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Ann Thorac Surg 1999;68:2074-2080
© 1999 The Society of Thoracic Surgeons
a Division of Cardiovascular Surgery, Department of Surgery, The Toronto Hospital, University of Toronto, Toronto, Ontario, Canada
Address reprint requests to Dr Li, General Division, The Toronto Hospital, CCRW 1-815, 101 College St, Toronto, ON, Canada M5G 2C4;
e-mail: rli{at}torhosp.toronto.on.ca
Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.
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Abstract |
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Methods. A transmural scar was produced in the left ventricular free wall of adult rats by cryoinjury. The left atrial appendage was harvested, and the atrial heart cells were cultured and their number expanded ex vivo. Three weeks after cryoinjury, either a cell suspension (2 x 106 cells, n = 12 rats, transplant group) or culture medium (n = 10 rats, control group) was injected into the scar. Rats having a sham operation (n = 5) did not undergo cryoinjury or transplantation with cells or culture medium.
Results. Five weeks after injection, ventricular function was evaluated in a Langendorff preparation, measuring systolic, diastolic, and developed pressures over a range of intraventricular balloon volumes. Systolic and developed pressures were greater in the transplant group than in the control group (p = 0.0001). Rats with a sham operation had the greatest systolic, diastolic, and developed pressures (p = 0.0001). Histologic studies demonstrated survival of the transplanted heart cells within the scar. The area of the scar was smaller (p = 0.0003) and its thickness greater (p = 0.0003) in rats in the transplant group. Left ventricular chamber volume was smaller in the transplant group (p = 0.043).
Conclusions. Transplantation of autologous cultured adult atrial heart cells limited scar thinning and dilatation and improved myocardial function compared with results in control hearts. This technique may lead to a novel therapy to prevent scar expansion after a myocardial infarction and prevent the development of congestive heart failure.
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Introduction |
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Successful transplantation of cultured cardiomyocytes into normal myocardium was reported in the early 1990s [3, 4]. In 1996, we [5] reported that transplanted fetal cardiomyocytes improved heart function after cardiac injury. Cultured fetal cardiomyocytes were injected into the scarred area 4 weeks after cryoinjury. Four weeks after transplantation, the transplanted cardiomyocytes formed cardiac tissue, limited scar expansion, and improved ventricular function.
Several concerns have been expressed about the use of fetal cardiomyocytes. First, fetal cells are subject to rejection because they are either allografts [6] or xenografts [7], thus necessitating extensive immunosuppression; this limits the clinical utility of the technique. Second, the use of human fetal cardiomyocytes raises major ethical concerns. Transplantation of cells from established cell lines such as AT-1 cardiomyocytes [4] or C2C12 myoblasts [8] may carry an additional potential risk of continued, uncontrolled hyperplasia of the transformed cells. The use of autologous adult cells may avoid both the problems of immunorejection and the risk of uncontrolled growth. Autologous adult heart cells are available from biopsy specimens, and their numbers can be expanded in vitro. They may be an ideal source of cells for transplantation. This study was undertaken to investigate the feasibility of transplantation of autologous cultured adult heart cells.
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Material and methods |
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Anesthesia and postoperative care of rats
Adult rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg) followed by an intramuscular administration of ketamine hydrochloride (22 mg/kg). The anesthetized rats were endotracheally intubated. Positive-pressure ventilation was maintained at a rate of 60 cycles per minute with a tidal volume of 3 mL with room air supplemented with oxygen (2 L/min) using a Harvard ventilator (Harvard Apparatus model 683). Penlong XL (penicillin G benzathine, 150,000 U/mL, and penicillin G procaine, 150,000 U/mL) was given intramuscularly (0.4 mL per rat) preoperatively. The rats were monitored for 4 hours postoperatively and then returned to cages with filter tops.
Myocardial scar formation
With the animal under general anesthesia as just described, a 2-cm left lateral thoracotomy was made. The pericardium was opened, and the heart was exposed. The left atrial appendage was ligated at its base with 5-0 silk suture, excised, and stored in cold culture medium for cell isolation. An elliptic metal probe 8 x 10 mm in diameter was cooled to -190°C by immersion in liquid nitrogen and applied to the left ventricular free wall (LVFW) between the left anterior descending coronary artery and the posterolateral branch for 1 minute to produce a cryoinjury. This procedure was repeated ten times. The thoracotomy was closed in layers with 2-0 silk running sutures. For the group having a sham procedure, the left atrial appendage was removed and the chest incision was closed without scar generation by cryoinjury.
Cell culture and preparation for transplantation
Heart cells from the harvested left atrial appendage (wet weight, 12.9 ± 4.3 mg) of each rat were isolated and cultured by methods we [9] have previously described. In brief, the cells were washed in phosphate-buffered saline solution (PBS) (in millimoles per liter: NaCl, 136.9; KCl, 2.7; Na2HPO4, 8.1; KH2PO4, 1.5; pH 7.3) three times to remove any blood and clots. The left atrial appendage was minced with fine scissors and incubated in PBS containing trypsin (0.5%), collagenase (0.1%), and glucose (0.02%) for 30 minutes at 37°C. The myocardial cells were separated by repeated shaking of the digested myocardial tissue.
The cells in the supernatant were transferred into a tube containing 20 mL of culture medium (Iscove’s modified Dulbecco’s medium [GIBCO Laboratory, Life Technologies, Grand Island, NY] containing 10% fetal bovine serum, 0.1 mmol/L ß-mercaptoethanol, 100 U/mL of penicillin, and 100 µg/mL of streptomycin sulfate). Then the tube was centrifuged at 600 g for 5 minutes. The cell pellet was resuspended in 20 mL of culture medium, evenly plated on two dishes 100 mm in diameter, and incubated at 37°C in 5% carbon dioxide and 95% air. The culture medium was changed every 3 to 4 days.
The cells increased in number and became confluent in the culture dish 10 to 14 days after seeding. The cells were then detached from the dish with 1 mL of 0.05% trypsin in PBS solution and plated on six dishes 100 mm in diameter. On the day of transplantation, the confluent cells were detached from the six culture dishes as described, and the cell pellet was resuspended in the culture medium at a concentration of 2 x 106 cells in 0.05 mL.
Heart cell transplantation
Three weeks after cryoinjury, rats underwent general anesthesia, and their hearts were exposed through a median sternotomy. At the time of operation, animals were randomly selected for transplantation with cultured atrial heart cells or injection with medium control. For the transplant group (n = 12), a suspension of cultured autologous atrial cells in 0.05 mL of culture medium (2 x 106 cells) was injected into the center of the scar tissue of the LVFW using a tuberculin syringe. During the injection procedure, the puncture sites were sealed with drops of cryoprecipitate and thrombin solutions. In the control group (n = 10), 0.05 mL of cell culture medium was injected in the same manner. The chest was closed in layers with 2-0 silk sutures. The group having a sham procedure underwent chest opening but no injection of cells or culture medium.
Determination of cultured cell purity
The purity of the cardiomyocytes after 3 weeks in cell culture was evaluated using a monoclonal antibody against myosin heavy chain (Rougier Bio-Tech Ltd, Quebec, PQ, Canada). The cultured cells were washed with PBS and fixed with 100% methanol at -20°C for 15 minutes. The cells were washed with PBS three times and incubated with the antibody against myosin heavy chain at 37°C for 45 minutes. The cells were then washed three times with PBS for 15 minutes each at room temperature and exposed to a rabbit anti-mouse immunoglobulin G conjugated with fluorescein isothiocyanate for 45 minutes at 37°C in the dark. The cells again were washed three times with PBS, mounted, and then photographed using light and ultraviolet microscopes. The purity of the cardiomyocyte cultures was determined by counting the percentage of stained cells in ten random fields per dish.
Identification of transplanted cells
Transplanted cells within the cardiac scar were identified by immunocytochemical labeling of the cultured cells in vitro with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) (n = 4). When the cultured cells reached 50% confluence, 25 µL of 0.4% BrdU solution was added to the culture dishes, and they were incubated for 48 hours. The efficiency of BrdU incorporation into the cultured cells was determined by counting the percentage of BrdU–stained cells in ten random fields per dish. The BrdU–labeled cells were transplanted as previously described. At the end of the study, a monoclonal antibody against BrdU was used to localize the labeled transplanted cells in the scar [10].
Myocardial function study
Five weeks after transplantation, heart function in the three groups was evaluated in a Langendorff preparation [11]. The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (26 mg/kg) followed by intramuscular administration of ketamine hydrochloride (88 mg/kg) and heparin sodium (200 units) administered intravenously. The hearts were quickly excised and perfused in a Langendorff apparatus with filtered Krebs-Henseleit buffer (in millimoles per liter: NaCl, 118; KCl, 4.7; KH2PO4, 1.2; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25; glucose, 11; pH 7.4) equilibrated with 5% carbon dioxide and 95% oxygen.
A latex balloon was inserted into the left ventricle through the mitral valve and connected to a pressure transducer (model p10EZ; Viggo-Spectramed, Oxnard, CA), a transducer amplifier, and a differentiator amplifier (model 13-6615-50 and model 11-G4113-01, respectively; Gould Instrument System Inc, Valley View, OH). After 30 minutes of stabilization at 37°C, coronary flow was measured by taking the mean value of 2-minute timed collections of the buffered solution in an empty beating state. The balloon volume was initially set at 0.04 mL and then increased in 0.02-mL increments to 0.48 mL by the stepwise addition of saline solution. The systolic and diastolic pressures, the maximal rate of myocardial contraction, and the maximal rate of myocardial relaxation were recorded at each balloon volume, and the developed pressure was calculated as the difference between peak systolic pressure and end-diastolic pressure. After the measurements were completed, the heart was arrested by injection of 10 mL of a 20% solution of KCl into the aortic root.
Measurement of left ventricular remodeling
After arrest of the heart, the ventricular volume was measured as described by Pfeffer and associates [12]. The left ventricle was distended to a balloon pressure of 30 mm Hg, and the volume required was designated the ventricular chamber volume. The heart was fixed with 10% phosphate-buffered formalin solution in the distended state. Two days after fixation, the balloon was removed, and the atria and great arteries were excised. The heart was weighed. The epicardial and endocardial surface areas of the LVFW and those of the cryoinjury-derived scar tissue on the LVFW were measured by planimetry as previously described [5]. Briefly, the heart was cut into slices 3 mm thick. Each heart yielded five slices. On both the apical and basal surfaces of the slices, the areas and the lengths of the myocardium and the scar were traced on transparencies and then quantified using computed planimetry (Jandal Scientific, Sigma-Scan, Corte Madera, CA). The total surface area of the scar tissue and that of the LVFW were measured as the sum of the epicardial length and the endocardial length times the slice thickness (3 mm).
The cryoinjury-created lesion was defined as the area of the LVFW covered by epicardial scar. The mean wall thickness (MWTh) of this lesion was calculated as follows: 
The mean wall thickness was calculated on each slice of the heart specimen except the most apical or the most basal slice. All the mean wall thicknesses were then averaged to yield the total mean wall thickness of the lesion.
Histologic studies
The heart sections were fixed in 5% glacial acetic acid in methanol, embedded in paraffin, and sectioned to produce specimens 10 µm thick. The sections were stained with hematoxylin and eosin as described in the manufacturer’s specification (Sigma Diagnostics, St. Louis, MO).
Data analysis
All data are expressed as the mean ± the standard deviation, unless otherwise indicated. Data from the studies of ventricular function were analyzed with the SAS software package for Windows v. 6.12 (SAS Institute Inc, Cary, NC) and the other data, with the SPSS software package for Windows v. 8.0 (SPSS Inc, Chicago, IL). Comparisons of continuous variables between the three groups were performed by a one-way analysis of variance, and differences were specified by Duncan’s multiple range test. A p value of less than 0.05 was considered significant.
Indices of ventricular function were evaluated by an analysis of covariance, using intraventricular balloon volume as the covariate and systolic pressure, diastolic pressure, developed pressure, maximal rate of myocardial contraction, and maximal rate of myocardial relaxation as dependent variables. Group, volume, and interaction between group x volume were examined. If no significant interactive effect was observed, only the main effects were modeled. When the analysis of covariance identified a significant difference, multiple pairwise comparisons were performed to specify which groups were different.
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Results |
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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The major drawback of fetal cardiomyocytes as a cell source for cell transplantation is the inevitable immunorejection of allogenic or xenogenic transplants. We [6] found that rejection resulted in the gradual elimination of all the transplanted fetal cardiomyocytes despite immunosuppression with cyclosporin A (5 mg · kg-1 · d-1). Fetal cardiomyocyte transplantation therefore delayed but did not prevent the ultimate thinning and dilatation of the cryoinjury-produced scar. In an attempt to avoid this rejection phenomenon, we have undertaken several studies of autologous heart cell transplantation.
Skeletal satellite cells have been used as a source of autologous cells for transplantation into the heart [16, 17]. Chiu and colleagues [16] transplanted cultured autologous satellite cells into the left ventricle of a canine heart immediately after cryoinjury. They showed histologic evidence that implanted satellite cells coupled with each other by intercellular junctions resembling gap junctions or intercalated discs. Taylor and associates [17] transplanted satellite cells into the cryoinjured myocardium of rabbits and documented survival of the transplanted cells and improvement in ventricular function. However, the isolation and culture of a sufficient number of satellite cells for transplantation can be difficult, as their population in the muscle tissue is low, and the duration of maintenance of these cells in culture must be less than 3 days to prevent early differentiation into skeletal muscle cells [16].
In this study, we investigated the efficacy of transplanted cultured autologous adult atrial heart cells in the improvement of ventricular function after myocardial cryoinjury. The left atrial appendage was selected as the source of these autologous heart cells because the harvesting of the left atrial appendage is technically easy and its removal is unlikely to affect ventricular function. Atrial cardiomyocytes cultured in the presence of fetal serum resembled immature cardiomyocytes more than cultured ventricular cardiomyocytes [18], which may indicate a greater capacity to proliferate than cultured ventricular cardiomyocytes. Culture of atrial cardiomyocytes may be more technically straightforward than that of ventricular cardiomyocytes.
In the present study, we found that the transplanted cultured heart cells survived in the scar tissue for 5 weeks. The transplanted cells modified the remodeling process of the left ventricle, preventing left ventricular chamber volume dilatation, preserving wall thickness, and minimizing expansion of the cryoinjury-produced scar. The transplant group generated greater developed pressure and a higher maximal rate of myocardial contraction at any balloon volume than did the control group. The smaller left ventricular chamber volume in the transplant group may have been the major contributor to the improvement in ventricular function. Further studies are required to establish the mechanisms by which transplantation of adult atrial heart cells may improve heart function, including potential angiogenesis or release of growth factors.
The future clinical applications of cardiomyocyte transplantation are intriguing. In patients who have sustained a myocardial infarction but who are not candidates for standard surgical therapies, heart cells could be obtained from the atrial appendage during a port access procedure. The cells could be expanded in vitro for several weeks and then injected into the infarct region through a minimally invasive approach. This cell transplantation approach may prevent thinning and dilatation of the injured region and maintain cardiac function. In addition, transfection of these cultured heart cells may allow expression of specific transgenes to induce angiogenesis, hypertrophy, or other therapeutic effects [19, 20].
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Acknowledgments |
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Ren-Ke Li is a Research Scholar of the Heart and Stroke Foundation of Canada. This research was supported by his research grants from the Medical Research Council of Canada (MT-13665) and The Hospital for Sick Children Foundation (XG 98-063).
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