(Circulation. 2000;102:III-359.)
© 2000 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Development of a Novel Method for Cell Transplantation Through the Coronary Artery
From the Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Medicine at the Heart Science Centre, Harefield Hospital, Middlesex, UK.
Correspondence to Professor Sir Magdi H. Yacoub, Department of Cardiothoracic Surgery, Harefield Hospital, Harefield, Middlesex, UB9 6JH, UK. E-mail Ken.Suzuki{at}harefield.nthames.nhs.uk
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Abstract |
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Background—Cell transplantation is a promising strategy to treat end-stage heart failure. At present, a popular method to deliver cells into the heart is direct intramuscular injection. This method, however, may not be efficient in spreading cells globally into the myocardium. We have developed a novel method for cell transplantation using intracoronary infusion.
Methods and Results—An L6 rat skeletal muscle cell line
expressing ß-galactosidase (ß-gal) was generated by gene
transfection and clonal selection. These cells (106 in 1 mL
medium) were infused into explanted rat hearts through the
coronary artery, followed by heterotopic heart transplantation
into the abdomen of recipients. Control hearts were infused with
cell-free medium. According to ß-gal activity measurements,
5x105 grafted cells per heart existed on day 3,
increasing to 5x106 on day 28 in the cell-transplanted
hearts. At day 28, discrete loci positively stained for ß-gal were
observed throughout the cardiac layers of both left and right
coronary territories. Some of them differentiated into
ß-gal–positive multinucleated myotubes that aligned with the cardiac
fiber axis and integrated into the native myocardium,
whereas others formed colonies consisting of undifferentiated
myoblasts. Connexin 43, a cardiac gap junction protein, was expressed
between grafted cells and native cardiomyocytes. No
reduction in cardiac function was observed in a Langendorff perfusion
system.
Conclusions—We have developed a unique method for efficient cell transplantation based on intracoronary infusion. This method, potentially applicable in the clinical setting during cardiac surgery, could be useful to globally supply cells to the heart.
Key Words: cells • transplantation • heart failure
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Introduction |
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Although death from heart disease has shown a steady decline for the past decade, the prevalence of congestive heart failure continues to increase. At present, the effect of drug treatment is limited, and thus heart transplantation is the only established way to efficiently treat patients with end-stage heart failure. There are some serious disadvantages, however, such as complications from immunosuppressive agents and high cost.1 2
Cell transplantation is a promising strategy to treat end-stage heart failure. The ability to augment the number of cardiomyocytes could be of therapeutic value if the new myocytes functionally integrated with preexisting myocardium. Several types of cells have been used as a graft in cell transplantation models, demonstrating successful long-term survival in the mammalian myocardium.3 4 5 6 7 8 9 10 11 12 13 Recently, it has also been reported that cell transplantation could improve cardiac function of damaged heart.4 5 6 7 The popular method for cell delivery into the heart described in the reports is direct intramuscular injection, in which 0.5 to 10x106 cells can be infused into the myocardium through a small thoracotomy.4 5 6 7 8 9 12 13 This method enables one to transplant cells selectively into either intact or infarcted parts of the myocardium. One cannot, however, avoid mechanical injury and induction of an inflammatory response, resulting in myocardial damage to some extent. In addition, this grafting system may not be advantageous in spreading cells globally into the myocardium. Cells infused by this method usually produce localized islet-like formations,4 5 6 7 8 9 12 13 resulting in limitation of the cell-to-cell interaction between grafted cells and native cardiomyocytes. We speculate that this may limit integration of the grafted cells into native myocardium, restricting the efficiency of cell transplantation. We have, in the present study, developed a unique system for global dissemination of cells into the myocardium by using intracoronary infusion.
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Methods |
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Animal Care
All studies were performed with the approval of the institutional ethics committee for animal research. The investigation conforms to the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
Cell Culture and Generation of ß-Galactosidase–Expressing L6
Skeletal Myoblasts
The L6 rat skeletal muscle cell line (American Type Culture
Collection) was maintained with Dulbecco’s modified Eagle’s medium
(DMEM, Sigma) supplemented with 2 mmol/L glutamine, 50 IU/mL
penicillin, 50 µg/mL streptomycin, and 10% FCS. LacZ
reporter gene was transfected into L6 myoblasts with
MFGnlslacZ retrovirus-mediated gene transfection as
described before.14 Approximately 50% of cells
expressed nuclear ß-galactosidase (ß-gal). Clonal cells expressing
ß-gal were selected by following 2 rounds of limiting dilution at a
concentration of 0.2 cell/well. Cells were incubated at 37°C in a
humidified chamber equilibrated with 5% CO2 in
air. The culture was passaged before reaching 80% confluence to
maintain the undifferentiated state and used before the 12th passage.
Evaluation of ß-Gal Expression
ß-gal expression in the cells was confirmed with ß-gal
staining in vitro and Western blotting. For ß-gal staining, cells
were incubated on 8-well chamber slides (Nunc). After fixation in 2%
formaldehyde and 0.2% glutaraldehyde, cells were
washed and incubated with 1 mg/mL
5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside, 5
mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide,
and 2 mmol/L MgCl2 for 24 hours at
37°C.3 14 15
For Western blotting, cells were harvested by scraping confluent 60-mm plates in 500 µL of 1% SDS containing 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL leupeptin, and 5 µg/mL aprotinin and then homogenated. After 30-second sonication, 25 µg of protein was loaded onto an SDS 10% polyacrylamide gel. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane. After blocking nonspecific binding sites, the membrane was immunoreacted with a 1:1000 dilution of anti–ß-gal mouse monoclonal antibody (Sigma) for 8 hours at 4°C. The blots were then incubated with a 1:1000 dilution of horseradish peroxidase–conjugated rabbit anti-mouse IgG antibody (Dako) for 1 hour. Blots were visualized with the use of a chemiluminescence detection system (Amersham).
Intracoronary Infusion of Skeletal Myoblasts
The myoblasts expressing ß-gal were harvested with the use of
trypsin, resuspended in serum-free DMEM at a concentration of
2x106/mL, and stored at 4°C until infusion to
the heart (<30 minutes). The hearts of Sprague-Dawley rats (250 g)
were arrested with cold crystalloid cardioplegic solution and removed
under anesthesia with sodium pentobarbital (50 mg/kg IP)
and anticoagulation with heparin (200 USP units IV). Just before
infusion, the cells were filtered through a 20-µm membrane filter
(Millipore) and adjusted to a concentration of
1x106 cell/mL in serum-free DMEM. The hearts
were infused with 1x106 cells through the
coronary artery, with the venae cavae, pulmonary
arteries, and veins ligated (group CTx). For the control group, the
same volume of cell-free DMEM was infused. After incubation under
increased intracoronary pressure on ice for 10 minutes, the
pulmonary artery was incised and the coronary
circulation flushed with 10 mL of cold PBS to remove excess myoblasts.
The hearts were then transplanted into the abdomens of recipient rats
(350 g) of the same strain.16 17 These hearts were
collected on days 0 (10 minutes after cell infusion without heart
transplantation), 3, 7, 14, and 28 after cell transplantation.
Functional Assessment of the Heart in a Langendorff Perfusion
System
At the selected time, recipient rats were anticoagulated by
intravenous injection of heparin under terminal
anesthesia. The transplanted hearts were quickly excised
from the abdomen and perfused with modified Krebs-Henseleit buffer
(120.0 mmol/L NaCl, 4.5 mmol/L KCl, 20.0 mmol/L
NaHCO3, 1.2 mmol/L
KH2PO4, 1.2 mmol/L
MgCl2, 1.25 mmol/L
CaCl2, and 10.0 mmol/L glucose; gassed with
95% O2+5% CO2 at 37°C)
at a 1 m H2O pressure with a Langendorff
apparatus. A thin-wall balloon was inserted into the left
ventricle to monitor left ventricular pressure. After 20
minutes of stabilization, functional parameters were
measured with left ventricular diastolic
pressure stabilized at 10 mm Hg.16
Assay for ß-Gal Activity
Assay for ß-gal activity was performed to evaluate the number
of transplanted cells existing in the heart.14 15 After
Langendorff perfusion, the hearts (n=7 at each point for each group)
were immediately frozen in liquid nitrogen and homogenized
in 0.25 mol/L Tris-HCl (pH 7.8). The homogenates were
centrifuged at 3500g for 5 minutes and then
12 000g for a further 5 minutes. Thirty microliters of the
supernatant was mixed with 66 µL of 4 mg/mL ONPG
(O-nitrophenyl-ß-D-galactopyranoside;
Sigma) dissolved in 0.1 mol/L sodium phosphate (pH 7.5), 3 µL of 4.5
mol/L ß-mercaptoethanol dissolved in 0.1 mol/L
MgCl2, and 201 µL of 0.1 mol/L sodium
phosphate. The mixture was incubated 37°C for 30 minutes and the
reaction was stopped by adding 500 µL of 1 mol/L
Na2CO3. OD was read on a
spectrophotometer at a wavelength of 420 nm. The value was divided by
protein concentration measured with the Bradford
assay.15
Standard Scale of ß-Gal Activity
A standard scale was produced to evaluate the number of
myoblasts existing in the heart from the data of ß-gal activity.
Hearts were removed from nontreated rats under terminal
anesthesia, perfused with a Langendorff
apparatus to wash out blood, and mixed with known numbers
(1x104, 1x105,
1x106, 5x106, or
1x107) of ß-gal–expressing L6 myoblasts. The
mixtures (5 samples in each group) were then homogenated to
measure ß-gal activity of the samples as described above.
In Situ Staining for ß-Gal and Connexin 43
The remaining hearts from both groups (n=5 at each point for
each group) were frozen in an embedding medium with liquid nitrogen for
in situ ß-gal staining. The embedded samples were cut into 10-µm
sections, fixed, and stained as above. For immunohistochemical study of
connexin 43 (Cx43), after ß-gal staining, the sections were blocked
with 5% FCS and incubated for 1 hour in a 1:50 dilution of anti-Cx43
mouse monoclonal antibody (Chemicon) at room temperature. After
washing, these were followed by 1 hour incubation in a 1:100 dilution
of biotinylated goat anti-mouse IgG antibody (Dako) at room
temperature. Coloring was performed with a kit (StreptABComplex/HRP;
Dako) followed by counterstaining with 1% neutral red for 10
minutes.
Statistical Analysis
All values are expressed as mean±SEM. Statistical comparison of
the data for ß-gal activity was performed with ANOVA for repeated
measures followed by Bonferroni’s test to individual significant
difference. The differences in the data for cardiac function were
determined with the Student’s t test. A value of
P<0.05 was considered statistically significant.
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Results |
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Skeletal Myoblasts Genetically Engineered to Express Nuclear ß-Gal
To identify grafted skeletal myoblasts from native heart cells, L6 cells were genetically labeled to express ß-gal in the nucleus.14 ß-gal staining in vitro demonstrated that all the cells expressed enough nuclear ß-gal to be stained (Figure 1A
). Western blotting confirmed ß-gal
expression in the genetically manipulated cells, whereas no ß-gal
expression was observed in the wild-type L6 cells (Figure 1B).
Expression of nuclear-targeted ß-gal did not affect myogenic
differentiation ability in vitro. In a differentiation medium
containing 2% horse serum instead of 10% FCS,18 the
genetically labeled L6 myoblasts could be induced to undergo
myodifferentiation and form multinucleated myotubes (hematoxylin and
eosin staining, Figure 1C).
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Cell Transplantation to the Heart Through the Coronary
Artery
One million ß-gal–expressing L6 skeletal myoblasts were infused
into the heart through the coronary arteries, followed by a
10-minute incubation under increased intracoronary pressure (50
to 80 mm Hg) and heterotopic heart transplantation. The overall
operative mortality rate was 6%, mainly related to ileus and
intra-abdominal infection.
Functional Assessment of Hearts After Cell Transplantation
Functional assessment was performed to assess the myocardial
damage caused by the cell transplantation procedure. At no selected
time point after cell transplantation did we observe any functional
reduction in cell-transplanted hearts as compared with control-treated
hearts in terms of heart rate, maximum dP/dt, minimum dP/dt, and
coronary flow. The data for day 28 is shown in the
Table. Additionally, no
dysrhythmic effects caused by infused cells were noted.
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Time Course of Grafted Cell Survival
Assay for ß-gal activity with ONPG was done to evaluate graft
cell survival after cell transplantation. First, the standard samples
that were created by mixing isolated hearts with a known number
(1x104, 1x105,
1x106, 5x106, or
1x107) of ß-gal–expressing cells were
analyzed. As a result, ß-gal activity was 3.4±0.4,
17.2±3.5, 57.8±6.2, 120.6±15.7, or 173.1±20.5
OD420/g protein, respectively. The time course of
ß-gal activity after cell transplantation is shown in Figure 2. Using the standard scale on the right
of the graph, we estimated that 9x105 cells
were present in the heart 10 minutes after infusion, decreasing to
5x105 cells on day 3. The ß-gal–expressing
myoblasts quickly increased in number to
2.5x106 on day 7 with a slow rise between day
7 and day 28, finally up to 5x106 cells. The
values for the control hearts were <10 OD420/g
protein throughout the postoperative period.
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Histological Findings of Engrafted Skeletal
Myoblasts
The transplanted hearts were collected at the selected time points
and stained for ß-gal. Ten minutes after cell transplantation,
grafted cells were found throughout the both left and right
coronary distributions in all cardiac layers, where they
appeared to be entrapped within the lumina of small capillaries,
occasionally permeating into the myocardial interstitium (Figure 3). At day 28 after cell transplantation,
under a low-power magnification, discrete positively stained loci were
observed to be widely distributed throughout the cardiac layers of left
and right coronary territories (Figure 4A). At some of these loci, surviving
cells formed colonies composed of the ß-gal–positive myoblasts that
appeared to be undifferentiated (Figure 4B). It was, in
contrast, observed that at other loci, surviving myoblasts had
completely differentiated into ß-gal–positive multinucleated
myotubes that aligned with the cardiac fiber axis and had integrated
into the native myocardium (Figure 4C).
Histological evidence of myocardial thrombosis or
infarction was not identified.
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Expression of Cx43
Cardiomyocytes are electrically coupled to adjacent cells by
specialized gap junctions, composed of the hexamers of the protein
Cx43, which allow the exchange of ions and small molecules between
adjacent cells.19 20 A linear pattern of staining for Cx43
at apparent cardiomyocyte-cardiomyocyte
interfaces was observed throughout myocardium.
Additionally, we identified Cx43 expression at the interfaces between
skeletal muscle–derived cells forming myotubes and native
cardiomyocytes, as shown in Figure 4D
.
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Discussion |
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Intracoronary infusion of cells provides theoretical merits over direct intramuscular injection in cellular cardiomyoplasty. It is advantageous not only in producing less myocardial damage during engraftment but also in disseminating cells globally into the myocardium.3 11 Previously, Robinson and colleagues3 examined an arterial delivery method of skeletal myoblasts by injection into the mouse left ventricular cavity and evaluated that 50 000 myoblasts were entrapped widely in the heart. Taylor and coworkers11 suggested the possibility of catheter-associated selective coronary infusion of skeletal myoblasts into the rabbit heart. The grafting efficiency, however, was not adequate: Only 700 surviving cells were detected 1 week after engraftment.3 The translocation of entrapped cells from coronary capillaries to the myocardial interstitium can be postulated to be a major limiting factor of graft survival. Willerson’s group (Mar et al21 ) previously reported on a system for intracoronary infusion of fetal cardiomyocytes into a limited area of the canine heart through a catheter, in which they interrupted coronary arterial flow for 1 to 5 minutes after cell infusion. In the present work, we have demonstrated a novel cell-grafting system by intracoronary infusion to the whole heart by applying increased intracoronary pressure (50 to 80 mm Hg) during the 10-minute incubation time on ice. This unique situation might help the cells entrapped in the coronary bed to permeate into the myocardial interstitium through the coronary endothelial barrier, though the precise mechanism of translocation of grafted myoblasts into the extravascular space remains unclear. Additionally, cooling may be useful in reducing graft cell damage as well as myocardial injury during incubation.
Coronary embolism leading to myocardial infarction is a major concern in arterial cell delivery to the heart. When 1x107 or 108 cells were infused into the heart by use of the same system in our pilot study (n=4 each), the engrafted heart developed a large myocardial infarction just after reperfusion and stopped beating immediately, with concurrent extreme swelling and hemorrhage (data not shown). As we have demonstrated, however, at least 1 million cells can be successfully grafted with the use of this method, with little risk of coronary embolism and subsequent myocardial dysfunction. We passed cells to be grafted through a 20-µm membrane filter just before infusion. This might be useful in removing clumps of cells, which are likely to be a major cause of coronary embolism. Further, although we did not use any immunosuppressive reagents, our histological results did not demonstrate any findings that suggested significant immunorejection over the 1-month period after cell transplantation.
With the use of this cell grafting system, it has been demonstrated
that 90% of the total number of 1 million infused myoblasts were
entrapped in the lumina of small capillaries or had migrated into the
myocardial interstitium at day 0. This observation was supported by our
unpublished data that the number of myoblasts flushed into
coronary effluent before heart transplantation was only 3% to
5% of the initial number of cells infused. The graft cell survival was
50% on day 3. Subsequently, the ß-gal–expressing cells quickly
increased in number to 2.5x106 on day 7, with
a slow rise from day 7, finally up to 5x106 on
day 28. Discrete loci of grafted cells were observed to be widely
distributed throughout cardiac layers of both left and right
coronary territories on day 28. No histological
evidence of large myocardial infarction or functional reduction was
identified after cell transplantation in any hearts analyzed.
No ß-gal–stained cells were observed in the liver, kidney, spleen,
and lung after cell transplantation (data not shown). We therefore
consider that this cell-grafting system could disseminate cells
globally into the myocardium with reasonable success. This
system for intracoronary infusion followed by heterotopic heart
transplantation would be relevant to a clinical situation in which the
failing heart is arrested by cardioplegic solution and infused with
cells through the intracoronary route with coronary
sinus occlusion followed by circulatory support with a
ventricular assist device until heart function has
improved.
In the present study, the function of the treated normal heart was not improved. Although one might expect that infusion of a larger number of skeletal myoblasts might improve function of the normal heart, it is not known how many cells would be required to achieve this. In addition, improvement of cardiac function after skeletal myoblast transplantation is affected not only by cell number infused but also by many factors such as the survival, proliferation, integration, and differentiation of grafted cells. The main aim of our study was to clarify the feasibility of intracoronary cell transplantation and to investigate the behavior of infused skeletal myoblasts. We therefore used normal hearts as myoblast recipients and have demonstrated that this method did not damage cardiac function. We shall study whether the number of skeletal myoblasts infused with the present method is enough to improve the damaged heart’s function by using some heart failure models in future work.
As a semiquantitative indicator of cell number surviving in the myocardium, we used ß-gal activity. Stably transfected clonal cells can generally be expected to express ß-gal constantly on the whole for an extended period, though the level of expression of an individual cell may be affected by cell cycle and circumstance to some extent. Therefore, one can expect the ß-gal activity to be directly proportional to the number of the cells (proliferation).14 15 Using this measurement, we observed that the number of ß-gal–expressing cells existing in the heart rapidly increased up to day 7, with a gradual rise thereafter. This observation suggested that the surviving myoblasts proliferated in the early period and began to differentiate on reaching a particular cellular concentration. The mechanism of switching from the proliferation to the differentiation state with cell cycle withdrawal is likely to involve cyclin-dependent kinases and their inhibitors,22 23 which can be modulated by certain growth factors.24 Further study, however, is necessary to clarify this issue. We have also shown in these experiments that surviving ß-gal–positive myoblasts behaved in two different ways: Some myoblasts fully differentiated into multinucleated myotubes and integrated into the native myocardium; others formed undifferentiated colonies. It is unknown why some remained in an undifferentiated state whereas others differentiated. Even though the differentiation ability of the ß-gal–expressing myoblasts was observed in vitro, it might not be sufficient when transplanted into the myocardium in vivo. To solve this problem, it would be useful to enhance differentiation potential by genetically engineering grafted myoblasts to overexpress certain beneficial proteins such as Cx43.25
Intercellular communication between grafted myoblasts and native cardiomyocytes is another concern in skeletal myoblast transplantation to the heart. Cardiac cells are electrically coupled to adjacent cells by specialized gap junctions, composed of hexamers of the protein Cx43.19 20 25 Exchange of ions and small molecules between cells also occurs across these junctions. Gap junctions are found within the intercalated disk and at sites of side-to-side contact between cardiac cells.19 20 It still remains controversial whether grafted skeletal myoblasts could form sufficient gap junctions with native cardiomyocytes.3 4 8 9 We identified Cx43 expression at the interfaces between skeletal muscle–derived cells forming myotubes and native cardiomyocytes, suggesting gap junction formation between these cells.
In conclusion, we have developed a novel method for efficient cell transplantation to the heart by using intracoronary infusion. This method, which is applicable to the clinical setting in cardiac surgery, could be useful to globally disseminate cells into the heart with little myocardial damage.
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