Despite the recent remarkable progress in medical and surgical treatments for heart failure, end-stage heart failure has been still a major cause of death worldwide. After myocardial infarction, the myocardium is capable of a limited regenerative capacity and no medication or procedure used clinically has shown efficacy in regenerating myocardial scar tissue with functioning tissue. Thus, there is a need for new therapeutics to regenerate damaged myocardium.
Recent developments in tissue engineering show promise for the creation of functional cardiac tissues without the need for biodegradable alternatives for the extracellular matrix (1). And we reported that cardiomyocyte sheets have been developed by using temperature-responsive culture dishes and these sheets survived in the back of nude rats and showed a spontaneous contraction over a long period of time (2). Recent reports suggested that cardiomyocyte sheets integrated with the impaired myocardium and improved cardiac performance in a rat model of ischemic myocardium (3).
And more recently, in the aim of clinical application, nonligature implantation of skeletal myoblast sheet regenerated the damaged myocardium and improved global cardiac function by attenuating the cardiac remodeling in the rat ligation model (4) and dilated cardiomyopathy hamster model (5). This cell delivery system by using cell sheets implantation showed better restoration of damaged myocardium compared with needle injection (4, 5). Moreover, grafting of skeletal myoblast sheets attenuated cardiac remodeling and improved cardiac performance in pacing-induced canine heart failure model (6).
Given this body of evidence, we hypothesized that the autologous skeletal cell (SC) sheet implantation might remodel the chronic heart failure caused by ischemic injury.
Therefore, this preclinical study using Swine model was designed to test therapeutic effectiveness.
MATERIALS AND METHODS
Myocardial Infarction Model
“Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resource and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). This animal experiment was approved by the Animal Care Committee of Osaka university graduate school of medicine. We induced acute myocardial infarction of 10 swine (20 kg, KEARI, Japan) by the following method. Swine were preanesthetized by intramuscular injection of ketamine hydrochloride 20 mg/kg (Ketalar, Sankyo, Japan) and xylazine 2 mg/kg (Seractar, Bayer). Animals were positioned spine and a 22-gauge indwelling needle (Surflo F&F, Terumo, Tokyo, Japan) was inserted in the central vein of the auricle. A three-way cock (Terufusion TS-TR2K, Terumo, Tokyo, Japan) was attached to the external cylinder of the indwelling needle, and an extension tube was connected for continuous anesthetic injection. The animals were intubated with an endotracheal cannula (6 Fr, Sheridan) using a pharyngoscope and then connected to an artificial respirator (Harvard, USA) by the cannula. Artificial respiration was implemented at a stroke volume of 200 to 300 mL/stroke and a stroke frequency of 20/min. The animals were continuously drip injected with propofol 6 mg/kg/hr (Diprivan, AstraZeneca) and vecuronium bromide 0.05 mg/kg/hr (Musculux, Sankyo Yell Yakuhin Co., Ltd., Japan) using a syringe pump (Terufusion TE-3310N, Terumo, Japan). The animal was then fixed in a recumbent position, so that the left thorax was exposed, and the outer layer of skin and muscles between the third and fourth ribs were dissected. After confirming the cutting into the thoracic cavity, the distance between the third and fourth ribs was widened with a rib spreader to allow a direct view of the left auricle and the LAD coronary artery. The pericardium was dissected along the LAD from the upper part of the left auricle (∼6 cm) to expose the myocardium around the LAD. LAD on the proximal side below the left auricle from the myocardium was exfoliated for approximately 1 cm, and then a small amount of lidocaine hydrochloride jelly (Xylocaine jelly, AstraZeneca) was applied to allow for anesthetizing the area. An ameroid constrictor (COR-2.50-SS, Research Instruments) was then fit using No. 1 or 2 suture. The chest cavity was closed to end the procedures. The animals were randomly divided into two treatment groups: the first received autologous SC sheet implantation (SC group, n=5). For control, we have performed sham operation (C group, n=5).
Preparation of Skeletal Cell Sheets for Grafting
One week after implantation of ameroid constrictor on LAD, skeletal muscle weighing approximately 5 g was removed from the pretibial region with the porcine under general anesthesia. Following the addition of trypsin-ethylenediaminetetraacetic acid (Gibco, Grand Island, NY), excessive connective tissue was carefully removed to minimize the content of contaminating fibroblasts, and the muscle tissue was minced until the fine pieces formed a homogeneous mass. The specimens were then incubated at 37°C in shaker bath with 0.5% type 1 collagenase (Gibco) in Dulbecco's modified Eagle's medium (Gibco). After brief placement, the fluid was collected, and the same volume of culture medium, SkBM (Cambrex, Walkersville, MD) supplemented with fetal bovine serum (Thermo Trace, Melbourne, Australia), was added to halt the enzymatic digestion process. The cells were collected by centrifugation, and the putative SCs were seeded into 150 cm2 polystyrene flasks after removal of fibroblasts by sedimentation for a few hours and cultured in SkBM at 37°C. During the culture process, we maintained cell densities at less than 70% confluence by carrying out passaging of cells for one time to prevent SCs from premature differentiation and fusion process resulting in myotubes formation. When the cells become approximately 70% confluent after 10 to 11 days cultivation, the cells were dissociated from the flasks with trypsin-ethylenediaminetetraacetic acid and reincubated on 100 mm temperature-responsive culture dishes (Cellseed, Tokyo, Japan) at 37°C with the cell numbers adjusted to 1×107 per dish. More than 90% of these cells were desmin positive (Fig. 1). After 4 days, the dishes were removed to refrigerator set at 20°C, and left there for approximately 30 min. During that time, the SC sheets detached spontaneously from the surfaces. Each sheet had a diameter of 30 to 40 mm and consisted of layers of SCs; the sheets were approximately 100-μm thick in cross-sectional views (Fig. 1). Approximately 10 sheets were obtained from the 5 g of skeletal muscle.
Implantation of Skeletal Cell Sheets
Autologous SC sheet implantation was performed in the swine 4 weeks after LAD ligation. Swine were anesthetized as mentioned above. The swine were exposed through the sternum. The infarct area was identified visually on the basis of surface scarring and abnormal wall motion. In the SC group, we implanted 10 SC sheets into the infarcted myocardium. The control group was treated similarly but received no SC sheets. Because pilling up four or more sheets caused the central necrosis of the myoblasts presumably because the lack of in oxygen supply, we decided to pile two or three layers of the SC sheet over the broad surface of the impaired heart.
Measurement of Cardiac Function
Swine were anesthetized as mentioned above. Cardiac ultrasonography was performed with a commercially available echocardiograph, SONOS 5500 (PHILIPS Electronics, Tokyo, Japan). A 3-MHz annular array transducer was placed on a layer of acoustic coupling gel that was applied to the left hemithorax. Swine were examined in a shallow left lateral decubitus position. The heart was first imaged in the two-dimensional mode in short-axis views at the level of the largest left ventricle (LV) diameter. The calculation of the LV volume was based on the LV short-axis area using AQ system (7). And fractional area shortening (FAS) of the LV diastolic was calculated as follows:
These data are presented as the average of measurements of two or three selected beats.
Quantification of Regional Diastolic and Systolic Function by Color Kinesis
Diastolic CK images were obtained using a commercially available ultrasound system (SONOS 5500, Philips Medical Systems) from the LV midpapillary short-axis view for the determination of wall motion asynchrony as previously reported (8). CK examined every image pixel within the region of interest, which was drawn around the LV cavity, classifying it as blood or tissue based on integrated backscatter data. During diastole, each pixel was tracked into the next frame, and pixel transitions from endocardium to blood were detected and interpreted as diastolic endocardial motions. These pixel transitions were encoded using a color hue specific to each consecutive video frame, so that each color represents the excursion of that segment during a 33-ms period of time. The sites of regional LV diastolic wall motion or regions of interest were set on the basis of standard segmentation models: anterior, lateral, posterior, inferior, anteroseptal wall. The CK diastolic index was defined as the LV segmental filling fraction during the first 30% of the diastolic filling time (LV segmental cavity area expansion during the first 30% of diastole, divided by the segmental end-diastolic LV cavity area expansion, expressed as a percentage). We introduced the use of color kinesis method that displays endocardial motion in real time to evaluate the regional systolic function (8).
LV myocardium specimens were obtained 6 months after the SC sheet implantation. Each specimen was fixed with 10% buffered formalin and embedded in paraffin. A few serial sections were prepared from each specimen and stained with hematoxylin-eosin (H&E) stain and elastica Masson-Goldner for histological examination or with Masson's trichrome stain to assess the collagen content.
To label vascular endothelial cells so that the blood vessels could be counted, immunohistochemical staining of factor VIII-related antigen was performed according to a modified protocol. Frozen sections were fixed with a 2% paraformaldehyde solution in phosphate-buffered saline (PBS) for 5 min at room temperature, immersed in methanol with 3% hydrogen peroxide for 15 min, then washed with PBS. The samples were covered with bovine serum albumin solution (DAKO LSAB Kit DAKO CORPORATION, Denmark) for 10 min to block nonspecific reactions. The specimens were incubated overnight with an Enhanced Polymer One-Step Staining (EPOS)-conjugated antibody against factor VIII-related antigen coupled with horseradish peroxidase (DAKO EPOS Anti-Human Von Wille brand Factor/HRP, DAKO, Denmark). After the samples were washed with PBS, they were immersed in diaminobenzidine solution (0.3 mg/mL diaminobenzidine in PBS) to obtain positive staining. Ten different fields at 200× magnification were randomly selected, and the number of the stained vascular endothelial cells in each field was counted under a light microscope. The result was expressed as the number of blood vessels per square millimeter.
The following antibodies against smooth muscle cells and skeletal myosin (slow) were used to evaluate the existence of SCs: primary antibodies, antismooth muscle actin (clone 1A4, DAKO) antiskeletal myosin (slow) (clone NOQ7.5.4D, Sigma); secondary antibodies, anti-mouse Ig biotinylate (DAKO).
Picro-sirius red staining for the assessment of myocardial fibrosis or periodic acid-Schiff staining for that of cardiomyocyte hypertrophy was performed as described (9).
Positron Emission Tomography Procedure
We performed positron emission tomography (PET) studies on pigs which were transplanted SC sheets and control by using 150-water and 18F-FDG. The pigs were anesthetized by the introduction of pentobarbital followed by continuous inhalation of propofol (4 mg/kg/hr) and were placed supine on the bed of the scanner. PET was performed using a HEADTOME-III tomograph (Shimadzu, Kyoto, Japan) and data were analyzed as described elsewhere (10).
To evaluate arrhythmia we used Holter electrocardiography (ECG) for 24 hours. We checked arrhythmia by checking the number of ventricular premature beat after SC sheet implantation in myocardial infarction porcine (n=3).
Data are expressed as means ± SEM and subjected to multiple analysis of variance (ANOVA) using the StatView 5.0 program (Abacus Concepts, Berkeley, CA). Echocardiographic data were first analyzed by two-way repeated measurement ANOVA for differences across the whole time course, and one-way ANOVA with the Tukey-Kramer posthoc test was used to verify the significant for the specific comparison at each time point. To assess the significance of the differences between individual groups concerning other numeral data, statistical evaluation was performed with an unpaired t test. Statistical significance was determined as having a P value less than 0.05.
Characteristics of Myoblast Sheet
We obtained monolayered myoblast sheets by lowering the temperature, which released them from the Poly(N- isopropylacrylamide)-grafted polystyrene. Its size is approximately 3 cm×2 cm2 (Fig. 1a). H&E staining demonstrated that SC sheet contained a lot of SCs and SC sheets had an appearance of homogenous tissue, which thickness of one SC sheet was approximately 100 μm (Fig. 1b). Some smooth muscle cells are detected in the SC sheets, but those cells are not majority (Fig. 1c).
H&E staining demonstrated that transplanted SC sheets were attached in the epicardium (Fig. 1d) and oval-shaped cell that showed positive for eosin in cytoplasm were detected in the SC group microscopically in some layers over epicardium (Fig. 1e). Elastica Masson-Goldner showed that oval-shaped cells that supposed to origin from skeletal tissue exist in the transplantation site (Fig. 1f). These cells were not seen in the control group. And the SC group demonstrated decrease in the cross-sectional LV area compared with the C groups (Fig. 2a). Masson's trichrome staining showed that clustered SCs were detected in the center of the scar, whereas clustered SCs were not detected in the C group (Fig. 2a, b). Many clusters of well-developed smooth muscle cells exist in the center of the whole scar in the SC group, whereas in the C group, smooth muscle cells which formed vasculature exist in the scar (Fig. 2c–f). Although slow-type myosin-positive cells exist only on the endocardium and epicardium, those cells were not detected in the center of scar (Fig. 2g,h). So these figures depict that the skeletal muscle cells that exist in the center of the scar is not residual myocyte after infarction.
Quantification of Histopathology
In the SC group, vascular density was found to be significantly higher than in the C groups (SC vs. C=217.1±30.2 vs. 114.2±18.2 /field; P<0.05) (Fig. 3b).
Picro-sirius red staining demonstrated that % fibrosis was significantly reduced in the SC group compared with the C group (SC vs. C=1.6±0.2 vs. 3.1±0.3%; P<0.05) (Fig. 3b). Periodic acid-Schiff staining showed that cell diameter was significantly shorter in the SC group than the C group (SC vs. C=10.7±0.3 vs. 18.3±1.4 μm; P<0.05) (Fig. 3b).
These histological findings were universally identified in the native myocardial tissue without distinction of distance from the grafted region.
Functional Assessment of the Infarcted Myocardium
The FAS and LV end-ESA scores at baseline were not significantly different between the two groups.
Three months after the implantation, two-dimensional echocardiography showed significant improvement of the FAS (Fig. 4a) in the SC group compared with the C group (SC vs. C=49.5±2.8 vs. 24.6±2.0%, P<0.05). These functional improvements were preserved 6 months after implantation (SC vs. C=50.8±6.4 vs. 25.3±2.8%, P<0.05). The ESA was significantly smaller in the SC group than in the C group 3 months after the implantation (SC vs. C=4.3±0.5 vs. 9±1.3 cm2, P<0.05) (Fig. 4b). These attenuation of LV dilatation were preserved 6 months after implantation (SC vs. C=4.9±0.8 vs. 7.5±1.4 cm2, P<0.05). During this long-term observation, all SC sheet-treated animals were alive and exhibited no malignant arrhythmia assessed by 24-hour Holter ECG once a week (data not shown).
Before treatment, diastolic dysfunction was observed in the infarction area of myocardium and the regional delayed relaxation was detected in the remote site of infarction by color kinesis. After 3 months after implantation, CK-diastolic index in the lateral (SC vs. C=61.7±6.4 vs. 43.7±4.8%, P<0.05), anterior (SC vs. C=57.4±8.6 vs. 30.2±4.7%, P<0.05), and anteroseptal (SC vs. C=59±6.6 vs. 38.4±6.6%, P<0.05) segment were significantly ameliorated in the SC group compared with the C group, and regional systolic function in transplanted site was significantly improved in the SC group while not in the C groups (SC vs. C: lateral, 59.8±3.3 vs. 43.6±5.4%, P<0.05; anterior, 58.5±4.5 vs. 35.4±6.6%, P<0.05; anteroseptal, 59.8±3.3 vs. 43.6±5.4%, P<0.05), respectively (Fig. 5).
We could detect no ventricular premature beat for 24 hr by the Holter ECG in three myocardial infarction porcine received SC sheets.
Regional Myocardial Blood Flow and Residual Myocardial Tissue
PET study by using 150-water showed that the myocardial water-perfusable tissue fraction and myocardial blood flow were higher in the anterior wall where SC sheets were implanted compared with the myocardium receiving no sheets. These data depict that myocardial blood flow was better and microcirculation in the infarcted myocardium was preserved in the SC sheets implanted myocardium. PET study by using 18F-FDG revealed that more viable myocardial tissues were preserved in the skeletal sheet implanted myocardium compared with the myocardium receiving no sheets. Coronary angiography revealed that LAD was occluded by the ameroid constrictor in both cases (Fig. 6).
Over the past several years, increasing awareness of the shortcomings of heart transplantation and left ventricular assist system implantation has led cardiovascular surgeons to consider alternative means of treating end-stage heart failure. In clinical setting, cellular cardiomyoplasty has been reported to have the potential of fundamental regenerative capability and has already been introduced in clinical trials with skeletal myoblast (11) or bone marrow mononuclear cells (12), and results suggest that it is a relatively feasible and safety therapy as a therapeutic angiogenesis. In this setting, cardiac tissue implantation was proposed to the treatment of end-staged heart failure as a new concept of regenerative therapy and experimentally some groups depicted it's effectiveness in the damaged myocardium (13, 14). We also reported that cell sheets have great impacts on restoration of damaged myocardium in the rat infarction model (3, 4) and dilated cardiomyopathy hamster (5). To convince the effectiveness of cell sheets in preclinical trial, we examined whether autologous SC sheets implantation might become one of the armamentarium of regenerative therapy for chronic heart failure caused by myocardial infarction in the porcine model.
The potential added advantages of the cell sheet implantation method include the implantation of a high number of cells with minimum cell loss. In contrast, the injection method is associated with a high loss of cells or surface proteins due to the trypsin treatment. Despite a high number of cell loss in needle injection, the cell sheet implantation method might provide the advantages of a higher number of cell implantation without cellular community destruction, leading the more improvement of cardiac performance rather than cell injection method (4). In case of needle injection, inflammation accompanied with destruction of myocardium induced by needle injection promotes graft death after cell transplantation (15).
To examine the effects of the SC sheet implantation therapy, we analyzed cardiac function and performed a histological assessment of the infarcted heart after SC sheet transplantation in a swine infarction model. SC sheet implantation therapy significantly induced angiogenesis, reduction of fibrosis histologically. And cell diameter of host myocyte was significantly attenuated its hypertrophy compared with the no treatment group. PET study revealed the better regional blood perfusion and better regional myocardial viability in the myocardium receiving cell sheets compared with the myocardium receiving no sheets.
Moreover, SC sheet implantation induced functional recovery of damaged myocardium. Especially, we demonstrated that the regional diastolic and systolic dysfunction was well recovered in the sheet implanted group. Before treatment, diastolic dysfunction of infarcted area and regional delayed relaxation of noninfarcted site were detected by color kinesis in the porcine infracted myocardium. After treatment, diastolic dysfunction of infarcted site was significantly recovered and the phenomenon of regional delayed relaxation in noninfarcted site was not seen. Presumably, implanted elastic myoblast sheets and a large quantity of well-developed smooth muscle cells, which are detected in the center of the scar, improved the regional diastolic dysfunction of implanted site. Although SC sheet can not contract in vivo after implantation, this recovery of diastolic disassociation of LV might result in the recovery of systolic dysfunction.
To the best of our knowledge, this is the first report in which tissue-engineered SC sheets implantation was successfully used to improve cardiac performance in a large animal model of ischemic myocardium according to the Laplace's theory.
The mechanisms of the restoration of damaged myocardium by SC sheet implantation might be complicated and many pathways might affect the recovery of ischemic myocardium. Recent reports depict that cell sheets enhance the recruitment of hematopoietic stem cells through the release of stromal-derived factor 1 (4). The fact of thicker anterior wall and the improvement of regional function might depend on both the recruitment of cytokine releasing stem cells, survival of grafted cells, and well-developed smooth muscle cells. And these cells might have good elasticity and these elastic cells and tissues softened the stiffness of anterior wall in association with the attenuating fibrosis even in the infarct area. This reduced stiffness of anterior wall might lead to the improvement of the diastolic dysfunction. Transplanted SCs cannot differentiate into cardiomyocyte anymore, but regional systolic function improved in the transplanted site. Probably, the improvement of regional diastolic function due to elastic cells might be responsible for the restoration of regional systolic dysfunction. Recent reports demonstrated that regional left ventricular myocardial relaxation was closely related to regional myocardial contraction (16) and the improvement of regional myocardial relaxation leads to the recovery of global diastolic function (17). Moreover, the improvement of regional systolic function is closely related to global systolic function (18). We assume that this theory about the relationship between diastolic and systolic function is one of the mechanisms about the improvement of diastolic and systolic function in the cell sheet transplanted myocardium.
Question is why the well-developed smooth muscle cells exist in the center of the scar in the SC sheet group after transplantation despite a small quantity of smooth muscle cells in the SC sheet? Does a small quantity of smooth muscle cells in the SC sheet proliferate after transplantation? Do progenitor cells in the SC sheet differentiate to smooth muscle cells? Do progenitor cells or smooth muscle cells in the host myocardium migrate to the implanted site and proliferate? To the regret, there is no data to answer these questions exactly in this article and more detailed studies are needed to elucidate this important question.
Some reports depicted that the expression of hepatocyte growth factor (HGF) in the myoblast sheet transplanted ischemic myocardium is higher compared with the nontransplanted ischemic myocardium (4). HGF has an antifibrotic activity both through the activation of a matrix degradation pathway (19), restoration of cytoskeletal proteins on cardiomyocyte (20), and induce angiogenesis in the ischemic myocardium (21). Our study demonstrated that % fibrosis was significantly reduced in the SC sheet transplanted group. This paracrine secretion of HGF from SC sheets might attribute the reduction of % fibrosis. In our study, much more factor VIII-positive cells are detected in the SC sheet transplanted myocardium. This might be induced by paracrine secretion of HGF and angiogenesis might rescue the ischemic host cardiomyocyte and bring about the improvement of the distressed function of host cardiomyocyte. The distressed cytoskeletal proteins on the cardiomyocyte in the ischemic myocardium might be reorganized by the HGF secreted from skeletal sheet and the restoration of cytoskeletal proteins might lead to the improvement of cardiac function. And some reports demonstrated that myoblast sheets maintain the distressed cytoskeletal proteins on the host cardiomyocyte in the dilated cardiomyopathy hamster model (5). Consequently, cell sheet treatment is appropriate for recovery of ischemic cardiomyopathy. Recent research works demonstrated that several regenerative factors such as insulin-like growth factor-1 (22) and Thymosin b4 (23) were expressed in the rat ischemic myocardium model after myoblast sheet implantation by reverse-transcriptase polymerase chain reaction analysis (data not shown). After myoblast sheet transplantation to ischemic myocardium, several regenerative factors are expressed in the transplanted site, and these long-term and low-dosed expressed regenerative factors might cooperatively restore the damaged myocardium.
We could find no ventricular premature beat analyzed by Holter ECG after SC sheet implantation. We have already proved that in the rat infarction model, arrhythmia is less in the SC sheet implantation group compared with the needle injection group and this work represented that more monocyte chemotactic protein-1-positive cells and CD11b (macrophage marker)-positive cells were detected in the needle injection group compared with SC sheet implantation (data not shown). We speculate that needles destroy the myocardium and this destroyed myocardium may induce the inflammation and this inflammation may induce the arrhythmia. Conversely, SC sheet implantation technique normally doses not destroy the myocardium when they are implanted to recipient heart. Moreover, SC sheet will survive on the epicardium and electrical wave originated from implanted myoblasts may not deliver to the recipient myocardium directly. But when we implant myoblasts by needle injection, implanted myoblasts survive in the center of the myocardium and electrical wave will deliver to the myocardium directly, leading to the arrhythmia.
In conclusion, we have preclinically demonstrated SC sheets produced histologically and functionally apparent prevented the deterioration of the impaired myocardium in the swine model. These data provide a basis for attempting clinical cell sheet implantation in ischemic disease as the armamentarium to promote the regeneration of chronic heart failure caused by myocardial infarction.
The authors thank Shigeru Matsumi and Masako Yokoyama for their excellent technical assistance.
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