Transplantation of cardiotrophin-1-expressing myoblasts to the left ventricular wall alleviates the transition from compensatory hypertrophy to congestive heart failure in Dahl salt-sensitive hypertensive rats.
ABSTRACT We investigated whether autologous transplantation of skeletal myoblasts (MB) transferred with cardiotrophin-1 (CT-1) gene could retard the transition to heart failure (HF) in Dahl salt-sensitive (DS) hypertensive rats.
Although MB is a therapeutic candidate for chronic HF, little is known about the efficiency of this strategy when applied in nonischemic HF. Cardiotrophin-1 has potent hypertrophic and survival effects on cardiac myocytes. We hypothesized that transplantation of CT-1-expressing myoblasts could provide cardioprotective effects against ventricular remodeling in DS hypertensive rats.
The DS rats were fed a high salt diet for 6 weeks and developed left ventricular (LV) hypertrophy at 11 weeks. At this stage, animals underwent MB to the myocardium with skeletal myoblasts transferred with CT-1 gene using retrovirus (transplantation of CT-1-expressing myoblasts [MB + CT], n = 31) or myoblasts alone (MB, n = 31). The sham group rats were injected with phosphate-buffered saline (n = 24).
At 17 weeks, MB and MB + CT groups showed a significant alleviation of LV dilation and contractile dysfunction compared with the sham group. The degree of alleviation was significantly greater in the MB + CT group than the MB group (LV end-diastolic dimension: sham 7.06 +/- 0.14 mm, MB 6.51 +/- 0.16 mm, MB + CT 6.24 +/- 0.07 mm; fractional shortening: sham 32.1 +/- 1.4%, MB 38.5 +/- 1.5%, MB + CT 43.2 +/- 0.8%). Histological examination revealed that the myocyte size was 20% larger in the MB + CT group at 17 weeks than in the age-matched sham group. Upregulation of renin-angiotensin and endothelin systems during the transition to HF was attenuated by myoblast transplantation, and this effect was enhanced in the MB + CT group.
Transplantation of skeletal myoblasts combined with CT-1-gene transfer could be a useful therapeutic strategy for HF.
- SourceAvailable from: nih.gov[show abstract] [hide abstract]
ABSTRACT: Intracardiac grafts comprised of genetically modified skeletal myoblasts were assessed for their ability to effect long-term delivery of recombinant transforming growth factor-beta (TGF-beta) to the heart. C2C12 myoblasts were stably transfected with a construct comprised of an inducible metallothionein promoter fused to a modified TGF-beta 1 cDNA. When cultured in medium supplemented with zinc sulfate, cells carrying this transgene constitutively secrete active TGF-beta 1. These genetically modified myoblasts were used to produce intracardiac grafts in syngeneic C3Heb/FeJ hosts. Viable grafts were observed as long as three months after implantation, and immunohistological analyses of mice maintained on water supplemented with zinc sulfate revealed the presence of grafted cells which stably expressed TGF-beta 1. Regions of apparent neovascularization, as evidenced by tritiated thymidine incorporation into vascular endothelial cells, were observed in the myocardium which bordered grafts expressing TGF-beta 1. The extent of vascular endothelial cell DNA synthesis could be modulated by altering dietary zinc. Similar effects on the vascular endothelial cells were not seen in mice with grafts comprised of nontransfected cells. This study indicates that genetically modified skeletal myoblast grafts can be used to effect the local, long-term delivery of recombinant molecules to the heart.Journal of Clinical Investigation 02/1995; 95(1):114-21. · 12.81 Impact Factor
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ABSTRACT: Cellular cardiomyoplasty (CCM), or introduction of immature cells into terminally injured heart, can mediate repair of chronically injured myocardium. Several different cell types, ranging from embryonic stem cells to autologous skeletal myoblasts, have been successfully propagated within damaged heart and shown to improve myocardial performance. However, it is unclear if the functional advantages associated with CCM depend upon the use of myogenic cells or if similar results can be seen with other cell types. Thus, we compared indices of regional contractile (systolic) and diastolic myocardial performance following transplantation of either autologous skeletal myoblasts (Mb) or dermal fibroblasts (Fb) into chronically injured rabbit heart. In vivo left ventricular (LV) pressure (P) and regional segment length (SL) were determined in 15 rabbits by micromanometry and sonomicrometry 1 week following LV cryoinjury (CRYO) and again 3 weeks after autologous skeletal Mb or dermal Fb transplantation. Quantification of systolic performance was based on the linear regression of regional stroke work and end-diastolic (ED) SL. Regional diastolic properties were assessed using the curvilinear relationships between LVEDP and strain (epsilon) as well as LVEDP and EDSL. At study termination, cellular engraftment was characterized histologically in a blinded fashion. Indices of diastolic performance were improved following CCM with either Mb or Fb. However, only Mb transplantation improved systolic performance; Fb transfer actually resulted in a significant decline in systolic performance. These data suggest that both contractile and noncontractile cells can improve regional material properties or structural integrity of terminally injured heart, as reflected by improvements in diastolic performance. However, only Mb improved systolic performance in the damaged region, supporting the role of myogenic cells in augmenting contraction. Further studies are needed to define the mechanism by which these effects occur and to evaluate the long-term safety and efficacy of CCM with any cell type.Cell Transplantation 01/2000; 9(3):359-68. · 4.42 Impact Factor
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ABSTRACT: Gene transfer into human hematopoietic stem cells with expression targeted to the maturing myelomonocytic progeny has applications for gene therapy of genetic diseases affecting granulocytes and macrophages. We hypothesized that promoters of myeloid-specific genes that are upregulated with myelomonocytic differentiation would also upregulate expression of an exogenous gene in a retroviral vector. Moloney murine leukemia virus (MoMuLV)-based retroviral vectors using promoters from hematopoietic genes (CD11b, CD18, and CD34) were compared with vectors with viral promoters (MoMuLV long terminal repeat [LTR], cytomegalovirus [CMV], and simian virus 40 [SV40]). Human glucocerebrosidase (GC) cDNA was the reporter gene. HL60 cells were transduced with these vectors and vector-derived GC activity was compared in undifferentiated HL-60 cells and the same cells differentiated into granulocytes using dimethyl sulfoxide or monocyte/macrophages using phorbol myristate acetate. In undifferentiated HL-60 cells, vector-derived GC activity was the highest when it was controlled by the MoMuLV LTR. In HL-60 cells differentiated into granulocytes, vector-derived GC activity transcribed from the CD11b, MoMuLV LTR, and CMV promoters was equivalent to 1.7, 1.5, and 1.5 times the normal endogenous GC activity, respectively, and 0.8, 2.0, and 3.6 times the normal GC activity, respectively, in those differentiated into macrophages. With granulocytic differentiation, the CD11b promoter showed maximal induction in GC activity (8-fold); with macrophage differentiation, the CD11b promoter showed a fourfold induction in GC expression. The CD11b promoter also generated significant levels of GC activity in the myelomonocytic progeny of transduced CD34+ cells. Expression from the CD11b promoter, unlike that from the CMV or the MoMuLV LTR promoters, was relatively myelomonocyte-specific, with minimal expression observed in Jurkat T cells or HeLa carcinoma cells. The induction of expression from the CD11b promoter with differentiation in HL-60 cells correlates with the developmental regulation of the CD11b gene. Retroviral vectors using the CD11b promoter have potential utility for gene therapy of disorders affecting the myelomonocytic lineage.Blood 11/1995; 86(8):2993-3005. · 9.06 Impact Factor
Transplantation of Cardiotrophin-1–Expressing
Myoblasts to the Left Ventricular Wall
Alleviates the Transition From Compensatory
Hypertrophy to Congestive Heart
Failure in Dahl Salt-Sensitive Hypertensive Rats
Ryuji Toh, MD,* Seinosuke Kawashima, MD, PHD,* Miki Kawai, MD, PHD,*
Tsuyoshi Sakoda, MD, PHD,† Tomomi Ueyama, MD, PHD,* Seimi Satomi-Kobayashi, MD,*
Sonoko Hirayama, MD, PHD,* Mitsuhiro Yokoyama, MD, PHD*
Kobe and Nishinomiya, Japan
We investigated whether autologous transplantation of skeletal myoblasts (MB) transferred
with cardiotrophin-1 (CT-1) gene could retard the transition to heart failure (HF) in Dahl
salt-sensitive (DS) hypertensive rats.
Although MB is a therapeutic candidate for chronic HF, little is known about the efficiency
of this strategy when applied in nonischemic HF. Cardiotrophin-1 has potent hypertrophic
and survival effects on cardiac myocytes. We hypothesized that transplantation of CT-1–
expressing myoblasts could provide cardioprotective effects against ventricular remodeling in
DS hypertensive rats.
The DS rats were fed a high salt diet for 6 weeks and developed left ventricular (LV)
hypertrophy at 11 weeks. At this stage, animals underwent MB to the myocardium with
skeletal myoblasts transferred with CT-1 gene using retrovirus (transplantation of CT-1–
expressing myoblasts [MB ? CT], n ? 31) or myoblasts alone (MB, n ? 31). The sham
group rats were injected with phosphate-buffered saline (n ? 24).
At 17 weeks, MB and MB ? CT groups showed a significant alleviation of LV dilation and
contractile dysfunction compared with the sham group. The degree of alleviation was
significantly greater in the MB ? CT group than the MB group (LV end-diastolic
dimension: sham 7.06 ? 0.14 mm, MB 6.51 ? 0.16 mm, MB ? CT 6.24 ? 0.07 mm;
fractional shortening: sham 32.1 ? 1.4%, MB 38.5 ? 1.5%, MB ? CT 43.2 ? 0.8%).
Histological examination revealed that the myocyte size was 20% larger in the MB ? CT
group at 17 weeks than in the age-matched sham group. Upregulation of renin-angiotensin
and endothelin systems during the transition to HF was attenuated by myoblast transplan-
tation, and this effect was enhanced in the MB ? CT group.
CONCLUSIONS Transplantation of skeletal myoblasts combined with CT-1-gene transfer could be a useful
therapeutic strategy for HF.(J Am Coll Cardiol 2004;43:2337–47) © 2004 by the
American College of Cardiology Foundation
Despite medical and surgical advances, heart failure (HF) is
still a major cause of death. Because mature cardiac myo-
cytes cannot re-enter the cell cycle and the adult heart lacks
functional repair mechanisms, myogenic cell transplantation
into the damaged myocardium is a promising approach to
the treatment for end-stage HF (1). Recent experimental
studies have demonstrated that intramyocardial skeletal
myoblast transplantation improves cardiac function after
myocardial infarction (2–5). Clinical trials with skeletal
myoblast transplantation are also on the way (6,7). How-
ever, little is known about the efficiency of this strategy
when applied in global HF of nonischemic causes (8–10).
The Dahl salt-sensitive (DS) hypertensive rats undergo
the transition from compensatory hypertrophy to congestive
HF (11). When they are fed a high salt diet after the age of
6 weeks, they develop systemic hypertension and concentric
left ventricular (LV) hypertrophy at 11 weeks, followed by
marked LV dilation and contractile dysfunction at 15 to 20
weeks (11). Thus, they have been used as an animal model
for nonischemic hypertension-based HF. We investigated
whether autologous transplantation of skeletal myoblasts
(MB) could retard the transition from compensatory hyper-
trophy to HF in DS hypertensive rats.
Skeletal myoblast transplantation is also useful as a tool
for cell-mediated gene therapy, providing sustained local
expression of recombinant proteins in the heart (12,13).
Cardiotrophin-1 (CT-1), a member of the interleukin-6
superfamily, induces hypertrophy and prolongs survival of
cardiac myocytes in vitro (14–16). It has been reported that
CT-1 expression in the myocardium is upregulated in
chronic HF (17,18). However, the pathophysiologic signif-
From the *Division of Cardiovascular and Respiratory Medicine, Department of
Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan; and
the †Department of Internal Medicine, Cardiovascular Division, Hyogo College of
Medicine, Nishinomiya, Japan. Supported by grants-in-aid for the research from the
Ministry of Health and Welfare of Japan (2002 to 2003) and from the Ministry of
Education, Science, Sports, and Culture of Japan (2001 to 2002).
Manuscript received October 23, 2003; revised manuscript received December 30,
2003, accepted February 3, 2004.
Journal of the American College of Cardiology
© 2004 by the American College of Cardiology Foundation
Published by Elsevier Inc.
Vol. 43, No. 12, 2004
icance of CT-1 in the transition from compensated to
decompensated HF is not fully elucidated (18). We accord-
ingly analyzed the feasibility and efficiency of transplanting
skeletal myoblasts that are transferred with CT-1 gene using
retrovirus in preserving cardiac function of DS hypertensive
rats. We hypothesized that this strategy could provide
cardioprotective effects against ventricular remodeling in
combination with functional benefits of skeletal myoblast-
All animal experiments were performed in accordance with
the guidelines for animal experimentation at Kobe Univer-
sity Graduate School of Medicine. Male DS rats were
obtained from Japan SLC Co. Ltd (Hamamatsu, Japan).
After weaning, the rats were fed a low salt diet (0.3% NaCl)
until the age of six weeks; thereafter, a high salt diet (8%
NaCl) was started. The myoblast culture process was per-
formed according to the method reported previously (2). In
brief, skeletal muscles were harvested from hind limb
skeletal muscle of DS rats, and then they were minced and
seeded on polystyrene plates. Tissue pieces were incubated
at 37°C for 48 h in Dulbecco’s Modified Eagle’s Medium
(Sigma, St. Louis, Missouri) supplemented with 10% fetal
bovine serum (FBS), 100 ?g/ml streptomycin, and 500
?g/ml penicillin (all from Sigma) to allow primary satellite
cell (myoblast) isolation. When cells began to migrate out of
tissue, tissue pieces were removed. Myoblast growth densi-
ties were maintained at ?70% to avoid the differentiation
into the myotube (2).
Retrovirus-mediated CT-1–gene transfer. The full-length
complementary deoxyribonucleic acid of the rat CT-1 was
obtained from total ribonucleic acid in neonatal rat ven-
tricular cells using reverse transcriptase-polymerase chain
reaction. Sense and antisense primers for rat CT-1 were
and 5?-TATTCAGGCAACGCCCCCTGGCAC-3?, re-
spectively (14). The polymerase chain reaction fragment was
confirmed by deoxyribonucleic acid sequencing and then
inserted into pLN plasmid (generously provided by Dr.
Noriyuki Kasahara, USC Institute for Genetic Medicine, Los
Angeles, California) (19) to construct a retroviral transfer
acid sequence (pLN-CT-1). Retroviral vector encoding CT-1
(19,20). Briefly, a 100-mm dish of nonconfluent 293T cells
were transfected with 15 ?g of pLN-CT-1, 15 ?g of pHIT 60
(i.e., cytomegalovirus [CMV] gag pol encoding plasmid), and
10 ?g pHIT 123 (CMV ecotropic envelope) using the calcium
phosphate coprecipitation method (21); 16 h after transfer, the
media were adjusted to a final concentration of 10 mM in
sodium butyrate. After 8-h incubation, cells were washed and
incubated in fresh medium without sodium butyrate. Condi-
tioned medium containing retrovirus was harvested 16 h later
and filtered through 0.45-?m Millipore-HA (Millipore Co.,
Bedford, Massachusetts). For transfer of CT-1 gene into the
myoblasts, cells were infected overnight with a dilution of virus
stock in cultured medium supplemented with 8 ?g of poly-
brane per ml. Infections were performed twice for efficient
transfer into myoblasts.
Cell transplantation. At the age of 11 weeks, animals
underwent thoracotomy under general anesthesia with in-
traperitoneal sodium pentobarbital (50 mg/kg) and autolo-
gous transplantation to the myocardium with CT-1-
transferred myoblasts (MB ? CT group, n ? 31) or
myoblasts alone (MB group, n ? 31). Cells (1 ? 106) were
harvested and resuspended in 0.15 ml of phosphate-buffered
saline, followed by intramyocardial injection 10 to 15 times
into the anterior aspects of the LV free wall with a 26G
needle. Accordingly, approximately 1 ? 105cells were
injected in each site. In the sham group (n ? 24), the same
volume of phosphate-buffered saline was injected. The
surgical wounds were repaired, and the rats were returned to
the cages to recover.
Echocardiographic and hemodynamic studies. Systolic
blood pressure and heart rate were measured by a tail-cuff
method (Muromachi Kikai, Japan).
At the age of 11, 15, and 17 weeks, transthoracic
two-dimensional echocardiography (SONOS 5500, Philips
Medical Systems Corp., Andover, Massachusetts) was per-
formed under light anesthesia with sodium pentobarbital. A
12-MHz Ultraband Sector Transducer (Philips Medical
Systems Corp.) probe was used. Left ventricular end-
diastolic dimension (EDD), end-systolic dimension (ESD),
and LV posterior wall thickness (PWT) were determined
from the M-mode tracing based on the short-axis view of
the LV at the papillary muscle level. Left ventricular percent
fractional shortening (%FS) was calculated as: [(EDD ?
ESD)/EDD] ? 100.
At the age of 17 weeks, animals underwent direct cardiac
catheterization via subdiaphragmatic approach to measure
LV pressure under light anesthesia. The catheter was
connected to a pressure transducer, and continuous mea-
surements of LV pressure and heart rate were recorded
using a Maclab system (Bioresearch Center, Nagoya, Ja-
pan). Animals were allowed to breathe spontaneously dur-
Abbreviations and Acronyms
MB ? CT ? transplantation of cardiotrophin-1–
? posterior wall thickness
? angiotensin II
? Dahl salt-sensitive
? end-diastolic dimension
? percent fractional shortening
? heart failure
? left ventricle or left ventricular
? transplantation of skeletal myoblasts alone
2338 Toh et al.
CT-1–Myoblast Transplantation for HF
JACC Vol. 43, No. 12, 2004
June 16, 2004:2337–47
ing the pressure recording. Maximal rate of pressure rise
(dP/dtmax) and LV end-diastolic pressure were determined
from tracings of LV pressure, and averaged on 100 consec-
utive cardiac cycles.
Western blot analysis. At the age of 13 weeks, three hearts
from each group were collected to assess CT-1 expression in
the heart. The isolated LV free wall was cut into small
pieces and homogenized with a Polytron homogenizer
(Kinematica Inc., Cincinnati, Ohio). Homogenates were
centrifuged, and the supernatants were collected. For detec-
tion of secreted CT-1 from the transferred myoblasts,
myoblasts were cultured in DMEM supplemented with
10% FBS, and conditioned media were collected after 72 h
incubation. The expression of CT-1 protein was determined
by Western blot analysis using an anti-human CT-1 poly-
clonal antibody (Pepro Tech EC Ltd., London, United
Kingdom). The results were quantified by scanning
Histological analysis. Left ventricular specimens were ob-
tained at the age of 17 weeks (n ? 5 for each group).
Specimens were frozen with liquid nitrogen and sectioned
to 8-?m-thick slices. The slices were stained with
hematoxylin-eosin. The slices also underwent immunohis-
tochemical staining for skeletal-specific myosin heavy chain
by MY-32 monoclonal antibody (Sigma-Aldrich Inc., St.
Louis, Missouri), and for CT-1.
In the hematoxylin-eosin–stained sections, the cross-
sectional area of cardiac myocytes that was cut transversely
and showed nuclei in the center was measured in the free
wall and the septum of the LV, respectively. In each side of
LV wall, approximately 50 cells were counted per each
animal. Before myoblast transplantation, the myocyte size
was measured at the age of 6 and 11 weeks (n ? 5 for each
stage). A total of 100 cells in random areas of LV, including
both the free wall and the septum, were counted per each
animal, and the average was used for analysis.
Measurements of plasma angiotensin II (Ang II) and
endothelin-1 (ET-1) levels. At the age of 13 and 17
weeks, blood was collected in a polypropylene tube contain-
ing aprotinin (300 kallikrein-inhibiting units/ml) and
ethylenediamine-tetraacetic acid (1 mg/ml) and then cen-
trifuged at 3,000 rpm for 15 min at 4°C. The plasma thus
obtained was stored at ?80°C until assayed. The plasma
levels of Ang II and ET-1 were measured by SRL, Inc.
Effects of transferred-gene–derived CT-1 in vitro.
Myoblasts were cultured in DMEM supplemented with
0.1% bovine serum albumin, ITS (10 ?g/ml insulin, 10
?g/ml transferrin, and 10 ng/ml selenious acid), and con-
ditioned media were collected after 72-h incubation. Pri-
mary culture of neonatal rat cardiac myocytes was prepared
as previously described (21). Cardiac myocytes were treated
with either DMEM supplemented with 0.1% bovine serum
albumin and ITS (control), the media from myoblasts with
or without CT-1–transfer, or 1 nM recombinant human
CT-1 (Pepro Tech EC Ltd.) for 48 h. Then cellular
morphology was examined and photographed under light
Proliferation and survival tests in myoblasts were per-
formed by use of C2C12 myoblasts (American Type Cul-
ture Collection, Manassas, Virginia). Briefly, 5,000 cells
were plated in 96-well dishes and grown for 24-h in media
containing 10% FBS. The media were then changed to 0%
or 10% serum-media, and cell number was determined by
the absorbance of the WST-8 reagent (Dojindo Co.,
Kumamoto, Japan) at 0, 8, 24, and 48 h after media
Statistical procedures. All values were expressed as mean
? SEM. The serial measurements of echocardiography
were assessed using two-way analysis of variance for re-
peated measures. The differences at specific stages among
groups were analyzed by one-way analysis of variance,
followed by Bonferroni’s multiple-comparison t test. Paired
t test was used to assess significant differences in myocyte
size between areas in each group. Statistical analyses were
performed using StatView (version 5.0, SAS Institute Inc.,
Cary, North Carolina). Values were considered statistically
significant at p ? 0.05.
Functional assessment after cell transplantation. The
DS rats who were fed a high salt diet developed systemic
hypertension (?220 mm Hg) at the age of 11 weeks, which
continued until the age of 17 weeks. There was no signifi-
cant difference in systolic blood pressure among three
groups throughout the experiment (Table 1). At the age of
14 to 17 weeks, two rats in the sham group and one rat in
the MB group deceased. These animals showed labored
respiration with a loss of activity before they died. There-
fore, the cause of their death seemed to be congestive heart
failure. At the age of 17 weeks, LV end-diastolic pressure
increased, and LV dP/dtmaxdecreased in the sham group,
reflecting congestive HF (Table 2). In contrast, LV end-
diastolic pressure was not increased, and LV dP/dt was
relatively preserved in both the MB and MB ? CT groups.
Moreover, LV dP/dtmaxwas significantly higher in the MB
? CT group than in the MB group (p ? 0.01) (Table 2).
Representative M-mode echocardiograms of the LV at
the papillary muscle level were shown in Figure 1A. At the
age of 11 weeks, the DS rats developed concentric LV
hypertrophy, and there were no differences in preoperative
data among the three groups (Fig. 1). At the age of 17
weeks, the sham group showed marked LV dilation and
global hypokinesis (Fig. 1A). From the age of 11 to 17
weeks, a marked decrease in %FS and an increase in EDD
occurred in sham group, which was associated with a
reduction in PWT (%FS, 50.9 ? 0.4 vs. 32.1 ? 1.4; EDD,
5.68 ? 0.02 vs. 7.06 ? 0.14 mm; PWT, 2.12 ? 0.05 vs.
1.68 ? 0.02 mm) (Fig. 1B). In contrast, LV dilation was
attenuated, and contractile function was maintained signif-
icantly in both the MB and MB ? CT groups at the age of
JACC Vol. 43, No. 12, 2004
June 16, 2004:2337–47
Toh et al.
CT-1–Myoblast Transplantation for HF
17 weeks compared with the age-matched sham group (Fig.
1). Moreover, %FS, EDD, and PWT were more preserved
in the MB ? CT group than in the MB group at the age of
17 weeks (%FS 43.2 ? 0.8 vs. 38.5 ? 1.5; EDD 6.24 ?
0.07 vs. 6.51 ? 0.16 mm; PWT 1.79 ? 0.02 vs. 1.73 ? 0.02
mm, p ? 0.05, respectively) (Fig. 1B). On the other hand,
B-mode echocardiogram did not exhibit asymmetrical LV
wall motion after cell transplantation in both the MB and
MB ? CT groups (data not shown).
We also performed Holter electrocardiogram in some
animals at the age of 17 weeks, which revealed no lethal
arrhythmias after cell transplantation (n ? 3 in the MB
group and n ? 2 in the MB ? CT group, data not
Grafted myoblasts in the myocardium. Serial sections of
the transplanted area after cell transplantation were shown
in Figure 2. Graft survival was identified at six weeks after
transplantation (the age of 17 weeks) by hematoxylin-eosin
staining and immunohistochemical staining for skeletal-
specific myosin heavy chain, by MY-32 mAb. Multinuclear
elongated structure was identified in H-E staining, which
indicates that myoblasts had differentiated into myotubes
(Fig. 2). These muscular structures were positively stained
with MY-32 (Fig. 2), whereas no cells were stained in the
PBS-injected hearts. Positive staining for skeletal myosin
heavy chain revealed the presence of myotubes. Surviving
cells aligned with the cardiac fiber axis within the native
myocardium. On the other hand, accumulation of inflam-
matory cells was hardly detected around the transplanted
area at day 0 and two weeks, four weeks, and six weeks after
transplantation in both MB and MB ? CT groups. As
reported previously, the fibrosis was found mainly in
perivascular regions of the arterioles (11), and there were no
differences in the extent of fibrosis among the three groups.
CT-1 expression in the myocardium after cell transplan-
tation. The secretion of transferred gene-derived CT-1
was confirmed in vitro by Western blot analysis (Fig. 3A).
Immunohistochemical staining of the transplanted area for
CT-1 is shown in Fig. 3B. Myotubes positively stained for
CT-1 were detected in the MB ? CT group at six weeks
after transplantation, whereas no myotubes were stained in
the MB group (Fig. 3B). These data suggest that local
expression of CT-1 in CT-1–transfected cells was sus-
tained. Positive staining of myocardium for CT-1, although
slightly, was also detected, indicating that endogenous
CT-1 was expressed in the myocardium. Western blot
analysis revealed that tissue expression of CT-1 in the LV
free wall of the MB ? CT group significantly increased
compared with sham group at two weeks after transplanta-
tion (Fig. 3C) (2.3 ? 0.5-fold, p ? 0.05).
Morphometry of the ventricular myocytes. The cross-
sectional area of LV myocytes markedly increased from the
age of 6 to 11 weeks (Fig. 4B). Then the sham group
exhibited a slight decrease of myocyte size, although statis-
tically not significant, from the age of 11 to 17 weeks (Fig.
4B). In contrast, the myocyte size in both the free wall and
the septum wall of LV increased in the MB ? CT group,
from the age of 11 to 17 weeks (Fig. 4B). At the age of 17
weeks, although no difference in myocyte size was found
between the sham and MB groups, the myocyte size in the
free wall was 20% larger in the MB ? CT group than in
sham group (p ? 0.05) (Figs. 4A and 4B). Furthermore, the
myocyte size in the free wall at the cell-injected site was
significantly larger than that of septum wall at the remote
site of cell injection in the MB ? CT group at this stage (p
? 0.05) (Fig. 4B). On the other hand, the myocyte size in
the free wall did not significantly differ from that of the
septum wall in both the sham and MB groups. We also
demonstrated that the conditioned media from CT-1–
transferred myoblasts induced cardiac myocyte hypertrophy
in vitro (Fig. 4C). These data suggest that CT-1 secreted
from grafted cells in the MB ? CT group induced hyper-
trophy of the adjacent myocardial cells in a paracrine
Table 1. Systolic Blood Pressure and LVW/BW Ratio
nSBP (mm Hg) BW (g)HW (mg) LVW (mg)LVW/BW (mg/g)
MB ? CT
214 ? 10
241 ? 24
333 ? 6
337 ? 18 1,468 ? 831,221 ? 693.7 ? 0.4
12214 ? 8
221 ? 13
331 ? 3
326 ? 166 1,514 ? 46 1,199 ? 693.7 ? 0.1
11201 ? 12
233 ? 20
329 ? 5
331 ? 54 1,575 ? 321,330 ? 304.0 ? 0.1
Values are presented as mean ? SEM.
BW ? body weight; CT ? cardiotrophin; HW ? heart weight; LVW ? left ventricular weight; MB ? myoblasts; SBP ?
systolic blood pressure; W ? the age (weeks).
Table 2. Hemodynamics at the Age of 17 Weeks
(n ? 4)
(n ? 6)
MB ? CT
(n ? 7)
LVEDP, mm Hg
887.8 ? 85.21,252 ? 35† 1,849 ? 168†‡
14.0 ? 2.96.9 ? 2.3*6.8 ? 1.1*
Values are presented as mean ? SEM. *p ? 0.05 and †p ? 0.01 vs. sham group; ‡p
? 0.01 vs. MB group by ANOVA and Bonferroni’s multiple-comparison t test.
CT ? cardiotrophin; dP/dt ? maximal dP/dt; LVEDP ? Left ventricular
end-diastolic pressure; MB ? myoblasts.
2340 Toh et al.
CT-1–Myoblast Transplantation for HF
JACC Vol. 43, No. 12, 2004
June 16, 2004:2337–47
Neurohumoral regulation during the transition to con-
gestive HF. In this DS rat model, it has been demon-
strated that the activation of local renin-angiotensin and
endothelin systems in the heart contributes to the transition
to heart failure (22,23). Indeed, serum Ang II levels in-
creased at the congestive heart failure stage compared with
the LV hypertrophy stage in sham group (Fig. 5A). How-
ever, these changes were attenuated in both the MB and
MB ? CT groups, and the degree of attenuation was
greater in MB ? CT group than the MB group (p ? 0.05)
(Fig. 5A). The serum ET-1 levels were also upregulated
during the transition to congestive heart failure in sham
group, but remained unchanged in both the MB and MB ?
CT groups (Fig. 5B). We also found that upregulation of
angiotensinogen, angiotensin-converting enzyme, prepro-
ET-1, and ET-converting enzyme messenger ribonucleic
acid in the LV during the transition to congestive heart
failure were all attenuated by myoblast transplantation,
using semiquantitative reverse transcriptase-polymerase
chain reaction (data not shown).
The effect of CT-1 on myoblast survival in vitro. To
examine the effect of CT-1 on myoblast survival in vitro, we
Figure 1. (A) Representative tracings of left ventricular M-mode echocardiograms of the sham group at the age of 11 weeks (upper left) and 17 weeks
(upper right), the myoblast transplantation (MB) group (lower left), and the transplantation of CT-1–expressing myoblasts (MB ? CT) group (lower
right) at the age of 17 weeks. Left ventricular dilation and contractile dysfunction were attenuated in both MB and MB ? CT groups at the heart failure
stage (six weeks after transplantation). (B) Serial measurements of echocardiography in the sham, MB, and MB ? CT groups. A p value by two-way analysis
of variance: group ?0.001; time course ?0.001; group/time course interaction ?0.001 for each parameter. *p ? 0.05 and **p ? 0.01 vs. sham group; †p
? 0.05, and ††p ? 0.01 vs. MB group at same stage by Bonferroni’s multiple-comparison t test. Values are means ? SEM. EDD ? end-diastolic diameter;
FS ? fractional shortening; PWT ? posterior wall thickness; W ? the age (weeks).
JACC Vol. 43, No. 12, 2004
June 16, 2004:2337–47
Toh et al.
CT-1–Myoblast Transplantation for HF