Mature myocardial tissue does not possess the ability

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1 Intracardiac Transplantation of Skeletal Myoblasts Yields Two Populations of Striated Cells In Situ B. Zane Atkins, MD, Cleveland W. Lewis, MD, William E. Kraus, MD, Kelley A. Hutcheson, BS, Donald D. Glower, MD, and Doris A. Taylor, PhD Departments of Medicine, Surgery, and Biomedical Engineering, Duke University Medical Center, Durham, North Carolina Background. Adult heart lacks stem cells and cannot effectively regenerate. In contrast, skeletal muscle is constantly undergoing repair. We proposed to transplant immature skeletal myoblasts into injured myocardium. Methods. Approximately soleus skeletal myoblasts were expanded in vitro from adult New Zealand White rabbits (n 23) whose posterior left ventricle was cryoinjured to create a transmural lesion. Autologous myoblasts (n 18) or saline (n 5) was transplanted into the central cryolesion at the time of injury (n 6) or 1 week later (n 12). Hearts were harvested 2 weeks after injection. Results. Myoblast transfer did not incur further morbidity. After cryolesion, grossly, a 1.6-cm epicardial hemorrhagic lesion could be seen. Histologically, the transmural lesion contained inflammatory cells and active scarring but no viable cardiomyocytes. Electron microscopy demonstrated a predominance of collagen and fibroblasts. Nine hearts contained multinucleated cells within the cryolesion that covered approximately 75% of the central cryolesion in 17% of animals. Immunohistochemical analysis confirmed their skeletal muscle origin. At the periphery of the lesion, isolated clusters of nonskeletal muscle cells could be visualized (n 12) that resembled immature cardiocytes. Conclusions. Autologous skeletal myoblasts can regenerate viable striated tissue within damaged myocardium. Myoblast transfer warrants further investigation as a new method for improving myocardial performance within infarcted myocardium. (Ann Thorac Surg 1999;67:124 9) 1999 by The Society of Thoracic Surgeons Mature myocardial tissue does not possess the ability to effectively regenerate after ischemic injury. As a result, the normal progression of injured myocardium involves development of fibrous, noncontractile scar tissue, leading to end-stage heart failure in approximately 400,000 patients in the United States each year [1]. Pharmacologic intervention is aimed at improving the function of the remaining myocardium. However, investigators have recently attempted delivery of skeletal myoblasts, fetal cardiocytes, or embryonic stem cells to damaged myocardial tissue to either improve recovery or to regenerate functional tissue within the damaged muscle [2 10]. This technique, transplanting reserve cells into an infarcted region of myocardium, has been labeled cellular cardiomyoplasty [2]. A model of myocardial injury in which transplanted cells can be evaluated requires a reproducible lesion in an animal model that mimics human physiology. Rabbits, in particular, provide several unique advantages over other animal models of myocardial damage. Unlike mouse, rat, and canine heart, the rabbit myocardium expresses contractile protein isoforms similar to human heart [11], and it lacks silent collateral vessel growth [12] often present in the dog [13]. In addition, rabbit myocardium has many properties in common with human heart, including prominent post-extrasystolic potentiation and Accepted for publication July 10, Address reprint requests to Dr Taylor, Duke University Medical Center, Box 3327, Durham, NC ( dataylor@duke.edu). a positive force frequency relation [14]. Furthermore, unlike rats and dogs, the rabbit cardiovascular system also resembles that in humans with regard to myocardial contractile protein gene expression. Rabbits may also be subjected to cellular and genetic manipulation more easily than dogs. In the present study, we directly transplanted autologous primary skeletal muscle myoblasts into previously damaged rabbit myocardium, with the ultimate goal of repopulating the damaged region to augment function in the diseased state. By histologic evaluation of the grafts, we demonstrate that transplanted myoblasts can differentiate in situ, survive, and populate a significant portion of the damaged region. Material and Methods All animals were studied under guidelines published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHHS publication No. [NIH] revised 1985) and approved by the Institutional Animal Care and Use Committee at Duke University Medical Center. Skeletal Myoblast Biopsy and Isolation After premedication with intramuscular ketamine (25 mg/kg body weight) and xylazine (4 mg/kg), 23 New Zealand White rabbits (4 to 6 kg) were prepared for isolation and harvest of a skeletal muscle biopsy specimen subsequent to local anesthesia (lidocaine, 1999 by The Society of Thoracic Surgeons /99/$20.00 Published by Elsevier Science Inc PII S (98)

2 Ann Thorac Surg ATKINS ET AL 1999;67:124 9 INTRACARDIAC MYOBLAST TRANSPLANTATION mg/kg). An oblique incision was made on the lateral surface of the left hindlimb, just distal to the knee. Dissection was carried through the first two glycolytic muscle layers to reveal the oxidative soleus muscle. An excisional biopsy of the soleus was performed, and the tissue was immediately placed in a conditioning solution (growth medium) on ice. The incision was repaired in layers, and the animals were allowed to recover for 2 weeks. The resulting biopsy specimen was immediately transferred to a sterile chamber for in vitro isolation of skeletal myoblasts. To isolate skeletal myoblasts, the biopsied tissue was first mechanically dissected and then washed, resuspended, and plated in growth medium composed of Dulbecco s Modified Eagle Medium (Gibco, Grand Island, NY) with 20% (vol/vol) horse serum (Hyclone, Logan, UT) and 0.5% (vol/vol) gentamicin (Gibco). Beginning on the third day after biopsy and every subsequent 2 days, one-half of the growth medium was removed and replaced with fresh medium. Myoblasts were expanded for 14 to 21 days and passaged as necessary. To prevent premature differentiation before transplantation, myoblasts were grown at densities less than 70%. When total cell count approximated , myoblasts were harvested, counted, and directly transplanted into cryoinjured myocardium. At the time of intracardiac injection, myoblasts at passage number 2 or 3 were harvested from culture plates with 0.5% trypsin/edta (ethylenediaminetetraacidic acid) (Gibco). Trypsin was inactivated by the addition of growth medium containing horse serum, and recovered myoblasts were washed twice to remove horse serum. Approximately myoblasts were resuspended in a minimal volume of serum-free Dulbecco s Modified Eagle Medium, and the suspension was stored on ice (typically less than 20 minutes) until delivery into the myocardium. Surgical Preparation After premedication with intramuscular ketamine (50 mg/kg) and xylazine (5 mg/kg), 23 New Zealand White rabbits (4 to 6 kg) were anesthetized with intravenous fentanyl citrate (10 g/kg intravenously). Intravenous lidocaine (1 mg/kg) and succinylcholine (5 mg/kg) were administered after induction. The animals were intubated with a 2.5F endotracheal tube and mechanically ventilated (Bird Products Corp, Palm Springs, CA) at a rate of 32 breaths per minute and a normalized tidal volume of 10 to 15 ml/kg. A sterile left thoracotomy through the fifth intercostal space was performed, and the pericardium was divided to expose the heart. To create cryoinjury, a 1-cm-diameter cryoprobe cooled to 70 C by continuously circulating nitrous oxide (Frigitronics of Cooper Surgical, Shelton, CT) was placed on the epicardial surface of the left ventricle, between the left circumflex coronary artery and the posterior interventricular groove for approximately 3 minutes. During cryoinjury, standard electrocardiographic leads were monitored to detect ventricular irregularity and S-T wave alteration. The injured area was observed for several minutes during thawing. After thawing, the harvested myoblasts were injected into the center of the cryolesion, with care taken to avoid leakage of the cell supernatant. The chest was closed in layers, and the animals were allowed to recover for 2 weeks before each heart was harvested and prepared for histologic examination. Histologic Analyses At study completion, each animal underwent anticoagulation (heparin sulfate, 500 Units intravenously) and euthanasia as outlined by Duke University Medical Center institutional animal care guidelines. The heart was explanted, rinsed in saline, and conditioned at 4 C for 24 hours in a 30% sucrose phosphate-buffered saline solution. Each heart was sectioned into 5-mm cross-sectional pieces along the longitudinal axis and frozen in liquid nitrogen. Thin sections (8 m) of each frozen slice were stained with hematoxylin and eosin for visualization of muscle, Masson s trichrome to delineate fibrous tissue, and antibody to the skeletal muscle specific transcription factor, myogenin (Santa Cruz, Santa Cruz, CA), to identify transplanted differentiated myoblasts. All infarcts were examined histologically to verify that the control animals exhibited a transmural, myocyte-free defect. Results Adult skeletal muscle is composed of intact myofibers surrounded by quiescent immature reserve satellite cells [15]. On damage to the muscle, these cells proliferate to form myoblasts, migrate, and fuse to damaged muscle or other myoblasts to regenerate new muscle fibers. We derived skeletal myoblasts from rabbit soleus muscle (see Material and Methods). Figure 1A depicts undifferentiated biopsy-derived skeletal myoblasts at 80% to 90% confluence after approximately 6 days in vitro. After reaching approximately 70% confluence, myoblasts begin to chemically differentiate and become committed to fuse and form mature multinucleated skeletal myotubes (Fig 1B) similar to those seen in intact muscle. These elongated myotubes then coalesce in vivo to form myofibers. In 6 animals, approximately myoblasts were implanted at the time of cryolesion; however, in 12 animals, myoblasts were transplanted 1 week after cryoinjury to determine whether inflammation at the time of injury affected myoblast uptake and survival. Five shaminjected rabbits received only saline at 1 week after cryolesion. Animals were allowed to recover for approximately 2 weeks. No increase in mortality was associated with myoblast transplantation. At2 weeks after transplantation, hearts were harvested, examined grossly, and subjected to histologic analysis. Gross demarcation of the cryoinjured region was obvious on the epicardial surface of the heart, where an approximately 1.6-cm hemorrhagic lesion could be visualized. Histologically, the cryolesion was seen to arise from the epicardial surface but was transmural at its center (Fig 2A). At 2 days to 6 weeks after injury, the damaged area in saline-injected hearts contained inflammatory cells and active scarring but no viable cardiomyocytes (Fig 2B, 2 weeks). The extent of inflammation, however, as indicated by the number of inflammatory cells per high-

3 126 ATKINS ET AL Ann Thorac Surg INTRACARDIAC MYOBLAST TRANSPLANTATION 1999;67:124 9 Fig 1. (A) Unstained undifferentiated primary rabbit soleus skeletal myoblasts in vitro (20 magnification). (B) Multinucleated myotubes ( 20 magnification before reduction) formed within an in vitro preparation by fusion of differentiated myoblasts. power field, was maximal at 2 to 7 days after cryoinjury and subsequently subsided. On sectioning of the myoblast-injected hearts, gross regions resembling intact muscle could be seen within the cryodamaged area (Fig 2C, arrows). By light microscopic examination after hematoxylin and eosin staining in 13 animals, elongated structures closely resembling multinucleated skeletal myotubes covered up to 75% of the central cryolesion, as indicated in Figure 2C. In 8 animals injected at 1 week after cryoinjury and in 1 animal injected at the time of cryoinjury, the engrafted structures extended up to 1 cm in diameter. However, growth and expansion of the engrafted cells did not proceed beyond the borders of the cryoinfarct in any rabbit, so that in 100% of the hearts where successful myotube engraftment occurred, the myotubes were surrounded by a distinct region of inflammatory cells and scarring, as demonstrated in Figure 2C. Immunohistochemical staining and visual examination of cells within the grafted hearts indicated two populations of apparent muscle cells within the damaged myocardium. At the center of the graft, a strong correlation existed between the elongated structures depicted in Figure 2D (arrow at same site as in Fig 2C) and immunohistochemical staining for the skeletal muscle specific transcription factor myogenin (Fig 2G, open arrow at same site as in Fig 2C) demonstrating the skeletal myoblast origin of these myotubes. However, at the periphery of the lesion in all injected animals, smaller clusters of isolated myogenin-negative cells were visualized (Figs 2E, 2F, arrows). The visual appearance of these cells is similar to that of immature cardiocytes both in terms of striations and centrally located nuclei. Comment Implantation of fetal cardiac myocytes into adult heart has received attention but is not readily applicable to human disease, primarily because of a lack of donor tissues and the need for recipient immunosuppression. Two groups [4, 10] introduced cardiomyocytes immortalized with the viral oncoprotein SV40 large T antigen into the hearts of syngeneic mice and discovered cardiomyocyte proliferation within the myocardium. However, these newly elaborated cells do not form normal syncytial connections characteristic of cardiomyocytes. In addition, these cells have been shown to form tumors when transplanted into genetically immunocompromised mice, although cardiac tumors have not been reported [16]. Skeletal muscle, which comprises approximately 24% of body mass by weight [17], constantly undergoes regeneration and repair [15, 18, 19]. The ability to isolate skeletal myoblast cell lines has enabled transplantation of these cells into injured heart. Koh and colleagues [20] transplanted myoblasts from the C 2 C 12 immortalized cell line into the hearts of syngeneic mice and observed that the cells formed multinucleated myotubes characteristic of mature skeletal muscle. However, these immortalized myoblasts also fail to form syncytial relationships with cardiomyocytes and eventually form tumors in immunocompromised mice. Recently, Robinson and associates [21] reported the introduction of C 2 C 12 myoblasts into syngeneic mouse myocardium through intracavitary and intracoronary injection. The C 2 C 12 cells maintained long term in a cardiac environment appear to change expression of musclespecific proteins in a manner that indicates conversion of the transplanted cells from a fast skeletal to a slow skeletal fiber type. The myotubes appear to align parallel to the contraction axis of the myocardial cells. Their data demonstrate that immortalized myoblasts can be transplanted into syngeneic or immunosuppressed animals but do not demonstrate that myoblasts can be transplanted autologously. In addition, these studies do not address transplantation of cells into diseased myocardium. The ability to harvest immature skeletal myoblasts in vitro has enabled multiple investigators to examine them as potential candidates for reintroduction in dystrophic disease states or for genetic manipulation and reintroduction in deficiency states. However, to date, this myoblast transfer has been done with human lymphocyte antigen matched donors and has proved inefficient and relatively unsuccessful because of immunorejection [22, 23, 24]. More recentlyour laboratory and others have begun to transplant autologous skeletal myoblasts into cryoinjured myocardium of larger animals [2, 9]. By using primary autologous myoblasts from the skeletal muscles of adult animals, we have eliminated the need for fetal tissue and syngeneic donors or for chronic immunosuppression. Chiu and colleagues [2] reported the successful engraftment of autologous myoblasts into canine myo-

4 Ann Thorac Surg ATKINS ET AL 1999;67:124 9 INTRACARDIAC MYOBLAST TRANSPLANTATION 127 Fig 2. (A) Hematoxylin and eosin staining of cryoinjured rabbit myocardium at 2 weeks, demonstrating a transmural myocardial lesion in the posterolateral wall of the left ventricle. (B) Higher power ( 40 magnification before reduction) of hematoxylin and eosin stained cryoinjured myocardium depicting the inflammatory infiltrate and fibrous strands of elastin and collagen. (C) Hematoxylin and eosin staining ( 4 magnification before reduction) of an injected heart containing engrafted myotubes within approximately 75% of a transmural cryoinjury in a typical animal 2 weeks after transplantation. Closed and open arrows depict the regions marked with similar arrows and displayed at higher magnification in Figure 2 (D) and (G), respectively. (D) Higher power ( 20 before reduction) magnification of hematoxylin and eosin-stained myotubes corresponding to the region of the same heart marked by the closed arrow in Figure C. (E) Higher power ( 20 before reduction) hematoxylin and eosin staining of a section at the periphery of a typical cryolesion, showing isolated normal myocardium at the left, the sharp demarcation of the injury, and myogenin-negative cells (arrow) within the periphery of the lesion. (F) High-power magnification ( 40 before reduction) of the region (at the site of the arrow) containing myogenin-negative cells depicted in E. The morphologic appearance of these cells is very different than that of the elongated multinucleated structures within the center of the cryolesion. (G) Comparative myogenin antibody immunoreactivity ( 20 before reduction) within the region depicted in D. Brown staining indicates myogenin immunoreactivityprimarily within nuclei of the elongated myotubes. Open arrow delineates the same region as that of the open arrow in C.

5 128 ATKINS ET AL Ann Thorac Surg INTRACARDIAC MYOBLAST TRANSPLANTATION 1999;67:124 9 cardium, whereas our laboratory reported a rabbit model of cryoinjury [9], thereby making it possible to evaluate the feasibility of cellular cardiomyoplasty in a pathologic environment. Although several laboratories have transplanted autologous myoblasts into cryoinjured myocardium [2, 5], our data differ from those previous reports in several important ways. First, Chiu and colleagues [2] transplanted skeletal myoblasts into channels created within the left ventricle in the cryoinjured canine myocardium. Although dogs have been used extensively for myocardial analyses, their intrinsic collateral formation, which is unlike that of humans, may unduly influence the outcome of investigations. Rabbits, in contrast, are more similar to humans both in terms of collateral vessel formation and contractile protein gene expression. In addition, although channels may localize myoblasts to the injured region, they prevent assessment of the intrinsic ability of the cells to integrate with surrounding myocardium. Finally, Chiu and associates [2] incorporated sutures to close and seal the artificial channels within the cryoinjury used to localize myoblasts to the region. The presence of synthetic suture within the cryoinjury may exacerbate the host inflammatory response and lead to increased clearing of myoblasts. More recently, Murry and colleagues [7] injected neonatal myoblasts into the hearts of immunologically inbred rats and demonstrated myoblast survival and integration within the scar. However, our observation that skeletal myoblasts can survive and generate intact myotubes within up to 75% of the scar extends their data. In addition, our ability to deliver autologous myoblasts into damaged cardiac tissue with no increased mortality suggests that the technique does not intrinsically increase morbidity. These data taken together suggest that myoblast transfer may offer new possibilities for myocardial repair. Although the effect of the transplanted myoblasts on myocardial function was not evaluated in the present study, the morphology of the transplanted cells, their positive immunoreactivity for myogenin, and the presence of striations all suggest that the myocytes are intact and potentially functional. The further observation that these structures can be detected in animals up to 16 weeks after transplantation (data not shown) also suggests their viability. It is interesting to speculate therefore that the myotubes my respond to the intrinsic mechanical stretch encountered during diastole with a passive mechanical contraction. Future studies to evaluate this possibility are needed. In every animal examined, the transmural cryolesion was filled with inflammatory cells and scarring. One potential explanation for the lack of intact myotubes in five rabbits injected with myoblasts at the time of cryoinjury is that the injected cells were rapidly cleared by the inflammatory response to the injury [25]. This finding is supported by the observation that in two-thirds of the animals injected 1 week after cryolesion, myotubes could be detected, but only one heart injected at the time of cryoinjury contained myotubes. Although cryoinjury differs from ischemic injury in that cryoinjury arises from the epicardium and is of a reproducible size, it has characteristics similar to those seen after an acute myocardial infarction, including decreased blood flow, inflammation, and myocyte death and clearing. These characteristics make cryoinjury a reasonable model in which to evaluate cellular cardiomyoplasty, which is likely to be most relevant clinically after an acute myocardial infarction. In all nine animals where myotube engraftment was obvious, the myotubes were surrounded by regions of cellular necrosis and scarring. Thus, no evidence for syncytial interactions of myotubes with cardiocytes was seen. Although this electrical isolation could prove a hindrance ultimately to improve myocardial performance, it is also likely that isolation protects both the myocardium and the myotubes. The effects of this isolation on myoblast contraction remain to be determined. Myogenin-negative cells localized at the periphery of the cryolesion clearly do not histologically resemble myotubes or differentiated myoblasts. In fact, they more closely resemble cardiocytes than they do skeletal myoblasts or myotubes in that approximately 30% of them have a single, centrally located nucleus, whereas none of them express myogenin. Although they cannot be specifically classified, their origin from damaged myocardium or from skeletal myoblasts would be equally interesting because, to our knowledge, no generation of immature muscle cells has been reported as a result of myocardial injury. It is of interest that these cells are seen in closer proximity to the periphery of the lesion where electrical and vascular influences from the surrounding normal myocardium are more likely to be seen in combination with injury. It is possible that the electrical and mechanical activity of the adjoining myocardium, combined with the microenvironment within the injury, influenced the phenotype differentiation of the injected cells or the cells at the periphery of the myocardium. Further investigation as to the origin and phenotype of these cells is needed. Future studies to improve the reproducibility of engraftment and the integration of myotubes with surrounding myocardium are needed. In addition, developing reliable techniques for the long-term marking of myoblasts before transplantation is necessary to definitively establish the origin of the mononucleated myogenin-negative cells within the periphery of the infarct. Nonetheless, the present data provide evidence that autologous skeletal myoblasts can regenerate both elongated, striated structures composed of myogeninpositive cells surrounded by scarring along with regions of apparently more immature and structurally unconnected but viable striated tissue within a significant portion of damaged myocardium. In this report, large regions of engrafted cells were accompanied by a second population of apparently less mature cells within the injured heart. These findings suggest that cellular cardiomyoplasty warrants further investigation as a method for improving myocardial performance within infarcted myocardium. References 1. Carpentier A, Chachques JC. Clinical dynamic cardiomyoplasty: method and outcome. Semin Thorac Cardiovasc Surg 1991;3: Chiu RC-J, Zibaitis A, Kao RL. Cellular cardiomyoplasty:

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Muscle Nerve 996;19: INVITED COMMENTARY This report by Atkins and colleagues is the first independent confirmation of our earlier observation [1] that skeletal myoblasts transplanted into the heart may undergo milieu-dependent transdifferentiation to exhibit cardiac muscle phenotype. The clinical advantage of this approach in therapeutic cell transplantation for myocardial repair is obvious because autologous myoblast implantation avoids both the ethical and logistic difficulties, as well as the immunosuppression required when fetal cardiac myocytes are used as donor cells. The idea that skeletal myoblast is capable of transdifferentiating into a cardiac myocyte remains controversial because it contravenes the prevailing paradigm of embryogenesis. In the early embryo, fetal cardiomyocytes differentiate before the progenitor mesenchymal cells give rise to skeletal myoblasts. Findings presented in the present report suggest that the microenvironment, such as cell-to-cell contacts, may modulate the differentiation pathway of myoblasts to exhibit either skeletal or cardiac muscle phenotypes. Further confirmation of such observations and elucidation of the mechanisms involved are warranted because they will have major impact on our understanding of the biology of muscle differentiation. Clinically, the functional potential of such implants, as recently reported by present authors in [2] may herald a new approach to the management of myocardial damage and heart failure. Ray C.-J. Chiu, MD, PhD Division of Cardiothoracic Surgery The Montreal General Hospital 1650 Cedar Ave, Room C9-169 Montreal, Quebec PQ H3G 1A4 Canada References 1. Chiu RCJ, Zibaitis A, Kao RL. Cellular cardiomyoplasty with satellite cell implantation. Ann Thorac Surg 1995;60: Taylor DA, Atkins BZ, Hungspreugs P, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998;4: by The Society of Thoracic Surgeons /99/$20.00 Published by Elsevier Science Inc PII S (98)