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TRANSLATIONAL AND CLINICAL RESEARCH

Cellular Cardiomyoplasty: Improvement of Left Ventricular Function Correlates with the Release of Cardioactive Cytokines

^aDepartment of Medicine III and
^bInstitute of Physiological Chemistry, Martin Luther University, Halle, Germany;
^cMax Planck Institute for Heart and Lung Research, Bad Nauheim, Germany;
^dInstitute for Cell Culture Technology, University of Bielefeld, Bielefeld, Germany

Key Words. Infarction • Stem cells • Myocytes • Cytokines

Correspondence: Thomas Braun, M.D., Ph.D., Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany. Telephone: 49-6032-705-401; Fax: 49-6032-705-419; e-mail: thomas.braun{at}kerckhoff.mpg.de

Received June 20, 2006; accepted for publication September 8, 2006.
First published online in STEM CELLS EXPRESS   September 14, 2006.

	ABSTRACT

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A growing number of studies are reporting beneficial effects of the transplantation of alleged cardiac stem cells into diseased hearts after myocardial infarction. However, the mechanisms by which transplanted cells might help to promote repair of cardiac tissue are not understood and might involve processes different from the differentiation of transplanted cells into cardiomyocytes. We have compared the effects exerted by skeletal myoblasts (which are not able to form new cardiomyocytes) and ESC-derived cardiomyocytes after implantation into infarcted mouse hearts by echocardiographic follow-up and histological analysis and related these effects to the release of cardioactive cytokines. We found that both cell types led to a long-lasting improvement of left ventricle function and to an improvement of tissue architecture. Since no relevant amounts of myoblast-derived cells were present in infarcted hearts 28 days after transplantation, we investigated the release of cytokines from implanted cells both before and after transplantation into infarcted hearts. ESC-derived cardiomyocytes and myoblasts secreted substantial amounts of interleukin (IL)-1, IL-6, tumor necrosis factor-ß, and oncostatin M, which strongly supported survival and protein synthesis of cultured cardiomyocytes. We postulate that the beneficial effects of the transplantation of myoblasts and cardiomyocytes on heart function and morphology only partially (if at all) depend on the integration of transplanted cells into the myocardium but do depend on the release of a complex blend of cardioactive cytokines.

	INTRODUCTION

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Myocardial infarction and the resulting loss of contractile heart muscle is a frequent cause of heart failure and death. Mammalian cardiomyocytes are terminally differentiated cells, which lose their proliferative potential shortly after birth, so that the adult heart is unable to replace dead or damaged cardiomyocytes after myocardial injury [1]. At present, it is not clear whether putative native cardiac precursor cells, which have been identified recently, contribute to a significant degree to the replacement of myocardial cells in particular since they are present only at very low numbers in the adult heart [2]. Therefore, the transplantation of different types of progenitor or stem cells into diseased hearts remains a promising concept to treat heart insufficiency and myocardial infarction (MI).

In recent years, not only experimental studies in animals but also several clinical trials have shown beneficial effects of cell transplantations to improve cardiac repair after MI [3, 4]. However, the mechanisms that cause these effects are still enigmatic. In particular, the acclaimed formation of new cardiomyocytes from noncardiac stem cells [5] has been questioned and is a matter of fierce debate [6]. Alternatively, it might be envisaged that implanted progenitor cells stimulate vasculogenesis or angiogenesis, inhibit apoptosis, activate endogenous repair mechanisms, or otherwise support cells of the diseased host tissue. Some of these effects might be mediated by the release of cytokines and growth factors either from implanted cells or by the reacting host tissue, thereby establishing autocrine and/or paracrine loops. Cytokines have been demonstrated to affect myocardial functions in a complex manner; these functions are strongly context dependent. The relative contribution of activated signaling pathways and the physiological milieu might evoke either stimulatory or depressant contractile responses [7]. Tumor necrosis factor- (TNF-), for example, confers resistance to hypoxic injury of adult mammalian cardiac myocytes [8], cardiotrophin-1 shows a protective effect against nonischemic cell death of cardiomyocytes [9], and insulin-like growth factor (IGF)-I has well-known anabolic effects on skeletal muscle cells and acts as a survival factor for the myocardium and other tissues [10]. The effect of other cytokines, such as oncostatin M (OSM), interleukin (IL)-1, and IL-6, on adult cardiomyocytes, however, has been studied only poorly so far.

In our experiments, we compared effects of the transplantation of two different cell types (myoblasts and ESC-derived cardiomyocytes) into infarcted mouse hearts with respect to the improvement of cardiac function and morphology. These two cell types differ fundamentally in their potential to form new cardiomyocytes. Skeletal myoblasts have been used as cardiac transplants for a long time [11] but are unable to form new cardiomyocytes or to integrate functionally into the host myocardium. ESC-derived cardiomyocytes [12], on the other hand, might be considered "ideal transplants," since they have the potential to integrate into the host myocardium. Surprisingly, we found that both cell types improve cardiac function and morphology to approximately the same degree after experimental MI in mice, although myoblasts were not present in hearts 28 days after transplantation. Since both cell types secrete an overlapping set of cytokines that inhibit apoptosis and stimulate cardiomyocyte protein synthesis and cell growth in vitro, we suggest that the release of cardioactive substances might explain the beneficial effects of noncardiac stem cells on diseased hearts.

	METHODS

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Myocardial Infarction, Cell Transplantations, and Determination of Infarct Size
The investigation conforms to the NIH Guide for the Care and Use of Laboratory Animals (NIH publication number 85-23, revised 1996). Adult female CD-1 mice underwent left anterior descending coronary artery (LAD) ligation as described [13]. Briefly, mice were anesthetized with isoflurane and ventilated mechanically, left sided thoracotomy was performed in the fourth intercostal region, and the LAD was ligated proximal to its main bifurcation. In sham-operated animals, thoracotomy was performed without LAD ligation. Seven days after MI, before transplantation of different cell preparations, all animals were subjected to echocardiography to determine the size of the infarcted myocardium. Animals that turned out to have small MIs were excluded from the study (5 of 42 mice). All remaining mice received injections of 3 x 10⁵ ESC-derived cardiomyocytes or myoblasts in phosphate-buffered saline (PBS) or PBS alone directly into infarcted hearts using a Hamilton microsyringe. Injections were targeted to the border zone of the infarcted region, which was readily visible due to its pale color. At the end of the experiments, a retrograde perfusion of the heart with 4% paraformaldehyde in diastole was performed to reliably assess histomorphological endpoints. Four sections dividing the space between apex and mitral valve into five equal parts were selected for determination of infarct size as described [14]. The infarct size was calculated based on the following equation: Infarct size (%) = (circumference of infarcted left ventricle [LV])/(total circumference of LV) x 100.

Histological Analysis of Cardiac Remodeling and Immunohistochemical Stainings
Wall thickness was determined on the same four slides per heart that were used for infarct size measurements (trichrome staining). Myocyte cross-sectional area (MCSA) and interstitial collagen fraction were measured by fluorescence microscopy using either fluorescein-conjugated peanut agglutinin or rhodamine-labeled lectin I from Griffonia simplicifolia, respectively, as described [15]. To monitor myofibrillogenesis, cells on chamber slides were fixed with 4% formaldehyde and stained for F-actin with rhodamine-phalloidin and a monoclonal antibody against sarcomeric -actinin (clone EA-53; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) as described previously [16].

Echocardiography
Echocardiography in spontaneously breathing mice was performed under anesthesia with 1.5% isoflurane as described using a 10-MHz Toshiba ultrasound probe (Toshiba, Neuss, Germany, http://www.toshiba-medical.de) [17]. Two-dimensional images and M-mode tracings were recorded from the parasternal short axis view at midpapillary level to determine left ventricular wall thickness (e.g., thickness of left ventricular posterior wall at diastole) and for quantification of left ventricular dimensions (left ventricular internal diameter in diastole [LVIDD] and left ventricular internal diameter in systole [LVIDS]). Left ventricular pump function was analyzed by independent calculation of both fractional shortening (FS) (one-dimensional estimation of LV function) and fractional area change (two-dimensional estimation of LV function).

Cell Culture
Skeletal myoblasts from CD-1 mice were prepared as described previously [18]. ESC-derived cardiomyocytes were obtained from embryonic stem cells following published procedures [12]. Briefly, genetically engineered embryonic stem cells carrying a fusion gene consisting of the -major histocompatibility complex-promoter driving the aminoglycoside phosphotransferase (neomycin resistance) were aggregated into EBs, inoculated into stirred suspension cultures, and differentiated for 9 days before selection of cardiomyocytes by the addition of G418. Throughout the culture period, EBs and viable cell numbers were measured. In addition, flow cytometric analysis was performed to monitor sarcomeric myosin (a marker for cardiomyocytes) expression. Based on myosin heavy chain (MyHC) staining, the purity of cardiomyocytes was >99% before transplantation. Directly before transplantation, both myoblasts and ESC-derived cardiomyocytes were labeled with the fluorochrome DiI according to instructions of the manufacturer. Viability of the cells was determined in parallel by propidium iodide (fluorescence-activated cell sorting; supplemental online Fig. A) and trypan blue exclusion [19]. Ventricular cardiac myocytes of 2–3-month-old male Sprague-Dawley rats were isolated, cultured, and labeled as described [16].

Detection of Cytokines
Cytokines secreted by the cells used for transplantations were determined in cell culture supernatants using the mouse cytokine array 3.1 (RayBiotech, Norcross, GA, http://www.raybiotech.com) according to the manufacturer's instructions. Expression levels were normalized to total protein concentrations in supernatants and to the intensity of reference spots included on each array. All supernatants were compared with culture medium to exclude contaminations of serum additives. After transplantation of cells into infarcted hearts, expression of cytokines were detected by Western blot analysis. Tissue samples were homogenized by sonication and then heated for 1 minute at 99°C. Twenty µg of protein samples were resolved on 4%–12% SDS polyacrylamide gradient gel (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and blotted onto nitrocellulose. Bound antibodies were visualized using Quentix and the Femto detection kit (Perbio Science, Bonn, Germany, http://www.perbio.com) according to instructions of the manufacturer. Antibodies against cytokines were all purchased from R&D Systems (Minneapolis, http://www.rndsystems.com).

	RESULTS

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Transplantation of Skeletal Myoblasts or Embryonic Stem Cell-Derived Cardiomyocytes Improves Cardiac Function After MI in Mice
To compare putative beneficial effects of two different cell types (myoblasts and cardiomyocytes) on diseased hearts, we generated a cohort of CD-1 mice (n = 47) with a ligation of the LAD. This procedure induced a large MI that led to a severe impairment of LV function (Table 1). Although sham-operated animals showed a stable LV geometry and function, animals with LAD ligation displayed a significant dilation of the LV (LVIDD and LVIDS), as well as a reduced systolic function as measured by FS (echocardiographic one-dimensional estimation of left ventricular pump function) and fractional area change (echocardiographic two-dimensional estimation of left ventricular pump function) 7 days after LAD ligation. Animals that received a mock treatment (injection of PBS) were characterized by a progressive enlargement of the LV and a reduction of systolic function. In most animals, we observed maximal dilatation and minimal systolic pump function 35 days after MI.

View larger version (26K): [in this window] [in a new window]   Figure 1. Infarct expansion is restricted by myoblasts or ESC-derived cardiomyocytes. Echocardiographic measurements of FS (A) and LVIDD (B). Implantation of myoblasts or cardiomyocytes both improved cardiac function compared with controls. , p < .05 PBS versus myoblasts; #, p < .05 PBS versus cardiomyocytes. See text and Table 1 for further details. (C): Echocardiographic measurement of the end diastolic thickness of the LVPWD. Seven days after myocardial infarction, only a moderate reduction of LVPWD in all groups was visible, since the posterior wall is not directly affected by left anterior descending coronary artery ligation. In PBS-treated animals, thinning of the LVPWD progressed with time. A reduced thinning was seen in animals that received ESC-derived cardiomyocytes or myoblast transplantation. *, p < .05 myoblasts versus PBS; #, p < .05 cardiomyocytes versus PBS. (D): Histological quantification of the infarct size 28 days after transplantations revealed a restriction of infarct expansion after transplantation of myoblasts or ESC-derived cardiomyocytes in comparison with PBS-treated animals. p values relate to a comparison of the treatment groups versus PBS. Abbreviations: FS, fractional shortening; LVIDD, left ventricular end diastolic diameter; LVPWD, left ventricular posterior wall at diastole; PBS, phosphate-buffered saline.

Transplantation of Myoblasts and ESC-Derived Cardiomyocytes Led to Different Effects on Long-Term Cardiac Remodeling but Both Mitigated Ventricular Wall Thinning After MI
Myocyte hypertrophy and interstitial collagen deposition are hallmarks of chronic remodeling processes after MI. We therefore analyzed whether and to what extent transplantation of myoblasts and ESC-derived cardiomyocytes affected these parameters. As shown in Figure 2A, transplantation of either myoblasts or cardiomyocytes reduced the hypertrophic response of the remote myocardium in the interventricular septum (indicated by a reduced MCSA). In addition, transplantation of cardiomyocytes decreased the reactive collagen deposition in the interventricular septum (Fig. 2B). Surprisingly, myoblast engraftment did not reduce interstitial fibrosis after MI, although the infarct size in myoblast-treated animals was diminished significantly in comparison with the PBS control group.

View larger version (22K): [in this window] [in a new window]   Figure 2. Transplantation of myoblasts or ESC-derived cardiomyocytes limits adverse cardiac remodeling. Effects of cell transplantations on remodeling of the remote myocardium in the interventricular septum were determined by the extent of MCSA (A) and interstit. collagen deposition (B). p values relate to a comparison of the treatment groups versus PBS. Thickness of the interventricular septum (C) and of free left ventricular walls (D–F) was analyzed histologically 28 days after cell transplantations. Transplantation of myoblasts or ESC-derived cardiomyocytes constrained the reduction of the thickness of left ventricular free walls after infarction. #, p < .05 in comparison with noninfarcted hearts (sham). p values correspond to the comparative treatment with PBS. Abbreviations: interstit., interstitial; LV, left ventricle; MCSA, myocyte cross-sectional area; PBS, phosphate-buffered saline.

Improvement of Cardiac Function After Cardiomyoplasty Did Not Depend on the Fate of Transplanted Cells
In principle, it might be possible that the improvement of cardiac function and the suppression of adverse cardiac remodeling in animals that have received cellular transplants is caused by an integration of contractile cells into the remaining myocardium (coupled or uncoupled), which results in improved contraction force. Alternatively, it might be envisioned that transplanted cells nourish or support the diseased myocardium in a paracrine manner that is not dependent on the contractile status of transplanted cells. To distinguish between these two possibilities, we followed the fate of transplanted cell after MI using DiI labeling. We chose DiI labeling since the expression of enhanced green fluorescent protein impairs contractile functions of muscle cells [20] and might even lead to cardiomyopathy [21]. Twenty-eight days after transplantation, we detected several regions in the border zone of the MI, which contained islets of new muscle cells derived from cardiomyocytes, as indicated by DiI fluorescence (Fig. 3). "New" cardiomyocytes were isolated from the host myocardium by fibrous tissue, making it unlikely that the majority of transplanted cardiomyocytes became part of the functional myocardial syncytium (Figs. 3F, 4 A–4C). High-resolution laser scan microscopy revealed that all transplanted cells expressed MyHC (Fig. 4A–4C). Moreover, we found that transplanted cardiomyocytes expressed connexin 43, the major gap junction protein of cardiomyocytes. Although the expression pattern of connexin 43 was not as regular as in the endogenous myocardium, we concluded that the transplanted cardiomyocytes were able to couple functionally to each other (Fig. 4A–4F). We excluded, however, a coupling of transplanted cells with the remaining host myocardium, since all transplanted cardiomyocytes were well separated from endogenous cardiomyocytes by fibrous tissue. None of the animals that received differentiated embryonic stem (ES)-derived cardiomyocytes showed any signs of tumor formation.

View larger version (98K): [in this window] [in a new window]   Figure 3. ESC-derived cardiomyocytes, but not myoblasts, are present in host hearts 28 days after transplantation. The fate of DiI-labeled transplanted ESC-derived cardiomyocytes (A, C, F) and myoblasts (B, D) in recipient hearts was analyzed 2 days (A, C, E) and 28 days (B, D, F) after transplantation. (A–E): Fluorescence microscopic pictures: red, DiI (cell tracker); blue, nuclear staining (Hoechst 33258). After transplantation of ESC-derived cardiomyocytes, DiI-labeled cells were easily located 2 days (A) and 28 days (B) after engraftment. Skeletal myoblasts were present only after 2 days (C). Virtually no DiI-positive myoblasts were found 28 days after transplantation (D). No DiI-labeled cells were found in PBS-treated animals (E). Islets of ESC-derived cardiomyocytes 28 days after transplantation embedded in fibrous tissue (trichrome staining) (F). Scale bars = 75 µm. Abbreviation: PBS, phosphate-buffered saline.

View larger version (115K): [in this window] [in a new window]   Figure 4. Transplanted ESC-derived CMs and primary skeletal muscle cells are separated by scar tissue from the remaining myocardium. Shown is fluorescence labeling for sarcomeric myosin (MF20 antibody [green in (A, C, G, H, J, L); red in (D, F)], connexin 43 [green staining in (E, F)], DiI [red staining in (B, E, H, K)], and DAPI [blue nuclear staining in (A–L)]). White lines in (A–L) indicate the border zone of the infarcted myocardium. All transplanted ESC-derived CMs stained positive for MyHC but were separated from the remaining myocardium by scar tissue (A–C). ESC-derived CMs expressed connexin 43 suggesting coupling to each other but not to the remaining myocardium (D–E). Insets in (F) reveal expression of connexin 43 in healthy myocardium (left) and in transplanted ESC-derived CMs (right). Two days after transplantation, large numbers of primary skeletal muscle cells, which expressed MyHC, were present in the infarcted hearts (G–I). After 28 days, only a very few skeletal muscle-derived cells remained in the infarcted area (arrows in [J–L]). Note the stable labeling of transplanted cells by DiI without transfer to neighboring cells, which are also negative for MyHC. Abbreviations: CM, cardiomyocyte; DAPI, 4,6-diamidino-2-phenylindole; MyHC, myosin heavy chain; SM, skeletal myoblast.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining revealed only a small number of apoptotic myoblasts (Fig. 5E–5H) but a considerably higher number of apoptotic cells derived from ESC-derived cardiomyocytes (Fig. 5A–5D). Our findings suggest that most myoblasts initially engrafted into the host myocardium without signs of massive apoptosis but later disappeared from the tissue, probably by nonapoptotic cell death.

View larger version (32K): [in this window] [in a new window]   Figure 5. Skeletal myoblasts and ESC-derived CMs show a different degree of apoptosis 2 days after transplantation into infarcted hearts. Apoptotic cells were detected using the TUNEL assay 48 hours after transplantation of ESC-derived CMs (A–D) and primary skeletal muscle myoblasts (E–H). Blue (A, E), nuclear staining (Hoechst 33258); red (B, F), DiI (cell tracker); green (C, G), TUNEL-positive cells. (D, H): merged images. Only a very few transplanted skeletal muscle cells (red staining in [F]) were TUNEL-positive (arrow in [G]), whereas many more transplanted CMs underwent apoptosis (green staining in [C]). Scale bars = 25 µm. Abbreviations: CM, cardiomyocyte; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

View larger version (56K): [in this window] [in a new window]   Figure 6. Skeletal myoblasts and ESC-derived CMs but not primary fibroblasts release a mixture of different cytokines after transplantation into infarcted hearts. Western blot analysis of extracts prepared from the infarct area of hearts 2 (short) or 21 days (long) after injection of phosphate-buffered saline (PBS) (CON), ESC-derived CMs, MBs, and primary FBs. Left: CON hearts did not express IL-6, OSM, or TNF-ß. CM transplantation resulted in a robust presence of OSM, IL-6, and TNF-ß 2 days after injection. The expression of IL-6 and TNF-ß declined after 21 days. MB transplantation led to an expression of IL-6 and OSM but not of TNF-ß. Staining for pan-actin was used to assure equal loading. Right: Transplantation of FBs did not result in an expression of OSM, IL-6, or TNF-ß hearts 2 days (short) or 21 days (long) after injection and was undistinguishable from PBS treatment. The position of OSM (and all other cytokines analyzed) within the gel matched the expected molecular weight for each individual molecule. Abbreviations: CM, cardiomyocyte; CON, control; FB, fibroblast; IL, interleukin; MB, myoblast; OSM, oncostatin M; TNF, tumor necrosis factor.

View larger version (55K): [in this window] [in a new window]   Figure 7. Cytokines released from skeletal myoblasts and ESC-derived cardiomyocytes stimulate survival and protein synthesis of adult cardiomyocytes. Fluorescence labeling for actin (red) and striated muscle-specific -actinin (green) of adult rat cardiomyocytes after stimulations with various growth factors (B, C, E–L) and in nontreated controls (A, D) at day 10. All cultures contained 2% fetal calf serum. The insets in (D–F) and (J–L) show higher magnifications to reveal the cross-striations in cultured cells. Total [3H]phenylalanine incorporation (sum of protein synthesis and degradation) of rat adult cardiomyocytes pulse labeled for the last 12 hours of culture (M). Values for phenylalanine incorporation are expressed relative to DNA content (for normalization) as percentage of untreated control cells. Scale bars = 80 µm. Abbreviations: con, control; IGF, insulin-like growth factor; IL, interleukin; OSM, oncostatin M; TNF, tumor necrosis factor.

	DISCUSSION

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Our experimental set-up was based on the usage of two completely different cell types, which intentionally are either unable to form cardiomyocytes (skeletal myoblasts) or ES-derived cardiomyocytes that are already differentiated along the "correct" cellular lineage and hence do not depend on reprogramming or (trans)differentiation. Our results show that despite the different origin of the cells a similar improvement of cardiac function was achieved, although some significant differences were noted.

Skeletal myoblasts have been proposed to improve heart function after myocardial infarction in several animal models and in humans [11, 22, 23]. However, the exact mechanisms that eventually lead to functional improvements are still under debate. Although initial reports favored the idea of the formation of cardiomyocyte-like cells that might be implemented in the concerted process of systolic contraction [24], recent studies clearly demonstrated that skeletal myoblasts remain functionally isolated from the host myocardium. Only a very few transplanted myoblasts fuse with residing cardiomyocytes and are therefore able to contribute to "synchronized" contractions [25].

As anticipated, we were able to corroborate the positive effects of myoblast transplantations after MI. Both echocardiography and histological analysis revealed an improved heart function after myoblast engraftment. Surprisingly, this effect occurred even in the absence of a reasonable number of stable long-term grafts. Furthermore, we found that transplanted cells (both cardiomyocytes and skeletal muscle cells) were always well separated from the remaining intact myocardium by scar tissue preventing functional coupling of transplanted cells. Engraftment of myoblasts was accompanied by several characteristic changes of organ function and morphology that differed from effects exerted by ESC-derived cardiomyocytes. (a) Improvement of LV function (better: reduction of LV decline) occurred rapidly. Myoblasts seemed to exert their beneficial effects immediately after engraftment, whereas protective effects of the transplantation of cardiomyocytes became apparent after 14 days. Morphologically this effect was reflected by an (early) inhibition of infarct expansion, which might, at least partially, be explained by the release of survival-promoting growth factors and cytokines. Since the number of skeletal muscle cells decreased considerably over time, it seems reasonable to assume that the reduced presence of myoblasts went along with incremental lower concentrations of secreted molecules, further strengthening the argument that the cytokines released from skeletal muscle cells acted at early stages of MI-induced cardiac remodeling. (b) In contrast to ESC-derived cardiomyocytes, only a very few skeletal muscle-derived cells were still present in the host myocardium 4 weeks after transplantation, although a large number of MyHC-positive skeletal muscle cells were detected shortly after transplantation into recipient hearts. The lasting improvement of LV function, despite the disappearance of myoblasts from the host tissue, clearly indicated that a therapeutic intervention by cardiomyoplasty with myoblasts during the first days or weeks after MI sufficed to prevent adverse cardiac remodeling and to establish long-lasting effects. Despite the virtually complete disappearance of myoblasts from host myocardium, the number of apoptotic cells was higher in animals that received cardiomyocytes, which suggests that myoblasts disappeared by various routes, probably including nonapoptotic modes of cell death. (c) Transplantation of myoblasts led to an enhancement of interstitial fibrosis of the remote myocardium. Obviously, the containment of infarct expansion (and eventually infarct size) by grafted myoblasts, which resulted in a "mechanical advantage," did not result in a reduction of interstitial fibrosis of the IVS. It seems that profibrotic effects exerted by transplanted myoblasts overcame the potential mechanical benefits, which should have alleviated the fibrotic response. The detection of several proinflammatory and profibrotic cytokines secreted by myoblasts nicely supported this notion. It should be pointed out that the profibrotic effects of myoblasts might not necessarily be harmful or unfavorable, since the formation of stable scar tissue in LV walls might inhibit progressive wall thinning and adverse LV remodeling.

Several groups have reported an improvement of cardiac function after transplantation of ESC-derived cardiomyocytes (a recent review is given in [26]). Since ESC-derived cardiomyocytes show most of the characteristics of native primary cardiomyocytes, it was generally assumed that transplanted ESC-derived cardiomyocytes directly contribute to the contractile force of recipient hearts, although the extent of tissue colonization by grafted cells did not always match the improvement of cardiac function. We have also observed a clear improvement of LV function after transplantation of ESC-derived cardiomyocytes into infarcted hearts although new cardiomyocytes were always clearly isolated from the host myocardium by fibrous tissue. Echocardiographic monitoring of transplanted animals indicated that beneficial inputs of predifferentiated cardiomyocytes were delayed in comparison with myoblast treatments but reached a similar level after 28 days. In agreement with this observation, ESC-derived cardiomyocytes seemed less effective in restricting infarct expansion but were more successful in preventing adverse chronic remodeling of the heart, indicated by the deposition of collagen and fibrosis. Our data suggest that at least some effects of ESC-derived cardiomyocytes were achieved by the release of cardioactive growth factors and cytokines.

In vitro IGF-I, OSM, IL-1, IL6, and TNF-ß all improved cardiomyocyte survival. Similar observations had been made for CT-1, IL-6, and TNF-, which confer cytoprotective effects to adult cardiac myocytes [8, 9], whereas others, such as insulin and IGF-I, stimulate the metabolism of cardiomyocytes [27, 28]. Interestingly, the same proinflammatory cytokines, such as granulocyte colony-stimulating factor [29], TNF-, and IL-6, which were released by ESC-derived cardiomyocytes and/or myoblasts, have profound effects on LV contractility, although this effect strongly depends on the concentration of cytokines and exposition time of the heart (reviewed in [7]). In addition to an improvement of cardiomyocyte survival, several of the cytokines and growth factors released by ESC-derived cardiomyocytes and myoblasts do have effects on macrophages and mesenchymal stem cells [30] and modulate the inflammation reaction, interstitial matrix composition, scar formation, and other processes related to cardiac remodeling, which might explain the reduced level of adverse remodeling after cardiac body engraftment.

Taken together, our experiments demonstrate that transplantation of either skeletal myoblasts or ESC-derived cardiomyocytes, most probably by using overlapping paracrine signaling pathways, improves heart function. A careful delineation of the effects of individual cytokines (or certain cytokine combination) on post-MI remodeling might lead to the formulation of growth factor cocktails that do not rely any longer on potentially "hazardous" cells. Such cocktails when applied locally to the diseased myocardium possibly with the help of recently developed nanofibers [31] or other carriers might eventually be implemented into future MI treatment strategies to achieve maximal protection of cardiomyocytes from apoptosis and optimization of scar composition and cardiac remodeling.

	DISCLOSURES

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The authors indicate no potential conflicts of interest

	ACKNOWLEDGMENTS

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This work was supported by the Max Planck Society, Deutsche Forschungsgemeinschaft, SFB 598, the Myores program of the European Commission, and the Wilhelm Roux Program for Research of the Martin Luther University.

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