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Mending Broken Hearts One Cell at a Time
J Mol Cell Cardiol 34, 87–89 (2002) doi:10.1006/jmcc.2001.1509, available online at http://www.idealibrary.com on
Mending Broken Hearts One Cell at a Time W. Robb MacLellan The Cardiovascular Research Laboratories, Departments of Medicine and Physiology, UCLA School of Medicine, Los Angeles, California, USA (Received 26 November 2001, accepted for publication 27 November 2001)
Since the adult heart lacks the capacity to regenerate itself, patients surviving a myocardial infarction are left with an area of irreversibly damaged myocardium. To compensate for the increased hemodynamic stress resulting from this damage, the left ventricle (LV) undergoes hypertrophy and dilation. Unfortunately, this compensatory response is ultimately detrimental and leads to progressive LV dysfunction. Present day therapies are aimed at slowing the rate of LV remodeling by blocking the actions of neurohumoral factors; however, despite this pharmacological intervention the disease remains progressive and the mortality of patients with congestive heart failure (CHF) remains unacceptably high. Options when pharmacological therapy fails are limited but include heart transplantation or insertion of mechanical assist devices. However, these approaches are invasive and not without significant morbidity and mortality themselves. In response to these shortfalls, several investigators have been developing techniques to transplant cells into the area of damaged myocardium to prevent the progression of CHF. Many cell types have been successfully transplanted into damaged myocardium, including fetal cardiomyocytes,1 skeletal myoblasts,2 smooth muscle cells,3 embryonic stem cells,4 and bone marrow derived stromal5 and hematopoietic stem cells.6 While these studies have documented the feasibility of engrafting cells into the myocardium, each cell type has unique properties particularly with respect to availability and differentiation potential. Initial
studies using allogenic cardiac myocytes were successful but over time, the transplanted cells were progressively lost, presumably secondary to immunorejection, which led to concerns over the longterm stability of engrafted cells.7 This prompted speculation that long-term cell transplantation would require autologous cells. Since performing autologous cardiac myocyte transplantation in adults is not feasible, skeletal myoblasts have been the primary cell type studied. Skeletal myoblasts (also known as satellite or skeletal muscle stem cells) are easily isolated and cultured even from adult skeletal muscle. They engraft stably and differentiate into adult skeletal myotubes within damaged myocardium.8 More importantly, myoblasts have been repeatedly demonstrated to improve ventricular function in several models of myocardial injury and across species.8–10 Autologous skeletal myoblasts hold several advantages over other cell types for myocardial repair. Since they are easily obtained in adult subjects they circumvent the need for immunosuppression and are not associated with the ethical concerns that surround the use of fetal or embryonic cell types. Their limited proliferative potential, although a theoretical concern when attempting to expand myoblast populations to obtain adequate cells for transplantation, means there is less likelihood of tumor formation after transplantation. Consequently, skeletal myoblasts were chosen as the first cell type for human studies. Autologous skeletal myoblasts have been successfully and safely injected into damaged human
Please address all correspondence to: W. Robb MacLellan, Cardiovascular Research Laboratories, UCLA School of Medicine, 675 C.E. Young Dr., MRL 3-645, Los Angeles, California, 90095-1760, USA. Tel: (310) 825-2556; Fax: (310) 206-5777; E-mail: [email protected]
0022–2828/02/020087+03 $35.00/0
2002 Elsevier Science Ltd.
88
W. R. MacLellan
myocardium at the time of coronary artery bypass surgery with encouraging effects on LV function.11 These results have prompted the initiation of several US and European Phase I clinical trials of myoblast transplantation for patients with depressed LV function. Despite the exciting progress that has been made in this field over the last ten years, many questions remain to be resolved with the use of skeletal myoblasts for cell therapy. For instance, the precise mechanism of their beneficial effect remains uncertain, the necessity and ability of engrafted cells to transdifferentiate is controversial, the length of graft survival is unknown, and the optimal timing of implantation post-injury remains to be determined. While on the surface these questions might seem like trivial technical issues, the answers to them will be critical when designing future clinical trials to evaluate the efficacy of cell transplantation if we wish to maximize the potential for therapeutic benefit and minimize possible complications. The accompanying article by Reinecke et al.12 is the most careful and comprehensive attempt to date to resolve one question associated with the use of skeletal myoblasts, namely, do they have the capacity to transdifferentiate into a more cardiac-like cell when engrafted into adult myocardium, as has been suggested in previous studies.8,13–16 This question is of interest to both biologists and clinicians. Not only would it be a novel model of cardiac differentiation to explore experimentally but it would represent a potential source of cardiac myocytes for which up until recently6,17 no stem cell population was thought to exist. Likewise, if transplanted skeletal satellite cells have the capacity to transdifferentiate into cardiac muscle as has been suggested, then they would be capable of electrically coupling with the endogenous myocytes, which could benefit contractility, but also be a potential source of arrhythmias. The concept that transplanted skeletal satellite cells could have the capacity to transdifferentiate into cardiac muscle has been strengthened by recent data documenting the plasticity of many adult stem cell populations. However, as correctly pointed out by the authors of the present study, previous papers employed neither unambiguous methodology to track transplanted cells nor definitive molecular probes to characterize the transdifferentiated cells. Therefore, to address these deficiencies, Reinecke et al. labeled skeletal muscle satellite cells in vitro with bromodeoxyuridine (BrdU), a thymidine analogue that is stably integrated into the cell’s DNA. These labeled cells were then grafted into normal hearts of syngeneic rats. At 4 and 12 weeks after trans-
plantation the engrafted cells overwhelmingly differentiated into mature skeletal muscle, and were multinucleated with cross-striated myofibers expressing fast skeletal myosin heavy chain (MHC). Double immunostaining for BrdU and cardiac-specific markers revealed that virtually no BrdU positive cells expressed cardiac markers such as -MHC, cardiac troponin I, atrial natriuretic peptide or intercalated disk proteins N-cadherin or connexin43. However, the authors acknowledged that four BrdUlabeled cells out of the thousands examined expressed either -MHC or troponin I. Do these few questionable cells represent transdifferentiation or are they a result of limitations of the assay method? If we accept that these cells are false positives, does this study definitively resolve the question of whether skeletal myoblasts retain the ability to transdifferentiate into cardiac myocytes or is the applicability of the results limited by technical issues such as the use of BrdU as the tagging agent or the model itself (normal v injured myocardium)? Use of BrdU as a tracking agent has been criticized because of the tendency of the agent to be diluted with progressive cell cycles leading to an underestimation of the total number of transplanted cells. In contrast, use of genetically modified myoblasts that express LacZ or GFP, and do not suffer from this limitation, is preferable for longterm tracking of transplanted cells. However, while this might affect estimates of total myoblasts engrafted it does not invalidate measurements of the percentage of cells that actually transdifferentiated. Although the authors did not report the actual percentage of myoblasts transdifferentiating, a rough estimate would be <0.1%. Therefore, in this model it is highly unlikely that significant transdifferentiation occurs. While it is possible, although unlikely, that grafting into models of injured myocardium might result in higher rates of transdifferentiation, proof of this will require both definitive labeling and identification of cardiac-specific markers in engrafted cells. If skeletal myoblasts truly do not transdifferentiate then it is unlikely they couple electrically or directly contribute to myocardial contraction despite numerous studies documenting their efficacy. While the mechanism for the beneficial effect of transplanted cells may be uncertain, it is more than an academic question. If the transplanted cells contribute directly to LV contractility then transplantation at any time after injury should be beneficial. Alternatively, if prevention of infarct expansion and attenuation of adverse LV remodeling is the primary mechanism for its beneficial effects, then injection of cells may need to be per-
Mending Broken Hearts One Cell at a Time
formed in the early post-infarct period for full benefit. Hence, present clinical studies being performed in patients with end-stage cardiomyopathies are less likely to be successful. Likewise, if prevention of remodeling is the primary mechanism for benefit, then future efforts to optimize efficacy need to address this. These questions only highlight the complex issues that need to be resolved when basic science is translated to clinical usage and the necessity for well designed and executed studies such as that described by Reinecke et al. Whatever the answer to these questions, it is likely that cell transplantation will open a new era in therapy for patients with myocardial dysfunction.
References 1. K GY, S MH, K MG, P HP, C BJ, Z DP, F LJ. Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs. J Clin Invest 1995; 96: 2034–2042. 2. M D, D C, A M, K RL, C RC. Cell transplantation for myocardial repair: an experimental approach. Cell Transplant 1992; 1: 383–390. 3. L RK, J ZQ, W RD, M F, M DA. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol 1999; 31: 513–522. 4. K MG, S MH, K GY, F LJ. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98: 216–224. 5. W JS, S-T D, G J, C E, E N, C RC. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J Thorac Cardiovasc Surg 2000; 120: 999–1005. 6. O D, K J, C S, J I, A SM, L B, P J, MK R, N-G B, B DM, L A, A P. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410: 701–705.
89
7. L RK, M DA, W RD, M MK, Z J, R V, L G, M F, J ZQ. Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 1997; 96: II179–186. 8. T DA, A BZ, H P, J TR, R MC, H KA, G DD, K WE. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998; 4: 929–933. 9. M CE, W RW, S SM, H SD. Skeletal myoblast transplantation for repair of myocardial necrosis. J Clin Invest 1996; 98: 2512– 2523. 10. A BZ, H MT, M JM, C MJ, H KA, T DA. Myogenic cell transplantation improves in vivo regional performance in infarcted rabbit myocardium. J Heart Lung Transplant 1999; 18: 1173–1180. 11. M P, H AA, S M, P B, D M, D D, S K, V JT, M JP. Myoblast transplantation for heart failure. Lancet 2001; 357: 279–280. 12. R H, P V, M CE. Skeletal muscle stem cells do not transdifferentiate into cardiomyocytes after cardiac grafting. J Mol Cell Cardiol 2002; 34: 241–249. 13. C RC, Z A, K RL. Cellular cardiomyoplasty: myocardial regeneration with satellite cell implantation. Ann Thorac Surg 1995; 60: 12–18. 14. A BZ, L CW, K WE, H KA, G DD, T DA. Intracardiac transplantation of skeletal myoblasts yields two populations of striated cells in situ. Ann Thorac Surg 1999; 67: 124–129. 15. Y PD, K RL, M GJ. Myocardial regeneration. Transplanting satellite cells into damaged myocardium. Tex Heart Inst J 1995; 22: 119–125. 16. D J, D M, Z A, P MP, S-T D, L C, C RC. Myocardial tissue engineering with autologous myoblast implantation. J Thorac Cardiovasc Surg 1998; 116: 744–751. 17. J KA, M SM, W H, P J, H CJ, M MW, E ML, M LH, H KK, G MA. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001; 107: 1395–1402.
Mending Broken Hearts One Cell at a Time W. Robb MacLellan The Cardiovascular Research Laboratories, Departments of Medicine and Physiology, UCLA School of Medicine, Los Angeles, California, USA (Received 26 November 2001, accepted for publication 27 November 2001)
Since the adult heart lacks the capacity to regenerate itself, patients surviving a myocardial infarction are left with an area of irreversibly damaged myocardium. To compensate for the increased hemodynamic stress resulting from this damage, the left ventricle (LV) undergoes hypertrophy and dilation. Unfortunately, this compensatory response is ultimately detrimental and leads to progressive LV dysfunction. Present day therapies are aimed at slowing the rate of LV remodeling by blocking the actions of neurohumoral factors; however, despite this pharmacological intervention the disease remains progressive and the mortality of patients with congestive heart failure (CHF) remains unacceptably high. Options when pharmacological therapy fails are limited but include heart transplantation or insertion of mechanical assist devices. However, these approaches are invasive and not without significant morbidity and mortality themselves. In response to these shortfalls, several investigators have been developing techniques to transplant cells into the area of damaged myocardium to prevent the progression of CHF. Many cell types have been successfully transplanted into damaged myocardium, including fetal cardiomyocytes,1 skeletal myoblasts,2 smooth muscle cells,3 embryonic stem cells,4 and bone marrow derived stromal5 and hematopoietic stem cells.6 While these studies have documented the feasibility of engrafting cells into the myocardium, each cell type has unique properties particularly with respect to availability and differentiation potential. Initial
studies using allogenic cardiac myocytes were successful but over time, the transplanted cells were progressively lost, presumably secondary to immunorejection, which led to concerns over the longterm stability of engrafted cells.7 This prompted speculation that long-term cell transplantation would require autologous cells. Since performing autologous cardiac myocyte transplantation in adults is not feasible, skeletal myoblasts have been the primary cell type studied. Skeletal myoblasts (also known as satellite or skeletal muscle stem cells) are easily isolated and cultured even from adult skeletal muscle. They engraft stably and differentiate into adult skeletal myotubes within damaged myocardium.8 More importantly, myoblasts have been repeatedly demonstrated to improve ventricular function in several models of myocardial injury and across species.8–10 Autologous skeletal myoblasts hold several advantages over other cell types for myocardial repair. Since they are easily obtained in adult subjects they circumvent the need for immunosuppression and are not associated with the ethical concerns that surround the use of fetal or embryonic cell types. Their limited proliferative potential, although a theoretical concern when attempting to expand myoblast populations to obtain adequate cells for transplantation, means there is less likelihood of tumor formation after transplantation. Consequently, skeletal myoblasts were chosen as the first cell type for human studies. Autologous skeletal myoblasts have been successfully and safely injected into damaged human
Please address all correspondence to: W. Robb MacLellan, Cardiovascular Research Laboratories, UCLA School of Medicine, 675 C.E. Young Dr., MRL 3-645, Los Angeles, California, 90095-1760, USA. Tel: (310) 825-2556; Fax: (310) 206-5777; E-mail: [email protected]
0022–2828/02/020087+03 $35.00/0
2002 Elsevier Science Ltd.
88
W. R. MacLellan
myocardium at the time of coronary artery bypass surgery with encouraging effects on LV function.11 These results have prompted the initiation of several US and European Phase I clinical trials of myoblast transplantation for patients with depressed LV function. Despite the exciting progress that has been made in this field over the last ten years, many questions remain to be resolved with the use of skeletal myoblasts for cell therapy. For instance, the precise mechanism of their beneficial effect remains uncertain, the necessity and ability of engrafted cells to transdifferentiate is controversial, the length of graft survival is unknown, and the optimal timing of implantation post-injury remains to be determined. While on the surface these questions might seem like trivial technical issues, the answers to them will be critical when designing future clinical trials to evaluate the efficacy of cell transplantation if we wish to maximize the potential for therapeutic benefit and minimize possible complications. The accompanying article by Reinecke et al.12 is the most careful and comprehensive attempt to date to resolve one question associated with the use of skeletal myoblasts, namely, do they have the capacity to transdifferentiate into a more cardiac-like cell when engrafted into adult myocardium, as has been suggested in previous studies.8,13–16 This question is of interest to both biologists and clinicians. Not only would it be a novel model of cardiac differentiation to explore experimentally but it would represent a potential source of cardiac myocytes for which up until recently6,17 no stem cell population was thought to exist. Likewise, if transplanted skeletal satellite cells have the capacity to transdifferentiate into cardiac muscle as has been suggested, then they would be capable of electrically coupling with the endogenous myocytes, which could benefit contractility, but also be a potential source of arrhythmias. The concept that transplanted skeletal satellite cells could have the capacity to transdifferentiate into cardiac muscle has been strengthened by recent data documenting the plasticity of many adult stem cell populations. However, as correctly pointed out by the authors of the present study, previous papers employed neither unambiguous methodology to track transplanted cells nor definitive molecular probes to characterize the transdifferentiated cells. Therefore, to address these deficiencies, Reinecke et al. labeled skeletal muscle satellite cells in vitro with bromodeoxyuridine (BrdU), a thymidine analogue that is stably integrated into the cell’s DNA. These labeled cells were then grafted into normal hearts of syngeneic rats. At 4 and 12 weeks after trans-
plantation the engrafted cells overwhelmingly differentiated into mature skeletal muscle, and were multinucleated with cross-striated myofibers expressing fast skeletal myosin heavy chain (MHC). Double immunostaining for BrdU and cardiac-specific markers revealed that virtually no BrdU positive cells expressed cardiac markers such as -MHC, cardiac troponin I, atrial natriuretic peptide or intercalated disk proteins N-cadherin or connexin43. However, the authors acknowledged that four BrdUlabeled cells out of the thousands examined expressed either -MHC or troponin I. Do these few questionable cells represent transdifferentiation or are they a result of limitations of the assay method? If we accept that these cells are false positives, does this study definitively resolve the question of whether skeletal myoblasts retain the ability to transdifferentiate into cardiac myocytes or is the applicability of the results limited by technical issues such as the use of BrdU as the tagging agent or the model itself (normal v injured myocardium)? Use of BrdU as a tracking agent has been criticized because of the tendency of the agent to be diluted with progressive cell cycles leading to an underestimation of the total number of transplanted cells. In contrast, use of genetically modified myoblasts that express LacZ or GFP, and do not suffer from this limitation, is preferable for longterm tracking of transplanted cells. However, while this might affect estimates of total myoblasts engrafted it does not invalidate measurements of the percentage of cells that actually transdifferentiated. Although the authors did not report the actual percentage of myoblasts transdifferentiating, a rough estimate would be <0.1%. Therefore, in this model it is highly unlikely that significant transdifferentiation occurs. While it is possible, although unlikely, that grafting into models of injured myocardium might result in higher rates of transdifferentiation, proof of this will require both definitive labeling and identification of cardiac-specific markers in engrafted cells. If skeletal myoblasts truly do not transdifferentiate then it is unlikely they couple electrically or directly contribute to myocardial contraction despite numerous studies documenting their efficacy. While the mechanism for the beneficial effect of transplanted cells may be uncertain, it is more than an academic question. If the transplanted cells contribute directly to LV contractility then transplantation at any time after injury should be beneficial. Alternatively, if prevention of infarct expansion and attenuation of adverse LV remodeling is the primary mechanism for its beneficial effects, then injection of cells may need to be per-
Mending Broken Hearts One Cell at a Time
formed in the early post-infarct period for full benefit. Hence, present clinical studies being performed in patients with end-stage cardiomyopathies are less likely to be successful. Likewise, if prevention of remodeling is the primary mechanism for benefit, then future efforts to optimize efficacy need to address this. These questions only highlight the complex issues that need to be resolved when basic science is translated to clinical usage and the necessity for well designed and executed studies such as that described by Reinecke et al. Whatever the answer to these questions, it is likely that cell transplantation will open a new era in therapy for patients with myocardial dysfunction.
References 1. K GY, S MH, K MG, P HP, C BJ, Z DP, F LJ. Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs. J Clin Invest 1995; 96: 2034–2042. 2. M D, D C, A M, K RL, C RC. Cell transplantation for myocardial repair: an experimental approach. Cell Transplant 1992; 1: 383–390. 3. L RK, J ZQ, W RD, M F, M DA. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol 1999; 31: 513–522. 4. K MG, S MH, K GY, F LJ. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98: 216–224. 5. W JS, S-T D, G J, C E, E N, C RC. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J Thorac Cardiovasc Surg 2000; 120: 999–1005. 6. O D, K J, C S, J I, A SM, L B, P J, MK R, N-G B, B DM, L A, A P. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410: 701–705.
89
7. L RK, M DA, W RD, M MK, Z J, R V, L G, M F, J ZQ. Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 1997; 96: II179–186. 8. T DA, A BZ, H P, J TR, R MC, H KA, G DD, K WE. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998; 4: 929–933. 9. M CE, W RW, S SM, H SD. Skeletal myoblast transplantation for repair of myocardial necrosis. J Clin Invest 1996; 98: 2512– 2523. 10. A BZ, H MT, M JM, C MJ, H KA, T DA. Myogenic cell transplantation improves in vivo regional performance in infarcted rabbit myocardium. J Heart Lung Transplant 1999; 18: 1173–1180. 11. M P, H AA, S M, P B, D M, D D, S K, V JT, M JP. Myoblast transplantation for heart failure. Lancet 2001; 357: 279–280. 12. R H, P V, M CE. Skeletal muscle stem cells do not transdifferentiate into cardiomyocytes after cardiac grafting. J Mol Cell Cardiol 2002; 34: 241–249. 13. C RC, Z A, K RL. Cellular cardiomyoplasty: myocardial regeneration with satellite cell implantation. Ann Thorac Surg 1995; 60: 12–18. 14. A BZ, L CW, K WE, H KA, G DD, T DA. Intracardiac transplantation of skeletal myoblasts yields two populations of striated cells in situ. Ann Thorac Surg 1999; 67: 124–129. 15. Y PD, K RL, M GJ. Myocardial regeneration. Transplanting satellite cells into damaged myocardium. Tex Heart Inst J 1995; 22: 119–125. 16. D J, D M, Z A, P MP, S-T D, L C, C RC. Myocardial tissue engineering with autologous myoblast implantation. J Thorac Cardiovasc Surg 1998; 116: 744–751. 17. J KA, M SM, W H, P J, H CJ, M MW, E ML, M LH, H KK, G MA. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001; 107: 1395–1402.