International Journal of Cardiology 173 (2014) 5564

Contents lists available at ScienceDirect

International Journal of Cardiology

journal homepage: www.elsevier.com/locate/ijcard

In vitro and in vivo characteristics of connexin 43-modied human

skeletal myoblasts as candidates for prospective stem cell therapy for the
failing heart
T.J. Kolanowski a,1, N. Rozwadowska a,1, A. Malcher a, E. Szymczyk b, J.D. Kasprzak b,
T. Mietkiewski c, M. Kurpisz a,,2
a
b
c

Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland

Department of Cardiology, Medical University of Lodz, Poland
Traumatology and Orthopaedics Department, District Hospital of Wielkopolska, Poznan, Poland

a r t i c l e

i n f o

Article history:
Received 5 April 2013
Received in revised form 26 September 2013
Accepted 8 February 2014
Available online 20 February 2014
Keywords:
Human skeletal myoblasts
Connexin 43
Cell therapy
Genetic modication
Post-infarction model

a b s t r a c t
Background: Previously, connexin 43-modied skeletal myoblasts (MbCx) were shown to reduce the proarrhythmic effect during the regeneration of heart tissue in an animal model of infarction. To increase the
relevance to clinical implementation, in this study, we introduced connexin 43 into human myoblasts
using a highly safe non-viral vector and demonstrated that their transplantation had a positive effect on
the function of the injured heart.
Methods and results: Myoblasts were efciently transfected with a pCiNeo-GJA1 plasmid (65.72%). qPCR
analysis revealed over 32-fold higher expression of the connexin 43 gene in the MbCx cell population compared to native controls. The susceptibility of the myoblasts to oxidative stress conditions (p b 0.001) and
the fusion index (p b 0.01) were increased in the MbCx cells. Additionally, we observed changes in the
MYOG and MYH2 gene expression levels in the GJA1-modied myoblasts. Finally, we observed a signicant
improvement in the post-infarction echocardiographic parameters after intervention using MbCx cells
compared with non-transfected myoblasts (MbWt) and the control (0.9% NaCl), wherein a signicant decrease in the left ventricular area change in the short axis (SAX AC%) was observed at the two-month
follow-up (p b 0.05 and p b 0.01, respectively).
Conclusions: We demonstrated the positive effect of connexin 43 overexpression on the biology and function of human skeletal myoblasts in the context of their potential clinical applications. Our preclinical studies using a mouse infarction model indicated the positive effect of MbCx implantation on the function of the
injured heart.
2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Regenerative therapy of the infarcted heart has been extensively studied for several years. Numerous stem-cell types have been examined
to improve an injured heart's function [15]; however, the effects of the
The study was supported by grants: R13006506 from the National Centre for Research
and Development and 2011/01/N/NZ1/04454 from National Centre of Science of Poland.
TJK declares that he is a scholarship holder within "Scholarship support for PH.D. students
specializing in majors strategic for Wielkopolska's development", Sub-measure 8.2.2
Human Capital Operational Programme, co-nanced by European Union under the
European Social Fund.
Corresponding author at: Institute of Human Genetics Polish Academy of Sciences,
Strzeszynska 32, 60-479 Poznan, Poland. Tel.: +48 61 6579 202/212; fax: +48 61 8233
235.
E-mail address: kurpimac@man.poznan.pl (M. Kurpisz).
1
These authors contributed equally to this study.
2
This author takes responsibility for all aspects of the reliability and freedom from bias
of the data presented and their discussed interpretation.

http://dx.doi.org/10.1016/j.ijcard.2014.02.009
0167-5273/ 2014 Elsevier Ireland Ltd. All rights reserved.

applied cell therapies remain controversial. Among the tested cells,

human skeletal myoblasts (SkM) have also been used. Because they already have a contractile mechanism, as well as a high proliferative potential and easy access (muscle biopsy), they were considered promising
candidates for autologous cell transplantation [6]. Moreover, skeletal
myoblasts' commitment to the myogenic lineage gives them physiological characteristics closely related to those of the myocardium and reduces the risk of uncontrolled development. Although experiments
provided evidence that the transplanted myoblasts were able to repopulate scar tissue, it appeared that the improvement of the heart's
haemodynamic parameters and the inhibition of cardiac remodelling
were the most benecial effects of implanting stem cells into an injured heart [7]. Unfortunately, during a number of clinical trials, limitations of skeletal myoblast-based therapy were also observed. The
most disputed limitation was the inability to create a functional syncytium
with the recipient cardiomyocytes, which could cause life-threatening arrhythmia [8]. This constraint originated from the down-regulation of

56

T.J. Kolanowski et al. / International Journal of Cardiology 173 (2014) 5564

connexin 43 expression in myoblasts undergoing differentiation into

myocytes [9], as opposed to cardiomyocyte membrane structures in
which connexons are permanently present [10].
Connexin 43 (GJA1; Gene ID: 2697) is a member of the connexin
family, which includes transmembrane proteins that form gap junctions
between adjacent cells. Gap junctions consist of two hemichannels
consisting of hexamers of these molecules located in the membranes
of adjoining cells [11]. Direct communication through gap junctions
has been proven to be fundamental for coordinating cardiac cell function and the immediate transfer of action potentials and small molecules [12].
In recent years, several studies concerning skeletal myoblast
cardiomyocyte coupling in regenerative therapies of the injured
heart have been published. The most popular strategy to ensure
communication with the transplanted cell grafts was to induce the
overexpression of connexin 43 using genetic engineering; however,
the alternative approaches of oscillating pressure treatment [13] and
mechanical preconditioning [14] were also tested. The studies of Roell
et al., who used connexin 43-modied mouse myoblasts, not only
showed the elimination of the arrhythmic potential but also the prevention of post-infarction arrhythmia by increasing cell-to-cell coupling
[15]. The hypothesis that connexin 43 has an anti-arrhythmic potential
was bolstered in recent studies using a pig model [16]. However, in
experiments performed by Fernandes et al. using Wistar rats, despite
evidence of proper electrical coupling, no anti-arrhythmic potential
was observed for genetically modied myoblasts transplanted into injured hearts [17].
In this study, we focused on characterising a connexin-43 modied
human skeletal myoblast population to conrm the improvement of
heart function as a consequence of stem cell engraftment and to

evaluate the effect of GJA1 overexpression on the biological functions

and myogenic features of the myoblasts. These effects were manifested
in the expression levels of muscle-specic transcription factors as well
as in the biological properties induced by GJA1 overexpression.
2. Materials and methods
2.1. Myoblast isolation and culture
The tissue collection protocol was approved by the Local Ethical Committee of the
Medical University of Poznan. Fragments of the skeletal muscle tissue were harvested
from patients under local anaesthesia during anterior cruciate ligament resection. After removing the remnants of adipose tissue from the obtained muscle fragments, the specimens were subjected to digestion using a 0.2% solution of collagenase 1 (Sigma-Aldrich,
St. Louis, MO, USA) for 45 min in a water bath (33 C) with constant orbital shaking.
Subsequently, the cell suspensions were ltered through an 80 m mesh, centrifuged
and seeded in 75 cm2 culture asks (Becton Dickinson, Franklin Lakes, NJ, USA) coated
with 0.1% gelatine (Sigma-Aldrich). Myoblasts were isolated using the pre-plating technique, as previously described [18]. The culture medium comprised 20% FBS (Lonza,
Basel, Switzerland), 1% UltraGlutamine (Lonza), high-glucose DMEM (Lonza) and the appropriate growth factors, as previously described [19]. After they achieved approximately
70% conuence, the cells were harvested using a trypsin-EDTA solution (Sigma-Aldrich),
centrifuged and seeded in culture asks at a cell density of approximately 7 103/cm3.
To induce myogenic differentiation, medium containing 2% horse serum and lacking
growth factors was provided to cultures at 100% conuence, which were cultured for a
week, after which time properly fused multinucleated myotubes were easily observed.
2.2. Plasmid preparation and transfection
The pCiNeo plasmid (Promega, Fitchburg, WI, USA) containing the coding sequence
for the connexin 43 gene (GJA1, NM_000165.3) and the pEGFP-C1 plasmid, for estimation
of the electroporation efciency (Promega), were prepared as previously described [20].
Briey, the coding sequence of GJA1 was amplied using a cDNA template obtained
from commercially available human heart RNA (Life Technologies, Carlsbad, CA, USA)
and cloned into the pCiNeo vector. The primers used included sequences recognised by

Fig. 1. Evaluation of transfection efcacy. A, B ow cytometry cytograms of GFP overexpression in transfected (B) and control human myoblasts (A). C, D images of GFP-transfected cells under
a light microscope (C) and in the green uorescence channel (D).

T.J. Kolanowski et al. / International Journal of Cardiology 173 (2014) 5564

57

Fig. 2. Overexpression of connexin 43 at the RNA and protein levels. A, B quantitative PCR results showing the increased expression of the GJA1 gene in modied human myoblasts
MbCx (A) and myocytes after differentiation (B). C, D results of Western blot analysis. Connexin 43 (protein) overexpression (C) did not affect the level of desmin expression (D). All of
the results were normalised to -actin gene expression in both assays. Asterisks indicate statistical signicance (*p b 0.05, **p b 0.01, ***p b 0.001). Abbreviations: MbWt non-modied
myoblasts, MbCx connexin 43-transfected myoblasts, MbPCiNeo mock-transfected myoblasts, Kasumi lymphoblastoid cell line.

the BamHI and XhoI restriction enzymes to enable subsequent amplicon cloning. Cells
were electroporated (Gene Pulser Xcell, Biorad, Hercules, CA, USA) according to the developed protocol (square wave, 160 V, 15 ms, 1 pulse and 0.2 gap cuvette) and subsequently
seeded onto culture dishes. We used 16 g of plasmid DNA for 3 106 cells. For further experiments, cells incubated for 24 h in standard culture medium were used.

2.3. FACS analysis

The ow cytometry technique was used at least twice. First, it was used to quantify the
efcacy of the transfection procedure based on the GFP expression level (Fl1) in myoblasts
modied using the control plasmid compared to the negative control.
To evaluate their response to an oxidative stress, the studied cell populations were
simultaneously plated in culture asks. We induced oxidative stress by a 24 h incubation
with H2O2 (500 M), whereas the control cells were cultured under standard cell culture
conditions [21]. Subsequently, analyses were performed on all of the cell groups. An
Annexin-V-FITC kit was used to assess the number of viable and apoptotic cells in all of
the investigated myoblast populations (Beckman Coulter, Brea, CA, USA), following
the manufacturer's protocol. The cells were subdivided into 3 populations (namely: viable,
apoptotic and necrotic) based on their staining with Annexin-V-FITC (Fl1) and propidium
iodide (Fl3), according to the asymmetry and integrity of the cellular membranes.
For each experiment, the background uorescence was estimated using unlabelled
control cells. All of the measurements were performed using a Cell Lab Quanta ow
cytometer (Beckman Coulter).

2.4. Western blotting

Semi-quantitative analysis of the selected proteins in the genetically modied and wildtype cells was conducted using Western blotting. The expression levels of connexin 43 and
desmin in reference to -actin protein expression were evaluated in these assays. Briey,
proteins were isolated from cell pellets using the Cell Lytic Reagent (Sigma-Aldrich), and
an equal amount of proteins (100 g) was subjected to IPA gel electrophoresis followed by
an overnight transfer (4 C) to nitrocellulose membranes (Biorad). The appropriate
antigen-specic antibodies were applied (see Supplementary Table 1), and an ECL Western
Blotting System was used for subsequent chemiluminescent antibody detection (GE
Healthcare, Little Chalfont, UK). Semi-quantitative analysis was performed using a Molecular
Imager ChemiDoc XRS+ System (Biorad, USA) and Image Lab software (Biorad, USA). The
measurements were normalised to the -actin expression (ACTB).

2.5. Immunouorescence
For immunouorescent staining, the cells were xed in 4% paraformaldehyde (in PBS)
for 15 min. After washing with PBS (3 5 min), the samples were permeabilised for 5 min
using 0.2% Triton X-100 and again washed with PBS. Subsequently, after being blocked
with 5% FBS in PBS for 30 min, the samples were incubated overnight with primary antibodies against desmin or connexin 43 (at 4 C), washed with PBS and incubated with secondary antibodies for 1 h in the dark. Finally, approximately 100 l of UltraCruz mounting
medium containing DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for
nuclei counterstaining. Observations were made using an Axio Imager 1 uorescence
microscope (Zeiss, Oberkochen, Germany). The antibodies and the applied dilutions are
listed in Supplementary Table 1.
2.6. Fusion index
To estimate the myoblast differentiation potential, in vitro culture was conducted
using the differentiation protocol (described above). We determined the fusion index
(FI) of both populations wild-type myoblasts and connexin 43-overexpressing myoblasts. FI is dened as the ratio of the number of nuclei present in differentiated myotubes
(Nd) to the number of nuclei in undifferentiated cells (Nud), and is expressed as follows:
FI

Nd
:
Nud

To determine the fusion index, the differentiated cell populations were xed in freezing
methanol:acetic acid (3:1) for 15 min, washed with 1 PBS and stained using a 10% Giemsa
solution (Merck, Darmstadt, Germany) for 30 min. To avoid excessive staining, the samples
were washed several times in distilled water containing 0.005% acetic acid. Photographs
were taken using a standard light microscope. At least 500 nuclei were counted.
2.7. qPCR
The total RNA was isolated from the in vitro cultured cells using the TRI Reagent
(Sigma-Aldrich). The RNA yield and quality were analysed using a Nanodrop 2000
(Thermo Scientic, Waltham, MA, USA) and standard 1.5% agarose gel electrophoresis.
A 10 g aliquot of isolated RNA was treated with oligo(dT)-Dynabeads (Life Technologies)
to obtain the mRNA fraction, which was subsequently reverse transcribed using SuperScript III
reverse transcriptase (Life Technologies). The resultant cDNA was diluted (4) and used for
the quantitative PCR reactions employing the iQ SYBR Green Supermix and an iCycler, both

58

T.J. Kolanowski et al. / International Journal of Cardiology 173 (2014) 5564

Fig. 3. Immunouorescent staining of wild-type and connexin 43-overexpressing human myoblasts. Desmin protein was observed in MbWt cells (A) as well as in MbCx (D) cells,
conrming their myogenic characteristics. Anti-connexin 43 staining revealed the presence of GJA1 protein in both cell populations under study (B; MbWt, E; MbCx); however, in
MbCx cells, more discrete signals appear to be displayed (C; MbWt, F; MbCx). White bars indicate 40 m, whereas red bars represent 13 m. Abbreviations: MbWt non-modied myoblasts, MbCx connexin 43-transfected myoblasts, DES desmin, GJA1 connexin 43.

purchased from Biorad. All of the measurements were performed based on the coefcient obtained using the appropriate standard curves, and the standard Ct method was used for
further calculations. Preparation of the standard curves required amplicon cloning into the
pSC-A plasmid (Agilent Technologies, Santa Clara, CA, USA) and serial dilution of the plasmid
to 102 to 108 copies per reaction. We chose the ACTB gene as the housekeeping gene with
which all of normalisation calculations were performed [22]. All of the primers used are
shown in Supplementary Table 2.

2.8. Scrape loading

Scrape loading is a test for gap junction function in vitro. The uorescent dye used in
this assay can be passed from cell to cell only through functional connexons. The initial
dye loading of the cells was achieved by mechanically rupturing the cell membranes.
To estimate the level of gap junction connections, cells seeded on culture plates were
grown to approximately 100% conuence, and then, the medium was removed and the
cultures were washed three times with 1 PBS. Subsequently, a working solution
of 0.05% Lucifer Yellow (Sigma-Aldrich) in PBS was added precisely at the site where an
incision using a surgical blade had been made. After a 3 min incubation, this solution
was removed, and the preparation was washed three times with 1 PBS to remove any
background staining [23]. After applying the cover slip, the cells were immediately observed under an Axio Imager 1 uorescence microscope (Zeiss). To perform the

quantitative analysis, the number of cell layers perpendicular to the incision that exhibited
a uorescent signal was counted.

2.9. Myocardial infarction and echocardiography

The research protocol was approved by the Local Ethical Committee for Animal Research
in Poznan. The experimental group consisted of 14 post-infarction NODSCID mice (mean
weight of 27.09 3.38 g) divided into the 3 following subgroups: transplanted with
connexin 43-modied myoblasts (MbCx, n = 7), transplanted with wild-type myoblasts
(MbWt, n = 3) and the sham operated group, in which an equivalent volume of 0.9%
NaCl was injected (NaCl, n = 4). We used also an additional control group (Contr, n = 5,
mean weight of 26.94 1.23 g) that consisted of mice that were not subjected to LAD
ligation.
The procedure of left coronary artery ligation was performed as follows [24]: the
animals were pre-anaesthetised with isourane, intubated and subsequently ventilated (MiniVent, Harvard Apparatus, USA) under general anaesthesia (2% isourane).
First, an incision in thoracic region was made, and the pectoral muscles were secured. In
the next stage, a left thoracotomy was performed, and subsequently (after removal of
the pericardium), the left coronary artery was ligated. In the nal phase, the thorax was
closed, and at least three sutures were applied to the skin. After the surgical procedures
were completed, the mice were ventilated until spontaneous respiration recommenced.

Fig. 4. Scrape-loading assay. Comparison of Lucifer Yellow dye transfer in genetically modied human myoblasts (MbCx) and the control cell population (MbWt). A. More intense dye
transfer in the MbCx population can be observed (over a 2.5 larger area occupied by MbCx cells containing LY), which is an indirect proof of the presence of functional gap junctions.
The red bar indicates the site of the incision using a surgical blade; yellow arrows direction of dye propagation; white dashed line area of signal spreading. The white bars represent
40 m. B. Quantitative results of the gap junction functional assay. The number of cells that contained the uorescent dye after 3 min of incubation. Asterisks indicate statistical signicance
(p b 0.001). Abbreviations: MbWt non-modied myoblasts, MbCx connexin 43-transfected myoblasts, LY Lucifer Yellow uorescent dye.

T.J. Kolanowski et al. / International Journal of Cardiology 173 (2014) 5564

59

assessed as the control animals for the baseline values. We measured the left ventricular
end-systolic and end-diastolic areas in the short axis (LVESAS and LVEDAS) to determine
the change in the left ventricular area ratio (SAX AC%), using the following equation [25]:

SAX AC% 


LVEDASLVESAS
 100%:
LVEDAS

2.10. Statistical analysis

The data are expressed as the means SEM or as frequencies. The quantitative
PCR results were assessed using one-way ANOVA together with Bonferroni's multiple
comparison test. The differentiation potential was evaluated using the t-test. The cell
viability and echocardiographic results were analysed using two-way ANOVA and
Bonferroni's multiple comparison test. Statistical signicance levels are appropriately
indicated. All of the calculations were performed using GraphPad Prism 5 software
(GraphPad Software, La Jolla, CA, USA).

3. Results
3.1. Overexpression of Cx43 and transfection efcacy in primary human
skeletal myoblasts

Fig. 5. Analysis of the viability of the studied populations of human myoblasts and their
susceptibility to oxidative stress. Increased cell death under normal in vitro cell culture
conditions as well as after oxidative stress in the transfected cell populations (MbCx and
MbPCiNeo) was observed (panel A). A signicant decrease in cell viability after oxidative
stress was observed in both the MbCx and MbPCiNeo populations (panel B); however, in
the case of the MbCx population, the observed changes tended to be smaller. Asterisks indicate statistical signicance (*p b 0.05, ***p b 0.001, ns non signicant). Abbreviations:
MbWt non-modied myoblasts, MbCx connexin 43-transfected myoblasts,
MbPCiNeo mock-transfected myoblasts, ox stands for oxidative stress conditions as
described in the text.

Cell transplantation was conducted 1 month post-surgery. The cells to be used for transplantation were harvested from standard in vitro cultures using trypsin and centrifuged.
The cells were counted and prepared in DMEM lacking phenol red that was supplemented
with 1% BSA (bovine serum albumin). The animals were subjected to the transplantation
procedure according to the protocol applied for infarction induction. The cells were
injected into the infarction border zone in three different points (3 7 l, 7 105 cells
per injection). After myoblast transplantation, the muscle and skin were sutured, and
the animals were allowed to recover from the isourane administration.
Echocardiographic image acquisition was performed using a GE Vivid 7 highresolution ultrasound scanner equipped with a M12L linear transducer (GE Healthcare,
USA). Assessment of the heart parameters was conducted at three time points, 24 h before
cell transplantation and 30 and 60 days after cell transplantation. Additionally, a nontreated group of healthy individuals without infarction or cell transplantation was

The transfected cell population was evaluated using ow cytometry

48 h after electroporation. In all of the experiments, we obtained a
transfection efcacy of 65.72 3.89% (Fig. 1) based on the GFP expression level.
The content of connexin 43 in the genetically modied cells (MbCx)
was measured at both the RNA and protein levels (Fig. 2). For the quantitative PCR analysis, we used myoblasts after in vitro propagation (Mb),
as well as differentiated myocytes (Mc). The MbCx population exhibited
a 32.80 6.21 times greater expression of the gene encoding connexin
43, and this expression remained augmented in differentiated McCx by
3.24 0.39 times. In both cases, we observed statistically signicant
differences, conrming the acquisition of the GJA1 gene by this cell population (Fig. 2A, B). Additionally, an increased amount of connexin
(15) was demonstrated by Western blotting (Fig. 2C). All of the measurements were compared with those of non-transfected human myoblasts (MbWt) or myocytes (McWt). As a negative control for GJA1 gene
expression, we used Kasumi cells, a lymphoblastoid cell line.
In addition, we performed immunouorescent assays to evaluate
the myogenic character (desmin expression) of the investigated cell
populations (genetically modied and native myoblasts). Immunouorescent anti-connexin 43 staining was also conducted (Fig. 3). No
signicant differences were noted in the cell populations under
study. GJA1 protein was observed in MbCx cells as well as in MbWt

Fig. 6. Analysis of the differentiation potentials of human myoblasts. The images illustrate the potential of the McWt (A) and McCx (B) cell populations. Bars indicate 50 m. Signicant
differences between the populations under study were observed (C). Asterisks indicate a signicance level of p b 0.01. Abbreviations: McWt non-modied differentiated myocytes,
McCx connexin 43-transfected differentiated myocytes.

60

T.J. Kolanowski et al. / International Journal of Cardiology 173 (2014) 5564

cells; however, in the population that was transfected with connexin

43, the observed signal revealed a more discrete localisation, suggesting that this protein was organised in clusters (Fig. 3F).
3.2. In vitro test for functional connexons (scrape loading)
After conrmation of GJA1 gene and protein overexpression, it was
necessary to verify its potential to create functional connexons
that could improve myoblastcardiomyocyte coupling in the infarcted
recipient myocardium. To demonstrate this phenomenon, a scrapeloading assay was performed (Fig. 4). More efcient transport of the
uorescent dye (b1 kDa) between adjacent cells was observed in the
connexin 43-transfected population (MbCx) (6.17 0.79 cells near
the incision) compared with wild-type myoblasts (MbWt; 2.01 0.36
cells). Therefore, we conrmed the functional impact of the introduced
genetic modication on physiological characteristics of myoblasts.
3.3. Analysis of cell viability and susceptibility to an oxidative stress
condition
When characterising the transfected cells, a justied concern is
whether transmembrane channels introduced into the myoblast cells
could potentially affect their viability through both apoptotic signal
propagation and changes in membrane integrity that deviate from the
physiological state. To test this assumption, we performed a cell membrane integrity-based assay to estimate cellular apoptosis and necrosis.
In addition to the standard comparison between wild-type and connexin
43-modied cells, to evaluate whether the transfection process itself
affected cell viability, we used control myoblasts transfected with a
non-coding vector (MbPCiNeo). The susceptibility to the oxidative stress
condition of each cell population was measured simultaneously (Fig. 5).
A statistically signicant difference between the percentage of viable
cells in the MbWt (88.30 1.27) and MbCx (71.77 .82) populations
was observed (p b 0.001), indicating that the transfected cells were
more prone to cell death under standard culture conditions. Moreover,
a similar disparity (p b 0.001) was observed when comparing MbWt
(88.30 1.27) and MbPCiNeo (72.18 2.60) cells. No statistically signicant differences were observed between the viability in the MbCx
and MbPCiNeo populations. In the cell populations tested for their susceptibility to oxidative stress, a similar trend was observed. However,
with respect to the cells treated with H2O2 vs. the non-treated cells,
only the differences between the MbCx and MbPCiNeo populations
were found to be signicant (p b 0.05 and p b 0.001, respectively). All
of these results are included in Supplementary Table 3.
3.4. Differentiation potential
Considering the results of the apoptosis evaluation assay, we subsequently employed standard tests to examine the differentiation potential of the myoblast populations under investigation. A signicant
difference between the fusion indices (FI) of the MbWt (4.05 0.20)
and MbCx (5.16 0.05) cells was observed (Fig. 6). The analysis performed revealed that the connexin 43-modied myoblasts differentiated
more intensively and that the process of fusion was more frequent in
these cells than in their non-modied counterparts.
3.5. Myogenic gene expression analysis
For further evaluation of the effect of connexin 43 overexpression on
myoblast character, an analysis of myogenic gene expression was conducted. We chose genes crucial to the process of stemness maintenance
in myoblasts (MYOD, MYOG, and MYF5), as well as a gene that is thought
to be upregulated during differentiation into myocytes (MYH2).
In undifferentiated myoblasts, the levels of most of the evaluated
genes, namely MYOD, MYF5, and MYH2, exhibited stable expression
(Fig. 7A). Signicant differences among the studied cell populations

appeared only in respect to the MYOG expression level (p b 0.001). A

higher level of MYOG expression was observed in the MbPCiNeo population compared with that in the MbWt (p b 0.05) or MbCx (p b 0.01) population. The opposite tendency was observed in the MbCx population, in
which the level of myogenin expression was lower than that in the control populations of MbWt (p b 0.05) or MbPCiNeo cells (p b 0.01).
Moreover, analysis of expression in the differentiated myocytes
(McWt, McCx and McPCiNeo cells) by quantitative PCR was conducted
in a similar way. In this case, however, a large increase in the levels of
MYOG and MYH2 mRNA was observed only in the myocytes overexpressing connexin (p b 0.05) (Fig. 7B). The expression levels of other
genes were not signicantly different. All of the values are included in
Supplementary Table 4.
3.6. Echocardiographic evaluation of in vivo cell function
To assess the effect of cell transplantation on the post-infarction heart,
we examined changes in heart function parameters using echocardiography prior to and after cell transplantation (Fig. 8). We observed that
after transplantation of genetically modied myoblasts (MbCx), the left
ventricular area in the short axis (SAX AC%) was not signicantly modied
during the two months of the experiment. During the rst month after
transplantation, we did not observe any changes in the animals treated
using wild type myoblasts; however, in the follow-up period, a tendency
of deterioration in cardiac function was noticed (p b 0.05). These data
are in clear contrast to those of the control animal group (infarction without cell transplantation), in which a signicant decrease in the measured
parameters during the experimental period was observed (p b 0.01). A
signicantly diminished SAX AC % in the control group between the 1st
and the 2nd month after MbWt cell transplantation (p b 0.05) was also
observed. A complete set of the results is presented in Supplementary
Table 5.
4. Discussion
In previous studies, we focused on the effects of connexin 43 overexpression in mouse skeletal myoblasts together with genetic modications
of human skeletal myoblasts [20,21], which have been extensively used
for several years in cell therapy of the infarcted heart [26,27]. As reported
earlier, due to their inability to create proper cell-to-cell coupling
with cardiomyocytes, skeletal myoblasts can cause life-threatening arrhythmias [28]. Fortunately, previous studies have already explored the
functional effects of SkM cells on the injured myocardium in different animal models, demonstrating that Cx43-modied myoblasts are able to
generate grafthost coupling and even an anti-arrhythmic effect [15,17].
In this study, we explored an approach for clinical trials using genetically
modied human stem cells by investigating the effects of connexin 43
modication on their biological features together with indirect evidence
for improving the homing of connexin 43-modied myoblasts to the
host myocardium.
To genetically modify suspended isolated primary cells, we used the
pCiNeo plasmid. Optimisation of the electroporation technique in our
laboratory resulted in an improved efcacy of the transfection process,
which is currently at the level of ~65%. Until now, connexin 43 overexpression in myoblast cell lines was successfully obtained using lentiviral
[17], retroviral [29] or adenoviral [30] vectors, as well as using plasmidbased [31] transfection. To our knowledge, none of the published papers
has presented a clinically applicable method of connexin 43 modication of SkM derivatives. Fernandes et al. reported that their lentivirusbased technique led to a 50% modication efcacy and only a 2.5-fold
higher expression of connexin 43 overall, which did not indicate a
very efcient protocol (although they observed myoblastcardiomyocyte
coupling, the antiarrhythmic effect of the transfected cells was not
emphasised by this group) [17]. In experiments conducted using other
types of virus-derived vectors, other vector-related problems arose. As reported by Reinecke et al., adenoviral transduction led to an increased rate

cC

eo

C
iN

cP

0.01

M
cC

iN
eo

cP
C

bC

eo

iN

bP

bW

bP

bC

eo

bW
t
iN

um
i

as

Relative expression

MYOG
*

MYH2
*
t

0.01

M
cW

0.01

cW

0.1
i

10

0.1
um

0.01

1
as

0.1

um

K
as

Relative expression

eo

bC

iN

0.1

*
Relative expression

bC

bP

bW
t

as
u

Relative expression

cC

eo

um
i

as

**

eo

iN

bP

Relative expression

M
cC

10

eo

10

iN

iN

cP

bW

um

as

Relative expression

10

cP
C

cW

um

as

Relative expression

cW

um

as

Relative expression

T.J. Kolanowski et al. / International Journal of Cardiology 173 (2014) 5564

61

MYOD

10

0.1

0.01

MYH2
MYF5

10

0.1

0.01

MYOG
MYOD

10

0.1

0.01

10

MYF5

0.1

0.01

62

T.J. Kolanowski et al. / International Journal of Cardiology 173 (2014) 5564

Fig. 8. Evaluation of the functional effect of cell intervention using the SAX AC% parameter
in the heart post-infarction animal model. Asterisks indicate statistical signicance
(*p b 0.05, **p b 0.01). Abbreviations: MbWt non-genetically modied myoblasts
cell intervention. MbCx-connexin 43-modied human myoblast cell intervention,
NaCl sham intervention with an equal volume of physiological saline (0.9% sol),
Contr group of animals without infarction, Tx cell transplantation by thoracotomy, SAX AC% left ventricle area change ratio in the short axis.

of cell death [30], most likely due to the high transduction rates; the application of retroviral gene delivery (based on incorporation into the host
genome) has always been hazardous, frequently leading to oncogenic activation and an insertional mutagenesis phenomenon [32]. Moreover, it
has been noted that in general, clinical application of virus-derived
vectors has always raised ethical concerns. In terms of safety, plasmidderived vectors can be regarded as one of the most interesting candidates
for clinical applications. In an intriguing study, Suzuki et al. induced the
over-expression of connexin 43 in myoblasts using a plasmid similar to
ours, which led to an expression level corresponding to that obtained in
this study (approximately 15-fold higher compared with that of the native control), as documented by Western blotting and by IF staining. The
difference between those two reports was that we used suspended primary human cell and no clonal selection was performed, which is a critically important in restricting the potential of cell-population doubling
and consequently, future therapy using these autologous cells. The increased expression of connexin 43 in non-differentiated myoblasts,
which was sustained after myotube formation, should also be emphasised
(proven by qPCR, see Fig. 2). Thus, the coupling of implanted stem cells
with the host myocardium should be examined over a long-term period, not only during the rst phase of cellular graft implementation.
Functional gap junction assembly was veried using the scrape loading
assay. The observed positive differences in gap junction conductance
were in agreement with earlier reports showing enhanced LY propagation in modied myoblasts [31,33]. These data suggest that, keeping in
mind the safety of the clinical application, we managed to obtain a
connexin 43-modied human skeletal myoblast population by means
of a highly efcient method that preserved their desired biological
function.
The second phase of this study was related to the biological characteristics of the obtained MbCx cell population to differentiate their acquired changes from the features of the native non-transfected
human myoblasts. First, we investigated the effect of the genetic modication on the viability of the cells and their susceptibility to oxidative
stress because skeletal myoblasts are thought to have a high resistance

to stress conditions compared with cardiomyocytes [34]. Published data

concerning the effect of connexin 43 on apoptosis ranged from an effect
on the ability to propagate pro-apoptotic signals [35] to the antiapoptotic effect resulting from the increased stress resistance of
connexin 43-expressing cells [36]. Our results indicated a decrease in
the viability of the MbCx population both under normal in vitro cell culture conditions and after the induction of oxidative stress. However, the
observed phenomenon was most likely caused by electroporation itself
and was not specic to the induced connexin 43 overexpression (this
conclusion can be drawn on the basis of observations that the mocktransfected myoblast cell population exhibited effects similar to those
observed in MbCx cells) (Fig. 5). Suzuki et al. were the rst to demonstrate that connexin 43 expression could affect the differentiation process of myoblasts [31]; however, their study was conducted using a
rat myoblast cell line after clonal selection. Therefore, we provide here
evidence that suggests a mechanism that also operates in primary
human myoblast suspensions. As a nal outcome, we observed an increase in the fusion index (Fig. 6) of connexin 43-modied myoblasts
differentiating into myotubes (compared with that of the control
cells), with the concomitant up-regulation of myosin heavy chain 2
(MYH-2) expression which is a key protein in the highly organised contractile apparatus. This phenomenon demonstrated the increased tendency of MbCx cells to form differentiated cells. An additional interesting
aspect appeared upon evaluating the expression of myogenic factors,
particularly with respect to myogenin (MYOG) expression. In the
pCiNeo-transfected cells, the transient up-regulation of MYOG expression was observed (Fig. 7A), conrming the results obtained in
several of our myoblast-modication experiments (unpublished data),
in which this phenomenon was most likely caused by the electroporation
process itself. An opposite effect in the MbCx cell population was observed, when myogenin expression was down-regulated in the rst
phase after transfection with its subsequent up-regulation in differentiated myotubes compared with that in unmodied native cells.
In the last phase of this study, we tried to evaluate the effect of
connexin 43 (when overexpressed in myoblasts) on the functional parameters of the infarcted heart. In the experiments performed by Roell
et al. [15] and subsequently by Fernandes et al. [17], the electrical coupling of GJA1-expressing skeletal myoblasts in an animal model was elucidated. Functional assays, including electrophysiological recordings
and programmed electrical stimulation performed in vivo, conrmed
the crucial role of connexin 43 in reducing the pro-arrhythmic features
of skeletal myoblasts [15]. Although anti-arrhythmic inuence was
emphasised as well, it has not yet been conrmed by other authors
[17,32]. In our experiments, we focused on monitoring the effect of
human primary MbCx cell suspensions on the physiological parameters
of the hearts of the post-infarction model of NODSCID mice. Echocardiographic evaluation of the parameter of the left ventricular area
change in the short axis (SAX AC%) revealed that in the control group
with no cell intervention, this parameter worsened with time during
the in vivo follow-up of the heart. Only in the group that received the
MbCx cell intervention did we not observe a signicant decrease of
the SAX AC% and progressive heart remodelling (Fig. 8). These data conrm the results reported by Shiba et al. [37]; however, their model was
restricted to a one-month follow-up, whereas we found that in the second month after the in vivo experiment, the negative effect on heart
functioning was even more intensied in the control post-infarction
group compared with the MbCx-treated animals (Fig. 8). A prolonged
effect of myogenic stem cells on the injured myocardium together

Fig. 7. Expression of myogenic genes in human myoblast (A) and myocyte (B) populations. (A) Only with respect to the MYOG expression prole was a statistically signicant difference
observed. The results obtained showed that MYOG expression in the MbPCiNeo population was upregulated, in contrast to the MbCx cells, in which downregulation of the expression of
this gene was observed. (B) A statistically signicant upregulation of both the MYOG and MYH2 genes was observed in McCx cells compared with that of the native controls. Asterisks
indicate statistical signicance (*p b 0.05, **p b 0.01). In all of the cases, myogenic gene expression in the Kasumi lymphoblastoid cell line was signicantly lower than that in the
human myoblast populations, at least at the level of p b 0.05. Abbreviations: MbWt non-modied myoblasts, MbCx connexin 43-transfected myoblasts, MbPCiNeo mocktransfected myoblasts, Kasumi lymphoblastoid cell line. MYOG myogenin, MYOD myogenic differentiation antigen 1, MYH2 myosin heavy chain 2, MYF5 myogenic factor 5.

T.J. Kolanowski et al. / International Journal of Cardiology 173 (2014) 5564

with the observed relatively poor ability of myoblasts to migrate out of

myocardial tissue (unpublished data) is critical for their potential benets in regenerated organs. Overall, the results we have generated to date
could be treated as indirect evidence for the improved long-term impact
of connexin 43-modied myoblasts on heart parameters due to the
myoblasts anchoring to cardiomyocytes through the formation of gap
junctions.
Obviously, our study also has some limitations. First, we were not able
to precisely evaluate myoblastcardiomyocyte coupling in the in vivo
studies or estimate the impact of cellular xenografts on the electrical activity of the injured myocardium due to the lack of comparisons of the effect of genetically modied myoblasts in auto- or allo-transplantation
models. In this study, we only used a small animal model (mice), being
well aware of the concerns regarding the differences between the physiological characteristics of human and mouse hearts (e.g., the heart rate).
All in all, to conrm the benets of connexin 43 overexpression, human
SkM should be evaluated in a large animal model (pigs) with physiology
more closely related to that of the human heart.
To summarise, in this study, we generated skeletal myoblasts with
upregulated expression of connexin 43 for use in a functional, clinically
oriented model that avoided the application of virus-derived vectors or
pluripotent stem cells. Gap junction formation was conrmed at both
the molecular and functional level. To the best of our knowledge, this
is the rst documentation of the effect of connexin 43 overexpression
on the process of human primary skeletal myoblast differentiation.
Moreover, we observed changes in myogenin (MYOG) expression after
Cx43 gene transfection as well as after in vitro cell differentiation. In
the in vivo animal model, we observed the stabilisation of heart parameters for a long period after infarction longer than after intervention
using non-modied native myoblasts. This result could be treated as indirect evidence of the presence of MbCx cells in the post-infarction heart.
Connexin 43 has been demonstrated to play an important role in
strengthening skeletal myoblasthost cardiomyocyte coupling [38].
This observation led to the further development of regenerative therapy
for the infarcted myocardium using animal models. As for the other
stem cell therapies of the post-infarction myocardium, e.g., those
using pluripotent stem cell-derived cardiomyocytes [16,33], which are
still far from clinical application, there is a need to develop a protocol
that enhances the chances for patients' survival after ventricular ischaemic episodes by diminishing the risk of life-threatening arrhythmia and
strengthening the myocardium.
5. Conclusions
Connexin 43 modication of human skeletal muscle stem cells using
a plasmid vector was performed in an efcient manner. We observed
enhanced cell coupling and fusion in the process of in vitro myogenic
differentiation, together with changes in gene expression that suggested better cellular development. Moreover, transplantation of modied human myoblasts into an animal model of myocardial ischaemia
was found to have a positive effect on the infarcted heart in a longterm follow-up. Taken together, these ndings will contribute to the development of a clinical regenerative therapy for patients with a failing
heart.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ijcard.2014.02.009.
References
[1] Marsboom G, Janssens S. Endothelial progenitor cells: new perspectives and applications in cardiovascular therapies. Expert Rev Cardiovasc Ther 2008;6:687701.
[2] Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. Bone marrow stem cells
regenerate infarcted myocardium. Pediatr Transplant 2003;7(Suppl. 3):868.

63

[3] Rota M, Padin-Iruegas ME, Misao Y, et al. Local activation or implantation of cardiac
progenitor cells rescues scarred infarcted myocardium improving cardiac function.
Circ Res 2008;103:10716.
[4] Yang L, Soonpaa MH, Adler ED, et al. Human cardiovascular progenitor cells
develop from a KDR + embryonic-stem-cell-derived population. Nature
2008;453:5248.
[5] Zimmermann W-H, Didi M, Dker S, et al. Heart muscle engineering: an update on
cardiac muscle replacement therapy. Cardiovasc Res 2006;71:41929.
[6] Menasch P. Skeletal myoblasts as a therapeutic agent. Prog Cardiovasc Dis
2007;50:717.
[7] Jain M, DerSimonian H, Brenner D, et al. Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation 2001;103:19207.
[8] Mills WR, Mal N, Kiedrowski MJ, et al. Stem cell therapy enhances electrical viability
in myocardial infarction. J Mol Cell Cardiol 2007;42:30414.
[9] Leobon B, Garcin I, Menasche P, Vilquin J-T, Audinat E, Charpak S. Myoblasts
transplanted into rat infarcted myocardium are functionally isolated from their
host. Proc Natl Acad Sci U S A 2003;100:780811.
[10] Delmar M, Makita N. Cardiac connexins, mutations and arrhythmias. Curr Opin
Cardiol 2012;27:23641.
[11] Shl G, Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res
2004;62:22832.
[12] Goodenough DA, Paul DL. Beyond the gap: functions of unpaired connexon channels. Nat Rev Mol Cell Biol 2003;4:28594.
[13] Lee S-W, Kang H-J, Lee J-Y, et al. Oscillating pressure treatment upregulates
connexin43 expression in skeletal myoblasts and enhances therapeutic efcacy for
myocardial infarction. Cell Transplant 2009;18:112335.
[14] Neef K, Choi Y-H, Perumal Srinivasan S, et al. Mechanical preconditioning enables
electrophysiologic coupling of skeletal myoblast cells to myocardium. J Thorac
Cardiovasc Surg 2012;144:117684 [e1].
[15] Roell W, Lewalter T, Sasse P, et al. Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia. Nature 2007;450:81924.
[16] Greener ID, Sasano T, Wan X, et al. Connexin43 gene transfer reduces ventricular
tachycardia susceptibility after myocardial infarction. J Am Coll Cardiol
2012;60:110310.
[17] Fernandes S, Van Rijen HVM, Forest V, et al. Cardiac cell therapy: overexpression of
connexin43 in skeletal myoblasts and prevention of ventricular arrhythmias. J Cell
Mol Med 2009;13:370312.
[18] Siminiak T, Kalawski R, Fiszer D, et al. Autologous skeletal myoblast transplantation
for the treatment of postinfarction myocardial injury: phase I clinical study with
12 months of follow-up. Am Heart J 2004;148:5317.
[19] Rozwadowska N, Fiszer D, Siminiak T, Kalawski R, Kurpisz M. Human skeletal myoblasts in vitro for autotransplantation in infarcted myocardium. Kardiol Pol
2002;238.
[20] Rozwadowska N, Kolanowski T, Waclawska A, Fraczek M, Kurpisz M. Transgenic
line of C2C12 myoblasts with constitutive overexpression of connexin-43
(GJA 1). In: Kimchi Asher, editor. New Frontiers in Heart Disease. MEDIMOND;
2012. p. 18.
[21] Bialas M, Krupka M, Janeczek A, et al. Transient and stable transfections of mouse
myoblasts with genes coding for pro-angiogenic factors. J Physiol Pharmacol
2011;62:21928.
[22] Kubista M, Andrade JM, Bengtsson M, et al. The real-time polymerase chain reaction.
Mol Aspects Med 2006;27:95125.
[23] el-Fouly MH, Trosko JE, Chang CC. Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res
1987;168:42230.
[24] Korte T, Fuchs M, Guener Z, et al. In-vivo electrophysiological study in mice
with chronic anterior myocardial infarction. J Interv Card Electrophysiol
2002;6:12132.
[25] Szymczyk E, Lipiec P, Plewka M, et al. Feasibility of strain and strain rate evaluation
by two-dimensional speckle tracking in murine model of myocardial infarction:
comparison with tissue Doppler echocardiography. J Cardiovasc Med (Hagerstown)
2013;14:13643.
[26] Siminiak T, Fiszer D, Jerzykowska O, et al. Percutaneous trans-coronary-venous
transplantation of autologous skeletal myoblasts in the treatment of postinfarction myocardial contractility impairment: the POZNAN trial. Eur Heart J
2005;26:118895.
[27] Menasch P, Aleri O, Janssens S, et al. The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: rst randomized placebo-controlled study of
myoblast transplantation. Circulation 2008;117:1189200.
[28] Abraham MR, Henrikson CA, Tung L, et al. Antiarrhythmic engineering of skeletal
myoblasts for cardiac transplantation. Circ Res 2005;97:15967.
[29] Tolmachov O, Ma Y-L, Themis M, et al. Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes
in vitro. BMC Cardiovasc Disord 2006;6:25.
[30] Reinecke H, Minami E, Virag JI, Murry CE. Gene transfer of connexin43 into skeletal
muscle. Hum Gene Ther 2004;15:62736.
[31] Suzuki K, Brand NJ, Allen S, et al. Overexpression of connexin 43 in skeletal myoblasts: relevance to cell transplantation to the heart. J Thorac Cardiovasc Surg
2001;122:75966.
[32] Anson DS. The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated
gene delivery. Genet Vaccines Ther 2004;2:9.
[33] Stagg MA, Coppen SR, Suzuki K, et al. Evaluation of frequency, type, and function of
gap junctions between skeletal myoblasts overexpressing connexin43 and
cardiomyocytes: relevance to cell transplantation. FASEB J 2006;20:7446.

64

T.J. Kolanowski et al. / International Journal of Cardiology 173 (2014) 5564

[34] Gorbe A, Becker DL, Dux L, Krenacs L, Krenacs T. In differentiating prefusion myoblasts connexin43 gap junction coupling is upregulated before myoblast alignment
then reduced in post-mitotic cells. Histochem Cell Biol 2006;125:70516.
[35] Huang RP, Hossain MZ, Huang R, Gano J, Fan Y, Boynton AL. Connexin (Cx43) enhances chemotherapy-induced apoptosis in human glioblastoma cells. Int J Cancer
2001;92:1308.

[36] Nakase T, Fushiki S, Naus CCG. Astrocytic gap junctions composed of connexin 43 reduce apoptotic neuronal damage in cerebral ischemia. Stroke 2003;34:198793.
[37] Shiba Y, Fernandes S, Zhu W-Z, et al. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 2012;489:3225.
[38] Abraham MR, Henrikson CA, Tung L, et al. Antiarrhythmic engineering of skeletal
myoblasts for cardiac transplantation. Circ Res 2005;97:15967.