contacts and for direct physical
coupling of myofibroblast and cardiomyocytes in an in
vitro infarct border zone model.
ACKNOWLEDGMENTS
The authors thank P. O’Gara (Imperial College London,
London, United Kingdom) for the assistance with the isolation of
adult rat ventricular fibroblasts and M. Sikkel (Imperial College
London, London) and C. Mansfield (Imperial College London,
London) for generating rat models of myocardial infarction. This
work was supported by British Heart Foundation (RG/17/13/
33173 to J.G.), Medical Research Council (MR/L006855/1 to
J.G.), National Heart, Lung, and Blood Institute (NHLBI), U.S.
National Institutes of Health Grant-1R01HL141855-01 (to R.G.,
J.G. and A.A.-L.), Heart Research UK, and by Italian Ministry of
Education, Universities and Research (FFABR-MIUR-2017 to
M.M.). M.M. and J.G. jointly supervised this work. The authors
declare no conflicts of interest.
AUTHOR CONTRIBUTIONS
M. Miragoli and J. Gorelik conceived the study; F. Schultz,
P. Swiatlowska, A. Alvarez-Laviada, M. Miragoli, and J. Gorelik
designed experiments; F. Schultz, P. Swiatlowska,
A. Alvarez-Laviada, and M. Miragoli isolated, cultured, and
treated the cells; F. Schultz, P. Swiatlowska, and M. Miragoli
carried out scanning ion conductance microscopy (SICM)
experiments; A. Alvarez-Laviada and J. L. Sanchez-Alonso
carried out SICM/confocal experiments; A. Alvarez-Laviada
and E. Ongstad performed parachute assays; F. Schultz,
P. Swiatlowska, A. Alvarez-Laviada, Q. Song, and M. Miragoli
analysed the data; F. Schultz, P. Swiatlowska, and M. Miragoli
carried out Western blots; A. A. F. de Vries, D. A. Pijnappels,
V. M. M. Braga, E. Entcheva, and R. G. Gourdie provided
advice or supervision, assisted in analysis, provided reagents
and commented on the manuscript; and F. Schultz wrote
the manuscript with input from P. Swiatlowska, A. Alvarez-
Laviada, J. L. Sanchez-Alonso, V. M. M. Braga, E. Entcheva,
R. G. Gourdie, M. Miragoli, and J. Gorelik.
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Received for publication December 17, 2018.
Accepted for publication May 29, 2019.
SCHULTZ ET AL.