© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Gene transfer and cell transplant: an experimental approach to repair a ‘broken heart’
aCardiology Division, Soroka Medical Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel
bThe Institute of Genetic Medicine, Department of Medicine, Good Samaritan Hospital, University of Southern California, Los Angeles, CA, USA
cDepartment of Biochemistry and Molecular Biology, Good Samaritan Hospital, University of Southern California, Los Angeles, CA, USA
dThe Heart Institute, Good Samaritan Hospital, University of Southern California, Los Angeles, CA, USA
* Corresponding author. The Heart Institute, Good Samaritan Hospital, 1225 Wilshire Blvd., Los Angeles, CA 90017, USA. Tel.: +1 (213) 977 4050; fax: +1 (213) 977 4107.
Received 5 March 1997; accepted 27 May 1997
KEYWORDS Cardiomyocyte; Gene therapy; Myocardial infarction; Rat
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1 Introduction |
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 Top 1 Introduction 2 Post-infarction left...3 Gene transfer into...4 Molecular cardiomyoplasty:...5 Cell transplant6 Other strategies7 Unresolved issues and...8 Summary and future...References |
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Despite significant progress in prevention and therapy of ischemic heart disease, treating patients with heart failure after myocardial infarction remains a major therapeutic challenge. Adult cardiomyocytes cannot regenerate after injury. Therefore, cardiomyocyte loss due to myocardial infarction is irreversible. Currently, congestive heart failure is the only major cardiovascular disorder that is increasing in incidence and mortality [1]. Thus, there is still a need to develop alternative therapeutic strategies to prevent, arrest or reverse congestive heart failure after myocardial infarction.
Recent insights into the pathogenesis of myocardial disease and advances in molecular biology have opened up a new era of molecular and cellular therapies that target genes, molecules and peptides. Gene transfer as a therapeutic approach for the treatment of myocardial infarction and congestive heart failure has been suggested as a new treatment strategy for these serious disorders [2, 3]. The introduction, into injured myocardium, of recombinant transgenes that encode growth factors, could stimulate new vessel formation with increased collateral blood flow, accelerate healing and enhance myocardial performance. Today it is even possible to consider genetic manipulation leading to regeneration of myocytes within the infarcted myocardium.
An alternative experimental strategy to increase viability and augment ventricular function after myocardial infarction is cell transplantation [4]. Engrafted fetal cells might increase the number of functional myocytes in the infarcted myocardium, could serve as a potential source of growth factors and could be programmed for myocyte-based gene transfer.
The purpose of our review is to summarize recent advances in the attempts to develop molecular and cellular strategies for repairing a ‘broken heart’. The biological rationale for these new therapies and the potential limitations of gene therapy and cell transplant in treating myocardial infarction are discussed.
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2 Post-infarction left ventricular dysfunction |
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Top1 Introduction2 Post-infarction left...3 Gene transfer into...4 Molecular cardiomyoplasty:...5 Cell transplant6 Other strategies7 Unresolved issues and...8 Summary and future...References |
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Left ventricular remodeling following myocardial infarction refers to the process of early expansion and thinning of the infarcted segment that results in progressive dilation of the left ventricle (reviewed in [5, 6]). Left ventricular remodeling is also characterized by cardiac myocyte hypertrophy and matrix changes in the remote myocardium. This process is influenced by activation of local and systemic neurohormonal systems (e.g. the autonomic nervous system and renin-angiotensin system) and various cytokines. Initially, the remodeling process serves to restore stroke volume to normal but finally it leads to severe left ventricular dysfunction. Lengthening of the non-infarcted area also contributes to an increase in end-systolic volume and global dilatation. The heart cannot increase the number of myocytes. However, it can synthesize more sarcomeres and consequently increase the size of cardiac myocytes which leads to myocardial hypertrophy. The growth response of the heart is often accompanied by cardiac dilatation. Because prognosis after myocardial infarction is correlated with end-systolic volume, left ventricular remodeling is an important determinant of survival.
Current therapies to prevent or arrest left ventricular dilatation and hypertrophy following myocardial infarction include limitation of infarct size by early reperfusion, administration of angiotensin-converting enzyme inhibitors, new β-adrenergic blockers (e.g. carvedilol), and perhaps administration of new agents such as endothelin antagonists or cytokine antagonists such as vesnarinone. These interventions, however, are limited since they are unable to reconstruct the injured myocardium. In addition, these interventions should be started relatively early after myocardial infarction and are applied to selected populations only. Therefore, there is an urgent necessity to find new therapeutic strategies to repair irreversible and progressive myocardial damage.
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3 Gene transfer into the ischemic or infarcted myocardium |
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Top1 Introduction2 Post-infarction left...3 Gene transfer into...4 Molecular cardiomyoplasty:...5 Cell transplant6 Other strategies7 Unresolved issues and...8 Summary and future...References |
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The ability to perform gene transfer into the myocardium has only become feasible in recent years because of major advances in the field of molecular biology. The major advance has been the ability to isolate individual genes, or synthesize the analogous complementary DNA and to insert this DNA into plasmid expression vectors or recombinant viruses. The foreign DNA can then be introduced into the cell using a variety of delivery systems such as viruses or liposomes. Once the DNA enters the cell, it is taken to the nucleus where, depending on its mechanism of transport, it remains as an episome or becomes integrated into the host genome. Subsequently the foreign DNA is transcribed and the resulting mRNA is translated into the therapeutic protein.
Several strategies have been used for gene transfer into the myocardium (reviewed in [2, 3]) including direct injection of naked plasmid DNA [7], implantation of ex vivo genetically engineered transplanted cells, application of liposome–DNA complexes, or injection of recombinant viruses. The short-term results obtained by us and others have been encouraging.
One of the most promising techniques for gene transfer employs an adenovirus vector [8]. Although this vector delivers genes into the rat myocardium efficiently, there was a sharp decline in gene expression after 1 week associated with an inflammatory reaction in our studies [9]. This suggested an immunological mechanism behind this sharp decline. When we compared long-term results of adenovirus-mediated gene transfer into the myocardium between normal, immunocompetent rats and immunocompromised athymic nude rats, we found that the efficiency of recombinant gene expression assayed by production of a recombinant protein was sustained and vigorous in the immune-compromised rats, and that there was less inflammatory reaction. Our findings suggested that a host immune reaction against the adenovirus affects the expression of genes delivered by adenoviral vectors. In view of these results, we raised concerns about the feasibility of gene transfer into an ischemic or infarcted myocardium [9].
To begin to address this issue, Prentice et al. [10]subjected rats to 15 min or 60 min of coronary occlusion and 7 days of reperfusion, Fifteen minutes of ischemia does not cause cell death while 60 min produces myocardial necrosis. A DNA containing the luciferase reporter gene was injected into the ischemic area. Seven days after injection, significant levels of luciferase expression were obtained in hearts subjected to either 15 min or 60 min of ischemia. Thus, ischemic myocardium is capable of taking up and expressing foreign DNA.
In another series of experiments [10], we assessed the feasibility of using retroviruses to transduce myocardial scar tissue. Retroviruses are able to transduce only replicating cells. Thus, its efficacy for gene transfer into non-replicating adult cardiac myocytes is limited. However, granulation tissue after myocardial infarction is rich in proliferative fibroblasts, endothelial cells and macrophages. We hypothesized therefore that a retroviral vector could be used for in vivo gene transfer into granulation tissue. We used a canine model of myocardial infarction. Infarction was produced percutaneously by embolizing a helical coil into the left anterior descending coronary artery. After 6–11 days of coronary artery occlusion, retrovirus carrying the reporter gene β-galactosidase was injected directly into the infarcted area under direct visualization. After 7–12 days, hearts were examined for histology and the presence of the reporter protein by X-gal staining. Four out of five hearts with myocardial infarction that were injected with retrovirus stained positive for β-galactosidase. One dog that was not infarcted was negative for β-galactosidase. These experiments suggest that retroviral uptake, gene expression and recombinant protein expression are possible in the infarcted area and localize to the injection site in the vicinity of granulation tissue. Our findings suggest that ischemic or infarcted myocardium can be a target for gene transfer. These were the first studies showing that ischemic/reperfused myocardium is capable of taking up and expressing foreign genes.
Because of its high infectivity, adenovirus remains a promising tool for recombinant gene delivery to the myocardium. Based on our earlier studies [9], however, we hypothesized that following myocardial infarction, the efficiency of adenovirus-mediated gene transfer might be compromised by the intense inflammatory reaction in the necrotic area. To assess and measure the efficiency of adenovirus-mediated gene transfer and expression in infarcted myocardium, we produced myocardial infarction by subjecting rats to 60 min of coronary artery occlusion followed by sustained reperfusion [11]. Gene transfer into the infarcted area was performed using direct injection of a replication-defective adenovirus vector encoding the bacterial reporter gene, β-galactosidase. A total of 5.0x109 plaque-forming units of virus were injected into the infarcted myocardium either immediately or at 7, 22 or 30 days after reperfusion of rat hearts. Control rats received either saline or were not subjected to infarction and also received adenovirus carrying the β-galactosidase gene. All hearts were processed 7 days after cardiac injection and stained for β-galactosidase activity. Relative β-galactosidase activity was expressed as the % of the maximal area of β-galactosidase staining relative to the total area of the section examined (%±S.E.M.). The area of transgene expression in the non-infarcted hearts (28±7%) was significantly higher (p = 0.02) than at any time point studied in infarcted tissues (3.4±1.2%, 1.4±1.0%, 2.8±0.8% and 3.4±0.9% at reperfusion and at 7, 22 and 30 days after myocardial infarction, respectively (Fig. 1). β-Galactosidase gene expression was limited mainly to viable myocytes at the border of the myocardial infarction (Fig. 1 and Fig. 2). Hearts injected 7 days after infarction had significantly less transgene activity with 3 of 5 samples displaying no macroscopically visible β-gal activity. Following viral injection, an inflammatory response consisting of mononuclear cell infiltration was much less intense 7 days following injection in non-infarcted control rat hearts than at any of the time points examined for infarcted hearts. We concluded that gene transfer into infarcted myocardium, while feasible, was limited by low transfection efficiency when compared to non-infarcted normal myocardium. Still, the ability to introduce genes into viable peripheral cells might be a useful approach for enhancing neovascularization, collateral flow and healing [11]if expression of the transgene for a limited number of days might still lead to a therapeutic benefit.
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4 Molecular cardiomyoplasty: gene therapy with MyoD |
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Top1 Introduction2 Post-infarction left...3 Gene transfer into...4 Molecular cardiomyoplasty:...5 Cell transplant6 Other strategies7 Unresolved issues and...8 Summary and future...References |
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One of the more fascinating goals in the field of gene therapy for cardiovascular disease would be genetic manipulation leading to regeneration of myocytes after myocardial infarction. In skeletal muscle, the muscle-specific MyoD family of basic helix-loop-helix transcription factors functions as master genes that can induce the skeletal muscle differentiation program [12]. Most remarkably, members of the MyoD family can induce this program in a wide variety of cell types including fibroblasts.
Conversion of cardiac fibroblasts populating the scar of myocardial infarction into functional skeletal myocytes has the potential to contribute to cardiac contraction. Based on clinical experience with cardiomyoplasty in which the latissimus dorsi skeletal muscle was used to improve hemodynamic function in patients with heart failure [13], we hypothesized that MyoD-converted cells could be adapted to function as cardiac myocytes. Prentice et al. [14]investigated the feasibility of converting cardiac fibroblasts into skeletal muscle cells by forced expression of the MyoD gene. Fibroblasts isolated from rat hearts were infected with retrovirus – carrying the MyoD gene. Within a few days after transfection, fibroblasts became elongated and formed multinucleated myotubes morphologically resembling striated skeletal muscle myocytes (Fig. 3A,B). Immunofluorescent staining identified the presence of skeletal muscle-specific skeletal fast myosin heavy chain, protein markers of skeletal muscle differentiation (Fig. 3C,D). In similar experiments, Tam et al. [15]reported spontaneous contractions in a few clusters of converted myotubes.
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After proving the feasibility of forced expression of MyoD to convert fibroblasts into cells with a skeletal muscle phenotype in vitro, we used our canine model of myocardial infarction to evaluate the feasibility of this strategy in vivo. We produced myocardial infarction by percutaneous insertion of a helical coil into the left anterior descending coronary artery of dogs. Four infarcts were injected with β-galactosidase expressing retrovirus, carrying the regulatory gene MyoD, 6 or 10 days after occlusion. Seven to 12 days later, dogs were euthanized. Histochemical analysis with X-gal identified successful transfection in three hearts. In areas of positive β-galactosidase staining, specific antibody against skeletal fast myosin heavy chain identified rare cells that were stained positive for the myosin heavy chain (Fig. 4A,C). These cells were also stained positive for the muscle marker
-actinin (Fig. 4B,D). Our findings suggest that exogenous transfer of the MyoD gene can result in conversion of cardiac fibroblasts into cells which express skeletal fast myosin heavy chain. However, true morphologic myotubes were not observed. The extent of transfection and gene expression was limited and raised a doubt about the efficiency of this approach [14].
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In an attempt to test an alternative approach for MyoD gene transfer into myocardial infarction, we used the first-generation replication-defective adenovirus carrying another member of the MyoD family, the myogenic determination gene, myogenin. Similar to our previous studies with MyoD, we were able to convert fibroblasts to skeletal muscle cell phenotype in vitro (unpublished data). In the next experiment we injected adenovirus vector carrying either myogenin gene or reporter gene β-galactosidase into myocardial infarction of normal or athymic nude rats. Four days after injection, we detected islands of positive X-gal blue staining identifying successful transfection with β-galactosidase both in immunocompetent and immune-deficient nude rats. Immunofluorescent and immunohistochemistry staining for skeletal myosin heavy chain in those areas was negative (unpublished data). Morphologic skeletal myotubes were not observed. Based on these findings and our experiments on adenovirus-mediated gene transfer into myocardial infarction [11]we concluded that efficient gene transfer is feasible at the border of infarction but highly limited in the infarcted granulation tissue.
Recently, Murry et al. [16]reported successful transduction in vivo with MyoD gene transfer into myocardial granulation tissue of rats. They used a rat model of freeze–thaw myocardial injury. One week later, they injected 1010 pfu adenovirus carrying the MyoD gene or β-galactosidase. Rats were treated with cyclosporine to suppress the anticipated adenoviral immune response. One week after gene transfer, cells were identified that expressed both myogenin and embryonic skeletal myosin heavy chain. Northern blotting demonstrated MyoD mRNA in the injured regions of hearts receiving the MyoD virus, but not in control hearts receiving β-galactosidase. In several hearts they observed structures suggesting multinucleated myotubes. Similar to our studies [9, 11]Murry et al. [14]observed intense inflammatory reaction in the areas of high dose of adenovirus injections. The authors suggested that ‘MyoD gene transfer can induce skeletal muscle differentiation in healing heart lesions’. The authors did not evaluate the effect of this approach on the extent of myocardial injury or myocardial function. Their encouraging results need further investigation and confirmation.
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5 Cell transplant |
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Top1 Introduction2 Post-infarction left...3 Gene transfer into...4 Molecular cardiomyoplasty:...5 Cell transplant6 Other strategies7 Unresolved issues and...8 Summary and future...References |
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In view of the significant difficulties in delivering genes into infarcted myocardium [11], alternative approaches are warranted. A novel experimental strategy to increase viability and augment ventricular function after myocardial infarction is cell transplant [3]. This approach may provide a way to increase the number of functional cardiomyocytes in the infarcted myocardium [3]. In their pioneering studies, Field, Soonpaa, Koh and associates reported that fetal cardiomyocytes can be engrafted and integrated within the normal myocardium of mice [17]. Consequently, this group reported the formation of stable fetal cardiomyocyte grafts, as long as 10 weeks after engraftment, in the myocardium of dystrophic mice and dogs [18]. Whether this approach can be applied to the infarcted and failing myocardium is uncertain. In order to evaluate the feasibility of fetal myocardial tissue transplantation into myocardial infarction and whether the engrafted cardiomyocytes can survive, we subjected rats after myocardial infarction to 3 protocols of therapy [19]. In the first group, tissue fragments of cultured human fetal ventricles were injected into the scar 7–24 days after infarction. The rats were treated with intra-peritoneal injections of 12.5 mg/kg/day of cyclosporine. In the second protocol, fragments of cultured fetal rat ventricles were injected into the scar 9–17 days after infarction. A third group of myocardial infarction animals was treated with injection of saline into the scar (control). In protocol 1, engrafted tissue was detected in the infarcted myocardium, either by electron microscopy or
-actin staining, in 6 of 11 rats (55%) after 7 days and in 4 of 4 rats (100%) after 14 days. In the second protocol, electron microscopy or -actin staining detected the presence of engrafted fetal cardiac tissue 8 (1 of 2), 14 (1 of 2), 32 (1 of 1) and 65 days (2 of 2) after transplant. Overall, the graft survival rate in this protocol was 71%. The success of transplantation was independent of the timing after myocardial infarction. The engrafted fetal tissues stained for -actin (Fig. 5) which is unusual for the adult rat myocardium. In infarcted myocardium -actin staining was only positive within the vasculature.
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We were not able to prove that the implanted embryonic cardiomyocytes proliferated and differentiated. We did not assess the effect of the transplanted tissue on myocardial function. Thus, we concluded that fetal cardiomyocyte tissue can be implanted and survive in the infarcted myocardium. This experimental approach may eventually provide a therapeutic strategy for cardiomyocyte-based gene transfer for introduction of therapeutic proteins into myocardial infarction [19].
The study of Scorsin et al. [20]supported our findings, showing the feasibility of cardiac myocyte transplants into the border zone of myocardial infarction. In this model, cells were injected immediately after coronary artery occlusion. Cardiac myocytes were identified in the border zone of myocardial infarction 48 h after transplantation. In another preliminary report, Scorsin et al. [21]suggested that fetal cardiac myocytes injected into the border of myocardial infarction, created by 45 min of coronary artery occlusion, can improve myocardial function as assessed by two-dimensional echocardiography 1 month after myocardial infarction. The authors reported that compared with controls, cell transplant was associated with smaller infarcts. Our and other studies [19, 22], however, suggested that cell transfer into the necrotic myocardium is limited immediately after myocardial infarction, perhaps because of the intense inflammatory response following infarction. Li et al. [23]showed that fetal rat cardiac myocytes transplanted into the subcutaneous tissue of adult rat leg formed contractile cardiac tissue that contracted for at least 3 months. This provides hope for increasing the amount of contractile tissue in infarcted myocardium. In another experiment, Li et al. [24]used a rat model of myocardial cryoinjury. Four weeks after cryoinjury, cultured fetal rat cardiac myocytes or culture medium (control) were injected into the scar tissue. Four weeks after transplantation, scar size and heart function were measured. The authors found that scar and tissue in hearts subjected to cell transfer were smaller than controls. The transplanted cardiac myocytes had formed cardiac tissue that occupied 37% of the scar. Heart function as assessed with Langendorff preparation was better in the transplantation group than control. The authors suggested that cardiac myocytes formed cardiac tissue in myocardial scar and was associated with smaller infarct size, attenuation of infarct expansion and improved heart function compared with findings in the control hearts [24]. However, whether the implanted cells were directly contributing to regional contractions was not clear from either Li et al.'s studies [22–24]or that of Scorsin et al. [20, 21].
In a preliminary report, Mar et al. [25]showed that intracoronary or intra-pericardial delivery of cardiac myocytes is feasible in a canine model. Co-injection of basic fibroblast growth factor (bFGF) increased the efficiency of cell transplantation. This approach may open up new opportunities in the field of gene transfer into the myocardium [25].
Several mechanisms have been proposed for improved heart function following cardiac myocytes transplantation: direct contribution of the transplanted myocytes to contractility, attenuation of infarct expansion by the elastic properties of cardiac myocytes, angiogenesis induced by angiogenic factors secreted from the embryonic cells resulting in improved collateral flow, and augmentation of contractility by growth factor(s) secreted from the fetal cells. Based on our experience, we believe that attenuation of infarct expansion and secretion of therapeutic growth factors are the most likely mechanism rather than direct contribution of the transplanted cells to contractility. Such cellular grafting has emerged as a potential experimental approach for delivery of therapeutic peptides. The use of genetically modified cells for the delivery of recombinant molecules is a powerful approach for ex vivo gene transfer [18]. For example, the fetal graft could be engineered ex vivo to produce growth factors, as a useful strategy for enhancing angiogenesis to hasten myocardial healing and cellular viability.
An alternative strategy to increase the amount of contractile tissue in the infarcted myocardium is transplantation of skeletal muscle cells. There are several advantages of skeletal muscle cells which theoretically make them superior to cardiac myocytes for infarct repair. Unlike myocardium, skeletal muscle retains a regenerating capacity. Each mature skeletal myofiber bears a few myogenic cells known as satellite cells. These myogenic cells remain in an undifferentiated state. They are activated following skeletal injury, enter the mitotic cycle and later fuse with each other and with injured myofibers, thus restoring continuity and function of skeletal muscle. In addition, compared to cardiac myocytes, skeletal muscle cells are more resistant to ischemia.
Several groups have raised the hypothesis that transplanted myogenic cells obtained from skeletal muscle can undergo ‘milieu-influenced differentiation’ and thus become cardiac muscle cells. This phenomenon has been demonstrated during cardiomyoplasty in which the skeletal muscle latissimus dorsi is preconditioned with repeated electrical stimulation and converts from fast to slow (cardiac-like) twitch fibers [13]. The modified skeletal muscle is wrapped around the failing heart and serves to augment cardiac contraction.
Koh et al. [26]transplanted myoblasts from cell line C1C2 into hearts of normal synergistic mice. They showed that the engrafted myoblasts can differentiate into myotubes. The absence of tritiated thymidine incorporation suggests that the engrafted cell withdrew from the cell cycle. This group also demonstrated that C1C2 cells transfected with a plasmid encoding TGF-β could induce angiogenesis around the graft [18]. There was no coupling between the host cardiac myocytes and the engrafted skeletal cells [18].
Chiu et al. [27]showed that cultured satellite cells can be isolated from skeletal muscle of dog, can be implanted into the cryo-injured myocardium of the same dog and can differentiate into ‘cardiac-like’ myocytes and couple to each other by intracellular junctions resembling gap junctions or intercalated discs. The implanted cells were identified in the injured myocardium as late as 14 weeks. Yoon et al. [28]obtained similar results in a comparable model.
Most recently, Murry et al. [29]showed that neonatal skeletal myoblasts can be engrafted into cryo-injured rat heart. The engrafted myoblasts initially proliferate and after 3 days began to create multinucleated myotubes. The authors suggested that the myotubes differentiate into mature myofibers and later convert to slow twitch fibers (no longer expressing high level of fast myosin heavy chain). Furthermore, the authors suggested that the engrafted tissue could contract when stimulated ex vivo and perform cardiac-like contraction [29]. The authors concluded that skeletal myoblasts can establish new muscle tissue when grafted into injured hearts, and this new muscle may be suited to a cardiac work load.
Skeletal muscle cells, however, have electrophysiological properties that differ from cardiomyocytes. Thus, new muscle cells might create an arrhythmogenic substrate. This might also be a problem with engrafted cardiac myocytes. It is still uncertain whether the engrafted fetal cells can differentiate into mature functional cardiac myocytes that integrate with adjacent cells and enhance cardiac performance by directly contributing to contraction. Optimally, newly formed skeletal muscle within an infarcted scar should undergo electrical and mechanical adaptation and coupling to adjacent viable cardiac myocytes to provide synchronized mechanical support. This is a significant limitation, since skeletal myocytes do not synthesize gap junction proteins. It is possible that in the future, these cells could be engineered to produce these proteins.
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6 Other strategies |
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Top1 Introduction2 Post-infarction left...3 Gene transfer into...4 Molecular cardiomyoplasty:...5 Cell transplant6 Other strategies7 Unresolved issues and...8 Summary and future...References |
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Alternative strategies to ‘rejuvenate’ the heart include attempts to reverse the terminal differentiation of adult cardiac myocytes and to induce hyperplastic growth in existing cardiac myocytes [30, 31]. Another approach is to attempt to induce proliferation of cardiac myocytes by introducing genes that relieve cell cycle suppression. It has been shown that transfection with genes such as the SV40 large T antigen [32]can induce cardiac myocyte proliferation in embryonic and neonatal culture. Several studies suggested that expression of such genes can in fact induce proliferation of fetal or neonatal cells. Similar experiments using adult cardiac myocytes, however, failed to induce proliferation.
Another effort is to produce a permanent cardiac myocyte cell line that can be used as a resource of cell transplants. Klung et al. [33]recently reported generation of culture of cardiac myocytes from murine multipotent embryonic stem cells. They transfected into embryonic stem cells a fusion gene consisting of -cardiac myosin heavy chain promotor and a cDNA encoding for aminoglycoside phosphotransferase. They suggested that genetic manipulation can be used to select cardiac myocytes from differentiating embryonic stem cells. They demonstrated that these stem cell-derived cardiac myocytes were suitable for cardiac transplantation and formation of intracardiac grafts [33].
Down-regulation of β2-adrenoceptors and up-regulation of the β-adrenergic receptor kinase are among the abnormalities of the β-adrenergic signaling pathway associated with congestive heart failure. Thus, modulation of this pathway to improve cardiac contractility can be a new therapeutic approach to congestive heart failure. Lefkowitz and associates raised and tested the hypothesis that genetic modulation of the β-adrenergic signaling pathway can enhance cardiac function [34]. They showed that transgenic mice with cardiac overexpression of either the human β2-adrenergic receptor [34]or an inhibitor of β-adrenergic receptor kinase [35], an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial contractility. Most recently, they showed that gene transfer with adenovirus encoding either the β2-adrenoceptor or a peptide β-adrenergic receptor kinase enhances β-adrenergic signaling in cultured cardiac myocytes [36].
Finally, recent interest in growth factors as an alternative therapeutic strategy to alter the contractile properties of cardiomyocytes and to improve myocardial function and viability opens new frontiers in cardiovascular therapy (reviewed in [37]). Growth factors can provide adjuvant therapy with direct myocardial protection and increase the viability and function of the ischemic myocardium. The ability to introduce growth factor genes into vessel walls may be a useful strategy for therapeutic angiogenesis and prevention of restenosis, as well as to enhance healing and viability after vascular injury. Although growth factors have not yet become part of clinical practice, they have attracted a great deal of interest and are involved in several preliminary clinical trials [37].
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7 Unresolved issues and challenges |
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Top1 Introduction2 Post-infarction left...3 Gene transfer into...4 Molecular cardiomyoplasty:...5 Cell transplant6 Other strategies7 Unresolved issues and...8 Summary and future...References |
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Many challenges need to be overcome before MyoD gene therapy or cell transplant can be considered an efficient therapeutic tool for myocardial repair and regeneration. The efficacy of gene transfer into myocardial infarction needs considerable improvement. Better methods are needed to avoid the toxicity associated with injection of high titers of adenovirus and the immune reaction against the virus or the exogenous gene. The frequency of MyoD-converted cells in vivo is low and in our studies we were unable to document the formation of true myotubes in the in vivo setting. Better vectors or more potent master genes are needed.
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8 Summary and future research |
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Top1 Introduction2 Post-infarction left...3 Gene transfer into...4 Molecular cardiomyoplasty:...5 Cell transplant6 Other strategies7 Unresolved issues and...8 Summary and future...References |
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We reviewed novel experimental cardiac repair strategies: genetic modulation and cell transplant. These experimental strategies are attractive and some day may overcome many limitations of current therapies for heart failure after myocardial infarction.
Other experimental strategies for myocardial regeneration should be tested. These include attempts to produce hyperplastic growth of cardiac myocytes [30, 31], and to create a permanent cardiac myocyte cell line as a source of donor cells for transplant [33]. Another approach involves growth factor therapy for augmentation of myocardial healing, contractility, collateral circulation and viability. Genetic modulation of the β-adrenergic signaling cascade is another experimental strategy to enhance cardiac function. With the advances in our knowledge of the regulation of cardiac myosin isoforms, cloning of - and β-myosin heavy chain genes and new gene transfer techniques it may become possible to modulate cardiac myosin isoforms and to replace low activity V3 myosin with the high activity V1 isoform [38]. Stimulation of other genes under T3 control such as Na/K-ATPase or Ca2+-ATPAse of the sarcoplasmic reticulum might improve contractility of the failing myocardium [39]. Most recently, loss of cardiac myocytes due to programmed cell death (apoptosis) has been recognized as an important process that causes loss of cardiac myocytes and contributes to development of cardiac failure [40]. Prevention of apoptosis may become a new research target for prevention of cardiac myocyte loss and progressive myocardial dysfunction [41].
In summary, genetic modulation and cell transplant provide a revolutionary experimental approach for cellular cardiomyoplasty and tissue repair after myocardial injury. Current data derived from animal models suggest that it may be possible to treat heart failure by transferring genetic materials or cardiac myocytes into injured myocardium. These strategies, however, still face significant hurdles before they can become established therapeutic approaches. The progress of molecular medicine, the development of improved systems for in vivo gene transfer and cloning of new disease-related genes, give hope that gene therapy and cell transfer will one day evolve as a therapeutic tool to repair a ‘broken heart’.
Time for primary review 22 days.
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Acknowledgements |
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This work was supported in part by grant No. 95-00294/1 from the United States–Israel Binational Science Foundation (BSF).
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