In Vivo Survival and Function of Transplanted Rat Cardiomyocytes
Abstract Recent studies have demonstrated the feasibility of transplanting fetal mouse cardiomyocytes into the hearts of adult syngeneic mice. However, the function of the transplanted cardiomyocytes and their capacity to survive in fibrous connective tissue were not assessed. In the present study, we evaluated the viability and contractility of transplanted fetal and neonatal rat cardiomyocytes in the connective tissue of the adult rat hindlimb. Purified fetal or neonatal rat cardiomyocytes were cultured. These cells contained sarcomeres, formed junctions composed of desmosomes and fascia adherens, and contracted regularly and spontaneously. A fetal or neonatal cardiomyocyte suspension was injected into the subcutaneous tissue of adult rat hindlimbs. Cyclosporin A (5 mg/kg) was administered subcutaneously daily for the 3-month duration of the study, at which time the animals were killed. The transplanted cardiomyocytes formed ‘tissue’ in vivo that increased in size for the first 2 weeks and remained the same size at the third week. The tissue derived from the transplanted fetal cardiomyocytes contracted spontaneously at a rate of 73±12 bpm, and that from the neonatal cardiomyocytes contracted at a rate of 43±21 bpm. The electrocardiogram was similar to that seen in myocardium with an idioventricular rhythm. Histologically, the tissue appeared to be cardiac muscle with sarcomeres. Angiogenesis occurred in the cardiomyocyte graft. In summary, a cell suspension of cultured fetal and neonatal rat cardiomyocytes transplanted into the adult rat hindlimb formed contractile cardiac tissue in the subcutaneous connective tissue.
Myocardial tissue transplantation has been performed in a variety of animal species.1 2 3 Early studies showed that transplanted myocardial tissues survived in skeletal muscle of rats4 and in the ears of mice.5 Using a myocardial tissue transplantation model, Bishop et al6 studied fetal rat myocardial development and cardiomyocyte hypertrophy in the anterior eye chamber of adult rats. Jockusch et al7 compared contractile protein isomer composition in transplanted tissues from newborn rat atria and ventricles implanted into the anterior tibial muscle of nude mice. These studies showed that transplanted myocardial tissue can retain sarcomeres and contract in an adult host.
Cell transplantation into the myocardium offers certain advantages over transplantation of solid tissue: (1) Transplantation of one cell type is possible by injection of a cell suspension into normal or scarred myocardium. (2) Angiogenesis occurs earlier in cell transplantation than with tissue transplantation.8 A pioneering study in cardiomyocyte transplantation was performed by Soonpaa et al.9 They implanted cardiomyocytes isolated from transgenic fetal mouse hearts into the normal hearts of syngeneic mice and found that the transplanted cardiomyocytes formed junctions with host cardiomyocytes and survived in the host myocardium for 2 months. The clinical potential for cardiomyocyte transplantation, however, will require cell insertion into the scar tissue resulting from a myocardial infarction. In addition, clinical application will require that the transplanted cardiomyocytes maintain not only viability but also contractile function. Therefore, we designed the present study to determine whether cardiomyocytes could survive and contract in fibrous subcutaneous connective tissue. The primary cultured fetal or neonatal rat cardiomyocytes were implanted into the subcutaneous tissue of the adult rat hindlimb. Echocardiography was used to assess transplanted cell contractile function. We found that the transplanted cardiomyocyte suspension formed tissue that increased in size for the first 2 weeks and spontaneously contracted for the 3-month duration of the experiment. The tissue had the histological appearance of cardiac muscle, with sarcomeres and junctions composed of desmosomes and fascia adherens.
Materials and Methods
All procedures performed on animals were approved by the hospital’s animal care committee. Sprague-Dawley rats (Charles River Canada Inc, Quebec, Canada) were used. Male rats, weighing 200 to 250 g, were used as recipients. Cardiomyocytes from hearts of 18-day-gestation (fetal) and 5-day-old (neonatal) rats were used for transplantation.
Cardiomyocytes used for transplantation were isolated from donor hearts by enzymatic digestion and cultured as we have previously described.10 11 Twelve pregnant rats and six 5-day-old rats were anesthetized with ketamine (22 mg/kg body wt IM) and pentobarbital (30 mg/kg body wt IP). The hearts of the fetal and neonatal rats were excised and then washed with PBS (mmol/L: NaCl 136.9, KCl 2.7, Na2HPO4 8.1, and KH2PO4 1.5, pH 7.3). After removing the connective tissue, blood vessels, and the atria, the ventricles were minced and incubated in a PBS solution containing trypsin (0.2%), collagenase (0.1%), and glucose (0.02%) for 30 minutes at 37°C. The myocardial cells were then isolated by repeat pipetting of the digested myocardial tissue. The cells in the supernatant were transferred into a tube containing culture medium (Iscove’s modified Dulbecco’s medium containing 10% fetal bovine serum, 0.1 mmol/L β-mercaptoethanol, and 100 U/mL penicillin and 100 μg/mL streptomycin). The tube was centrifuged at 600g for 5 minutes at room temperature, and the cell pellet was resuspended in the culture medium for purification.
Cell Purification and Identification
Cardiomyocytes were purified by a preplating method,12 which takes advantage of the finding that cardiomyocytes require a longer time to attach to a cell culture dish than other cells in the myocardium, such as fibroblasts. The freshly isolated myocardial cells were plated on dishes and cultured for 2 hours at 37°C. The supernatant containing the suspended cells was transferred into another dish for further culturing.
To evaluate the purity of the cultured cardiomyocytes before transplantation, cardiomyocytes from the hearts of fetal rats (n=8) were separately isolated, purified, and cultured for 24 hours at 37°C in 5% CO2/95% air. The purity of the cardiomyocytes in culture was evaluated using a monoclonal antibody against myosin heavy chain (Rougier Bio-Tech Ltd).10 The cells were washed with PBS and fixed with 100% methanol at −20°C for 15 minutes. After they were washed with PBS three times, the cells were incubated with the antibody at 37°C for 45 minutes. The cells were then washed three times with PBS and exposed to a rabbit anti-mouse IgG conjugated with fluorescein isothiocyanate for 45 minutes at 37°C in the dark. After the cells were washed three times with PBS, a total of 1000 cells isolated from each fetal rat heart (n=8) were counted under ultraviolet light using an epifluorescent microscope (Nikon) with a blue filter.
Cell Preparation for Transplantation
Freshly isolated cardiomyocytes were purified and cultured for 24 hours. The cells were dissociated from the culture dishes with the trypsin solution and collected by centrifugation at 600g for 5 minutes at room temperature. After the cell number was determined, 4.2±0.9×106 cells (mean±1 SD) were resuspended in 1.0 mL saline, and 0.25 mL of the cell suspension was used for transplantation.
All procedures were performed with the rats under general anesthesia, which was achieved with ketamine (22 mg/kg body wt IM) and pentobarbital (30 mg/kg body wt IP). Once the rats were anesthetized, the subcutaneous tissue superficial to the quadriceps muscle was exposed through a 20-mm skin incision, and the cell suspension of fetal (n=12 rats) and neonatal (n=6 rats) cardiomyocytes was injected using a tuberculin syringe. The skin incision was closed with 5-0 silk. The other leg was used as the control and was injected with saline by following the same procedure. Cyclosporin A, at a dose of 5 mg/kg body wt per day, was administered subcutaneously, and the rats were housed in cages fitted with a filter. Antibiotics (benzathine penicillin G 37 500 U per rat and procaine penicillin G 37 500 U per rat) were administered intramuscularly every 3 days for 1 week after surgery, and analgesia (buprenorphine hydrochloride, 0.01 to 0.05 mg/kg body wt) was given subcutaneously every 8 to 12 hours for the first 2 days after surgery.
On days 7 and 14 after transplantation, the animals were anesthetized as previously described. Echocardiography (model 128 XP, with a 7-MHz linear phased-array probe, ACUSON) was used to evaluate the contractility of the transplanted cells along the long-axis length of the contractile tissue. On day 21, the transplanted area of the anesthetized animal was opened, contraction of the transplanted cells was visually observed, and then a videotape recording was performed. The long-axis length of contractile tissue at a relaxed state was measured, and the electronic activity of the transplanted cells was recorded with electrocardiography (Patient Care Management System, model 90303B, SpaceLabs) by placing a probe at each end of the transplanted tissue. The transplanted tissue was then collected for histological and electron microscopy studies. The animals were euthanized by injecting 1.5 mL sodium pentobarbital (540 mg/mL) intravenously.
In a separate 3-month study, fetal rat cardiomyocytes (n=4 preparations) were transplanted into adult rat hindlimbs as described above. After 3 months, the transplanted area was opened after the animals were anesthetized as previously described. The contractions of the tissue were observed, the size of contractile tissue measured, and the tissue collected for histological studies. The rats were then euthanized by injecting 1.5 mL sodium pentobarbital (540 mg/mL) intravenously.
Histology and Electron Microscopy
Tissue (0.5 cm3) in the transplantation site was collected 21 days after transplantation and fixed in 5% glacial acetic acid in methanol for histological study (n=4 samples). The samples were transferred to the Department of Pathology (The Toronto Hospital-Western Division) for further processing. The samples were embedded and sectioned to yield 10-μm-thick slices, which were stained with hematoxylin and eosin as described in the manufacturer’s specification (Sigma Chemical Co).
For immunocytochemical staining of vascular endothelial cells, the sample slices processed above were incubated with rabbit IgG against factor VIII–related antigen (Dimension Laboratory Inc) and then with goat anti-rabbit IgG conjugated with peroxidase as described in “Cell Purification and Identification.” The samples were then washed with PBS and treated with diaminobenzidine-H2O2 (2 mg/mL, 0.03% H2O2 in 0.02 mol/L phosphate buffer) solution for 15 minutes. After samples were washed with PBS, the stained capillaries (vascular endothelial cells) in the grafts (n=4) were photographed through a microscope.
For electron microscopy, the tissue was fixed in 1% glutaraldehyde in phosphate buffer and sent to the Department of Cardiovascular Pathology at the Hospital for Sick Children. The samples were then postfixed with 1% osmium tetroxide, embedded, sliced, and photographed.10
All results are presented as mean±1 SD. Student’s t test was used for comparison of the results.
Cardiomyocytes (Fig 1⇓), isolated from fetal and neonatal rats by digesting the minced myocardium, were purified by the preplating method. The cardiomyocyte purity of a 24-hour-old culture (immediately before transplantation) was 94±3.5% (n=8 cultures), as measured by immunofluorescent staining with monoclonal antibodies for myosin heavy chain (Fig 2⇓). The nonstained cells were thought to be fibroblasts and endothelial cells.
The purified cardiomyocytes were cultured in cell culture medium and contracted regularly. After 4 days of culturing, the fetal rat cardiomyocyte culture reached confluence. The cultured cells formed synchronously and spontaneously contracting myocardial tissue (Fig 3A⇓). Electron microscopy showed that the cardiomyocytes in culture formed sarcomeres and junctions composed of desmosomes and fascia adherens (Fig 3B⇓ and 3C⇓). The same results were observed for the neonatal rat cardiomyocytes.
The injection of the cardiomyocyte suspension into rat hindlimbs (n=11 rats) resulted in a mass with a long-axis length of 0.26±0.01 cm. Echocardiography demonstrated that contracting tissue formed as early as 7 days after transplantation and that the long-axis length of the tissue in a relaxed state increased to 0.62±0.03 cm on day 14 (P=.0003). No further growth was detected. The tissue contracted regularly and spontaneously in the subcutaneous tissue with a long-axis length of 0.60±0.01 cm in a relaxed state and 0.21±0.02 cm in a contracting state (a fractional shortening of 35%) on day 21 after transplantation (Fig 4⇓). No contractility was detected in the control hindlimbs (n=12 rats).
On day 21, the test and control transplantation sites were opened. In the sites transplanted with the fetal or neonatal cardiomyocytes, a spontaneously contracting tissue was apparent (Fig 5A⇓). In the control sites, nothing was observed. The rate of successful transplantation was 92% (11 of 12 animals transplanted) for the fetal cardiomyocytes and 50% for the neonatal cardiomyocytes (3 of 6 animals transplanted). Neither contractile cardiac tissue nor scar tissue was present in the unsuccessfully transplanted animals. The tissue derived from the transplanted fetal and neonatal cardiomyocytes beat spontaneously at rates of 73±12 and 43±21 bpm, respectively. The electrocardiographic recording of the contracting tissue (Fig 6⇓) was similar to that seen in hearts with an idioventricular rhythm. The tissue formed by the transplanted fetal rat cardiomyocytes was evaluated at 3 months in 4 animals. Contractile tissue was found in the transplanted areas in 3 of the 4 animals.
The cardiomyocytes in the tissue were elongated and formed a myocardium-like pattern (Fig 5B⇑). The cardiomyocytes contained organized sarcomeres, were interconnected, and formed junctions composed of fascia adherens and desmosomes (Fig 7⇓). Histological studies (Fig 5⇑) showed that blood vessels were present within the contractile tissue. Blood vessel endothelial cells were also identified by staining for factor VIII–related antigen (Fig 8⇓).
Successful transplantation of myocytes into the myocardium was first reported in the early 1990s. Researchers have implanted satellite cells,13 14 myoblasts,15 cardiomyocytes derived from myocardial tumors,16 and cardiomyocytes from transgenic fetal mouse hearts9 into the myocardium. In their cellular transplantation study, Koh et al16 transplanted AT-1 cardiomyocytes (a cell line isolated from transplantable subcutaneous tumors derived from the left atrium of the transgenic mouse) into syngeneic mouse hearts. The cells survived and proliferated in the myocardium. From the same research group, Soonpaa et al9 reported that cardiomyocytes isolated from fetal transgenic mice could be implanted into the myocardium of syngeneic mice and that the transplanted cardiomyocytes formed junctions with host cardiomyocytes. These studies demonstrated for the first time that transplanted cardiomyocytes survive in the myocardium. Contractile function of the transplanted cardiomyocytes, however, was not measured because of the contractions of the host heart. The ability of transplanted cardiomyocytes to survive in fibrous connective tissue has not been assessed. Since clinical application for cardiomyocyte transplantation requires that transplanted cells survive and function in fibrous tissue (myocardial scar tissue), we extended these observations by transplantation of cardiomyocytes into the fibrous subcutaneous tissue.
The present study found that cardiomyocytes isolated from normal fetal and neonatal rat myocardium grew in vitro to form a cardiac-like tissue in structure and function. Although the fibroblasts in cardiomyocyte culture also proliferated, the predominant cell type was the cardiomyocyte, as assessed by staining for myosin heavy chain. The cardiomyocytes contained organized sarcomeres and were linked together by junctions composed of desmosomes and fascia adherens. The cultured cardiomyocytes contracted regularly, spontaneously, and synchronously in vitro. These in vitro findings were in agreement with those reported by Kohtz et al17 and Goldmen and Wurzel,18 who used human fetal cardiomyocytes.
To assess the contractile function of the transplanted cardiomyocytes in vivo, we implanted a suspension of primary cultured cardiomyocytes isolated from fetal and neonatal rat hearts into the subcutaneous tissue of the hindlimb of allogeneic adult rats that were immunosuppressed with cyclosporin A. The transplanted cardiomyocytes survived in the connective tissue of the adult animals. The cardiomyocytes grew in vivo, organized their contractile proteins into sarcomeres, and linked with each other by junctions composed of desmosomes and fascia adherens to form a cardiac-like tissue. Echocardiography showed that the transplanted cardiomyocytes formed cardiac tissue as early as 7 days after transplantation. The cardiac tissue contracted spontaneously during electrocardiography in a manner similar to that seen in the myocardium with an idioventricular rhythm. The myocardium-like tissue in the subcutaneous tissue maintained contractile function for the 3-month duration of the study. Histological studies found that angiogenesis occurred in the cardiac tissue. This finding was similar to that reported by Koh et al,19 who showed the presence of blood vessels in the C2C12 myoblast graft in the myocardium. No connective tissue nodules were found in the sites of unsuccessful transplantations. Inaccurate injection of the cell suspension into the subcutaneous tissue and/or cell leakage could account for the unsuccessful transplantation.
The cardiac tissue formed by the implanted fetal rat cardiomyocytes enlarged 2.4 times in vivo but did not increase in size between 14 days and 21 days after transplantation. Further research needs to be done to determine the contributions of hyperplasia and hypertrophy in the enlargement of the graft.
Cell transplantation was more successful with fetal cardiomyocytes (92%) than with neonatal cardiomyocytes (50%). No morphological differences between the cultured fetal and neonatal cardiomyocytes were apparent. Aging has been shown by Komatsu et al20 to attenuate the induction of cardiomyocyte growth. Since the recipient adult male rats weighing between 200 and 250 g were randomly selected, it was unlikely that the difference in success in the present study between the fetal and neonatal cardiomyocytes could be explained by the age of the recipient animals. We attributed the difference in cell transplantation rates to the age of the donor animal. Bjorklund et al21 found that successful transplantation of neuronal cell suspensions was related to cell-donor age. We have found that transplanted cardiomyocytes isolated from young (22-day-old) and adult (32-day-old) rat hearts did not survive in the recipient adult rats.22 This difference between fetal and adult cardiomyocytes may relate to the stage of cardiomyocyte differentiation. The capacity of transplanted cardiomyocytes to stimulate angiogenesis must also be considered. Koh et al19 reported that growth factors were involved in angiogenesis in C2C12 myoblast cell transplantation. Different levels of growth factors in primary cultures of fetal and adult cardiomyocytes may be important in determining the success rate of cardiomyocyte transplantation. Without blood vessel development, transplanted cardiomyocytes could not form contractile cardiac tissue.
Cardiomyocyte transplantation could have potential clinical applications. Soonpaa et al9 showed that transplanted cardiomyocytes survived and were incorporated into the normal myocardium. We extended their observation by transplanting cardiomyocytes from normal fetal rat hearts into fibrous connective tissue. The transplanted primary cultured cardiomyocytes grew in vivo to form a cardiac-like tissue that contracted regularly and spontaneously for as long as 3 months. Although subcutaneous fibrous tissue differs in structure and composition from scar tissue of infarcted heart, the results suggested that cardiomyocytes could be successfully transplanted into myocardial scar tissue. Future studies will evaluate cardiomyocyte transplantation into myocardial scar tissue. Hopefully, the scarred myocardium can be rebuilt with contracting cardiomyocytes to preserve function of the heart after a myocardial infarction.
In summary, normal fetal and neonatal rat cardiomyocytes formed contractile myocardium-like tissue in vitro. These primary cultured cardiomyocytes transplanted by cell suspension into the connective tissue of adult rats survived and formed contractile cardiac tissue. The transplanted tissue enlarged and contracted regularly and spontaneously for the 3-month duration of the present study.
This study was supported by the Heart and Stroke Foundation of Ontario (grant A2604) and the Medical Research Council of Canada (grant MT-10392). We are indebted to B. Radii for the preparation of fetal rat hearts, M. Straits for the histological preparation, Dr G. Wilson for electron microscopy, and J. Graba for echocardiography.
- Received January 13, 1995.
- Accepted October 5, 1995.
- © 1996 American Heart Association, Inc.
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