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Eur J Cardiothorac Surg 2003;23:907-916
© 2003 Elsevier Science NL

Review

Cell transplantation to improve ventricular function in the failing heart

Gilbert H.L. Tang, Paul W.M. Fedak, Terrence M. Yau*, Richard D. Weisel, Alex Kulik, Donald A.G. Mickle, Ren-Ke Li

Division of Cardiovascular Surgery, Toronto General Hospital, Department of Surgery, University of Toronto, Toronto, Ontario Canada

Received 13 December 2002; received in revised form 26 February 2003; accepted 27 February 2003.

* Corresponding author. 13EN-239, Toronto General Hospital, 200 Elizabeth Street, Toronto, Ontario Canada M5G 2C4. Tel.: +1-416-340-4074; fax: +1-416-340-3803
e-mail: terry.yau{at}utoronto.ca


   Abstract
Top
 Abstract
1. Introduction
 2. Assessment of ventricular...
 3. Effect of cell...
 4. Implications and future...
 5. Conclusions
 References
 
Current therapies for congestive heart failure are limited in efficacy or in applicability. Cardiac cell transplantation offers a novel therapeutic approach to improve heart function. Although significant progress has been made over the past decade in the development of cell transplantation, only recently have investigators studied the changes in ventricular function following cell transplantation. This review article describes the latest research developments, evaluates recent studies of ventricular function after cell transplantation, and discusses the future directions of cell transplantation as a new therapy to ‘repair broken hearts’.

Key Words: Cell transplantation • Ventricular function • Congestive heart failure


   1. Introduction
Top
 Abstract
 1. Introduction
2. Assessment of ventricular...
 3. Effect of cell...
 4. Implications and future...
 5. Conclusions
 References
 
Congestive heart failure is a major cause of morbidity and mortality in developed countries [1]. Despite ongoing advances in medical therapy and surgical intervention to improve ventricular function, the prognosis of heart failure remains grim. Cell transplantation has been proposed as a novel therapy for congestive heart failure. Termed ‘cellular cardiomyoplasty’ by Chiu and colleagues, cells of myogenic ancestry or with the capacity to differentiate into myocytes can be transplanted into infarcted regions of the heart to prevent heart failure [2]. The transplanted cells may limit scar expansion and ventricular dilatation, and potentially increase regional contractility, thereby improving ventricular function. Recently, Anversa and colleagues have presented some intriguing preliminary evidence to suggest that the heart may be able to repair itself with resident cardiac stem cells and/or circulating bone marrow cells (BMCs), highlighting the possible utility of cell transplantation in treating heart failure [3]. The first clinical application of cardiac cell therapy was reported by Menasche and co-workers in a Phase I trial of skeletal myoblast transplantation [4]. All ten patients had evidence of cell engraftment with improvements in perfusion as well as regional and global function. These encouraging early results will warrant careful assessment in a Phase II trial.

The optimal design of clinical trials of cell transplantation will require a clear understanding of how implanted cells improve ventricular function. Over the past decade, research in cardiac cell transplantation has focused on studies of primarily histological endpoints. More recently, greater attention has been focused on the functional outcomes of cell transplantation. Although no review can be completely comprehensive in this intensely investigated area, we have attempted to describe and evaluate studies of ventricular function after cell transplantation, and to discuss the likely future directions in cardiac cell therapy.


   2. Assessment of ventricular function
Top
 Abstract
 1. Introduction
 2. Assessment of ventricular...
3. Effect of cell...
 4. Implications and future...
 5. Conclusions
 References
 
Current methods to study cardiac function involve both non-invasive and invasive approaches. To evaluate the effect of cell transplantation, attempts have been made to assess both global and regional function using different techniques. Each has its own merits and limits and will be discussed in brief.

2.1. Echocardiography
Echocardiography has the advantage of allowing non-invasive in vivo assessment of global ventricular function. End-systolic and end-diastolic dimensions are used to calculate the left ventricular end-diastolic volumes (LVEDV), end-systolic volumes (LVESV) and ejection fraction (EF). In cell transplantation experiments, however, the technique has some limitations. Measurements from M-mode tracings are taken from a limited view of the heart, which may lead to inaccuracies in assessing cardiac dimensions and performance. The alternative of using the single-plane method for volume calculations may underestimate the extent of infarction in a regional dysfunction model. More importantly, volume measurements in 2D echocardiography are based on an elliptical model of left ventricular geometry. While this simplification may be valid for studies of global heart failure such as in dilated cardiomyopathy, it may be less appropriate for myocardial infarctions (MI) where the left ventricle is not of uniform shape. To study regional function, acoustic quantification with color kinesis and tissue Doppler imaging (TDI) have been employed [5,6]. Assessment of diastolic performance with echocardiography is possible using Doppler or volume measurements, but these estimates have not been consistent in the setting of cell transplantation [7].

2.2. 99mTc-sestamibi single photon emission computed tomography
In a myocardial injury model, single photon emission computed tomography (SPECT) is a useful non-invasive approach to study global and regional function. It also provides information on myocardial perfusion, viability and wall thickness (Fig. 1 ). The advantage of SPECT methoxyisobutyl isonitrile (MIBI) over echocardiography is that SPECT does not make assumptions about left ventricular geometry, an important advantage when regional variability in function and perfusion are to be expected, as in MI.



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  		Fig. 1. Representative 3D views of reconstructed hearts from gated SPECT MIBI scans. The animals from control and transplanted groups were scanned 4 weeks after cell transplantation. From Tomita et al. [32].

 
The use of SPECT to study regional function may be limited by several factors. The finite resolution of the imaging system may underestimate count density from a structure that is too thin to be detected. A myocardial perfusion defect may be heterogeneous even when the distribution of 99mTc-sestamibi activity is uniform. Heart rate and contractility may play a role in the quantification of myocardial tracer concentration, affecting measurements of regional parameters. In our cell transplantation experiments, a semiquantitative method was used to grade regional parameters such as perfusion and wall motion, because absolute quantification is difficult to achieve. Also, SPECT was performed weeks after the initial infarct, thus allowing time to develop a permanent defect and minimize potential artifacts. Nevertheless, both echocardiography and SPECT MIBI are limited by the critical dependence of measured parameters on heart rate, preload, and afterload.

2.3. Conductance
To obtain load-insensitive measurements of cardiac function, more invasive approaches may be helpful. A conductance catheter and micromanometer can be inserted into the left ventricular apex, and pressure–volume relations measured at baseline and during caval occlusion (Fig. 2 ). Hemodynamic parameters including preload recruitable stroke work (PRSW)1, end-systolic elastance (Ees)2, and the end-diastolic pressure–volume relation can be derived from the resultant pressure–volume loops [8–10]. Parallel conductance is estimated by the hypertonic saline method and may affect the accuracy of volume measurements. In addition, the technique is unable to assess regional function after a MI.



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  		Fig. 2. Representative post-transplantation pressure–volume relations generated by conductance micromanometry. Following coil embolization of the left anterior descending coronary artery, this pig received autologous heart cell transplantation and had an improvement in ventricular function.

 
2.4. Sonomicrometry
Sonomicrometry can accurately quantify regional ventricular performance to evaluate the effect of cell transplantation in infarcted hearts. One or more pairs of ultrasonic transducers connected to a sonomicrometer are placed on the epicardial surface of the heart around the area of interest and a micromanometer is positioned in the left ventricular cavity. Regional dimensions and left ventricular pressure are measured at baseline and during inferior vena caval occlusion (Fig. 3 ). Based on a model evaluating global ventricular function, Atkins and colleagues reported the derivation of load-insensitive parameters of regional systolic and diastolic function, including PRSW, pressure–strain relation3, diastolic creep4, static5, and dynamic stiffness6 for cell transplantation studies (Fig. 4 ) [11]. Sonomicrometry is invasive and any calculation of systolic parameters such as regional PRSW assumes a normal, non-ischemic myocardial physiology [12]. Placement of the ultrasonic transducers is critical to measurement accuracy and may induce regional injury. To evaluate global function, serial placements of crystals or leaving them in situ may be necessary to determine chamber dimensions and extrapolation may be needed to yield global parameters. However, this approach often assumes an elliptical model of global LV geometry and may not be accurate in models of ischemia. In an attempt to overcome this limitation, DeAnda and co-workers employed myocardial markers and proposed a regional model to determine wall stress and global systolic performance [13].



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  		Fig. 3. Surgical preparation for sonomicrometry, showing ultrasonic dimension transducers on either side of the cryoinjured region of the anterolateral LV. A Fogarty balloon catheter is placed in the inferior vena cava at the time of data acquisition to allow controlled variation in preload. Inset: data acquisition configuration with LV intracavitary pressure transducer. From Atkins et al. [11].

 


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  		Fig. 4. Typical pressure (P) vs. EDSL curves post-cryoinjury (CRYO) and post-cellular cardiomyoplasty (CCM) with either skeletal myoblasts (Mb) or fibroblasts (Fb) showing dP/dL8 for comparison of dynamic stiffness between groups. From Hutcheson et al. [34].

 
2.5. Langendorff apparatus
Technical limitations may preclude the use of conductance catheter, sonomicrometry, or myocardial markers for evaluation of function in very small animals. A Langendorff preparation has frequently been employed as an alternative approach to evaluate global function in cell transplantation studies. Our group has employed the non-ejecting mode of the Langendorff preparation to study cell transplantation in rats. A balloon is passed into the left ventricle and hemodynamic parameters are measured ex vivo with each increment in balloon volume. Heart rate, systolic and diastolic left ventricular pressures, and maximal rate of change in left ventricular pressure (+dP/dt and -dP/dt) can be determined. Developed pressure, a measure of contractility, is calculated as the difference between systolic and diastolic pressures. Measurement of the passive diastolic pressure–volume relation is also possible with the heart arrested in diastole. The technique has the major advantage of controlling preload, afterload, heart rate, and perfusion pressure, which is very difficult in vivo in small animal models. However, the isolated heart may be subject to varying degrees of ischemia. In addition, hemodynamic parameters derived from specific ventricular volumes are not physiologic or load-insensitive. To overcome the above limitations in very small animals, Muller-Ehmsen and colleagues performed LV angiography and obtained similar but load-sensitive measurements in vivo to determine EF and segmental wall motion abnormalities [14].


   3. Effect of cell transplantation on ventricular function
Top
 Abstract
 1. Introduction
 2. Assessment of ventricular...
 3. Effect of cell...
4. Implications and future...
 5. Conclusions
 References
 
3.1. Studies of ventricular function in cardiac cell transplantation
A review of the current literature reveals over 30 published studies on the effect of cell transplantation on ventricular function in failing hearts. Allogeneic and autologous transplantation of skeletal myoblasts, smooth muscle cells, heart cells, and BMCs in various animal models have been reported. Both intramyocardial injection and intracoronary infusion have been employed to deliver cells to the infarct and peri-infarct zone, as well as to cardiomyopathic or normal hearts. Global and regional ventricular functions were evaluated either ex vivo or in vivo using the Langendorff apparatus, echocardiography, SPECT MIBI, conductance, or sonomicrometry. Most studies reported significantly improved systolic and/or diastolic function after cell transplantation in injured hearts. Representative studies are included in Tables 1–4 for reference, categorized by cell type and the animal model.


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  		Table 1. Representative studies on function using myogenic cells in a regional infarct model

 

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  		Table 2. Representative studies on function using BMCs in a regional infarct model

 

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  		Table 3. Representative studies on function comparing different cell types and using non-myogenic cells in a regional infarct model

 

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  		Table 4. Representative studies on function using myogenic cells in a global heart failure model

 
3.2. Studies of ventricular function in a regional infarct model
Animal models of regional ventricular dysfunction have utilized either cryoinjury or coronary occlusion to induce a focal injury. Cryoinjury results in a uniform, transmural scar and has the advantage of greater reproducibility, while coronary occlusion results in a more clinically relevant but variable infarct zone. A number of cell types have been assessed for their effect on ventricular function after transplantation.

3.2.1. Myogenic cells
(Table 1) Transplantation of allogeneic or autologous fetal, neonatal and adult cardiomyocytes, skeletal myoblasts, and smooth muscle cells has demonstrated improvement in systolic and/or diastolic performance compared to untreated controls. This effect was seen as early as 2 weeks after cell implantation, lasting up to 3–6 weeks, and in one study, as long as 6 months. The magnitude of improvement varied depending on the animal model and cell type but was generally significant. A majority of studies involved small animals and used echocardiography, a Langendorff apparatus or sonomicrometry to evaluate cardiac function. In studies using echocardiography, an increase of 20–35% in EF over the untreated group (control) was observed. With the Langendorff perfusion model, developed pressures and in some cases diastolic pressures were found to be significantly higher in the cell-treated hearts, although 20–35% lower than those of the sham-operated ones. Using LV angiography, a 10% absolute increase in EF and an improvement in regional wall motion with less dyskinesis were observed in the cell-treated group [14]. Atkins and colleagues demonstrated that skeletal myoblast-implanted rabbit hearts had significantly improved regional diastolic performance. One of their studies reported that cell-transplanted rabbits had a 66% increase in strain and 50% decrease in dynamic stiffness, compared to those without treatment [11]. The cell-treated animals also had preservation of static stiffness and diastolic creep, hence, maintaining regional geometry and improving compliance. We have transplanted autologous heart cells into infarcted porcine hearts and demonstrated an improvement in global ventricular function, including a 25% increase in PRSW compared to the control group [26]. SPECT MIBI also demonstrated greater regional wall motion and perfusion in the cell-treated animals. However, our coil occlusion model was limited by the variability of the induced infarct size. Smaller infarcts causing less severe LV dysfunction masked the effects of cell transplantation, while larger infarcts significantly increased mortality.

Of note, one study reported non-significant changes in function following cell transplantation. Using echocardiography, Etzion and co-workers did not find a significant increase in cardiac output 8 weeks following embryonic heart cell transplantation in rats, even though there was evidence of prevention of ventricular remodeling [21]. The non-significant improvement in function was partly attributed to the failure of myocytes to differentiate into mature myocardium during the study. In addition, the cell type, number of cells injected, and timing of implantation after the initial injury may be crucial determinants of the magnitude of the effect on ventricular remodeling and function.

3.2.2. Bone marrow cells
(Table 2) Recently, BMCs have undergone investigation as a potential donor cell type for cardiac cell therapy. Using a small animal model, Orlic and colleagues transplanted allogeneic adult murine Lin(-) c-kit(+) BMCs into viable myocardium adjacent to the infarct area and reported a significant improvement in systolic function [29]. Developed pressures in the BMC-transplanted mice were 33% higher than those in untreated mice, but 30% less than values in sham-operated animals. Transplanted cells occupied up to 68% of the infarct zone 9 days after the initial injection. Tomita and co-workers transplanted autologous 5-azacytidine-pretreated BMCs into porcine hearts and showed a significant improvement in PRSW, as well as regional perfusion, wall thickness and motion [32].

3.3. Which cell types improve function following an infarct?
(Table 3) Comparative studies with different cell types suggested that cells with myogenic properties are necessary to improve systolic function in a myocardial infarct model. Scorsin and colleagues reported a similar efficacy of cardiomyocyte and skeletal myoblast transplantation [35]. Hutcheson and co-workers demonstrated that while both skeletal myoblasts and fibroblasts improved diastolic function following cryoinjury in rabbit hearts, systolic improvement was observed only with skeletal myoblast transplantation [34]. Similar results from our group demonstrated that transplanted cardiomyocytes were superior to smooth muscle cells and fibroblasts in improving systolic and diastolic properties [33]. Endothelial cells induced regional angiogenesis but no improvement in ventricular performance [36]. We further showed that autologous transplantation of 5-azacytidine-pretreated BMCs and adult heart cells improved ventricular function, with BMCs demonstrating greater plasticity in in vivo differentiation into vascular progenitor cells [37]. These findings suggest that both contractile and elastic properties are important for implanted cells to improve function in damaged hearts.

3.4. Improvement of ventricular function in a global heart failure model
(Table 4) Systolic and diastolic LV function in both dilated cardiomyopathy and doxorubicin-induced heart failure have been found to improve significantly following skeletal myoblast, heart cell, and smooth muscle cell transplantation in small animals. In Scorsin's study, however, sex-mismatched engrafted fetal cardiomyocytes were not identified by Y-chromosome staining, and it was not possible to conclude that the improvement in function was due to engraftment of the transplanted cells [38]. Because of variations in experimental design and methodology, it is difficult to determine from current studies whether cell therapy results in a similar extent of improvement in regional vs. global left ventricular dysfunction. Further investigations will be necessary to compare the effect of cell transplantation in these two models of cardiac failure.

3.5. Is cardiac cell transplantation safe?
Although cell transplantation has consistently demonstrated improvement in ventricular function irrespective of the mode of injury, reports of its effect on mortality rate are rare and variable. Most published studies found a 15–30% mortality rate following myocardial injury, with two studies reporting no difference attributable to transplantation. Roell and co-workers recently demonstrated a clear short-term survival benefit in the transplantation of embryonic cardiomyocytes into cryoinjured myocardium [25]. A large group of mice (N=153) was studied and the cryoinjured, cell-treated group had no significant difference in survival when compared to the sham-operated, cell-treated group. More studies will be needed to evaluate the longer-term survival benefit of cell transplantation under varying circumstances. For now, it appears to have at least no detrimental effect on animals in these short-term studies.

3.6. Improvement of ventricular function in initial clinical experience
Most recently, cardiac cell therapy has made the transition from a laboratory to a clinical setting. Menasche et al. reported the first clinical application of autologous skeletal myoblast transplantation in a patient suffering from NYHA class III heart failure after a previous MI [4]. Autologous cells were injected in and around the infarct zone at the time of coronary artery bypass grafting (CABG) surgery. Significant improvement in both clinical symptoms and EF was observed 5 months post-operatively. The combined CABG-cell transplantation procedure was subsequently performed in ten patients in the Phase I trial. Although these results may be confounded by the effects of simultaneous coronary revascularization, 2 to 10-month post-operative follow-up demonstrated contractility of the previously akinetic, cell-transplanted area, with new-onset metabolic activity [42]. This finding has spurred ongoing investigation in skeletal myoblast transplantation as an adjunctive therapy for ischemic heart failure, and a randomized multi-center Phase II trial is currently under way.

Autologous BMCs have also been used clinically in post-MI patients. Strauer and co-workers reported that ten patients had improved regional perfusion, wall motion, and stroke volume index at 3 months after intracoronary infusion of mononuclear BMCs during percutaneous transluminal coronary angioplasty (PTCA) [43]. In a study by Assmus and colleagues, intracoronary infusion of either bone marrow-derived or circulating blood-derived progenitor cells during PTCA was associated with improved EF, regional wall motion, and myocardial viability during a 4-month follow-up [44]. Interestingly, there was no difference in benefit between the two types of progenitor cells. Stamm and colleagues injected mononuclear BMCs along the infarct border in six patients during CABG surgery and found significant improvements in EF and regional perfusion at 3–10 months post-treatment [45]. These Phase I trial results have so far demonstrated the safety and feasibility of cell transplantation as an adjunctive therapy to standard revascularization procedures. However, with a relatively small sample size and a lack of non-randomized controls, the clinical efficacy of cell therapy should be interpreted with some caution, as we await the results of larger, randomized multi-center trials.

In the initial trial of myoblast transplantation by Menasche and co-workers, four of the ten patients developed non-sustained ventricular tachycardias. A number of patients had an implantable defibrillator placed to protect against further arrhythmias [46]. Interestingly, cultured cardiomyocytes derived from pluripotent embryonic stem cells had been shown to possess arrhythmogenic properties [47]. It is, therefore, possible that engrafted cells of myogenic properties may act as an arrhythmogenic source, and the arrhythmias seen in the myoblast clinical trial could be a potential side effect of cell therapy. Further studies will be required to determine the influence of cell transplantation on arrhythmias.


   4. Implications and future research
Top
 Abstract
 1. Introduction
 2. Assessment of ventricular...
 3. Effect of cell...
 4. Implications and future...
5. Conclusions
 References
 
Although cell transplantation has thus far demonstrated fairly consistent improvement in function in failing hearts, several issues still need to be addressed in future studies. They include: (1) method of myocardial injury and evaluation of function, (2) choice of cell type, (3) technical refinements, and (4) mechanisms of benefit.

4.1. Method of myocardial injury and evaluation of function
Although cryoinjury is a reliable technique to create a MI in laboratory studies, the coronary occlusion method has greater clinical relevance. Given its similarity to humans in coronary anatomy and cardiac physiology, the porcine model may be most suitable for the evaluation of function and potential side effects of cell transplantation (e.g. ventricular arrhythmias). Sonomicrometry and conductance are currently the most accurate load-insensitive methods in the respective evaluation of global and regional ventricular function, but further refinements may increase their utility.

4.2. Choice of cell type
Allotransplantation requires immunosuppression to prevent rejection of the engrafted cells. Unresolved ethical issues surrounding the use of fetal cells make them unlikely candidates for cell transplantation. Autotransplantation of skeletal myoblasts alleviates this problem, and these cells have been shown to proliferate in infarcted myocardium and improve ventricular function. Because the number of satellite cells in normal skeletal muscle is low, the isolation and culture of a sufficient number of cells for transplantation have required careful optimization. The issue of whether the engrafted skeletal myoblasts communicate with the host myocardium is yet to be settled, due to the lack of gap junction formation between transplanted and native myocytes. Despite controversial suggestions that implanted skeletal myoblasts may transdifferentiate into a cardiac phenotype, such a process has not been observed in a labeling study by Reinecke and co-workers [48]. Nonetheless, the engrafted cells may contract in the presence of a non-electrical stimulus.

Ventricular and atrial heart cells have many characteristics of cardiomyocytes but do not beat spontaneously in situ. BMCs may offer an ideal alternative source of cells for transplantation. Cells can be easily aspirated, isolated, and rapidly expanded in culture. Although studies are not yet conclusive, intriguing data suggest that BMCs may differentiate into cardiomyocyte-like cells in vitro and in vivo after autologous transplantation given appropriate environmental cues [32]. In addition to myogenesis, BMCs may have the capacity to differentiate into vascular cells, such as smooth muscle and endothelial cells that are crucial components in angiogenesis and vasculogenesis. This greater plasticity makes BMCs promising candidates for optimal myocardial repair. Non-hematopoietic stem cells, such as those of cardiac or embryonic origin, may offer similar efficacy in repairing the damaged heart [44].

Although implanted BMCs appear to improve post-MI function, histological evidence is as yet inconclusive and their benefit may not be entirely due to cells of myogenic properties. Technical limitations and methodological heterogeneity may account for the uncertainty surrounding the transdifferentation of engrafted cells into other cell types, particularly cardiomyocytes. Spontaneous cell fusion, as suggested by Terada and co-workers, may also explain these observations [49]. The overall picture suggests that the synergistic effect of various cell types (myocytes, vascular smooth muscle, and endothelial cells) may provide the optimal benefit in ventricular remodeling and improvement in function in failing hearts. It is important to note that regardless of the cell type used for implantation, none has thus far been consistently shown to beat synchronously with the host myocardium. Isolated clusters of engrafted myogenic cells may, therefore, become a potential source of arrhythmogenesis. Future studies will be necessary to ensure that this therapeutic technique is safe and effective.

4.3. Technical refinements
There have been few investigations to determine the optimal timing of cell transplantation after a MI. Results from the above animal studies as well as a recent study by our group have suggested that cell treatment may be more beneficial after the acute inflammatory process has subsided but prior to the active phase of ventricular remodeling [50]. However, in two clinical trials using BMCs, a benefit in function was seen with transplantation occurring as early as hours after the infarct [43,44], and in the study by Orlic et al. transdifferentiation of BMCs to various phenotypes was also reported [29]. Given the pathophysiologic evolution of a MI, it is likely that the timing of BMC transplantation will have an effect on transdifferentiation in vivo. If transdifferentiation is ‘milieu dependent’ as some suggest, undifferentiated BMCs injected into scar may transdifferentiate into scar (fibroblasts) whereas early injection into an infarct zone with viable myocardium may induce the BMCs to undergo a transformation into muscle-like cells and therefore, contribute to myogenesis. BMCs may also respond to the host inflammatory response differently from myogenic cells and different cell types may have different optimal time periods of implantation to exert maximal benefit. Further studies will be necessary to resolve these uncertainties.

In addition to timing, the number of cells required to exert the optimal therapeutic effect has not been elucidated, although a recent study by Pouzet and colleagues demonstrated a linear relationship between improvement in EF and the number of autologous skeletal myoblasts injected into the infarcted area [23]. The same group also reported that pre-harvest muscle conditioning with bupivacaine significantly enhanced baseline myoblast cell yield prior to implantation, but cell culture remained essential for myoblast transplantation to be successful in rats with infarcted hearts [20]. Implanting a larger number of cells may have diminishing returns, however, as Muller-Ehmsen and co-workers showed a significant loss occurred within 24 h and a further loss between 4 and 12 weeks [51]. The authors attributed the initial loss to technical limitations and the latter loss to cell death. Further refinement in delivery technique and cell preconditioning will be necessary for future clinical applications. Muller-Ehmsen and colleagues also performed a longer-term study of cardiac cell transplantation and found a significant improvement in EF and decrease in infarct zone dyskinesis 6 months post-transplant, with survival of engrafted neonatal cardiomyocytes [23]. Similar results were observed by Ghostine et al. in a 1-year study on skeletal myoblast transplantation [52]. Continuing advances in cardiac cell transplantation suggest a long-term benefit of the novel therapy.

4.4. Mechanisms of benefit
With cardiac cell therapy now undergoing initial clinical application, understanding the mechanism of benefit is mandatory to optimize clinical trial design. There are at least five theories of how cell transplantation may affect the improvement in ventricular function that have been reported in multiple studies. First, contraction of the engrafted cells within the host myocardium may improve systolic function. Second, the augmented elastic properties of the transplanted region may improve regional diastolic function, limiting scar expansion and regional dilatation. Third, the engrafted cells may stimulate angiogenesis and provide a substrate for new vessel formation resulting in improved regional myocardial perfusion and increased contractility from hibernating myocardium. Fourth, the implanted cells may lead to scar stiffening and thickening, reducing wall stress, and preventing further cardiac decompensation. Finally, donor cell secretion of growth factors or cell-signaling peptides may limit both regional and global left ventricular remodeling. It is possible that donor cells may play a critical role in supporting the contractile apparatus and modulating the key events of cardiac remodeling including cell death, hypertrophy, and extracellular matrix reorganization, thereby preventing or delaying progressive ventricular dilatation and failure. There have been a number of investigations of these hypotheses, and future findings will likely elucidate the molecular mechanisms behind the benefit of cell transplantation.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Assessment of ventricular...
 3. Effect of cell...
 4. Implications and future...
 5. Conclusions
References
 
Current medical and surgical therapies have not solved the problem of heart failure. Cardiac cell transplantation represents a novel therapeutic option for patients with end-stage heart failure. Despite the limitations of different models and techniques in various studies, most reports have demonstrated significant improvement in systolic and diastolic function after cell transplantation into injured myocardium or in a global heart failure model.

Autologous cell transplantation is currently being evaluated clinically and offers the promise of a unique biological approach for myocardial revascularization and repair. Despite the large body of experimental data indicating its effectiveness, many issues will require resolution in the future. First, while preliminary clinical data suggests that cell transplantation is safe, the arrhythmogenic potential of this therapy must be rigorously investigated. Second, the optimal cell type, number, and timing of injection for a particular clinical problem remain to be determined. Third, the underlying mechanisms that lead to the benefits observed after cell transplantation need to be outlined and optimized. Notwithstanding these important challenges, cell transplantation for cardiac disease may offer a novel and effective therapeutic approach for patients with or at risk of heart failure.


   Footnotes
 
1 PRSW is represented by the slope of the linear relationship between stroke work (SW, defined as the area within a pressure–volume loop) and end-diastolic volume (EDV). The slope of the line (more than its position) reflects changes in contractility, which incorporate preload and afterload and are insensitive to heart rate and chamber size. An increase in slope indicates better ventricular contractility. Back

2 End-systolic elastance (Ees) is the slope of the linear relationship between end-systolic pressure (ESP) and end-systolic volume (ESV). Both the position and slope reflect ventricular contractile state insensitive to changes in heart rate, preload, and afterload. A left shift in the line's position or an increase in slope indicates better ventricular contractility. Emax (the slope of the linear relationship between maximal systolic pressure and ESV) very closely approximates Ees, and was used in our experiments to reflect Ees. Back

3 Regional strain ({varepsilon}) is the ratio of change in end-diastolic segment length (EDSL) to minimum EDSL. A greater strain corresponds to a greater change in EDSL ({Delta}EDSL) and indicates greater regional wall movement. In the pressure–strain curvilinear relation, a rightward shift or increased strain indicates increased diastolic compliance. Back

4 Diastolic creep is a sensitive indicator of myocardial injury and is measured by the minimal segment length (EDSL0). An increase in EDSL0 reflects regional dilatation and represents a decrease in compliance. Back

5 Regional static stiffness (Mstat) is the ratio of pressure to segment length [Mstat=(P-P0)/(EDSL0+{Delta}EDSL)]. Given a constant pressure, Mstat is dependent on EDSL0 (to a greater degree) and {Delta}EDSL. An increase in EDSL0 (diastolic creep), therefore, decreases Mstat and negatively alters regional geometry. Recall that an increase in {Delta}EDSL represents greater wall motion. While this may reflect an improvement in compliance (see Strain), an excessive increase in {Delta}EDSL indicates regional dilatation and will cause a decrease in Mstat. Back

6 Regional dynamic stiffness (dP/dL) is a measure of regional relaxation and is represented by the ratio of change in pressure to change in EDSL [dP/dL=d(P-P0)/d(EDSL)]. In a pressure–EDSL curvilinear relation, dynamic stiffness represents the slope of the tangent drawn at a normalized (P-P0). A decrease in dynamic stiffness (e.g. increase in dL) represents improved regional relaxation and compliance. Back


   References
Top
 Abstract
 1. Introduction
 2. Assessment of ventricular...
 3. Effect of cell...
 4. Implications and future...
 5. Conclusions
 References
 

  1. Hennekens C.H. Increasing burden of cardiovascular disease: current knowledge and future directions for research on risk factors. Circulation 1998;97:1095-1102.[Free Full Text]
  2. In: Kao R.L., Chiu R.C.J., eds. Cellular cardiomyoplasty: myocardial repair with cell transplantation. Austin, TX: Landes Bioscience, 1997.
  3. Anversa P., Nadal-Ginard B. Myocyte renewal and ventricular modelling. Nature 2002;415:240-243.[CrossRef][Medline]
  4. Menasche P., Hagege A.A., Scorsin M., Pouzet B., Desnos M., Duboc D., Schwartz K., Vilquin J.T., Marolleau J.P. Myoblast transplantation for heart failure. Lancet 2001;357:279-280.[CrossRef][Medline]
  5. Rajnoch C., Chachques J.C., Berrebi A., Bruneval P., Benoit M.O., Carpentier A. Cellular therapy reverses myocardial dysfunction. J Thorac Cardiovasc Surg 2001;121:871-878.[Abstract/Free Full Text]
  6. Gorcsan J., III, Strum D.P., Mandarino W.A., Gulati V.K., Pinsky M.R. Quantitative assessment of alterations in regional left ventricular contractility with color-coded tissue Doppler echocardiography: comparison with sonomicrometry and pressure–volume relations. Circulation 1997;95:2423-2433.[Abstract/Free Full Text]
  7. Pennock G.D., Yun D.D., Agarwal P.G., Spooner P.H., Goldman S. Echocardiographic changes after myocardial infarction in a model of left ventricular diastolic dysfunction. Am J Physiol 1997;273:H2018-H2029.
  8. Glower D.D., Spratt J.A., Snow N.D., Kabas J.S., Davis J.W., Olsen C.O., Tyson G.S., Sabiston D.C., Rankin J.S. Linearity of the Frank–Starling relationship in the intact heart: the concept of preload recruitable stroke work. Circulation 1985;71:994-1009.[Abstract/Free Full Text]
  9. Kono A., Maughan W.L., Sunagawa K., Hamilton K., Sagawa K., Weisfeldt M.L. The use of left ventricular end-ejection pressure and peak pressure in the estimation of the end-systolic pressure–volume relationship. Circulation 1984;70:1057-1065.[Abstract/Free Full Text]
  10. Kass D.A. Assessment of diastolic function: invasive modalities. Cardiol Clin 2000;18:571-586.[CrossRef][Medline]
  11. Atkins B.Z., Hueman M.T., Meuchel J., Hutcheson K.A., Glower D.D., Taylor D.A. Cellular cardiomyoplasty improves diastolic properties of injured heart. J Surg Res 1999;85:234-242.[CrossRef][Medline]
  12. Silvestry S.C., Taylor D.A., Lilly R.E., Atkins B.Z., Marathe U.S., Davis J.W., Kraus W., Glower D.D. The in vivo quantification of myocardial performance in rabbits: a model for evaluation of cardiac gene therapy. J Mol Cell Cardiol 1996;28:815-823.[CrossRef][Medline]
  13. DeAnda A., Komeda M., Moon M.R., Green G.R., Bolger A.F., Nikolic S.D., Daughters G.T., II, Miller D.C. Estimation of regional left ventricular wall stresses in intact canine hearts. Am J Physiol 1998;275:H1879-H1885.
  14. Muller-Ehmsen J., Peterson K.L., Kedes L., Whittaker P., Dow J.S., Long T.I., Laird P.W., Kloner R.A. Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function. Circulation 2002;105:1720-1726.[Abstract/Free Full Text]
  15. Li R.K., Jia Z.Q., Weisel R.D., Mickle D.A.G., Zhang J., Mohabeer M.K., Rao V., Ivanov J. Cardiomyocyte transplantation improves heart function. Ann Thorac Surg 1996;62:654-660.[Abstract/Free Full Text]
  16. Scorsin M., Hagege A., Marotte F., Mirochnik N., Copin H., Barnoux M., Sabri A., Samuel J.L., Rappaport L., Menasche P. Does transplantation of cardiomyocytes improve function of infarcted myocardium?. Circulation 1997;96(Suppl 2):188-193.
  17. Taylor D.A., Atkins B.Z., Hungspreugs P., Jones T.R., Reedy M.C., Hutcheson K.A., Glower D.D., Kraus W.E. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998;4:929-933.[CrossRef][Medline]
  18. Li R.K., Jia Z.Q., Weisel R.D., Merante F., Mickle D.A.G. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol 1999;31:513-522.[CrossRef][Medline]
  19. Sakai T., Li R.K., Weisel R.D., Mickle D.A.G., Kim E.J., Tomita S., Jia Z.Q., Yau T.M. Autologous heart cell transplantation improves cardiac function after myocardial injury. Ann Thorac Surg 1999;68:2074-2080.[Abstract/Free Full Text]
  20. Pouzet B., Vilquin J.T., Hagege A.A., Scorsin M., Messas E., Fiszman M., Schwartz K., Menasche P. Intramyocardial transplantation of autologus myoblasts: can tissue processing be optimized?. Circulation 2000;102(Suppl 3):210-215.
  21. Etzion S., Battler A., Barbash I.M., Cagnano E., Zarin P., Granot Y., Kedes L.H., Kloner R.A., Leor J. Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction. J Mol Cell Cardiol 2001;33:1321-1330.[CrossRef][Medline]
  22. Jain M., DerSimonian H., Brenner D.A., Ngoy S., Teller P., Edge A.S., Zawadzka A., Wetzel K., Sawyer D.B., Colucci W.S., Apstein C.S., Liao R. Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation 2001;103:1920-1927.[Abstract/Free Full Text]
  23. Pouzet B., Vilquin J.T., Hagege A.A., Scorsin M., Messas E., Fiszman M., Schwartz K., Menasche P. Factors affecting functional outcome after autologous skeletal myoblast transplantation. Ann Thorac Surg 2001;71:844-851.[Abstract/Free Full Text]
  24. Suzuki K., Murtuza B., Smolenski R.T., Sammut I.A., Suzuki N., Kaneda Y., Yacoub M.H. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation 2001;104(Suppl 1):207-212.
  25. Roell W., Lu Z.J., Bloch W., Siedner S., Tiemann K., Xia Y., Stoecker E., Fleischmann M., Bohlen H., Stehle R., Kolossov E., Brem G., Addicks K., Pfitzer G., Welz A., Hescheler J., Fleischmann B.K. Cellular cardiomyoplasty improves survival after myocardial injury. Circulation 2002;105:2435-2441.[Abstract/Free Full Text]
  26. Li R.K., Weisel R.D., Mickle D.A.G., Jia Z.Q., Kim E.J., Sakai T., Tomita S., Schwartz L., Iwanochko M., Husain M., Cusimano R.J., Burns R.J., Yau T.M. Autologous porcine heart cell transplantation improved heart function after a myocardial infarction. J Thorac Cardiovasc Surg 2000;119:62-68.[Abstract/Free Full Text]
  27. Tomita S., Li R.K., Weisel R.D., Mickle D.A.G., Kim E.J., Sakai T., Jia Z.Q. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100(Suppl 2):247-256.
  28. Kocher A.A., Schuster M.D., Szabolcs M.J., Takuma S., Burkhoff D., Wang J., Homma S., Edwards N.M., Itescu S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001;7:430-436.[CrossRef][Medline]
  29. Orlic D., Kajstura J., Chimenti S., Jakoniuk I., Anderson S.M., Li B., Pickel J., McKay R., Nadal-Ginard B., Bodine D.M., Leri A., Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701-705.[CrossRef][Medline]
  30. Fuchs S., Baffour R., Zhou Y.F., Shou M., Pierre A., Tio F.O., Weissman N.J., Leon M.B., Epstein S.E., Kornowski R. Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia. J Am Coll Cardiol 2001;37:1726-1732.[Abstract/Free Full Text]
  31. Kamihata H., Matsubara H., Nishiue T., Fujiyama S., Tsutsumi Y., Ozono R., Masaki H., Mori Y., Iba O., Tateishi E., Kosaki A., Shintani S., Murohara T., Imaizumi T., Iwasaka T. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 2001;104:1046-1052.[Abstract/Free Full Text]
  32. Tomita S., Mickle D.A.G., Weisel R.D., Jia Z.Q., Tumiati L.C., Allidina Y., Liu P., Li R.K. Improved heart function with myogenesis and angiogenesis following autologous porcine bone marrow stromal cell transplantation. J Thorac Cardiovasc Surg 2002;123:1132-1140.[Abstract/Free Full Text]
  33. Sakai T., Li R.K., Weisel R.D., Mickle D.A.G., Jia Z.Q., Tomita S., Kim E.J., Yau T.M. Fetal cell transplantation: a comparison of three cell types. J Thorac Cardiovasc Surg 1999;118:715-725.[Abstract/Free Full Text]
  34. Hutcheson K.A., Atkins B.Z., Hueman M.T., Hopkins M.B., Glower D.D., Taylor D.A. Comparison of benefits on myocardial performance of cellular cardiomyoplasty with skeletal myoblasts and fibroblasts. Cell Transplant 2000;9:359-368.[Medline]
  35. Scorsin M., Hagege A., Vilquin J.-T., Fiszman M., Marotte F., Samuel J.L., Rappaport L., Schwartz K., Menasche P. Comparison of the effects of fetal cardiomyocyte and skeletal myoblast transplantation on postinfarction left ventricular function. J Thorac Cardiovasc Surg 2000;119:1169-1175.[Abstract/Free Full Text]
  36. Kim E.J., Li R.K., Weisel R.D., Mickle D.A.G., Jia Z.Q., Tomita S., Sakai T., Yau T.M. Angiogenesis by endothelial cell transplantation. J Thorac Cardiovasc Surg 2001;122:963-971.[Abstract/Free Full Text]
  37. Yau T.M., Tomita S., Weisel R.D., Jia Z.Q., Tumiati L.C., Mickle D.A.G., Li R.K. Beneficial effect of autologous cell transplantation on infarcted heart function: direct comparison between bone marrow stromal cells and heart cells. Ann Thorac Surg 2003;75:169-176.[Abstract/Free Full Text]
  38. Scorsin M., Hagege A., Dolizy I., Marotte F., Mirochnik N., Copin H., Barnoux M., Le Bert M., Samuel J.-L., Rappaport L., Menasche P. Can cellular transplantation improve function in doxorubicin-induced heart failure?. Circulation 1998;98(Suppl 2):151-155.
  39. Yoo K.J., Li R.K., Weisel R.D., Mickle D.A.G., Li G., Yau T.M. Autologous smooth muscle cell transplantation improved heart function in dilated cardiomyopathy. Ann Thorac Surg 2000;70:859-865.[Abstract/Free Full Text]
  40. Yoo K.J., Li R.K., Weisel R.D., Mickle D.A.G., Jia Z.Q., Kim E.J., Tomita S., Yau T.M. Heart cell transplantation improves heart function in dilated cardiomyopathic hamsters. Circulation 2000;102(Suppl 3):204-209.
  41. Suzuki K., Murtuza B., Suzuki N., Smolenski R.T., Yocoub M.H. Intracoronary infusion of skeletal myoblasts improves cardiac function in doxorubicin-induced heart failure. Circulation 2001;104(Suppl 1):213-217.
  42. Menasche P., Vilquin J.T., Desnos M., Duboc D., Hagege A.A., Pouzet B., Sarateanu S., Scorsin M., Schwartz K., Marolleau J.P. Early results of autologous skeletal myoblast transplantation in patients with severe ischemic heart failure. Circulation 2002;105(Suppl 2):598.
  43. Strauer B.E., Brehm M., Zeus T., Kostering M., Hernandez A., Sorg R.V., Kogler G., Wernet P. Repair of infracted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002;106:1913-1918.[Abstract/Free Full Text]
  44. Assmus B., Schachinger V., Teupe C., Britten M., Lehmann R., Dobert N., Grunwald F., Aicher A., Urbich C., Martin H., Hoelzer D., Dimmeler S., Zeiher A.M. Transplantation of progenitor cells and regeneration enhancement of acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106:3009-3017.[Abstract/Free Full Text]
  45. Stamm C., Westphal B., Klein H.D., Petzsch M., Kittner C., Klinge H., Schumichen C., Nienaber C.A., Freund M., Steinhoff G. Autologous bone-marrow stem-cell transplantation for myocardial infarction. Lancet 2003;361:45-46.[CrossRef][Medline]
  46. Menashe P. Myoblast transplantation: feasibility, safety and efficacy. Ann Med 2002;34:314-315.[CrossRef][Medline]
  47. Zhang Y.M., Hartzell C., Narlow M., Dudley S.C., Jr Stem cell-derived cardiomyocytes demonstrate arrhythmic potential. Circulation 2002;106:1294-1299.[Abstract/Free Full Text]
  48. Reinecke H., Poppa V., Murry C.E. Skeletal muscle stem cells do not transdifferentiate into cardiomyocytes after cardiac grafting. J Mol Cell Cardiol 2002;34:241-249.[CrossRef][Medline]
  49. Terada N., Namazaki T., Oka M., Hoki M., Mastalerz D.M., Nakano Y., Meyer E.M., Morel L., Petersen B.E., Scott E.W. Bone marrow cell adopt the phenotype of other cells spontaneous cell fusion. Nature 2002;416:542-545.[CrossRef][Medline]
  50. Li R.K., Mickle D.A.G., Weisel R.D., Rao V., Jia Z.Q. Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury. Ann Thorac Surg 2001;72:1957-1963.[Abstract/Free Full Text]
  51. Muller-Ehmsen J., Whittaker P., Kloner R.A., Dow J.S., Sakoda T., Long T.I., Laird P.W., Kedes L. Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium. J Mol Cell Cardiol 2002;34:107-116.[CrossRef][Medline]
  52. Ghostine S., Carrion C., Souza L.C.G., Richard P., Bruneval P., Vilquin J.T., Pouzet B., Schwartz K., Menasche P., Hagege A.A. Long-term efficacy of myoblast transplantation on regional structure and function after myocardial infarction. Circulation 2002;106(Suppl 1):131-136.



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