Myocardial regeneration. Transplanting satellite cells into damaged ...

Denton A. Cooley's 50th Anniversary in Medicine

Myocardial Regeneration Transplanting Satellite Cells into Damaged Myocardium

Pyongsoo D. Yoon, MD Race L. Kao, PhD George J. Magovern, MD

This series in recognition of Dr. Cooley's 50th anniversary in medicine is continued from the December 1994 and March 1995 issues. Key words: Heart failure,

congestive; myocardium; regeneration; satellite cell

From: Cardiothoracic Surgical Research, AlleghenySinger Research Institute; Department of Surgery, Allegheny General Hospital; and Allegheny Campus, The Medical College of Pennsylvania; Pittsburgh, Pennsylvania 15212

Supported in part by Allegheny-Singer Research Institute and National Institutes of Health Grant HL 54286 Dr. Kao is now in the Department of Surgery, J. H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614. Section editors: Grady L. Hallman, MD Robert D. Leachman, MD John L. Ochsner, MD Address for reprints: George J. Magovern, MD, Department of Surgery, Allegheny General Hospital, 320 E. North Avenue, Pittsburgh, PA 15212 Texas Heart InstituteJournal

Millions of Americans suffer from chronic heart failure. Despite treatments with heart transplantation, cardiomyoplasty, and artificial assist devices, an ideal therapy is yet to be found. Since 1988, we have studied the transplantation of myogenic stem cells from skeletal muscle into injured myocardium in the hope that these cells would multiply and differentiate, thereby improving the function of the failing heart. We have achieved 2 goals thus far: the 1st was improving the culture technique to obtain high yield and purity of the satellite cells; the 2nd was successfully implanting cultured satellite cells in dog hearts and later identifying them as new myocardium. We share our findings here to encourage more study in this promising area. (Tex Heart Inst J 1995;22:119-25)

oday, many techniques are available for treating chronic heart failure, and many more are being studied. Heart transplantation has become an effective treatment, but the scarcity of donor hearts has limited its usefulness. 1.2 Mechanical assist devices are still in the experimental stage, with only a few of them approved as a bridge to transplantation.3 Skeletal muscle transformation by chronic electric stimulation has made clinical dynamic cardiomyoplasty a reality.2'4 However, muscle atrophy and loss of strength after electrical conditioning have limited the success of this remedy.5 Cardiomyoplasty using latissimus dorsi muscle shows some promise but is still in an experimental stage. Cardiomyoplasty has the obvious advantage of using autologous tissue, which avoids the problems that accompany immunosuppression due to the use of allogenic organs. However, this procedure has its disadvantages as well.6 Despite enormous advances made in the last 3 decades, there is currently no ideal treatment for patients with chronic heart failure. What is needed is a simple, minimally invasive procedure, which produces the least morbidity and mortality and avoids complicating factors such as immunorejection or use of foreign materials. The lack of regenerative capability in the myocardium of adult mammals is a well-established fact.79 Once the heart muscle cell is damaged, this damage is irreversible. Compensatory hypertrophy may occur, but the resulting decreased cardiac function is permanent and may produce chronic heart failure. Although cardiac myocyte does not regenerate, skeletal muscle does. Satellite cells were 1st described by Mauro in 1961.10 Their role in the regenerative capacity of skeletal muscle is well established."'' 2 Satellite cells (Fig. 1) have been shown to proliferate and differentiate into new myotubes (Fig. 2) in culture. Myoblasts also have the capacity to form new fibers or fuse to existing fibers when introduced directly into the skeletal muscle of a syngeneic host.'3'14 Discovery of satellite cells and their regenerative potential in skeletal muscle has led investigators to look into the possibility of regenerative potential in the cardiomyocyte.' '2' An immediate problem, however, is that cardiac myocytes are terminally differentiated cells and lose their ability to undergo cell division soon after the newborn period.'5"16 Therefore, the major work in cardiomyocytes so far has been in gene manipulation, such as the use of virally transfected mouse and rat cardiac myocytes.'7'20 Unfortunately, the unchecked proliferation of genemanipulated grafted cells limits the potential value of these grafts for myocardial T

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Fig. 1 Proliferation of satellite cells in a 3-day culture (orig. xlOO).

Fig. 2 Myotubes formed from cultured satellite cells forig. x200).

At Allegheny General Hospital, we have been interested since 1988 in an experimental treatment at the cellular level, which may be called "cellular cardiac transplantation" or "cellular cardiomyoplasty." This treatment embraces the concepts of both transplantation and cardiomyoplasty, because its basic goal is to transplant skeletal satellite cells into the damaged myocardium, which will then form differentiated intracardiac myocytes and improve cardiac function. Potentially, intracardiac skeletal grafts could become fatigue resistant when placed in the myocardial environment and stimulated rhythmically by the heart. They may even become fully differentiated into true cardiac muscle cells.

to generate new muscle in the injured myocardium, to improve the function of a failing heart. Cryoinjury Model. To test the feasibility of injecting cultured satellite cells into dog hearts and later identifying these, a variety of injuries were imposed on 16 dogs. The rapid freezing technique provided the best lesion. The myocytes were killed in the area of injury, whereas the peripheral blood-vessel and connective-tissue cells were reversibly damaged.22 This method produced an injury of well-controlled size and location, with a sharp visual demarcation between damaged and normal tissue. The retained vascularity was a necessity if cultured cells were to survive in this area. Development of a cryogenic injury model of myocardial dysfunction was therefore an important 1st step in testing the effects of satellite cell implantation in the heart. In a dog model, a localized transmural myocardial injury was produced by a 5-cm-diameter cryoprobe cooled to -160 °C by internally circulating liquid nitrogen. An obvious contrast between injured and normal tissue was observed grossly (Fig. 3), and uniformly destroyed cardiac myocytes and fibrous scar formation were documented histologically (Fig. 4). Cell Marking. The next step was to develop a reliable method of cell marking. Both 14C and 3H-thymidine have been used to label the dividing satellite cells in culture. Although successful labeling was attained by both radioisotopes, 3H-thymidine was selected for its significantly better resolution (Fig. 5). However, this labeling method suffered the limitation of losing labeling intensity as the cell divided. Our work then concentrated on increasing the quantity of cells and marking them with fluorescent beads (Fig. 6). Tibialis anterior muscle harvested from 19 dogs was minced, digested, spun, and plated. Many experiments were performed using

Materials and Methods Effects of Satellite Cell Implantation in Animals The goal of our study in the 1st year was to use myogenic stem cells (satellite cells) from skeletal muscle 120

Myocardial Regeneration

Fig. 3 Note the sharp demarcation between damaged and normal tissue after cryoinjury. Bulging of the cyanotic, edematous, and hypokinetic area can be clearly visualized.

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Fig. 4 Cryoinjured myocardium at 6 weeks after operation (Masson trichrome stain, orig. x200). Note dense connective tissue stained blue-green, and fibroblasts (reddish-brown) scattered in the field. A small blood vessel can also be observed at the lower-right corner,

dilutions of beads to media in ratios of 1:2, 1:5, 1:10, and 1:20. The 1:2 and 1:5 dilutions were too heavily labeled and resulted in cellular death. The 1:20 dilution incorporated too few beads. The 1:10 dilution appeared to be the best concentration, since it caused little cell death and a high percentage of labeling (>90%), with a significant number of beads per cell. The incubation intervals varied from 1 to 24 hours, but the 24-hour period proved to be the most efficacious. Transplantation of Satellite Cells. Cultured satellite cells carrying both 3H-thymidine and fluorescent

Fig. 6 Myotubes formed from satellite cells labeled with 3Hthymidine and fluorescent microspheres (orig. x400). Satellite cells were cultured and labeled as in Fig. 5. In addition, 10% of fluorescent microspheres (Polysciences; Warrington, PA) were included in all media used during day 4. The cells were allowed to form myotubes in fusion medium for 1 week before being fixed and photographed.

beads were liberated by trypsin or dispase treatment before being washed and transplanted into 3 groups of dogs (n=23 total). In Group I (n=10), 7-day cultures were injected into the subepicardial regions of cryolesions less than 1 hour old; in Groups II (n=7) and III (n=6), 4-day cultures were injected into the same locale, the difference being that in Group III the cultures were harvested from the dishes with dispase, to increase cell yield and minimize damage. The 4-day cultures were selected in the hope that the entire differentiation process would take place within the heart.

Obtaining Optimal Satellite Cell Culture

Fig. 5 Autoradiograph of myotube formed from satellite cells labeled with 3H-thymidine (orig. x400). The cells were cultured in proliferation medium for 4 days and exposed to 3H-thymidine on day 4 (10 uCi, 4 pulses, 20 minutes per pulse at 5-hour intervals). After each labeling period, fresh proliferation medium was used to wash away the free radioisotope. The cells were allowed to form myotubes in fusion medium for 10 days before being fixed for autoradiography. The sample was exposed for 2 weeks before development.

Texas Heart Institutejourtial

Data obtained from our initial studies made it obvious that both yield and purity of cultured satellite cells would have to be increased, and that an alternative system for long-term marking of the cells would be needed. Previous studies had resulted in a total yield of 7 x 107 cells from approximately 40 g of muscle, and there was a clear mixture of fibroblasts and myoblasts. The ability to produce large numbers of satellite cells in vitro is an absolute essential for determining the effects of satellite cell implantation into damaged myocardium, both in the short term (i.e., myoblast survival, extent of migration from the injection site, and differentiation) and in the long term (i.e., myosatellite cell survival and the functional consequences of its presence). We used a small initial sample size of 20 to 40 mg of skeletal muscle for obtaining satellite cells. This correlates well with the clinical situation, wherein a muscle biopsy technique will be used to harvest satellite cells for subsequent transplantation into damaged myocardium. Myocardial Regeneration

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Optimal Fibroblast Growtb Factor Concentration. A set of experiments was designed to find the optimal concentration of fibroblast growth factor (FGF) for improving the mitotic activity of satellite cells in culture. A clonal dilution of an MM14 miouse satellite cell line at 100 cells per plate in a volume of 3 mL was added to 6 plates. These plates were divided into 3 sets, with 2 in each set. One set received 40 ,uL FGF per plate (1 ig/mL stock FGF), the 2nd received 80, and the 3rd received 160. After 24, 48, 72, and 96 hours, the plates with 40 tL FGF had the greatest increase in cell numbers. Therefore, a concentration of 40 pL FGF per plate waS used for all Subsequent cultures. Cell Purity. A 2nd set of experiments was designed to improve satellite cell purity by decreasing fibroblast contamination. We began preplating cell suspensions before transfer to primary ancl secondary gelatin-coated plates. Since fibroblasts have a greater plating efficiency than myoblasts, these cells become preferentially attachecl to the culture dish and do not transfer easily to the coatecl plates. We then varied the collagenase treattment time during subculture. Mild collagenase treatment during the media transfer preferentially loosens satellite cells from the plate, leaving fibrobl.asts adhered. By clecreasing the collagenase treatment time from 10 min to 1.5 min, we removed fewer fibroblasts, ancl the resultant subcultures containecl at least 98% satellite cells ancl, in many cases, more than 99%. Maximal Ccell Yield. The last set of experiimients was designed to maximize the number of myosatellite cells obtained from 20- to 40-mg muscle samples without clepleting the cells' mitotic capability. In order to clo this, we felt that we had to maximize nuLtrients and the concentration of FGF available to the muscle cells. This was acco)mplished by keeping the plates on a rocker during incubation to prevent local depletion of FGF and to maintain high optimal nutrient and FGF concentrations via multiple feedings (media changes once a day and FGF additions twice a day).

muscle was identifiecl both grossly (Fig. 7) and by Masson trichrome stains (Fig. 8) in the imilplant chaninels. Juclged by the appearance of their mitoclhondria, glycogen, ancl intercalated disks (Fig. 9). the regenerated muscle cells displatyed morphologic chlaracteristics similar to those of cardiac imyocytes. The translocatecd skeletail mluXscle cells withiin the heart appeared to be viable and, in som11e cases, the newly formecd cells appeared to have developed specialized cell junctions either with adjacent cardiac cells or with eaclh other and to have taken on the appearance of cardioimiyocytes. In Group III (4-day CLlture withli dispase), cidta were available for 5 clogs at 6 weeks. Two control and 2 cell-implanted channels were madIe in eacl

Fig. 7 Gross specimen of the implanted channel. Muscle formed within the scar is sandwiched between dense connective tissue.

Results Effects of Satellite Cell Implantation in Animals From the 7-day (Group I) and 4-day (Group II) culture groups, data were available for implant clurations of 1 day (n=2), 1 week (n=4), 4 weeks (n=2), 6 weeks (n=3), and 8 weeks (n=2). All 13 clogs failed to show any histologic evidence of fluorescent beads, even in the 1-day test. These studies were batch-processedl in 2 lots. After viewing all slides, we postulated, and then proved with in vitro stuLdies, that the special system used for tissue processing had dissolvecd the beads. I)espite this problemi, viable 122


Fig. 8 Cross-section of the implanted channel, showing muscle formed from the implanted satellite cells (Masson trichrome stain, orig. x300). Control channels (culture medium) revealed scar tissue only (not shown). Some myocytes presented with peripheral nuclei and some with centrally located nuclei. Whether the location of the nuclei reflected the maturation of the myocytes as skeletal or as cardiac muscle cells cannot be concluded from available information.

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Fig. 10 A composite slide (x300) showing the radioactive nuclei (by autoradiography) and fluorescent microspheres in the muscle cells from the implanted satellite cells. This is done by superimposition of consecutive sections on the stained section (Fig. 8), to reveal radioactivity and fluorescence.

Fig. 9 Transmissional electron micrograph (x3000) showing regenerated muscle from implanted satellite cells with abundant mitochondria within myocytes and intercalated disks forming junctions between cells. Some collagen fibers can be seen between myocytes, and a cell most likely to be an undifferentiated satellite cell is also observed. At this magnification, we cannot clearly identify the cell as a satellite cell; however, the presence of the radioactive nuclei makes this most likely. The presence of intercalated disks is an important feature, enabling synchronized contraction of the newly formed fiber and existing myocardium. C = collagen fibers; ID = intercalated disks; MN = myonucleus; SC = satellite cell

cryoinjured area. Each dog showed the presence of fluorescent beads in the control channels into which media and beads had been injected. Also, the channels implanted with cells containing beads and labeled with 3H-thymidine showed that the cells were clearly viable. The muscle cells within the scar tissue containing fluorescent beads and radioactive nuclei (Fig. 10) suggested that these cells came from culture and not the native heart. The fact that only scar tissue was observed in control channels gave further support to the likelihood that muscle cells had originated in implanted channels. Thus the viability of transplanted skeletal myoblasts (satellite cells) has been established. What remains is the question of differentiation. This issue is important from a scientific viewpoint, but may not be critical to our practical goal of satellite cell implantation to improve cardiac function. It is clear from all the transformation studies of skeletal muscle, including dynamic cardiomyoplasty, that gene Texas Heart Institutejotirnal

expression is quickly altered when skeletal muscle cells are placed in a field of repetitive electrical stimulation. However, it is unknown whether or not transplanted satellite cells will differentiate into modified skeletal muscle cells that can develop fatigue resistance when placed in the myocardial environment and rhythmically stimulated. Even if this does occur, the development of special cell junctions with other cardiac myocytes will be necessary to have synchronized contraction for improvement of cardiac function.

Discussion It is estimated that 4 million Americans suffer from chronic heart failure, and 400,000 new cases are diagnosed each year,23 resulting in high morbidity and mortality, as well as tremendous cost to society.24 Despite improvement in medical management, success of cardiac transplantation, and the promise of cardiomyoplasty and artificial assist devices, the ideal treatment is yet to be found. Although transplantation of satellite cells into injured myocardium for improving cardiac function is at best in the earliest stages of development, it is theoretically promising as the ideal treatment of chronic heart failure. We have shown the viability of satellite cells, which in some cases had formed specialized cell junctions either with adjacent cardiac cells or with each other, after differentiation into muscle cells in the injured myocardium. We have also developed improved culture techniques, which has resulted in higher yields and purity. What is yet to be shown is whether the transplantation of satellite cells into injured myocardium will result in improved myocardial function. If so, what is the mechanism? In Myocardial Regeneration

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addition, the destiny of transplanted satellite cells needs to be ascertained. Do transplanted satellite cells differentiate into modified skeletal muscle cells that will develop fatigue resistance when placed in the myocardial environment and rhythmically stimulated by cardiac impulses, as transformed muscle does in dynamic cardiomyoplasty? Or do they differentiate into true cardiac cells? These are important questions for the future. Another issue that needs to be examined is that of the delivery of satellite cells. There may be methods of delivery other than direct injection into myocardium, such as catheter transmyocardial delivery.

6. Magovern JA, Magovern GJ Jr, Magovern GJ, Palumbi MA, OrieJE. Surgical therapy for congestive heart failure: indications for transplantation versus cardiomyoplasty. J Heart 7. 8.

9. 10. 11.

12.

Conclusion Recently, there was an enthusiastic article in Science called "New Cell Transplants May Mend a Broken Heart."25 Its author discusses a report26 regarding the transplantation of embryonal mouse heart muscle cells into adult mouse hearts. We share this enthusiasm, and have worked, experimentally, on using satellite cells to "mend a broken heart" since 1988. Our work with satellite cells has progressed in parallel with our program in dynamic cardiomyoplasty for augmentation of failing hearts. Two goals have been achieved: the 1st was the improved culturing technique that allowed us to obtain high yield and purity of satellite cells,27* and the 2nd was the success of implanting cultured satellite cells and later identifying them as neomyocardium in dog hearts.2&30* We share these experiences in the hope that more study of this subject can be initiated in the field of surgical research. We recognize that a significant amount of work needs to be done and that many questions need to be answered before new cell transplants may indeed mend a broken heart. This novel treatment may be a dream, but if successful, it will certainly be ideal for chronic heart failure.

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*These findings have recently been corroborated.3' 21.

References 22. 1.

First MR. Transplantation in the nineties. Transplantation

1992;53:1-11. Magovern JA, Magovern GJ Sr, Maher TD Jr, Benckart DH, Park SB, Christlieb IY, et al. Operation for congestive heart failure: transplantation, coronary artery bypass, and cardiomyoplasty. Ann Thorac Surg 1993;56:418-25. 3. Pae WE Jr. Ventricular assist devices and total artificial hearts: a combined registry experience. Ann Thorac Surg

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1993;55:295-8. 4. Carpentier A, Chachques JC, Acar C, Relland J, Mihaileanu S, Bensasson D, et al. Dynamic cardiomyoplasty at seven years. J Thorac Cardiovasc Surg 1993;106:42-54. 5. Lee KF, Wechsler AS. Dynamic cardiomyoplasty. In: Karp RB, Laks H, Wechsler AS, eds. Advances in cardiac surgery. St. Louis: Mosby Year Books, 1993;4:207-36.

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Lung Transplant 1992;11:538-44. Bugaisky LB, Zak R. Differentiation of adult rat cardiac myocytes in cell culture. Circ Res 1989;64:493-500. Kiortsis V, Koussoulakos S, Wallace H, eds. Recent trends in regeneration research. New York: Plenum Press, 1989. Rumyantsev PP, ed. Growth and hyperplasia of cardiac muscle cells. New York: Academic Press, 1991. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 1961;9:493-5. Snow MH. Myogenic cell formation in regenerating rat skeletal muscle injured by mincing. II. An autoradiographic study. Anat Rec 1977;188:201-17. Snow MH. An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats. Cell Tissue Res 1978;186:53540. Morgan JE, Watt DJ, Sloper JC, Partridge TA. Partial correction of an inherited biochemical defect of skeletal muscle by grafts of normal muscle precursor cells. J Neurol Sci 1988, 86:137-47. Watt DJ, Morgan JE, Partridge TA. Use of mononuclear precursor cells to insert allogeneic genes into growing mouse muscles. Muscle Nerve 1984;7:741-50. Rumyantsev PP. Interrelations of the proliferation and differentiation processes during cardiac myogenesis and regeneration. Int Rev Cytol 1977;51:186-273. Rumyantsev PP. Reproduction of cardiac myocytes developing in vivo and its relationship to processes of differentiation. In: Rumyantsev PP, ed. Growth and hyperplasia of cardiac muscle cells. New York: Academic Press, 1991:70159. Field LJ. Atrial natriuretic factor-SV40 T antigen transgenes produce tumors and cardiac arrhythmias in mice. Science 1988;239: 1029-33. Behringer RR, PeschonJJ, Messing A, Gartside CL, Hauschka SD, Palmiter RD, et al. Heart and bone tumors in transgenic mice. Proc Natl Acad Sci U S A 1988;85:2648-52. Katz EB, Steinhelper ME, Delcarpio JB, Daud Al, Claycomb WC, Field LJ. Cardiomyocyte proliferation in mice expressing alpha-cardiac myosin heavy chain-SV40 T-antigen transgenes. Am J Physiol 1992;262:H1867-76. Sen A, Dunnmon P, Henderson SA, Gerard RD, Chien KR. Terminally differentiated neonatal rat myocardial cells proliferate and maintain specific differentiated functions following expression of SV40 large T antigen. J Biol Chem 1988; 263: 19132-6. Koh GY, Klug MG, Soonpaa MH, Field LJ. Differentiation and long-term survival of C2C12 myoblast grafts in heart. J Clin Invest 1993;92:1548-54. Taylor CB, Davis CB Jr, Vawter GF, Hass GM. Controlled myocardial injury produced by a hypothermal method. Circulation 1951;3:239-53. Massie BM, Packer M. Congestive heart failure: current controversies and future prospects [introduction]. Am J Cardiol 1990;66:429-30. Schocken DD, Arrieta MI, Leaverton PE, Ross EA. Prevalence and mortality rate of congestive heart failure in the United States. J Am Coll Cardiol 1992;20:301-6. Nowak R. New cell transplants may mend a broken heart. Science 1994;264:31.

26. Soonpaa MH, Koh GY, Klug MG, Field LJ. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 1994;264:98-101. 27. Lutka F, Kao RL. Improved method for satellite cell isolation and culture [abstract]. FASEB J 1992;6:A1934.

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28. Kao RL, Rizzo C, Magovern GJ. Satellite cells for myocardial regeneration [abstract]. Physiologist 1989;32:220. 29. Kao RL. Regeneration of injured myocardium from im-

planted satellite cells [abstract]. Circulation 1991;84(Suppl II):II-386. 30. Kao RL, Magovern JA, Tong JY, Magovern GJ. Muscle regeneration of injured myocardium [abstract]. J Cell Biochem 1991;45(Suppl 15C):73. 31. Chiu RC-J, Zibaitis A, Kao RL. Cellular cardiomyoplasty: myocardial regeneration with satellite cell implantation. Ann Thorac Surg 1995. In press.

Editorial Comment The preceding paper by Yoon and colleagues is a promising progress report from Magovern's group on its pioneering work using cultured skeletal muscle cells to improve contractility in failing myocardium. Since there are no known satellite cells in the myocardium that could give rise to new cardiac muscle cells, the authors have been pursuing the very creative approach of using skeletal muscle satellite cells. There are many formidable obstacles, the first of which-the culture of essentially pure populations of satellite cells-appears to be nearly resolved. Because there are no biochemical markers for these cells at this time, the authors have had to rely on the functional end point of showing that they can select a population of cells that subsequently form multinucleated myotubes. The next question is, What happens to these cells if they are put into dis-

Texas Heart InstituteJournal

eased myocardium? The Magovern group has wisely chosen to implant these cells into well vascularized areas. The borders of injured areas typically are rich in growth factors and blood supply, and it is hoped that a good many of these cells will survive, but the data are not all in. The next problem is that these cells will have to link up with cardiac myocytes and with each other in order to contract synchronously. The body of information on this is still quite preliminary. Then it has to be hoped that these cells will undergo a switch to a slow-twitch type of muscle cell, which can contract repeatedly. The phenotype is characterized by lots of mitochondria and slow-twitch myosin isoforms. Other laboratories are picking up on this theme, some with the help of powerful molecular techniques, such as using genetic markers to follow the fate of these cells and other genes to immortalize them. There is little doubt that this is an exciting area of research and one that should have clinical application in the next 5 to 10 years. Ward Casscells, MD, Associate Director, Cardiology Research, Texas Heart Institute and St. Luke's Episcopal Hospital;

Chiefof Cardiology, University of Texas Medical School at Houston and Hermann Hospital, Houston


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