Improvements of cardiac electrophysiologic stability and ventricular fibrillation threshold in rats with myocardial infarction treated with cardiac stem cells.

Abstract

Arrhythmia is of concern after cardiac stem cell transplantation in repairing infarcted myocardium. However, whether transplantation improved the ventricular fibrillation threshold and whether severe malignant ventricular arrhythmia is induced in the myocardial infarction model are still unclear. We sought to investigate the electrophysiologic characteristics and ventricular fibrillation threshold in rats with myocardial infarction by treatment with allogeneic cardiac stem cells.
Prospective, randomized, controlled study.
University-affiliated hospital.
Male Sprague-Dawley rats.
Myocardial infarction was induced in 20 male Sprague-Dawley rats. Two weeks later, animals were randomized to receive 5 × 10(6) cardiac stem cells labeled with PKH26 in phosphate buffer solution or a phosphate buffer solution-alone injection into the infarcted anterior ventricular-free wall.
Six weeks after the cardiac stem cell or phosphate buffer solution injection, electrophysiologic characteristics and ventricular fibrillation threshold were measured at the infarct area, infarct marginal zone, and noninfarct zone. Labeled cardiac stem cells were observed in 5-μm cryostat sections from each harvested heart. The unipolar electrogram activation recovery time dispersions were shorter in the cardiac stem cell group compared with those at the phosphate buffer solution group (15.5 ± 4.4 vs. 38.6 ± 14.9 msecs, p = .000177). Malignant ventricular arrhythmias were significantly (p = .00108) less inducible in the cardiac stem cell group (one of ten) than the phosphate buffer solution group (nine of ten). The ventricular fibrillation thresholds were greatly improved in the cardiac stem cell group compared with the phosphate buffer solution group. Labeled cardiac stem cells were identified in the infarct zone and infarct marginal zone and expressed Connexin-43, von Willebrand factor, α-smooth muscle actin, and α-sarcomeric actin.
Cardiac stem cells may modulate the electrophysiologic abnormality and improve the ventricular fibrillation threshold in rats with myocardial infarction treated with allogeneic cardiac stem cells and cardiac stem cell express markers that suggest muscle, endothelium, and vascular smooth muscle phenotypes in vivo.

Full text

Page 1

Improvements of cardiac electrophysiologic stability and
ventricular fibrillation threshold in rats with myocardial infarction
treated with cardiac stem cells*
Shaoxin Zheng, MD; Changqing Zhou, MD; Yinlun Weng, MD; Hui Huang, MD; Hao Wu, MD;
Jing Huang, MD; Wei Wu, MD; Shijie Sun, MD; Jingfeng Wang, MD; Wanchun Tang, MD; Tong Wang, MD
I
tent stem cells capable of differentiating
n recent years, cell therapy has
emerged as a strategy for improv-
ing contractility of the diseased
heart (1, 2). Restorative therapies
consider the use of exogenous multipo-
into cardiomyocytes. These include em-
bryonic and bone marrow-derived stem
cells (3, 4) as well as intrinsic cardiac
stem cells (CSCs) that reside in the heart
(5–7) and are programmed to reconsti-
tute cardiac tissue (8). In vitro, CSCs can
differentiate into cardiomyocytes, endo-
thelial cells, vascular smooth muscle
cells, and fibroblasts (9), while in vivo,
CSCs can differentiate into cardiomyo-
cytes (10), and several animal studies
have shown promising results on im-
provement of left ventricular function
(11–13). Nevertheless, clinical applica-
tion of stem cells remains controversial
(14, 15); therefore, additional preclinical
investigations to further enhance the
beneficial effects of stem cell therapy are
advocated (16).
As with other stem cell therapies,
safety and proarrhythmia concerns have
arisen in relation to the potential risks of
cardiac stem cell therapy (17, 18) and
more specifically after intramyocardial
administration (19). This is related to the
formation of distinct cell clusters con-
taining donor-derived cells and accumu-
lated host-derived inflammatory cells in
the infarct border zone. Until now, no
study has focused on cardiac electro-
physiologic characteristics and ventricu-
lar fibrillation threshold (VFT) after CSC
transplantation. Safety and significant
improvement in VFT are important as-
pects for CSCs treatment in ischemic
heart disease and dysfunction. In the
present study, we investigated safety,
proarrhythmic effects and effects on VFT
of allogeneic CSCs used to treat myocar-
dial infarction induced by left anterior
descending coronary artery ligation in
rats. Our hypothesis was that cardiac
electrophysiologic stability and VFT
would be improved significantly in a rat
model of myocardial infarction treated
with allogeneic CSCs.
*See also p. 1222.
From the Sun Yat-sen Memorial Hospital of Sun
Yat-sen University, (SZ, CZ, YW, HH, WW, JW, WT, TW),
Guangzhou, China; Weil Institute of Critical Care Med-
icine (YW, SS, WT), Rancho Mirage, CA; and the Sec-
ond Affiliated Hospital of Guangzhou Medical College
(HW, JH), Guangzhou, China.
Supported, in part, by the National Natural Science
Foundation of China (81070125, 30700304, and
30973207) and the Natural Science Foundation of
Guangdong Province (8151008901000119).
The authors have not disclosed any potential con-
flicts of interest.
For information regarding this article, E-mail:
tongwang163@yahoo.com.cn
Copyright © 2011 by the Society of Critical Care
Medicine and Lippincott Williams & Wilkins
DOI: 10.1097/CCM.0b013e318206d6e8
Objectives: Arrhythmia is of concern after cardiac stem cell
transplantation in repairing infarcted myocardium. However,
whether transplantation improved the ventricular fibrillation
threshold and whether severe malignant ventricular arrhythmia is
induced in the myocardial infarction model are still unclear. We
sought to investigate the electrophysiologic characteristics and
ventricular fibrillation threshold in rats with myocardial infarction
by treatment with allogeneic cardiac stem cells.
Design: Prospective, randomized, controlled study.
Setting: University-affiliated hospital.
Subjects: Male Sprague-Dawley rats.
Interventions: Myocardial infarction was induced in 20 male
Sprague-Dawley rats. Two weeks later, animals were randomized
to receive 5 ? 106cardiac stem cells labeled with PKH26 in
phosphate buffer solution or a phosphate buffer solution-alone
injection into the infarcted anterior ventricular-free wall.
Measurements and Main Results: Six weeks after the cardiac
stem cell or phosphate buffer solution injection, electrophysiologic
characteristics and ventricular fibrillation threshold were measured
at the infarct area, infarct marginal zone, and noninfarct zone.
Labeled cardiac stem cells were observed in 5-?m cryostat sections
from each harvested heart. The unipolar electrogram activation re-
covery time dispersions were shorter in the cardiac stem cell group
compared with those at the phosphate buffer solution group (15.5 ?
4.4 vs. 38.6 ? 14.9 msecs, p ? .000177). Malignant ventricular
arrhythmias were significantly (p ? .00108) less inducible in the
cardiac stem cell group (one of ten) than the phosphate buffer
solution group (nine of ten). The ventricular fibrillation thresholds
were greatly improved in the cardiac stem cell group compared with
thephosphatebuffersolutiongroup.Labeledcardiacstemcellswere
identifiedintheinfarctzoneandinfarctmarginalzoneandexpressed
Connexin-43, von Willebrand factor, ?-smooth muscle actin, and
?-sarcomeric actin.
Conclusions: Cardiac stem cells may modulate the electro-
physiologic abnormality and improve the ventricular fibrillation
threshold in rats with myocardial infarction treated with alloge-
neic cardiac stem cells and cardiac stem cell express markers
that suggest muscle, endothelium, and vascular smooth muscle
phenotypes in vivo. (Crit Care Med 2011; 39:1082–1088)
KEY WORDS: arrhythmia; cardiac stem cells; electrophysiologic
characteristics; myocardial infarction; ventricular fibrillation
threshold
1082 Crit Care Med 2011 Vol. 39, No. 5

Page 2

METHODS
Adult male Sprague-Dawley rats weighing
350–450 g were obtained from the animal ex-
perimental center of our Sun Yat-Sen University
and were housed in a standard animal facility
with 12-hr on/off light conditions. All animals
were acclimatized for at least 1 wk before sur-
gery and allowed standard food and water ad
libitum. All procedures were in compliance with
the guidelines for the care and use of laboratory
animals and the ethical review process of our
institution and were approved by the institu-
tional animal care committee.
Isolation and Culture of Cardiac Stem
Cells. The 3-day-old Sprague-Dawley rats were
anesthetized by intraperitoneal injection of pen-
tobarbital (45 mg/kg) and then dipped into 75%
ethanol for 30 seconds for sterilization. Under
sterile conditions, the hearts were excised, put
into the aseptic culture dish, and minced into
1-mm3pieces. The pieces were then washed
twice with phosphate buffer solution (PBS) to
remove impurities. Then, 2–3 mL of 0.2% tryp-
sin was added for 5 min of digestion, followed by
2–3 mL of 0.1% collagenase II for 5 min for
additionaldigestion,andsubsequentelimination
of the digestive liquid. The procedure was then
repeated for an additional three times. The
pieces were then washed twice with complete
explant medium (supplemented with Iscove’s
modified Dulbecco’s medium, 10% fetal calf se-
rum, 100 units/mL penicillin G, 100 ?g/mL
streptomycin, 2 mmol/L L-glutamine, and 0.1
mmol/L 2-mercaptoethanol), then transferred
and dispatched with pipette into a 25-cm2flask,
and incubated with 1 mL of complete explant
medium at 37°C-humidified atmosphere with
5% CO2for 12 hrs. An additional complete
explant medium (3–4 mL) was then added.
At 90% confluence, the cells were trypsinized
(0.25% trypsin-ethylenediaminetriacetic acid,
catalog number 25–053-CI, Mediatech, Hern-
don, VA) and were passed into 25-cm2flasks at
1:2 ratios. Third-passage CSCs were used in all
experiments.
Cultured CSCs were analyzed by fluores-
cence-activated cell sorting (FACScan flow cy-
tometer, Becton Dickinson, Sparks, MD) as
reported previously (7, 11).
To monitor cell distribution in the heart,
on the day of implantation, suspended CSCs
were labeled with fluorescent dyes with the
use of a PKH26 red fluorescent cell linker kit
(Sigma Aldrich, St. Louis, MO), as reported
previously (20).
Rat Myocardial Infarction Model Prepara-
tion. Twenty male Sprague-Dawley rats weigh-
ing 350–450 g were fasted overnight except
for free access to water. The animals were
anesthetized by intraperitoneal injection of
pentobarbital (45 mg/kg). Additional doses (10
mg/kg) were administered at intervals of ap-
proximately 1 hr, or as required to maintain
anesthesia. The trachea was orally intubated
with a 14-g cannula mounted on a blunt nee-
dle with a 145° angled tip (Abbocath-T, Abbott
Critical Care Systems, North Chicago, IL) as
previously described (20). The animals were
mechanically ventilated with room air at a
tidal volume of 0.65 mL/100 g of body weight
and a frequency of 100 breaths/min. The elec-
trocardiogram lead II was continuously mon-
itored. A thoracotomy was performed via the
left fourth intercostal space. The pericardium
was incised, and the left atrial appendage was
elevated to expose the left anterior descending
coronary artery. The left anterior descending
coronary artery was ligated by using a 5/0
nylon suture. The chest was closed with a soft
tube in the cavity to withdraw air or blood.
Successful occlusion was confirmed electro-
cardiographically by the elevation of the ST
segment (20, 21). Animals were allowed to
recover from anesthesia and then were re-
turned into their cages for 2 wks. Postopera-
tion pain was controlled with intramuscular
injections of ketorolac (0.4 mg/kg).
Experimental Procedures. Two weeks after
surgical intervention, the animals were anes-
thetized and orally intubated as previously de-
scribed (20, 21). The animals were mechani-
cally ventilated with room air, and a new
thoracotomy was performed as described
above. The animals were randomized to be
subjected to injection of either 5 ? 106/0.1 mL
of CSCs labeled with PKH26 in PBS or PBS
alone as a placebo into the infarcted anterior
ventricular-free wall. Successful injection was
typified by the formation of a bleb covering the
infarct zone. The animals were allowed to re-
cover from anesthesia and returned to their
cages for another 6 wks. Postoperative pain
was controlled as described above.
Six weeks after CSCs or PBS injection, the
animals were reanesthetized. Additional doses
of pentobarbital were administered at hourly
intervals but not within 30 min preceding the
start of measurements. The trachea was orally
intubated as previously described. A thoracot-
omy was performed via the left fifth intercostal
space, and the heart was exposed. Three pre-
curved guide wires were then attached to the
infarct area, infarct marginal zone, and non-
infarct zone. The electrocardiogram lead II
was continuously recorded. A heat lamp was
used to maintain body temperature at
36.8°C ? 0.2°C throughout the experiment as
we described previously (22).
Measurements
All electrophysiologic testing in the cur-
rent study was performed by a blinded inves-
tigator who did not know to which group the
rat belonged.
Effective Refractory Period. The ventricu-
lar effective refractory period of the infarct
marginal zone and the noninfarct zone were
assessed by the programmed electrical stimu-
lation technique (DF-6A, Jiangsu, China). The
pacing protocol consisted of S1S2 and S1S2S3
(S1S2: Eight beats of basal stimuli in a cycle
length of 120 msecs and up to one extra stim-
uli; S1S2S3: Eight beats of basal stimuli in a
cycle length of 120 msecs and up to two extra
stimuli). Starting in late diastole, the last ex-
trastimulus-coupling interval was shortened
in 10-msec decrements until refractoriness
occurred. Electrocardiograms were recorded
with a frequency range from 0.05 to 500 Hz by
using a multichannel electrocardiogram am-
plifier and stored in a computer (Biopac Sys-
tems, Goleta, CA).
Unipolar Electrograms. The unipolar epi-
cardial electrograms were obtained from elec-
trodes placed on the ventricular epicardium
(the infarct area, the infarct marginal zone,
and the noninfarct zone) and standard bipolar
limb leads and recorded simultaneously. Elec-
trocardiograms were recorded with the appli-
cation of subcutaneous needle electrodes. The
signals were isolated, amplified, multiplexed,
and recorded by a computerized recording sys-
tem (Biopac Systems, Goleta, CA) with a fre-
quency range from 0.05 Hz to 500 Hz. Activa-
tion time, repolarization time, and activation
recovery time (ART), as a marker of local re-
polarization duration, were determined in
each epicardial lead as the minimum of the
potential time derivative during the QRS,
maximum of the potential time derivative dur-
ing ST-T, and the period between the two,
respectively. The ART dispersion (ARTd) was
defined as the difference between the maximal
and minimal ART durations in the infarct
area, the infarct marginal zone, and the non-
infarct zone. The correct ART (ARTc) were
calculated by using Bazett’s formula (ARTc ?
ART/square root from RR). The ARTc disper-
sion (ARTcd) was the difference between the
maximal and minimal ARTc durations (23).
Malignant Ventricular Arrhythmias In-
duction. The inducibility of the malignant ven-
tricular arrhythmias, including ventricular
tachycardia and ventricular fibrillation (VF), was
calculated as a ratio between the number of
induced arrhythmias and the number of at-
tempts for arrhythmia induction. The pro-
grammed electrical stimulation included S1S2,
S1S2S3, and the burst pacing (performed at cy-
cle lengths of 100/90/80/70/60/50 msecs for 1
sec) on the infarct area, the infarct marginal
zone, and the noninfarct zone. The end point
was the induction of ventricular tachycardia or
VF or completion of the protocol.
VFT. VFTs of the infarct area, the infarct
marginal zone, and the noninfarct zone were
determined by a homemade stimulator, using
a previously reported method (24). Briefly, the
hearts were stimulated with rectangular
pulses at a frequency of 30 Hz, an impulse
length of 10 msecs, and a stimulation duration
of 500 msecs. Current intensity was increased
in increments of 1 mA until VF was attained.
Two minutes after stabilization, another stim-
ulus was given with the same current inten-
sity. VFT was determined as the lowest current
intensity at which two consecutive stimuli
precipitated VF.
Immunohistochemistry. The animals were
euthanized, and the hearts were harvested.
1083Crit Care Med 2011 Vol. 39, No. 5

Page 3

Five-micrometer sections (HM 500 OM, Mi-
cron, Walldorf, Germany) were obtained from
each heart and fixed in acetone overnight at
4°C, blocked with 10% goat serum in PBS, and
used for immunohistochemistry with the pri-
mary antibody, Connexin-43 (Santa Cruz Bio-
tech, Santa Cruz, CA), von Willebrand factor
(Santa Cruz Biotech, Santa Cruz, CA),
?-smooth muscle actin (Abcam, San Fran-
cisco, CA), and ?-sarcomeric actin. The pri-
mary antigen-antibody reaction was detected
with goat anti-rabbit IgG conjugated with Al-
exa Fluor-488 (Invitrogen, Carlsbad, CA) for
Connexin-43, von Willebrand factor, and
?-smooth muscle actin and goat anti-mouse
IgM conjugated with fluorescein isothiocya-
nate (Santa Cruz Biotech, Santa Cruz, CA) for
?-sarcomeric actin. Nuclei were counter-
stained with 4?,6-diamidino-2-phenylindole.
The samples were examined by using a confo-
cal laser scanning microscope (TCS SP2
AOBS, Leica, Mannheim, Germany).
Statistical Analysis. All quantitative data
are described as mean ? SD. Basic comparative
statistics were performed by using Student’s
two-tailed t test. Differences in the frequency
of inducibility of ventricular arrhythmias were
tested by Fisher’s exact test. A value of p ? .05
was considered to be statistically significant.
RESULTS
High efficiency of staining (?90%)
was observed after PKH26 staining, as
shown in Figure 1.
No differences in ventricular effective
refractory period (programmed electrical
stimulation S1S2) were observed between
the CSC group and PBS group on the
infarct marginal zone (62.0 ? 6.3 msecs
vs. 64.0 ? 7.0 msecs, p ? .75) and the
noninfarct zone (65.0 ? 5.3 msecs vs.
63.0 ? 6.7 msecs, p ? .14).
No differences in ventricular effective
refractory period (programmed electrical
stimulation S1S2S3) were observed be-
tween the CSC group and PBS group on
the infarct marginal zone (66.0 ? 5.2
msecs vs. 63.0 ? 6.7 msecs, p ? .39) and
the noninfarct zone (64.0 ? 8.4 msecs vs.
66.0 ? 8.4 msecs, p ? .75).
There were also no significant differ-
ences in unipolar electrogram ART on the
infarct area between the CSC group and
PBS group. However, the ART was signif-
icantly longer on the infarct marginal
zone and the noninfarct zone in PBS
group compared with that in CSC group
(Table 1). Coincidentally, unipolar elec-
trogram ARTd were significantly greater
in the PBS group compared with that in
CSC group (Table 1). After correction, the
effects on the ARTc and ARTcd were sim-
ilar to those before correction (Table 2).
Malignant ventricular arrhythmias
were significantly (p ? .00108) less in-
ducible in the CSC group (one of ten)
than the PBS group (nine of ten). VFTs
were higher in the CSC group compared
with those in the PBS group (Table 3).
In the CSC group, CSCs were found to
be surviving in the infarct zone and the
infarct marginal zone, expressing the
Connexin-43 (Fig. 2), ?-sarcomeric actin
(Fig. 3), von Willebrand factor (Fig. 4),
and ?-smooth muscle actin (Fig. 5). CSCs
differentiated into cardiomyocytes, endo-
thelial cells, and vascular smooth muscle
cells in the cardiac microenvironment of
the infarct zone and the infarct marginal
zone and connected with native myocar-
dium by Connexin-43.
DISCUSSION
This study demonstrated the antiar-
rhythmic effects of intramyocardial allo-
geneic CSC transplantation after myocar-
dial infarction. Local injection of CSCs
can significantly improve cardiac electro-
physiologic stability and increase VFT in
rats with myocardial infarction treated
with CSCs.
Despite major improvements in med-
ical therapy, a significant proportion of
patients with ischemic heart disease re-
main symptomatic (25). The disease pro-
cesses lead to myocardial contractile dys-
function and heterogeneous electrical
instability that are associated with ven-
tricular arrhythmogenesis (15). Malig-
nant arrhythmias are, in fact, a major
concern in patients with advanced heart
disease. In recent years, cell therapy has
emerged as a potential new strategy for
patients with ischemic heart disease.
However, the major safety issue raised by
the use of stem cells for cardiac repair has
been the occurrence of ventricular ar-
rhythmias, including ventricular tachy-
cardia and/or VF (17). The proarrhythmic
condition following stem cell therapy
might be attributed to one or more of the
following: 1) intrinsic electrophysiologic
properties of stem cells; 2) modulated
graft-host or graft-graft electromechani-
cal coupling (or both); 3) changes in ion
channel function; 4) induced heterogene-
ity; 5) altered myocardial tissue architec-
ture, and 6) local injury or edema in-
duced by intramyocardial injection (18,
26). Accordingly, particularly after skele-
tal myoblast transplantation (17), even
though bone marrow cells seem less ar-
rhythmogenic than skeletal myoblasts,
their implantation could produce local de-
lays in propagation because of the interpo-
sition of electrically coupled but unexcit-
able cells with normal host cardiomyocytes
(18). On the other hand, embryonic stem
cells showed spontaneous activity, low dV/
dt, prolonged action potential duration,
and easily inducible triggered arrhythmias,
which may act as an unanticipated arrhyth-
mogenic source from any of the three clas-
sic mechanisms (reentry, automaticity, or
triggered activity) (27).
A functional integration of the grafted
cells such that they can safely and effi-
ciently contribute to increased pump
function requires that their phenotype
supports electrical propagation, electro-
mechanical coupling, and contraction
(18). There is compelling evidence that
cells recapitulating the cardiogenic dif-
ferentiation program are the best suited
for achieving this goal. CSCs, therefore,
represent a logical source to be investi-
gated in cardiac regeneration therapy be-
cause, unlike other adult stem cells, they
are intrinsically programmed to generate
cardiac tissue and to couple with host
cardiomyocytes (16). In support of the
above discussion, our study showed that
CSC injection increased the inducibility
threshold of malignant ventricular ar-
rhythmias and significantly improved
cardiac electrophysiologic stability. These
CSCs have also the advantage of being
able to be isolated and expanded in vitro
from small samples of myocardium (6).
Not only can the CSCs be transplanted
through intravascular injection (10, 12),
which is the clear prerequisite for clinical
translation, but they can also contribute
to improving the hemodynamics in rats
with myocardium infarction (10, 12, 28).
Increased heterogeneity of ventricular
repolarization facilitates and provides a
substrate for reentry and the develop-
ment of ventricular arrhythmias (29, 30).
Figure 1. Cardiac stem cells stained by PKH26
(?100 magnification).
1084 Crit Care Med 2011 Vol. 39, No. 5

Page 4

The ART from unipolar electrograms is a
valuable evaluation of global sequence
and dispersion of ventricular repolariza-
tion (23). In our study, ART, ARTd, ARTc,
and ARTcd were significantly shorter on
the infarct marginal zone and the nonin-
farct zone in the CSC group compared
with that in PBS group. Furthermore,
there were similar effects and tendency
on the infarct zone, although there were
no statistical significances. The results
suggest that CSCs transplanted in the
infarct zone may replace the fibroblasts
and therefore decrease the electrical het-
erogeneity between the infarct zone and
the noninfarct zone. In addition, VFTs on
the infarct zone, the infarct marginal
zone, and the noninfarct zone were im-
proved significantly in the CSC-treated
group compared with those in the PBS-
treated group. The results of the VFT
measurement were consistent with the
measurement of unipolar electrograms
and the ventricular arrhythmias induced
by PBS.
After myocardial infarction occurs,
there are several changes in the infarct
zone and the infarct marginal zone: 1)
the number of myocardiocytes decreases
due to necrosis and apoptosis; 2) inflam-
matory cells infiltrate combined with in-
flammatory factor exudation, and 3) myo-
cardial fibrosis following myocardial
infarction (31, 32). These changes lead to
ventricular heterogeneity, which plays an
important role on cardiac electrophysi-
ologic instability and ventricular arrhyth-
mias. The mechanisms of the significant
improvement in cardiac electrophysi-
ologic stability and VFT after CSC trans-
plantation are still unclear. The following
several mechanisms may be considered.
First of all, the CSCs, which reside in the
heart itself, can differentiate into cardio-
myocytes, endothelial cells, and vascular
smooth muscle cells in vitro and in vivo
(6, 7, 16) and reconstitute cardiac tissue
and electromechanical coupled with host
cardiomyocytes. In the current study,
CSCs expressed the markers, including
muscle, endothelium, and vascular
smooth muscle in the infarct zone and
infarct marginal zone, which means that
CSCs may differentiate into cardiomyo-
cytes, endothelial cells, and vascular
smooth muscle cells in the cardiac mi-
croenvironment, rather than phagocyto-
sed by the inflammatory cells. de Boer et
al (33) proved CSC-derived cardiomyo-
cytes had a homogeneous and rather ma-
ture electrical phenotype: Stable resting
membrane potential and near mature INa,
Ica-L, and IK1current densities. Therefore,
electrical heterogeneity and conduction
disturbances could be attenuated by ef-
fective cardiac regeneration strategies,
such as grafting of CSCs with suitable elec-
trophysiologic properties. Connexin-43
plays an important role in integrating the
electrical characteristics and engrafting for
the transplanted cells (33, 34). Rat models
have shown that loss of Connexin-43 ex-
pression greatly increases the risk of ven-
tricular tachycardia and sudden death (35).
Another study showed that in an infarcted
rat model, myoblast transplantation in-
duces electrical ventricular instability (36).
The in vitro study demonstrated that cocul-
ture of myoblasts and cardiomyocytes re-
sulted in reentrant arrhythmias and that
such arrhythmias could be limited by over-
expression of Connexin-43 (37). Con-
nexin-43 also contributes to mesenchymal
stem cell survival and improves therapeutic
efficacy in myocardial infarction (34). Re-
cent study demonstrated that mesenchy-
mal stem cells (MSCs) implanted into the
rats with myocardial infarction reduce vul-
nerability to ventricular arrhythmias; and
abnormal alterations of Connexin-43, in-
cluding reduction and lateralization in the
infarct marginal zone and noninfarct zone
were significantly attenuated by MSC treat-
ment (38). All of the above studies demon-
strated that Connexin-43 plays an impor-
tant role in integrating the electrical
characteristics and engrafting for cell
transplantation. The CSC-derived cardio-
myocytes had a homogeneous and rather
mature electrical phenotype, and Con-
nexin-43wastranslocatedtothecellborder
(33). Our study showed that the CSCs sur-
vived in the infarct zone and the infarct
marginal zone, and expressed the Con-
nexin-43 coupling to each other and the
neighboring cardiac myocytes. CSC trans-
plantation improved the electrical hetero-
geneity, and the reentry in the infarct zone,
the marginal infarct zone, and noninfarct
zone would be blocked. The results of the
Connexin-43measurementwereconsistent
with the measurement of unipolar electro-
Table 1. Influence on the unipolar electrogram activation recovery time and unipolar electrogram
activation recovery time dispersion
Groupn
Activation Recovery Time (msecs)
Activation
Recovery Time
Dispersion
(msecs)
Infarct
Area
Infarct Marginal
Zone
Noninfarct
Zone
Phosphate buffer solution
Cardiac stem cell
p
10
10
112.8 ? 37.0
91.5 ? 11.6
.0991
118.1 ? 24.8
94.5 ? 10.9
.0129
120.1 ? 26.0
94.0 ? 11.5
.0094
38.6 ? 14.9
15.5 ? 4.4
.000177
Values are means ? SD.
Table 2.
dispersion
Influence on the correct activation recovery time and correct activation recovery time
Groupn
Correct Activation Recovery Time (msecs)
Correct Activation
Recovery Time
Dispersion
(msecs)Infarct Area
Infarct Marginal
Zone
Noninfarct
Zone
Phosphate buffer solution
Cardiac stem cell
p
10
10
250.5 ? 67.4
217.1 ? 27.5
.1649
257.2 ? 40.8
223.2 ? 17.8
.0261
265.8 ? 50.0
220.7 ? 19.3
.0159
72.1 ? 23.2
33.3 ? 11.7
.00017
Values are means ? SD.
Table 3. Influence on the ventricular fibrillation threshold
Groupn
Ventricular Fibrillation Threshold (mA)
Infarct Area Infarct Marginal ZoneNoninfarct Zone
Phosphate buffer solution
Cardiac stem cell
p value
10
10
4.0 ? 1.6
8.4 ? 3.0
.00064
2.5 ? 0.8
6.4 ? 2.5
.00016
3.4 ? 1.0
7.0 ? 2.6
.00063
Values are means ? SD.
1085Crit Care Med 2011 Vol. 39, No. 5

Page 5

Figure 2. PKH26-marked cardiac stem cells (CSCs) expressed Connexin-43 in the infarct zone and the infarct marginal zone. Confocal laser scanning
microscope images (?1000 magnification) of fluorescent immunohistochemical staining of PKH26-labeled CSCs in the infarct zone and the infarct
marginal zone. A, PKH26-labeled CSCs (white arrows) in the infarct zone. B, Sections stained with antibody to gap junction marker Connexin-43; white
arrows point to CSCs transplant. C, Visualization of all the cell nuclei (4?,6-diamidino-2-phenylindole) in the infarct zone. D, Merged image of A–C shows
PKH26-labeled CSCs express Connexin-43 (white arrows). E, PKH26-marked CSCs in the infarct marginal zone. F, CSCs express Connexin-43. G,
Visualization of all the cell nuclei (4?,6-diamidino-2-phenylindole) in the infarct marginal zone. H, Merged image of E–G shows PKH26-marked CSCs
express Connexin-43 (white arrows).
Figure 3. PKH26-marked cardiac stem cells (CSCs) expressed ?-sarcomeric actin in the infarct zone and the infarct marginal zone. Confocal laser scanning
microscope images (?1000 magnification) of fluorescent immunohistochemical staining of PKH26-labeled CSCs in the infarct zone and the infarct
marginal zone. A, PKH26-labeled CSCs (white arrows) in the infarct zone. B, Sections stained with antibody to muscle marker ?-sarcomeric actin; white
arrows point to CSCs transplant. C, Visualization of all the cell nuclei (4?,6-diamidino-2-phenylindole) in the infarct zone. D, Merged image of A–C shows
PKH26-labeled CSCs express ?-sarcomeric actin (white arrows). E, PKH26-marked CSCs in the infarct marginal zone. F, CSCs express ?-sarcomeric actin.
G, Visualization of all the cell nuclei (4?,6-diamidino-2-phenylindole) in the infarct marginal zone. H, Merged image of E–G shows PKH26-marked CSCs
express ?-sarcomeric actin (white arrows).
1086Crit Care Med 2011 Vol. 39, No. 5