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N. AL ATTAR, A.B. RAZAK and M. SCORSIN
Department of Cardiac Surgery, Centre Cardiologique du Nord, St Denis, France
Keywords: Cellular transplantation, heart failure
End-stage heart failure results from the irreversible destruction of cardiomyocytes, which do not have the capacity to regenerate. Transplantation of myogenic cells into the damaged myocardium is an emerging therapeutic alternative in the management of this major public health problem. Experimental and clinical data suggest that cellular transplantation could improve ventricular function in ischaemic or dilated cardiomyopathies. Implantation of allogeneic and autologous cell types has been applied to induce cardiac myogenesis and, recently, other cell types have been tested for the induction of myocardial angiogenesis. The results of cellular transplantation are encouraging although the role of therapeutic angiogenesis remains to be clarified and the full potential of cellular transplantation to be determined
J.R.Coll.Edinb., December 2002, 47, 749-752
Heart failure is an important cause of morbidity and mortality and is a major public health problem. With the increasing longevity of the population, it is expected to become the dominant cardiac disease of this century. This high prevalence is due to the advances in the management of acute cardiac syndromes and the reduced mortality, especially from acute myocardial infarction. This has been labeled as an “ironic failure of success”. When heart failure becomes refractory to medical management, the available surgical options are limited and the only real effective treatment is cardiac transplantation. However, besides all the problems inherent to this procedure (namely organ shortage and immunological rejection), it cannot be used in all potential candidates. The implantation of circulatory assisting devices is still primarily considered as a bridge to transplantation in the most critically ill patients and is of marginal use. One interesting option is xenotransplantation, but this remains confined to the experimental stage because of the difficulty in adequately addressing major safety issues.
Less aggressive surgical techniques have been proposed, aiming to reshape the left ventricle back to its normal oval form from the rounded aspect of a dilated ventricle.1 While this is attractive from the Starling Law’s point of view, it does not correct the underlying disease which triggered the remodeling process, i.e., the death of the cardiomyocytes.
Dynamic cardiomyoplasty consists of wrapping the heart with skeletal muscle (the latissimus dorsi).2,3 It is used as an option to restore myocardial contractility; however, it has yielded rather inconsistent results basically due to atrophy of the skeletal muscle.
Myocytes
Given these limited options, cellular transplantation has recently emerged as an attractive alternative in the treatment of severe heart failure. This approach has been inspired by the fact that the normal myocardium can be successfully colonised by a variety of contractile cells. The engraftment of cardiomyocytes has been demonstrated by injecting cells taken from transgenic mice expressing the gene of ß-galactosidase which could then be identified by specific staining.4 Likewise, the presence of allogeneic dystrophin-positive cells in the heart of dogs suffering from Duchenne muscular dystrophy has brought additional evidence for the capacity of transplanted cells to be inserted into the recipient myocardium and integrated within the cardiac tissue, as demonstrated by the formation of intercalated discs between host and recipient cells.4,5 Finally, foetal cardiomyocytes were shown to be able to survive in the zone bordering of a myocardial infarct opening the possibility of colonisation of an ischaemic myocardium.6
Moreover, different studies have assessed whether cellular transplantation could effectively improve function of ischaemically damaged myocardium. A positive answer to this question has been produced by Li et al (1996) who have shown in cryoinjury-induced myocardial infarction in the rat, that the intramyocardial injection of foetal cardiomyocytes improved systolic and diastolic function up to two months after transplantation, as assessed by ex vivo Langendorff perfusion studies.7 To this end, Scorsin et al (1997) developed a protocol of echocardiography allowing accurate analysis of two-dimensional echocardiographic images recorded in these fast-beating animal hearts (approximately 300 beats/minute). In a reperfused infarcted area in rats, foetal cardiomyocytes were intramyocardially implanted and, one month later, function was found to be significantly improved (as reflected by higher values of ejection fraction and cardiac output) in transplanted animals, compared with controls that had only received an equivalent volume of culture medium alone.8
Another important issue was to determine whether the functional benefits of cellular transplantation, documented in a model of regional ischaemia, could be extended to the setting of global heart failure. To this end, in a mouse model of anthracyclineinduced toxic cardiomyopathy, it was possible to show that injected foetal cardiomyocytes also improved cardiac function one month after transplantation, as compared with control nontransplanted animals.9
The advantage of foetal cardiomyocytes as cellular grafts is their capacity to undergo progression through the cell cycle and develop connections with host cells, an essential condition for improvement of function. Nevertheless, issues relating to availability, ethical problems regarding the foetal source of these cells and the necessity for immunosuppressive therapy are real limitations for a potential clinical application of this allograft technique. Because of the above mentioned drawbacks associated with the use of foetal cells, the search for alternative cellular types have refocused on the clinical use of autografts. Among the cells with the greatest therapeutic potential, bone marrow stromal cells and skeletal myoblasts appear to be the most promising.
Bone marrow cells
Bone marrow contains multipotential progenitor cells (mesenchymal stem cells) in an indifferentiated state with a highly proliferative capacity. Differentiation into bone, tendon, fat and muscle can be achieved by chemical induction in vitro. In a complex culture procedure, in which cells were exposed to 5-azacytidine, 30% of these cells could change their initial stem form into cardiomyocyte-like cells with the presence of intercalated disks and myotubes.10 Another study showed that stromal bone marrow stem cells, transplanted in a normal myocardium without in vitro muscular differentiation, could undergo “milieu dependent” differentiation and express the cardiogenic phenotype.11 However, in a cryoinjury model of myocardial infarction in rats, only previously in vitro chemically-induced muscular differentiated cells were able to improve function two months later in a Langendorff preparation.12 Recently, implantation of bone marrow mononuclear cells was shown to provide angiogenesis and enhance regional function of the ischaemic myocardium in the pig.13 Despite the very interesting and promising option to restore myocardial viability, the use of bone marrow stem cells raises two important questions. Firstly, they have not yet shown the potential to multiply and produce a very large number of differentiated cells in vitro, the first condition to fully recolonise diseased myocardium and thus improving ventricular function. Secondly, the risk of the development of other types of tissue, especially if undifferentiated stem cells are used.
Skeletal myoblasts (satellite cells)
The precursors of skeletal muscle fibres, the myoblasts, are present in adult animals as quiescent cells and may become activated, proliferate and differentiate upon muscle injury (in vivo) or following tissue dissociation (in vitro) in culture.
Working on primary myoblast transplantations in a cryo-injury model of myocardial infarction in dogs, Chiu et al (1995) were able to characterise the donor cells in the myocardium 14 weeks after injection.15 In this study, they could identify a “milieu dependent differentiation” of the myoblasts since these cells lost their characteristic skeletal morphology, acquiring a cardiaclike phenotype. Murry et al (1996) observed the formation of myotubes and skeletal muscle fibres within the cardiac tissue but they were unable to identify cardiac specific markers within the tissue formed by the injection of myoblasts.16 Taylor et al (1998) assessed by sonomicrometry transplanted myoblasts in a model of rabbit heart cryo-infarction and observed a functional improvement of the skeletal muscle cells within the scar tissue following transplantation.17 Importantly, in this study, the functional improvement was only seen in those animals in which implanted cells were histologically identified, thereby bringing strong evidence for a causal relationship between the presence of engrafted cells and the functional outcome. On the other hand, Murry et al (1996) could not identify changes in the myoblasttransplanted phenotype.16 In a myocardial infarction model in rats created by coronary artery ligation, Scorsin et al (1998, 2000) showed that one month after myoblast transplantation the treated group displayed a significant improvement of function, primarily manifested as a limitation of postinfarction ventricular remodeling, compared with non-transplanted controls.18,19 Transplanted cells could be identified by a positive staining for embryonic myosin heavy chain (which is specific for skeletal myoblasts). No gap-junctions were detected between transplanted cells as demonstrated by the negative staining for connexin-43. In another experiment, the same group compared foetal cardiomyocytes and skeletal myoblasts and showed that the latter were as effective as foetal cardiomyocytes in improving function of the infarcted myocardium.19 This was an important finding because foetal cardiomyocytes were, until then, the standard cells in terms of restoring cardiac function. The fact that myoblasts were functionally equivalent opened the way to their use as an autograft to restore myocardial viability in humans.
A phase one trial concerning myoblast transplantation in patients with severe heart failure has started in France. The results in the first clinical case were very promising.20 After transplanting 8x108 cells in the inferior wall of a two-year old myocardial infarction scar (non-reversible and akinetic by dobutamine echocardiography, with absence of viability by positron emission tomography (PET)), the akinetic wall became contractile (echocardiographic positive response by one grade to dobutamine stress), improving the ejection fraction from 20 to 38%. This was associated with marked increase in metabolism in the previously nonviable area as assessed by PET.
The functional improvement, however, needs to be analysed with caution because of the confounding aspects produced by two associated bypass grafts on the left side of the heart, the significance of this report was the feasibility of a large scale production of autologous myoblasts and the successful surgical engraftment confirmed by the new-onset metabolic activity in the previously dead zone. However, many questions still need to be clarified: do these cells become fatigue resistant and what is their life span after transplantation? Do they form any kind of coupling with host cardiomyocytes or do they acquire a cardiac-like phenotype? It has already been shown that two to seven weeks after transplantation, myoblasts of fast fibre isoform convert into a slow twitch muscle that has a much greater resistance to fatigue.16 The fact that grafted myoblasts were able to convert the fibre isoform may indirectly indicate that they are contracting. However, they do not seem to form any cardiac-specific junctions, as showed in some experimental studies.16,19 One hypothesis proposed to explain this paradox is that they probably contract in reaction to a mechanical stimulus transmitted through the neighbouring myocardium rather than the natural electric stimulation through intercalated disks.
Another important issue to be stressed is the ideal number of cells to be transplanted. Based on an experimental study in rats, a direct relationship between the number of cells and the improvement of function has been demonstrated.21 In the French clinical case, (see above) it appears that to restore the viability of a medium-sized myocardial infarct (loss of 20-30% of the left ventricle mass) approximately one billion cells cultured for two weeks in a myogenic-specific culture medium are required, thus, precluding its use in emergencies.
Finally, looking at the natural history of heart failure and the severity of the remodeling process intensified over time, the optimal timing of cellular transplantation still needs to be determined. In all animal models, cellular transplantation has been used with success early after a myocardial infarction, before end-stage forms of heart dysfunction. Possibly, one of the main advantages of this therapy is to arrest the left ventricular remodeling process in a moderately dilated left ventricle, consequently allowing the hypertrophy of remaining viable cardiomyocytes in association with the new engrafted skeletal muscle to improve heart function.
While various donor cells have been studied to induce myogenesis, recent interest has arisen in promoting cardiac angiogenesis. Angiogenenic growth factors (as basic fibroblast growth factor and vascular endothelial growth factor) and genes encoding angiogenic factors are being delivered to the infarcted area to induce growth of new blood vessels.22-27 Increased perfusion has also been observed after endothelial cell transplantation even though this was not associated with improved ventricular function nor modification of ventricular remodeling suggesting that angiogenesis alone, without myogenesis, would be less beneficial in patients with extensive, transmural myocardial infarction.28
These techniques retain an important potential as an adjunct to cellular transplantation in inducing angiogenesis in injured myocardium when revascularisation cannot be achieved by standard techniques, namely percutaneous angioplasty and coronary artery bypass grafting.
Despite encouraging results, the exact mechanisms responsible for improved heart function after cellular transplantation remain undetermined. There are a number of hypotheses that have been put forward to try to explain these results.
• Induction of local angiogenesis: Injected cells could release growth and/or angiogenic factors that could partially explain engrafted cell survival. This has not been demonstrated to be significant and is confounded with the triggering of angiogenesis by epicardial-puncture alone.29
• “Scaffolding effect”: Engrafted cells might limit postinfarction ventricular expansion through their elastic properties. This scaffolding effect is supported by the reduced end-diastolic volumes observed in transplanted hearts compared with controls.
• Cell contractility: Transplantation of cells with inherent contractile properties suggest that their contractions would contribute to the improved function. Indeed, myotubes have been demonstrated in zones of injected cells up to one year after transplantation. Furthermore, these fibres express slow myosin. This change of phenotype suggests their ability to resist fatigue and sustain a cardiac workload.16 For this, the cells would have to contract in synchrony with the adjacent myocardium. The mechanism explaining the transmission and propagation of electrical impulses from the native myocardium to the engrafted cells has not been elucidated. Response to a mechanical stimulus exerted by surrounding cardiomyocytes could be responsible for inducing this contraction.
In summary, it is important to emphasise the consistency of the experimental data. Regardless of the species (rat, mouse, rabbit), of the type of contractile cells (foetal cardiomyocytes skeletal myoblasts), of the model used (coronary artery ligation, cryonecrosis, anthracycline-induced global cardiomyopathy) and of the method of assessment (Langendorff perfusion, pressurevolume loops, echocardiography), cellular transplantation has consistently resulted in a definite improvement of function, which provides a strong rationale for the utilisation of this technique in patients.6-9,17,19 A phase one clinical trial has been undertaken in France with the main purpose of demonstrating the feasibility and the safety of the procedure. Subsequently, a large multicentric study will follow to assess the real role of this therapy in the heart failure therapeutic armamentarium.
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Correspondence: Mr Al Attar, Cardiac Surgery, Centre Cardiologique
du Nord, 32-36 rue des Moulins Gémeaux, 93200 St Denis, France.
Email : alattar@ifrance.com