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Departments of 1Physiology and Biophysics, 2Surgery, and 3Pediatrics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 1X5; and 4Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
Submitted 10 October 2003 ; accepted in final form 19 May 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-galactosidase gene so that migration from the injection sites could be traced. Both freshly isolated CMs as well as CFs were found concentrated in the infarcted zone; these cells survived for at least 2 wk posttransplantation. Transplanted CMs were regularly striated and grew long projections that could form gap junctions with native CMs, which was evidenced by connexin43 labeling. In addition, CM transplantation resulted in increased angiogenesis in the infarcted areas. In contrast, transplanted CFs did not appear to make any gap junctional contacts with native CMs nor did they enhance local angiogenesis. Mbs cultured for 7 days and transfected Mbs were identified 7 days posttransplantation in the infarcted area. During that time and thereafter, Mbs proliferated and differentiated into myotubes that formed new, regularly striated myofibers that occupied most (50–70%) of the infarcted area by 2–3 wk. These newly formed myofibers maintained their Mb skeletal muscle origin as evidenced by their capacity to express myogenin and fast skeletal myosin. This skeletal phenotype appeared to downregulate with time, and Mbs partially transdifferentiated into a cardiac phenotype as indicated by labeling for cardiac-specific troponin T and cardiac myosin heavy chain. By the third week posttransplantation, new myofibers formed apparent contacts with the native CMs via putative gap junctions that expressed connexin43. Myocardial performance of animals that were successfully transplanted with Mbs was improved.
cellular cardiomyoplasty; myoblasts; ischemia; heart disease
Despite the increasing interest in this research field, many questions remain unanswered. Although some improvement of functional activity of infarcted hearts transplanted with skeletal Mbs has been reported (5, 11, 18, 26), there is little information available concerning the anatomical characteristics of transplanted cells into acutely infarcted tissues. Evidence of the functional connections of Mbs with surviving host CMs including phenotypical characteristics remains largely unknown. In the present study, we undertook to investigate 1) whether adult CMs or adult cardiac fibroblasts (CFs) can be successfully transplanted to reside in and assist with the repair of the injured myocardium, 2) how the effects of these implanted cells compare with those of grafted skeletal Mbs, and 3) the phenotypical properties of such Mbs after transplantation into the acutely infarcted hearts. Herein, we report that all three cell types can be successfully transplanted into a ventricular infarct as indicated by the presence of cells transfected with -galactosidase (-Gal). However, we identified that only skeletal Mbs developing into myotubes could repair a significant portion of the infarcted area; this was demonstrated by immunoconfocal microscopy.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Operative procedures. Animals were pretreated with 0.02 mg/kg sc atropine and sedated thereafter with 80 mg/kg ip ketamine. Preincision, the animals received a one-time-only dose of cefazolin (50 mg/kg im). Animals were endotracheally intubated with a modified piece of polyethylene-205 tubing that had a flanged end large enough to attach to the small-animal Harvard ventilator. Anesthesia was maintained with isoflurane for the remainder of the surgery. For postprocedure analgesia, the long-acting narcotic buprenorphine (0.05 mg/kg sc) was given after incubation. Three additional doses of buprenorphine were administered 8–12 h after the procedure described above. At the end of the procedure and after extubation, the nonsteroidal analgesic ketoprofen was given (1.25 mg/kg sc).
A lead-II ECG and pulse-pressure waveform analysis were monitored continuously throughout the coronary ligation experiments. Using sterile surgical techniques, we exposed the guinea pig pericardium via a limited left-sided thoracotomy at the fourth or fifth intercostal space. After a small pericardial incision was made to expose the left ventricle, a polypropylene monofilament suture ligature was passed around the ventral descending coronary artery (analogous to the left anterior descending artery in humans) just distal to its first diagonal branch (cf., Fig. 1). During arterial occlusion, myocardial cyanosis and ECG ST segment changes were observed. The infarcted area represented 20–25% of the left ventricular volume as estimated from the individual transmural serial sections (40 µm). Within a few minutes after the coronary ligation and using a 27-gauge needle in 70–80 µl of MEM media, all three types of the isolated cells were injected into a close vicinity of the infarct just distal to the arterial ligature. The number of animals that received each cell type at the time of injection is listed (see respective RESULTS). In the control animals, the myocardial ischemic infarct zone was injected with the same volume of culture media only. The thorax was closed in layers after this intervention, and any residual air was removed by manual evacuation.
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8–12 x 106 cells/animal), of which >80% were rod shaped and >90% were viable (as demonstrated by exclusion of Trypan blue stain). Transfection with -Gal adenovirus was at 100 multiplicity of infection (MOI) at 37°C for 3 h before transplantation.
CF preparation.
CFs were isolated from perfused hearts of adult guinea pigs together with CMs (see CM preparation). All cells were collected and cultured in Eagle's MEM with Earle's salts, were supplemented as described previously (19), and were cultured at 107 cells/dish (dish size, 100 x 20 mm). After 2 h, the dish was scraped to prevent adjusting of the CMs. After 24 h, the CMs were removed by washing the cells three or four times with PBS. After an additional 48 h, cells were harvested with 0.05% trypsin in PBS for 7 min at 37°C. Transfection with -Gal adenovirus was at 100 MOI at 37°C for 24 h. Cells were washed and incubated for an additional 24 h. After cells were harvested with 0.05% trypsin in PBS for 7 min at 37°C, they were pelleted for 7 min by centrifugation at 1,500 rpm. Cells were injected into the infarcted hearts at a concentration of 2 x 106 cells/70 µl of MEM.
Skeletal Mb preparation.
Soleus and external digitorum longus muscles were removed from one leg of anesthetized animals and cut into small pieces, and 300 mg of tissue were placed in beakers (80 mg/15 ml) that contained dissociating solution (DMEM; GIBCO), 4 U/ml protease (Sigma), and 100 U/ml collagenase II (Worthington). After dissociation for 2.5 h in a shaking bath at 37°C, cells were collected and centrifuged for 10 min at 1,000 rpm. Pellets that contained 16 x 106 cells (cf., cell count in CF preparation) were resuspended in Ham's F-12 growth medium (GIBCO), 20% FBS (GIBCO), penicillin (100 U/ml), and streptomycin (100 µg/ml) and were cultured on glass discs treated with laminin (1 µg/cm2) in a six-well Falcon dish (2 x 106 cells/well). Growth media were changed after 2 days. After an additional 2 days when the cells were 60–70% confluent, the growth medium was replaced by the differentiation medium, which contained DMEM, 7% horse serum, penicillin (100 U/ml), and streptomycin (100 g/ml). After 24 h, cells were transfected at 100 MOI with -Gal adenovirus for 24 h at 37°C. Cells were then rinsed and incubated at 37°C for 24 h, harvested with 0.05% trypsin in PBS, and centrifuged for 10 min at 1,000 rpm. Cells (106 cells/80 µl of MEM) were injected into a freshly occluded area. On the day of injection, cells were also fixed for immunohistochemistry and assayed with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal).
Amplification of adenoviral particles.
Adenovirus that expressed -Gal under the control of the cytomegalovirus promoter CMV5 was produced using -Gal DNA supplied by the manufacturer (Adeno-Quest Kit; Quantum Biotechnologies; Montreal, Quebec, Canada); the adenovirus was AdCMV5. -Gal was amplified by infecting QBI-293A cells through three passages (passage 1, 0.2 ml of viral particles per 1 x 105 cells; passage 2, 0.5 ml of passage 1 material per 5 x 106 cells; passage 3, 0.5 ml of passage 2 material per 1 x 107 cells), which resulted in 5 ml of viral particles (titer of 3 x 109 plaque-forming units/ml).
Tissue fixation for immunohistochemistry and -Gal assay.
Animals were anaesthetized with pentobarbital sodium (Somnotol; 160 mg/kg) and heparin (1,000 USP/300 g). Hearts were removed at times specified in the figures (1–6 wk) and rinsed with 0.15 M NaCl. The myocardium containing the infarcted area (1 cm2) was cut out and fixed for 2 h at 4°C in 2% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 and afterward was transferred into 30% sucrose in 0.1 M phosphate buffer for 24 h. Sections (40 µm) were cut transmurally parallel to epicardium on a Reichert Histostat cryostat microtome and collected in PBS. For immunohistochemistry, the sections were incubated for 2 h at room temperature with the exception of sections for Ki-67, which were incubated at 37°C in guinea pig serum that contained primary antibody, 0.2% Triton X-100, and 2% sheep or goat serum according to the host species of the secondary antibody (Table 1). Sections were washed and placed for 1 h in guinea pig serum that contained secondary antibodies, 0.2% Triton X-100, and 2% sheep or goat serum at room temperature. Control for specific labeling was provided by comparison of reactions with and without primary antibody. All secondary antibodies were obtained from Jackson Immunoresearch (West Grove, PA), and each was conjugated with either FITC, tetramethyl rhodamine isothiocyanate, or cyanine.
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-Gal assays were performed, sections were washed for 15 min with phosphate buffer (pH 7.4) that contained 0.02% Nonidet P-40 and 2 µM MgCl2. Staining for X-Gal was accomplished at 37°C for 16 h in solution that contained 1 mg/ml X-Gal, 4 µM magnesium chloride, 10 µM potassium ferrocyanide, 10 µM potassium ferricyanide, and 4.8% DMSO in phosphate buffer (pH 7.4). The transfection rate of these cells was established in cultures fixed at the time of intracardiac injection and was 30–50% in all three cell types investigated. Confocal microscopy. Tissues were examined with a Zeiss LSM 410 inverted laser-scanning microscope equipped with a krypton-argon (488, 568, and 647 nm) laser, a x40 magnification Zeiss Axiovert 100 microscope with 1.3 numerical objective, oil Zeiss Plan-Neofluar objective, and a x63 magnification, oil Zeiss Plan-Apochromat objective with 1.4 numerical objective. Simultaneous dual excitation by a double-band beam splitter for simultaneous excitation at 488 and 568 nm (DBSP 488/568 nm) and dual-channel emission detection by a DBS2 FT-560, which splits green and red emissions for simultaneous or separate detection with two photomultipliers, were used with FITC (band-pass filter, 515–540 nm) and rhodamine (band-pass filter, 575–640 nm). For each experiment, data were collected in each channel separately (to avoid overlap of the two signals) using the same microscope settings. Optical sectioning was done at a scanning speed of 4 s/frame and was averaged over four lines; image size was set at 512 x 512 pixels. Tissues were studied in this manner using 10–20 1-µm-thick sections. Tissue sections obtained from the confocal microscope were reconstructed by computer software into three-dimensional composite images such as those presented in the figures. There were no digital adjustments made to the images.
Echocardiographic recordings. Guinea pigs were sedated using isoflurane with an induction dose of 5% for 2 min and a subsequent continuous dose of 1.5%. The isoflurane was carried in 100% oxygen (1 l/min) and delivered by mask via a Bain circuit while the guinea pig breathed spontaneously. Once sedated, the guinea pig was shaved over the ventral thorax to create an echocardiographic window. ECG leads were placed for monitoring and data were recorded simultaneously with echocardiographic images. Echocardiography was performed with a 7-MHz multi-Hertz probe on an Acuson 120XP/10c system (Acuson; Mountain View, CA) that was configured for pediatric echocardiography, and data were stored on videocassette.
Two-dimensional echocardiography was performed in parasternal long- and short-axes and four-chamber views. M-mode echocardiography was also performed. From these views, biplane Simpson's measurements of ejection fraction and M-mode measurements of fractional shortening and chamber size were obtained. After data were acquired, the guinea pigs were allowed to recover while spontaneously breathing room air. Echocardiography was performed before and 5 wk after coronary artery ligation (control group, n = 5) and 3 and 5 wk after the implantation of myotubes into the vicinity of the occluded zone (experimental group, n = 4).
Data were statistically evaluated using paired t-test within each group. Comparisons between the preocclusion values and the values from the experimental group 3 wk after the grafting with skeletal myotubes were evaluated by unpaired t-test. Regarding the effectiveness of the echocardiographic measurements, the most limiting factor was the very high heart rate of the guinea pigs, which ranged from 350–450 beats/min. Thus, at the echocardiographic recording system's maximal rate of 50 Hz and a heart rate of 420 beats/min, we used 6 frames/heartbeat. The most difficult part of the echocardiographic recordings was due to the extremely small hearts of these animals, which often required repeated measurements in individual animals for up to 2 h.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Transplantation with adult CMs or CFs.
Our first objective in providing graft cells for cardiac repair of the infarcted tissue was to transplant the adult guinea pig ventricular CMs. To identify the transplanted cells in normal and infarcted tissue, we first transfected them with an adenovirus that expressed -Gal. Figure 2 represents the infarcted area in the absence (Fig. 2A) and presence (Fig. 2B) of the transplanted adult CMs. The transfected CMs were injected (105 myocytes in 70 µl of MEM) into close vicinity of the infarcted area at the time of the coronary ligation. During the first week postligation, the transplanted CMs (n = 4) remained viable and migrated mostly into the infarcted area (Fig. 2B). Some transfected CMs were also located at the border of the surviving myocardium and projected long processes (Fig. 2C) that formed gap junctions with the host CMs (Fig. 2D). These transfected CMs were identified throughout transmural infarctions 1–2 wk after transplantation. Their numbers decreased with time thereafter, and the CMs were no longer identified 3 wk after implantation. Within 3–6 wk postligation (n = 10), some CMs at the border zone projected fingerlike process into the infarcted area (Fig. 2, E and F). The border zone also exhibited a heavy network of neurites that surrounded the individual myofibrils and projected far into the infarcted zone (Fig. 2E). Neither of these reparative effects were observed in the control (n = 20) nontransplanted infarcted hearts (Fig. 2A). The most prominent effect of the CM transplantation was markedly increased angiogenesis in the infarcted area, which was indicated by increased positive immunolabeling to 
-smooth muscle actin antibody (Fig. 2G) vs. the neighboring intact myocardium (Fig. 2H).
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-Gal adenovirus in 70 µl of media per animal (n = 4). Many of these Mbs fused into multinucleated myotubes that were located mostly transmurally in the infarcted zone (Fig. 3B). Furthermore, these newly formed myotubes were aligned parallel to one another as well as to the adjacent surviving myofibrils. The fusion of Mbs into myotubes appeared to be a very active process especially during the first 2 wk after transplantation (Fig. 3, C and D). Thus, in some animals, >50% of the infarcted area was "repaired" with these new myofibers. During this period, many Mbs were still actively proliferating as indicated by positive immunolabeling of their nuclei with Ki-67 (Fig. 3, E and F). The Ki-67 protein was present in cells during all active phases of cell division but was absent from resting cells, which makes it an excellent proliferation marker (21). An additional population of round cells labeled positively for protein gene product (PGP) 9.5 (Fig. 3, E and F), which is considered to be a neuronal marker (27). In addition to labeling the cardiac neurites in the normal and infarcted zones, PGP 9.5 also labeled the myogenic cells in the injured regions (Fig. 3G). This is in agreement with the fact that it labeled cultured Mbs and myotubes in vitro (Fig. 4, A and B). The proliferation and differentiation process of the cultured Mbs and myotubes on day 7, i.e., immediately before they were harvested for transplantation and after 1 more day in culture is shown in Fig. 4, A and B, respectively. The latter culture is representative of the first day after administration of Mbs and myotubes into the heart.
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-actinin and myomesin. During the second week in vivo, these myotubes were often short and semicontracted, probably because they were not yet properly externally loaded by the surrounding myotubes (Fig. 4E). With time (cf., during the third week after transplantation), these myotubes grew in length and fused into long myofibers. In the stretched form, myotubes displayed regular cross-striations (Fig. 4F); 3 wk after transplantation, myotubes (the new striated myofibers) also started to express the gap junctional protein connexin43 (Cx43) along their entire surfaces at apparent contacts with the surviving CMs (Fig. 4, G and H). Such putative gap junctions were also observed in vitro in cocultures of adult guinea pig CMs and skeletal myotubes and were not present in cultured myotubes alone (data not shown). By the third postoperative week, the myotubes and myofibers had invaded 70% of the infarcted area (Fig. 5A). At this time, some myotubes still contained myogenin (Fig. 5C) although to a much lesser extent than at 2 wk (Fig. 5B). This immunomarker indicates the presence of skeletal muscle-specific transcription factor (12, 22). Some short myotubes also still expressed fast skeletal myosin (Fig. 5D). By 3 wk posttransplantation, progressive transdifferentiation into cardiac muscle phenotype was indicated by the fact that these cells expressed specific immunolabeling for cardiac troponin T (Fig. 5E) as well as for cardiac myosin heavy chain (Fig. 5F).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-Gal gene such that migration of all three cell types from the site of injection could be traced. The major findings are as follows. First, freshly isolated adult guinea pig CMs survive for at least 2 wk posttransplantation when administered into acutely occluded ventricular tissue. To the best of our knowledge, these results represent the first evidence of successful survival and maintenance of structural integrity of adult CMs after transplantation into the heart. We found that transplanted CMs not only survive but can grow long projections that can form gap junctions with the native CMs in the border zone of the MI. The absence of the transfected CMs after 2 wk was considered to be a result of immune response to this heterologous transplant. CM transplantation also resulted in increased angiogenesis within the infarcted area, an effect that was accompanied by enhanced growth of neurites into the infarct zone. Perhaps due to the combined effects of these responses, new outgrowth (100–200 µm) of CMs occurred in the infarcted zone (as evidenced by specific labeling for PGP 9.5) but not in control (nontransplanted) animals. Although PGP 9.5 is considered to be a neuronal marker (27), this marker was associated with all new myofibers at the initial stage of growth into the infarcted zone including the skeletal muscle myotubes (see below). Presently, we have no explanation for this phenomenon; because it was totally reproducible, it served as a convenient marker of newly formed myofibers. It should be noted that the infarcted zone also contained a number of PGP 9.5-positive structures, many of which were neurites or their fragments (see Fig. 2, A and E), but some appeared to be round cells (e.g., see Fig. 3, E–G). Overall, the histological effects of the transplanted CMs in the infarcted host heart were clearly identified, although they did not appear to substantially lessen the scaring process. Second, the transfected CFs were also found mainly in the infarcted zone during the first 2 wk after transplantation, some of which had fused into long (
100 µm) structures. The transfected CFs were presumably also destroyed later by the immune system because they disappeared with time. Unlike transplanted CMs, angiogenesis and native CM outgrowth into the infarcted area did not accompany CF implantation. In that respect, CFs were considered even less suitable for a successful grafting than CMs. Finally, adult guinea pig skeletal muscle Mbs presented the most successful implant in the MI and perhaps affected contractile performance in the ischemic zone. Most (95%) of these transfected cells were identified in the infarcted area 7 days posttransplantation.
Implanted myotubes proliferated and fused into myotubes and myofibers at a rate that was much faster than observed in vitro (unpublished data). It appears that two populations of Mbs that differ in their proliferative capacity can exist in vivo vs. in vitro as has been demonstrated in skeletal muscle (3). The population that proliferates rapidly under in vitro conditions (as represented by transfected cells) does not readily survive in vivo. The other consists of a relatively minor population in vitro that proliferates in vivo apparently stimulated by injured tissue. These newly formed myofibers occupied 50% of the MI area by 2 wk and 70% by 3 wk posttransplantation. The skeletal muscle origin of these newly formed, striated myofibers was evidenced by 1) expression of myogenin, which is a specific marker of skeletal Mb and myotube phenotype (12, 22); and 2) expression of fast skeletal myosin, which is not expressed by CMs. Expression of both of these markers decreased greatly from the second to the third week such that by the third week posttransplantation, the fast skeletal myosin was present only in short myofibrillar segments where apparently new proliferation and differentiation were occurring via segments fusing with the already-established myotubes (see Fig. 5). Thus it seems that their proliferation activity might downregulate with time.
Although the skeletal muscle phenotype was gradually downregulated over time, partial transdifferentiation of newly formed myotubes and myofibers to the cardiac phenotype occurred. This was evidenced by expression of 1) specific cardiac troponin T, and 2) cardiac myosin heavy chain. By the third week, these newly formed myofibers started to express gap-junctional protein Cx43 in a spotty pattern that included the entire points of contact with adjacent CMs. These putative gap junctions may not have been fully developed and may have exhibited distributions similar to those expressed during postnatal development (1a). Only at 90 postnatal days did the gap junctions reach the distribution identified in adult animals. Our confocal microscopic images indicate the possibility of electromechanical connections between the host CMs and the newly formed myofibers. It is important to note that these newly formed "gap junctions" were identified only at the proximal ends of the interface between long, multinucleated myofibers and surviving host CMs. Because the entire network of myotubes and myofibers appeared to be well interconnected, it may provide the anatomic substrate for conduction of an electrical impulse into the infarcted zone from its border. This hypothetical concept requires additional investigation in vivo.
Thus by using multiple immunohistochemical markers, we were able to demonstrate that transplanted skeletal muscle Mbs can proliferate and differentiate into myotubes that can eventually express the cardiac phenotype. It appears that these newly formed myofibers can form gap junctional contacts with the surviving host myocardium. These contacts may provide the basis of functional synchronization of the grafts with host myocardial tissue. Echocardiographic data indeed suggest that the functional status of the infarcted, grafted region may improve postgrafting.
Presently there are two main controversies concerning the suitability of skeletal muscle satellite cells as a graft for MI repair, including 1) the phenotypical characteristics of implanted cells and their transdifferentiation into cardiac-like myofibers, and 2) the existence of contacts with the surviving myocardium. A variety of conditions have been used to implant isolated Mbs and myotubes into myocardium that range from implanting cells into a healthy myocardium (8), an ischemic zone (2, 4), or partially (26) or even fully (10) scarred old MI tissue.
In injured skeletal muscle, Mbs actively migrate into the injured area. Mbs are capable of introducing normal copies of defective muscle genes only when the implantation site of choice is minimally fibrotic but is already undergoing necrosis at the time of implantation. This stimulates the proliferation and regeneration of the transferred Mbs (15, 16). From this it follows that similar conditions should be achieved when Mbs are transplanted into cardiac muscle. Grafting skeletal satellite cells into a healthy myocardium (19) may produce effects that are very different from those observed upon their transplantation into injured cardiac tissue. Certainly the stimulatory effects of chemicals liberated by injured tissue on their proliferation would be reduced as would their capacity to make contacts with the healthy myocardium. The other extreme is represented by a model in which autologous skeletal Mbs were injected into scar tissues of an old MI (10). Although some functional improvement was reported for some patients, several patients developed serious arrhythmias as a consequence that may be the result of heterogeneous proliferation and regeneration of the implanted Mbs, which could produce arrhythmogenic pathways through the scar tissues. Such arrhythmia formation can be observed in the presence of remodeled gap junctions (25). Integration of cell groups with poor electromechanical coupling and/or cell groups surrounded by the host scar tissue may cause a mosaic of cell-to-cell coupling and lead to arrhythmogenesis. Among implant studies (for review, see Ref. 11), the most promising results have been obtained after implantation of autologous Mbs into rabbit MI by Taylor and associates (2, 26). These investigators implanted the cells into an acute MI produced by cryoinjury or 1 wk post-MI. They produced very convincing data that demonstrate significant functional recovery after 2 wk of posttransplantation. Although limited immunohistology was used to provide evidence concerning the location and regeneration of the implants in the infarcted area, these authors indicated that implanted cells can retain skeletal and cardiac cell characteristics. Our data provide support for the double-phenotype properties of implanted Mbs as occurs posttransplantation within the acutely injured myocardium.
Our data support the possibility for successful use of skeletal Mb implants for cardiac MI repair particularly when applied in an acutely occluded heart. The importance of the timing of the implantation vs. the injury is supported by the fact that the skeletal muscle stem and satellite cells possess the capacity to activate diverse genetic programs in response to environmental stimuli (24). Our studies underestimated, if anything, the potential benefits of the cardiac repair by implanted Mb implantation because we employed heterologous cells. The most controversial issue to be investigated is the question of whether differentiated myofibers (grafts) can form appropriate electromechanical coupling with the surviving myocardium. Evidence from a number of sources supports this possibility. The myotubes can form gap junctions with adult CMs in vitro (18), which was also confirmed in our cocultures of adult guinea pig CMs with skeletal Mbs (unpublished data). Evidence obtained in vivo remains indirect (4, 26). The results presented herein indicate that the transplanted myotubes can express gap junctional protein Cx43 that is located at the contacts between native cardiac myofibrils and grafted skeletal myotubes and myofibers. At present, we do not know whether these contacts are functionally active; techniques for investigating this very important question are presently unavailable.
Perspectives.
Evidence presented herein indicates that skeletal muscle Mbs in vivo can proliferate, differentiate, and transdifferentiate in vivo into a viable, homogeneous network of myofibers within myocardium including the ischemic tissue after acute MI. We hope that these data will help stimulate additional research in this very promising field of cardiac repair.
The challenges remain to establish 1) at which stage Mbs and myotubes should be transplanted for optimal proliferation and differentiation in an infarcted zone; 2) what times after an MI occurs are optimally, maximally, and minimally effective for implantation; 3) what the optimal number of cells is that should be implanted; 4) whether sequential implantations would be more effective then a simple one; 5) whether such implantation could be achieved in a safe and efficient manner by catheter; and 6) how to identify electromechanical coupling between transplanted and host cells in vivo.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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