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J Thorac Cardiovasc Surg 2002;123:984-992
© 2002 The American Association for Thoracic Surgery
Evolving Technology (ET) |
From the Department of Cardiothoracic Surgery, Heart Science Centre, Harefield Hospital,a and Muscle Cell Biology Group, Hammersmith Hospital, Imperial College Faculty of Medicine,b London, United Kingdom.
This study was supported by the British Heart Foundation and the Harefield Research Foundation. Ken Suzuki is supported by the research fellowship from the Uehara Memorial Foundation.
Received for publication April 30, 2001. Revisions requested July 9, 2001; revisions received Oct 24, 2001. Accepted for publication Nov 7, 2001. Address for reprints: Professor Sir Magdi H. Yacoub, Department of Cardiothoracic Surgery, Harefield Hospital, Hill End Rd, Harefield, Middlesex, UB9 6JH, United Kingdom (E-mail: k.suzuki{at}ic.ac.uk).
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Abstract |
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Introduction |
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1-4 It has been demonstrated that skeletal myoblasts retain the ability to regenerate differentiated functional muscle and to improve cardiac function when transplanted in rabbit and rat heart failure models. 3,4 In addition, myoblasts can be isolated from patients with heart failure themselves and used as an autograft, obviating the need for immunosuppressive reagents. 5 Currently, direct intramyocardial injection of a skeletal myoblast suspension is the most popular method to deliver myoblasts into the heart. However, we consider that there remain some important issues that should be addressed to refine this therapeutic strategy for clinical application. One of these is the time-consuming and complex process required to prepare sufficient numbers of purified myoblasts from skeletal muscle. Millions of myoblasts are likely to be required to improve cardiac function by means of direct intramyocardial injection of a skeletal myoblast suspension, even in small animals. 3,4 There are concerns that repeated passaging of primary myoblasts in vitro might cause possible harmful effects, resulting in the alteration of important properties, such as the ability to fuse and differentiate. 6
Recently, we have established a simple, quick technique to isolate viable single fibers of rodent skeletal muscle. 6 A single fiber (here defined as muscle tissue separated into individual fibers), the minimal functional unit of skeletal muscle, retains myogenic precursors, skeletal myoblasts (satellite cells), between the basal lamina and plasma membrane. The myoblasts are normally mitotically quiescent, but when surrounding muscle is injured, they migrate from the fiber into the damaged area, proliferating and differentiating to regenerate myofibers. 7,8 In this study we investigated whether transplanted single fibers could work as a vehicle to deliver skeletal myoblasts-myotubes into the myocardium and whether this strategy leads to the improvement of cardiac function in heart failure induced by either doxorubicin administration or myocardial infarction.
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Methods |
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Isolation of single muscle fibers
Living single fibers of skeletal muscle were isolated according to the previously described method. 6,7 Briefly, the extensor digitorum longus of a male Lewis rat (80 g) was removed and digested in 0.2% type I collagenase (Sigma) in Dulbecco's modified Eagle's medium (DMEM). The muscle was transferred to a petri dish containing serum-free DMEM, in which single muscle fibers were liberated by means of gentle trituration. The individual, intact, viable muscle fibers (approximately 500 µm long and 20 µm in diameter) were collected and subsequently injected into the heart, as described below.
Characterization of single muscle fibers and derived myoblasts
When cultured in proliferation medium (DMEM with 20% fetal calf serum, 10% horse serum, and 0.5% chicken embryo extract), putative skeletal myoblasts were derived from the fibers and proliferated. Cells were grown to subconfluence in proliferation medium, which was then switched to differentiation medium (DMEM with 2% horse serum) to induce differentiation. 6 Cells were incubated to subconfluence to assess the purity of myoblast culture. After fixation and blocking, the samples were incubated in anti-
-sarcomeric actin monoclonal antibody (Dako) and then in fluorescein isothiocyanate-conjugated secondary antibody (Dako). Nuclei were counterstained with 4,6-diamino-2-phenylindole.
Generation of doxorubicin- or myocardial infarction-induced heart failure
Heart failure was induced by means of doxorubicin administration 9,10 or left coronary artery (LCA) occlusion 11 in male Lewis rats (200 g). Fifty rats were administered with doxorubicin hydrochloride (Sigma) in 6 equal injections (each containing 2.5 mg/kg in saline solution administered intraperitoneally) over a period of 2 weeks for a total dose of 15 mg/kg. Another 45 rats were anesthetized with sodium pentobarbital (50 mg/kg administered intraperitoneally) and mechanically ventilated. Through a left thoracotomy, the LCA was occluded 3 mm distal to its origin. The surgical wounds were repaired, and the rats were returned to their cages to recover. Aseptic surgical techniques were used throughout the procedure. Rats were observed for 4 weeks after the last injection of doxorubicin or LCA occlusion for their general appearance, behavior, and mortality.
Transplantation of syngeneic single fibers into the failing rat heart
Four weeks after the last doxorubicin injection or LCA occlusion, appropriately randomized rats were anesthetized and mechanically ventilated. Through a left thoracotomy, single fibers of a syngeneic donor (although not autografts) were injected into the left ventricular (LV) free wall (at 4 sites, each of which was injected with only one fiber in 3 µL of DMEM) with a glass micropipette (Drummond Scientific), the end of which was pulled to an extremely sharp point after heating (n = 20 for the doxorubicin model). For the LCA-occluded hearts (n = 18), fibers were injected into the border zone of infarcts (not into infarct scar). We believe that injection into the border zone has some potential advantages over the injection into the infarct scar, including easier injection and better blood supply for grafted cells. For the control group, the same volume of DMEM was injected (n = 20 for each model). After repairing surgical wounds, the rats were incubated for a further 4 weeks.
Histologic study
At 4 weeks after transplantation, hearts from each group were collected, and the left ventricle was cut into 4 segments and frozen in an embedding medium. After fixation, 6-µm cryosections were incubated with 0.6% H2O2 in 0.1% Triton X-100. After blocking, the sections were incubated with an anti-skeletal myosin heavy chain (MHC) antibody (Zymed laboratories), which reacts with skeletal MHC but not with cardiac or smooth muscle myosin. This was then incubated with a biotinylated secondary antibody (Dako). The sections were colored with a Dako streptABComplex/HRP kit, according to the manufacturer's instructions, and counterstained with hematoxylin and eosin. Sections were examined by blinded experienced histologists. The number of capillary vessels in the peri-infarct area was counted with a light microscope at 400 magnification by blinded experienced observers on the sections that included injection sites. 12 Five such high-power fields (HPFs) in each section were randomly selected, and the number of capillaries in each field was averaged and expressed as the number of capillary vessels per HPF (0.2 mm2).
Assessment of cardiac function and infarct size
At 4 weeks after single-fiber transplantation, rats (n = 8 in each group) were anesthetized again as above. After systemic heparinization, the hearts were quickly excised and perfused at 1 mH2O pressure with modified Krebs-Henseleit buffer by using a Langendorff apparatus, as described previously. 13 After 20 minutes of stabilization, heart rate, ventricular function, balloon volume, and coronary flow were measured with left ventricular end-diastolic pressure (LVEDP) stabilized at 10 mm Hg by using a thin-walled balloon. After perfusion, the hearts undergoing LCA occlusion were fixed in 3.7% neutral-buffered formaldehyde, and the left ventricle was cut into 5 segments parallel to the apex-base axis. A 10-µm section from each segment was stained with Masson trichrome and projected onto a screen for computer-assisted planimetry. The ratio of scar length to LV circumferences of the endocardium and epicardium was determined by blinded experienced histologists and expressed as a percentage to define infarct size. 11
Study for dynamics of original single fibers
ß-Galactosidase (ß-gal)-expressing single fibers of EDL muscle, obtained from a 3F-nlacZ-2E transgenic mouse, 14 were implanted into the hearts of male athymic (nude) rats (150-200 g, Charles River UK Ltd) 15 to further investigate the dynamics of grafted original fibers after implantation into the heart. The 3F-nlacZ-2E mouse, kindly provided by Dr M. Buckingham (Department of Molecular Biology, Institute Pasteur, France), carried regulatory elements from the locus of the fast myosin light-chain 2F/3F gene that drives a nlacZ reporter gene, and this transgene is expressed by all myonuclei in fast myofibers. 14 Such ß-gal-expressing single fibers and migrated myoblasts could be more clearly identified in the host myocardium by means of in situ staining for ß-gal. Nude rats were used as recipients to remove the need for immunosuppression. Isolation of single fibers and transplantation into the heart were performed exactly as described above. Heart samples were collected at 1 hour, 1 day, and 3 days after grafting (n = 3 at each time point); cut into 4 segments; and frozen in an embedding medium. The samples were cut into 10-µm cryosections, fixed in 2% formaldehyde and 0.2% glutaraldehyde, and then stained with 1 mg/mL 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside, 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, and 2 mmol/L MgCl2 for 12 to 16 hours at 37°C. 13 The sections were counterstained with hematoxylin.
Statistical analysis
All values are expressed as means ± SEM. Statistical comparison of the data was performed by using a Student t test with the Statview version 4.0 statistical package (Abacus Concepts Inc).
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Results |
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-sarcomeric actin (Figure 1, C), confirming the high purity of the myoblast culture. The cells were cultured in proliferation medium until subconfluence and then switched to differentiation medium to examine their myogenic differentiation ability. Consequently, the cells fused and differentiated into multinucleated myotubes within a week (Figure 1, D).
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, C and D). Such loci, consisting of several multinucleated myotubes positively stained for skeletal MHC, were also observed locally in the peri-infarct area of every LCA-occluded heart at 4 weeks after single-fiber transplantation at 20 of the 32 injection sites (Figure 3, B).
These differentiated, branching, multinucleated myotubes had aligned with the cardiac fiber axis within native myocardium, which is similar to that observed in the doxorubicin-treated hearts (Figure 3, C). Furthermore, angiogenesis induction with mature capillaries was observed in the surrounding area of the grafted myotubes in the single fiber-transplanted hearts (Figure 3, D). Local capillary density was significantly (P = .0046) higher in the single fiber-transplanted hearts compared with that in the control-treated hearts (4.1 ± 0.5 vs 2.3 ± 0.2 vessels/HPF, n = 8 for the single fiber-transplanted hearts and n = 6 for the control-treated hearts).
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, B) or 3 (Figure 4, C) samples.
At day 1 after grafting, a mass of ß-gal-positive debris, presumably originated from a destroyed single fiber, was observed (Figure 4, B). At day 3, such ß-gal-positive debris also disappeared, and instead, ß-gal-positive cells (putative skeletal myoblasts migrated from grafted fiber) were found (Figure 4, C).
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Discussion |
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6,7 Our study using implantation of ß-gal-expressing single fibers demonstrated that all the grafted original fibers are destroyed and subsequently removed after providing myoblasts into myocardium within 3 days after grafting. We have also described that, 4 weeks after syngeneic single-fiber transplantation, several discrete multinucleated myotubes are formed at each injection site where only one myotube (single fiber) had been implanted. These multinucleated myotubes were branching and appeared to be aligned with the cardiac fiber axis, showing a different appearance from the original single fiber. Because the mature myonuclei of the original fiber are postmitotic, these data indicates that skeletal myoblasts, which are derived from the parent fiber, are the source of the newly formed multinucleated myotubes.
Furthermore, we have shown that such single-fiber transplantation results in improvement of cardiac function in 2 different types of heart failure induced by either doxorubicin treatment or LCA occlusion, suggesting the feasibility of this strategy for treating heart failure. Both heart failure models used here have been established as stable and reliable models clinically relevant to idiopathic and ischemic cardiomyopathy, respectively. 9-11 Such consistent significant reduction in cardiac function at 1 month after doxorubicin administration and LCA ligation in comparison with our baseline data of normal intact hearts was confirmed in our own study. A limitation of using the Langendorff perfusion system with isolated hearts is the inability to measure the time course of functional changes in a single animal throughout the pretreatment and posttreatment period. Thus this system would not be optimal to study the degree of functional improvement after single-fiber transplantation compared with that before transplantation. In this study we therefore focused on the comparison of function between single fiber-treated hearts and sham-treated hearts at the same time point using randomized, diseased rats that had consistent myocardial damage caused by either doxorubicin administration or LCA ligation. As a result, we found a decrease in diastolic compliance in the control-treated hearts compared with that in the single fiber-treated rats in both heart failure models. This might have affected the data of systolic function to some extent; however, it is unlikely that such remarkable improvement both in terms of LVDP and maximum dP/dt, as demonstrated in our data, resulted from such a difference in diastolic function. Further study is needed to evaluate more detailed functional analysis after single-fiber engraftment, including ventricular chamber size and time course.
The use of single fibers could be advantageous over the conventional method of direct intramyocardial injection of a myoblast suspension in several respects. Foremost, the single-fiber protocol avoids the need for the prolonged in vitro processes required to derive an adequate number of myoblasts for transplantation. During this several-week period, repeated passaging is likely to be detrimental, and extended incubation carries risks of infection of the primary culture by means of bacteria, fungi, mycoplasma, and viruses. Furthermore, this prolonged in vitro culture might thus cause possible subtle changes in the myoblasts that render them either more visible to the host immune system or less competent for fusion and differentiation in vivo. In contrast, preparations of single fibers can be completed within only a few hours, avoiding the hazards of culture. This feature could be of great significance, particularly in clinical settings. Such easily harvested single fibers would also be promising as grafts in relatively urgent situations. In human patients the use of extensor digitorum longus might not be suitable because single-fiber preparation requires muscle from tendon to tendon. However, there are many other potential sites for harvesting smaller skeletal muscle fibers in human patients, including the digits, which would be clinically applicable. Another advantage of using single fibers is the lesser amount of medium required for injection. A single fiber can be suspended in only 2 to 3 µL of medium. One might expect a lesser injection volume of medium to induce less host myocardial damage as a result of inflammation after cell transplantation. A possible risk of cell transplantation is an embolism caused by grafts leaked into the systemic or coronary circulation. If grafted single fibers leaked, it could easily cause embolism because of their larger size. However, injection of single fibers is not more difficult than injection of cultured cells and requires a smaller injection volume, resulting in a possibly lower risk of leakage into the LV cavity or coronary vessels compared with that seen in cell suspension grafting.
In the present study we have shown that as few as 4 single fibers grafted into the heart can improve global LV function. Each single fiber from the Lewis rat extensor digitorum longus retains approximately 20 skeletal myoblasts (satellite cells) according to the results of immunostaining for M-cadherin, a marker for skeletal myoblasts (data not shown), suggesting that the myoblasts derived from these fibers not only survive at a high rate but also retain an adequate ability to proliferate and differentiate within myocardium. A similarly high degree of survival and differentiation is found when isolated mouse single fibers are transplanted into skeletal muscle (data from Heslop and associates, in preparation). Although further investigations are needed to prove this concept in detail, we speculate that using single muscle fibers as a vehicle for delivery of myoblasts might afford natural protective mechanisms to the myoblasts from environmental stress and subsequent adverse pathologic processes, particularly in the early phase after transplantation, which appears to be the most hazardous period for grafted myoblasts. 16,17 Original single fibers grafted into the myocardium are so sensitive to such environmental stress that they cannot survive for a long time after grafting. Such injury could, at the same time, signal the release of myoblasts, which we believe are the source of the newly formed myotubes observed in our in vivo study.
The report by Scorsin and colleagues 3 demonstrated that transplantation of 5 million skeletal myoblasts improved LV ejection fraction by 28.9% (measured by means of echocardiography) in rat ischemic hearts compared with that in medium-injected rats. In the present study transplantation of 4 single fibers improved maximum dP/dt by 11.4% (measured with Langendorff perfusion) in a similar model. This functional improvement seems to be less than half of the previous report; however, these data cannot be compared directly because several critical experimental conditions, including the timing of grafting, are different. In addition, one could expect that transplantation of more single fibers would lead to greater improvement in cardiac function. The extent of skeletal muscle engraftment and the number of skeletal myotubes has not been measured in this study. Although these measurements could be important in explaining enhanced myocardial function, we believe that the quality of the engrafted cells, including proliferation and differentiation capacity, contractile capacity, and gap junction formation ability, could be just as important in mediating improvement. The capacity to secrete growth hormones or cytokines is likely to contribute to improved angiogenesis, as found in our study, or delayed myocardial adverse remodeling, and thus this also might affect the functional result. The fact that the myoblasts have not been subjected to ex vivo culture and that the myoblasts localizing beneath the basal lamina of single fibers were protected from the hazardous environmental stress at the time of engraftment might explain the significant improvement in function after transplantation of only 4 single fibers in the rat heart. These issues need to be studied further.
We have also demonstrated that angiogenesis is induced in infarcted hearts after single-fiber transplantation and that this is associated with increased coronary flow. This finding has been observed in previous reports with direct intramyocardial injection of skeletal myoblasts. 12 In addition, other studies have shown that a nonspecific reaction caused by mechanical injury results in angiogenesis induction in the heart. 18 In our study infarct size was reduced after single-fiber transplantation in the hearts that had been subjected to LCA occlusion 1 month before grafting compared with that after medium injection. One month after LCA occlusion, the myocardial scar is mature, 11 and we would not expect grafting at this point to significantly reduce infarct size, although such angiogenesis might contribute to salvaging ischemic hibernating host myocardium to some extent. Apparent infarct size, as demonstrated by means of Masson trichrome staining, might have been reduced because of infiltration of the peri-infarct zone by surviving, newly generated, skeletal myoblast-derived myotubes.
It has been reported that transplantation of sliced skeletal muscle grafts into skeletal muscle itself results in extensive migration of the donor myoblasts with improved graft cell survival and fusion with host muscle. 19 Although this is a simple method for obtaining a source of myoblasts for transplantation, as far as the heart is concerned, this type of graft is unlikely to be suitable because of the greater physical constraints on graft size in the heart. This will even reduce the function without perfect integration within the heart. Minced skeletal muscle grafts might be an alternative source of myoblasts for transplantation that avoids the prolonged period of in vitro culture. However, transplantation of minced muscle has recently been shown to result in failure to improve postinfarction cardiac function, possibly as a result of an exaggerated inflammatory response caused by implantation of multiple chunks of minced tissue. 20 In contrast, we have demonstrated that direct intramyocardial injection of isolated, living single fibers results in formation of newly generated and differentiated myotubes within the host myocardium and improves cardiac function in both doxorubicin- and myocardial infarction-induced heart failure models. These data suggest that single-fiber transplantation could be potentially useful in treating heart failure.
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Acknowledgments |
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Footnotes |
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