Myoblast transplantation during cardiac surgery
1 Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery; University Paris-5, Faculty of Medicine; INSERM U 633, 20 rue Leblanc, 75015 Paris, France
2 Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiology; Faculty of Medicine, University Paris-Descartes, INSERM U 633, Paris, France
* Corresponding author.E-mail address: philippe.menasche@egp.aphp.fr
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Abstract |
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After a decade of experimental work, skeletal myoblast transplantation has now entered the clinical arena as a potentially new means of improving the function of the chronically infarcted heart through regeneration of fibrotic scars. Because the engrafted myoblasts do not connect with the host cardiomyocytes, it is increasingly considered that the functional benefits of myogenic cell transplantation are more related to limitation of adverse post-infarction remodelling and/or paracrine signalling on recipient tissue rather than to a synchronous contribution of the graft to the heart's systolic function. The initial clinical studies of intra-operative myoblast transplantation have primarily documented the feasibility of the procedure and unravelled a potential pro-arrhythmic risk that needs to be further characterized. It is now critical to assess whether the functional benefits observed in the laboratory setting translate into meaningful improvements in local and global contractility and ultimate patient outcomes. The results of ongoing randomized trials should soon help clarifying this efficacy issue. In parallel, experimental studies need to be actively pursued to address some remaining key issues including the means of optimizing myoblast delivery, engraftment and survival, and the development of a second-line generation of cells featuring the potential of a true electromechanical integration within the recipient myocardium.
Key Words: Skeletal myoblasts • Transplantation • Stem cells • Myocardial infarction • Heart failure
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Introduction |
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The changing referral patterns of coronary artery bypass grafting (CABG), due to the increasing use of percutaneous interventions, result in that most of the patients undergoing surgery now have a history of one or more myocardial infarcts. Except for some cases that are anatomically suitable for a restoration procedure, most of these scars are let untouched intra-operatively, as even though they may still be supplied by a patent, technically graftable coronary artery, the lack of viability may deter from revascularizing them since at most 10% of these non-viable segments are expected to recover some wall motion following revascularization of their supplying vessel. It has thus become intuitively appealing to repopulate these scars with surrogates or precursors for cardiomyocytes with the hope that these newly engrafted cells could, at least partly, regenerate them. So far, the cells that have been used clinically in combination with CABG to achieve this objective are skeletal myoblasts and bone-marrow-derived cells. The present review will focus on the former ones and present a critical appraisal of the available data, a summary of the major lessons drawn from these early experiences, and outline the major perspectives offered by this novel therapy.
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Review of current trials |
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TopAbstractIntroductionReview of current trialsLessons drawn from the...PerspectivesReferences |
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Chronologically, the first to enter the clinical arena, skeletal myoblasts feature several attractive characteristics including an autologous origin which overcomes problems related to immunogenicity and availability, a high scalability potential, an advanced commitment towards the myogenic pathway which should prevent from tumour formation, and a high resistance to ischaemia. A large experimental database, developed over the past decade, has documented the consistent engraftment of myoblasts into post-infarction scars and the related improvement in left ventricular function,1 thereby setting the stage for early clinical trials.
The first of them started started in June 2000, when we performed the first human transplantation of autologous myoblasts in a patient with severe ischaemic heart failure. This case was the first of a series of 10, which was primarily designed to test the feasibility and the safety of the procedure. In all cases, CABG was performed in ischaemic areas while the cell-implanted segments was not revascularized.2 The long-term follow-up (maximum: 5 years) of this initial cohort has recently been reported3 and can be summarized as follows: among the nine operative survivors (one patient died early post-operatively from a mesenteric infarction), one died 18 months after surgery from a stroke and the pathological examination of his heart demonstrated clusters of myotubes embedded in scar tissue,4 and the remaining eight patients have been symptomatically improved and the echocardiographic assessment shows a striking stability of end-diastolic volumes and ejection fraction (although there is a trend for the latter to have increased over baseline). Of note, systolic thickening of the once akinetic scarred segments transplanted with myoblasts is present in six patients. There has been a low incidence of rehospitalizations for heart failure (five episodes in three patients yielding a rate of 0.13 patient-years) and event recordings by the automatic defibrillators implanted in five patients document three arhythmic episodes at 6, 7, and 18 months post-operatively.
Three additional adjunct-to-CABG trials have been subsequently reported. They share in common with our study, the inclusion of patients with severe ischaemic left ventricular dysfunction (the ejection fraction is usually below 35%) and the presence of akinetic non-viable myocardial scars, but differ from our protocol by the systematic revascularization of the myoblast-implanted area. Herreros et al.5 reported on 12 patients who received an average of 221x106 cells previously cultivated in autologous serum. At a 3-month follow-up, ejection fraction had increased (from 35–53%) while regional wall motion had improved to a greater extent in segments receiving both cells and bypasses compared with those subjected to revascularization alone. One year later, Siminiak et al.6 reported the 4-month results of a series of 10 patients injected with variable amounts of cells (from 4x105 to 5x107). Left ventricular ejection fraction was also found to increase significantly over baseline values while four out of the 10 transplanted segments displayed improved kinetics. More recently, Dib et al.7 have reported the 4-year results of the US dose-escalating study and although the data are more difficult to interpret because of the heterogeneity of the case mix (some patients underwent a concomitant aneurysmectomy), the general trend is an improvement in both global and regional left ventricular function.
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Lessons drawn from the early trials |
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The first lesson pertains to the feasibility of the procedure. All these studies have consistently shown that myoblasts retrieved from a small biopsy could be efficiently grown under Good Manufacturing Practice conditions and that the subsequent injections of the final cell yield in multiple sites across the post-infarction scar could then be implemented without specific peri-operative complications. Regarding safety, these trials have also raised the concern of a potential pro-arrhythmic risk of myoblast transplantation. Although the exact mechanism of this risk is not yet fully elucidated, the prevailing hypothesis, primarily based on in vitro co-culture experiments,8 is that the electrical insulation of engrafted myoblasts subsequent to their failure to express gap junction proteins results in a slowing of impulse conduction velocity which, in turn, sets the stage for re-entry circuits. This assumption is to some extent supported by the observation that overexpression of connexin 43 by skeletal myoblasts increases conduction velocity, at least in the in vitro setting of co-cultures with cardiomyocytes, and reduces the prevalence of arrhythmic events. Other putative mechanisms of arrhythmias, however, have been considered, that include delayed repolarizations and the heterogeneity of calcium currents generated by the few chimeric cells formed by fusion of injected myoblasts with adjacent cardiomyocytes at the graft-host interface.9 Nevertheless, the assessment of this potential complication of myoblast transplantation should take into consideration a strong ‘background noise’, that is, the arrhythmogenicity intrinsic to heart failure. Thus, in the PATCH trial, which has randomized patients with severe left ventricular dysfunction (indeed very close to those included in cell therapy studies) to the prophylactic implantation of an internal cardioverter-defibrillator (ICD) at the time of CABG, the prevalence of sustained ventricular tachycardia and fibrillation recorded in the treated group (446 patients) was 5.8% and 3.4% (these values were 5.7% and 5.1%, respectively, in the non-implanted arm of the study).10 Therefore, in the ongoing randomized placebo-controlled double-blind myoblast autologous grafting in ischemic cardiomyopathy (MAGIC) trial (see in what follows), all patients are implanted with an ICD allowing event recordings so that it should ultimately provide a meaningful assessment of the causal relationship (if any) between myoblast engraftment and ventricular arrhythmias.
It should be stressed that these initial phase I trials have been neither designed nor powered to generate efficacy data. From this standpoint, they are thus fraught with several methodological inaccuracies (small numbers of patients, lack of appropriate controls and blinded assessment, confounding effect of concomitant revascularization among others), which should make us very cautious in the interpretation of functional outcomes. This issue of efficacy can only be addressed by prospective, randomized, placebo-controlled, double-blind studies. Therefore, it is this type of design which has been used in the MAGIC trial, which we have now completed in multiple centres across Europe. The results should be available by the end of 2006 and will hopefully provide some meaningful insights into the effects of engrafted myoblasts on regional and global left ventricular function. Should the proof-of-principle be demonstrated, the study should then be further expanded by a larger pivotal trial with robust clinical endpoints (like mortality and number of rehospitalizations for heart failure) as primary outcome measurements, so as to provide more definite clues about the risk-benefit and cost-effectiveness ratios associated with this procedure.
Even though efficacy is to be demonstrated clinically, it is fair to acknowledge that its mechanisms remain largely speculative. Because the engrafted myoblasts are physically disconnected from the neighbouring cardiomyocytes, it is unlikely that they contract synchronously with them11 and thus make a substantial contribution to the heart's pump function through their intrinsic contractile properties. Likewise, the donor–recipient chimeric cells formed by fusion are probably too scarce to achieve this objective. Limitation of left ventricular remodelling is another possibility that has been demonstrated experimentally, but is actually not supported by the clinical observations that end-diastolic volumes are unchanged by myoblast implantation. This is not really unexpected because most of these patients are referred for surgery late in the course of their disease and thus exhibit a fully completed left ventricular remodelling, which is unlikely to be reversible biologically. However, it cannot be excluded that by preventing further left ventricular dilatation, engrafted skeletal myoblasts contribute to reduce wall stress, and thus to improve function of remote host cardiomyocytes.12 Finally, an increasingly prevailing hypothesis is that the myoblasts could act paracrinally by releasing cytokines and growth factors like vascular endothelial growth factor or stromal-derived factor-1.13,14 This mechanism would provide a paradigm to explain observations where a definite improvement in function contrasts with the scarcity of sustained cell engraftment and is supported by the recent finding that intracerebral implantation of human mesenchymal stem cells in mice greatly increases proliferation of adjacent endogenous neural stem cells and, concomitantly, the expression of factors that promote neurogenesis.15 However, in the case of the heart, the paracrine hypothesis still needs to be confirmed by in vivo data providing a mechanistic link between gene upregulation, increased myocardial tissue expression of the corresponding proteins, and identification of their putative targets (stimulation of angiogenesis, extracellular matrix remodelling, limitation of apoptosis, recruitment of endogenous resident cardiac stem cells).
Genomic and proteomic studies currently under way should contribute to clarify this issue.
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Perspectives |
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Cell therapy is still in infancy and regardless of their ultimate outcome, the current trials with skeletal myoblasts (the same holds true for bone-marrow-derived stem/progenitor cells) should be viewed as particularly crude with regard to the techniques used for processing and transferring the cells to the target tissue. This observation, together with the data generated so far by the multiple experimental studies, leads to speculate that future improvements could probably be derived from three major strategies designed to enhance cell engraftment, cell survival, and cell functionality, respectively.
Cell engraftment
It should first be honestly recognized that current cell delivery methods are far from being ideal as, regardless of whether myoblasts are injected transepicardially during CABG operations or by a catheter-based approach, a substantial degree of leakage occurs, which correspondingly decreases the amount of cells remaining engrafted in the target area. This concern has been clearly illustrated by a study showing that injections of male cells in open-chest female recipients result in the identification of the Y chromosome in several extracardiac tissues (regardless of whether there is reperfusion or not).16 This leakage issue thus needs to be addressed by improvements in delivery techniques and devices. Of note, it has been shown experimentally that multiple small deposits of myoblasts were more effective than a limited number of punctures delivering larger amounts of cells.17 This study is important in that it shows that the technique of delivery can significantly affect the histological pattern of engraftment and the functional outcome of the procedure. This view is further supported by a recent experimental work showing that coverage of the infarcted area by a myoblast-seeded scaffold may be more effective than direct intramyocardial injections of the cells.14 It is clear that in the future, although intra-operative cell delivery may remain indicated in patients referred for an open-heart surgical procedure, the development of myoblast transplantation will require less invasive percutaneous approaches, among which the intravenous technique entailing catheterization of the coronary sinus system and direct in-scar cell delivery through a retractable needle seems particularly promising.18 These catheter-based techniques are particularly important if one keeps in mind that, although the bulk of myoblast studies have dealt with ischaemic models, recent laboratory data suggest that the benefits of these cells might extend to non-ischaemic globally dilated cardiomyopathies.19 If confirmed, these results would open new important therapeutic perspectives for these patients, who cannot currently be offered any other option than cardiac transplantation, but the corollary is the development of cell transfer methods suitable for diffusively damaged hearts.
Cell survival
Once the cells are where they are intended to be, the next objective is to enhance their survival. This objective is clinically relevant in view of the relationship which has been reported between the number of engrafted cells and the functional benefit of the procedure.20 Unfortunately, up to 90% of the injected myoblasts die within a few hours after transplantation and the proliferation of the surviving fraction cannot catch up the initial graft loss. This high rate of cell death results from the interplay of multiple factors including inflammation, apoptosis, hypoxia, and inadequate patterning of the transplanted cells relative to the extracellular matrix. The identification of these factors provides a convenient framework for designing interventions targeted at enhancing cell survival. In the perspective of clinically realistic applications, two strategies appear particularly appealing. The first consists of increasing the blood supply to the grafted area to address the ischaemic component of cell death. This can be accomplished through a variety of methods including direct revascularization by a CABG, co-injection of growth factors,21,22 transfection of the grafted myoblasts by genes encoding some of these factors,23 or co-transplantation of bone-marrow-derived cells featuring an angiogenic potential.24 The second strategy consists of providing cells with a three-dimensional environment as close as possible to the one they are used to. This can be accomplished, at least partly, by embedding them into bio-injectable scaffolds that have already yielded promising results regarding their capacity to promote cell survival, differentiation, and proliferation.25
Cell functionality
The third and last major commitment is to improve the functionality of the surviving cells that have successfully engrafted. We have previously stressed that the lack of physical connexions between skeletal myoblasts and host cardiomyocytes makes unlikely that the grafted cells may form a functional syncytium, which is the prerequisite for them to contribute to the heart's contractile function. Theoretically, this can be addressed by engineering myoblasts so as to make them overexpressing connexin-43, an approach which has indeed been shown experimentally to increase conduction velocity and reduce arrhythmic events in co-culture systems,8 but not yet to result in effective electromechanical coupling and the attendant improvement in function. Indeed, the key conceptual question here is to determine whether the contractile properties of the engrafted cells are mandatory for the procedure to be successful. If one considers that myoblasts can yet be protective by mechanisms independent from their intrinsic contractile properties and operating physically or biologically through changes in scar elasticity and paracrine effects, respectively, it may not be required to genetically modify them. Conversely, if the ultimate target is to repopulate the scar tissue with a sufficient number of newly formed cardiomyocytes (which we tend to believe), alternate approaches have probably to be considered, which primarily rely on three different sources of cells: (1) putative cardiac progenitors residing in extracardiac tissues like skeletal muscle, bone marrow, fat tissue, or umbilical cord, (2) resident cardiac stem cells, and (3) embryonic stem cells. So far, the cardiogenic potential of extracardiac progenitors has primarily been established in murine models and often at the cost of clinically irrelevant culture methods, so that the therapeutic applicability of this approach still remains elusive. The use of cardiac stem cells26 is also fraught with several hurdles that include the persisting ambiguity of their phenotype (at least four different markers have been described, which is a high number for an organ with a limited replicative capacity), the uncertainty as to whether they persist in adulthood, and the lack of conclusive evidence that they can be harvested, expanded, and re-injected without loosing their differentiation potential. The therapeutic use of embryonic stem cells also raises important concerns primarily related to availability, ethics, propagation, and immunogenicity (teratoma formation does not seem to be an issue as long as the injected cells have been appropriately precommitted towards a cardiac lineage), but it should be acknowledged that, so far, these cells are the only ones which have been convincingly shown in both small and large animal models of myocardial infarction27,28 and atrioventricular block29 to convert into cardiomyocytes and achieve a successful electromechanical integration within the transplanted heart.
Conflict of interest: A.A.H. and P.M. are consultants for Genzyme.
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