Cellular Cardiomyoplasty: Improvement of Left Ventricular Function Correlates wi - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:59 |只看该作者 |倒序浏览 |打印

作者：Henning Ebelta,b, Mirco Jungblutb, Ying Zhanga, Thomas Kubinc, Sawa Kostinc, Antje Technaub, Svetlana Oustaninab,c, Sylvia Niebrgged, Jrgen Lehmannd, Karl Werdana, Thomas Braunb,c 6 P. Y/ G( \9 ^$ c! Z
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      【摘要】" O9 i6 ?9 z+ b0 n; ]: G
   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-&szlig;, 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. # i7 `- P5 {/ C
      【关键词】 Infarction Stem cells Myocytes 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 . 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).; w1 S: }( c$ T
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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 . 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.. E# g9 @' x: v) l
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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 , 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.  J" y& H8 O2 O2 l8 A
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METHODS9 w1 @( Z& |* v- z- g' h

Myocardial Infarction, Cell Transplantations, and Determination of Infarct Size
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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 )/(total circumference of LV) x 100.% W" t" w& v* Q( ]0 l7 o
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Histological Analysis of Cardiac Remodeling and Immunohistochemical Stainings; K5 P( w" |* b6 \$ x

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 .
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Echocardiography* G& A/ D5 x$ _
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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) ). 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).9 r5 O* u; u# h: u4 f: C& _

Cell Culture
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Skeletal myoblasts from CD-1 mice were prepared as described previously .
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Detection of Cytokines
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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¡ãC. Twenty µg of protein samples were resolved on 4%¨C12% 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).
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RESULTS

Transplantation of Skeletal Myoblasts or Embryonic Stem Cell-Derived Cardiomyocytes Improves Cardiac Function After MI in Mice
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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.5 f: k0 b; J0 p) o9 _; e

Table 1. Morphometric and echocardiographic measurements of hearts 28 days after cell transplantations (more details are given in supplemental online Table A)

Transplantation of both skeletal myoblasts and ESC-derived cardiomyocytes significantly improved cardiac function (Fig. 1A; Table 1). Although both treatments failed to completely restore cardiac function, progression of chronic heart failure was slowed down leading to a significantly reduced LV dilation and improved LV function. Beneficial effects of skeletal myoblasts were already evident 7 days after transplantation, as indicated by a reduction of LV dilation (Fig. 1B, LVIDD). The effects of ESC-derived cardiomyocytes became apparent approximately 1 week later, 14 days after transplantation. In contrast to myoblasts, cardiomyocytes primarily improved the contractility of the heart (as measured by LVIDS and FS) rather than LV dilation (supplemental online Table A). Despite these initial differences, the echocardiographic parameters in both groups converged over time and were similar with respect to LV function 28 days after transplantation.
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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 1 n; ^+ u3 Y- D( W; _  k* B

Effects of Cell Transplantations on Infarct Expansion6 B% m& C& R8 i

It seemed possible that the improvement of LV function observed after myoblast and cardiomyocyte transplantation was caused by a reduction of infarct progression. In agreement with this hypothesis we found that hearts that had received myoblasts or cardiomyocytes showed a significantly reduced infarct expansion in comparison with PBS-treated animals (Fig. 1D). PBS-treated hearts were characterized by a progressive thinning of the left ventricular posterior wall at midpapillary level in echocardiography, although this region of the heart was not directly affected by LAD ligation (Fig. 1C). Histological examinations performed at the end of the experiment 28 days after transplantation revealed that a significant larger part of the LV of PBS-treated animals was occupied by scar tissue than hearts that had received myoblasts or cardiomyocytes.) `2 V/ r' I" Q9 X7 L

Transplantation of Myoblasts and ESC-Derived Cardiomyocytes Led to Different Effects on Long-Term Cardiac Remodeling but Both Mitigated Ventricular Wall Thinning After MI
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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.
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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¨CF) 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
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It is well known that due to LaPlace's law, thinning of the left ventricular walls after MI enhances adverse cardiac remodeling and LV dilatation. We therefore determined the thickness of the left ventricular free walls and the interventricular septum at the end of the experiment to analyze a potential contribution of ventricular wall thinning to adverse cardiac remodeling and LV dilatation. As expected, all animals that underwent LAD ligation and MI showed a hypertrophy of the interventricular septum (IVS). Interestingly, animals that received a PBS injection displayed a significantly increased thinning of the free left ventricular walls, whereas animals that were treated with either myoblasts or cardiomyocytes showed a only a moderate reduction of the diameter of the free left ventricular walls (Fig. 2C¨C2F). It seems likely that the reduced thinning of the free left ventricular walls accounts, at least in part, for the reduced LV dilatation after myoblast or cardiomyocyte treatment.+ K/ Q. P  D" k: J" _7 K

Improvement of Cardiac Function After Cardiomyoplasty Did Not Depend on the Fate of Transplanted Cells  b$ ?4 N# b$ n6 q3 d

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 . 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¨C4C). High-resolution laser scan microscopy revealed that all transplanted cells expressed MyHC (Fig. 4A¨C4C). 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¨C4F). 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." I+ N6 p) ?( j+ r
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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¨CE): 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.
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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 ). 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.' L6 l* U0 H% G9 _

Surprisingly, we only very rarely detected new muscle cells in hearts that had received skeletal myoblasts 28 days after transplantation (Figs. 3, 4J¨C4L). Although some hearts contained some DiI-labeled skeletal muscle cells, the number of surviving transplanted myoblast-derived cells was low in the vast majority of animals (Figs. 3, 4J¨C4L). To answer the question whether host animals initially received a sufficient number of viable myoblasts, which later disappeared from the host tissue during cardiac remodeling, we analyzed hearts at earlier time points after transplantation. Forty-eight hours after transplantation, the presence of myoblasts and ESC-derived cardiomyocytes were easily discernable by the DiI label (Figs. 3, 4G¨C4L). High-resolution confocal microscopy revealed that most skeletal muscle-derived cells expressed MyHC (Fig. 4G¨C4I) and differentiated in situ to myocytes. The number of contaminating fibroblasts in recipient hearts was very low (Fig. 4I), although we did not reach the purity of ESC-derived cardiomyocytes, which were derived by genetic selection.
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Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining revealed only a small number of apoptotic myoblasts (Fig. 5E¨C5H) but a considerably higher number of apoptotic cells derived from ESC-derived cardiomyocytes (Fig. 5A¨C5D). 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.4 [6 Y. R/ d+ |+ f3 L2 S: R; J
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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¨CD) and primary skeletal muscle myoblasts (E¨CH). 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 ). Scale bars = 25 µm. Abbreviations: CM, cardiomyocyte; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Myoblasts and ESC-Derived Cardiomyocytes Secrete a Complex Mixture of Growth Factors and Cytokines
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Since the transplantation of myoblasts led to a significant improvement of cardiac function after MI despite the absence of stable long-term grafts, we reasoned that myoblasts (and probably also cardiomyocytes) might activate endogenous signal cascades within the host tissue most likely by the release of cardioactive molecules. We therefore analyzed various cytokines and growth factors secreted by myoblasts and cardiomyocytes at the time of transplantation using a semiquantitative Western blot assay. As shown in Table 2, both skeletal myoblasts and ESC-derived cardiomyocytes released a complex, partially overlapping blend of growth factors and leukocyte-attracting cytokines. Cardiomyocytes secreted high amounts of proinflammatory cytokines, such as GRO, IL-6, MCPs, and TNF-, whereas myoblasts released mitogen-inducible gene, chemokine CXC motif ligand 9, stem cell factor, and IGF-I, which are known to promote cell motility and stimulate cardiomyocyte growth. Both types of cells produced vascular endothelial growth factor and platelet-derived growth factor-B, which stimulate angiogenesis; proinflammatory cytokines, such as IL-1, IL-4, TNF-ß, and macrophage colony-stimulating factor; and stimulants of cell motility and interstitial matrix remodeling (OSM and epidermal growth factor) (Table 2). It seems clear that such a complex composition of cytokines and growth factors will have profound effects on cardiomyocyte survival, remodeling of the myocardium, and cardiac function. To explore whether transplantation of myoblasts and ESC-derived cardiomyocytes resulted in a significant increase of cytokine levels within infarcted hearts in vivo, we performed a Western blot analysis. Extracts were prepared from the infarct area of hearts 2 or 21 days after injection of myoblasts, ESC-derived cardiomyocytes, and PBS. Hearts that received a PBS injection were devoid of IL-6, OSM, or TNF-ß expression both 2 and 21 days after injection (Fig. 6). In contrast, transplantation of cardiomyocytes resulted in a robust presence of OSM, IL-6, and TNF-ß 2 days after the injection. We observed a fading of IL-6 and TNF-ß signals 21 days after injection, whereas the concentration of OSM remained stable for up to 3 weeks (Fig. 6). Infarcted hearts that received myoblasts did contain significant amounts of IL-6 (which declined after 21 days) and OSM but lacked expression of TNF-ß. In addition, we found expression of IGF-I in infarcted hearts that received cardiomyocytes and myoblasts but also in mock-transplanted infarcted and healthy control hearts (data not shown). To address the question of whether expression of cytokines in the manipulated myocardium was an unspecific phenomenon resulting from implantation of any cell type, we also used primary fibroblasts for transplantation. Western blot analysis indicated that transplantation of fibroblasts did not result in a significant expression of OSM, IL-6, and TNF-ß in hearts 2 days (short) or 21 days (long) after injection (Fig. 6). Because of the low expression level of cytokines, however, which prevented an in situ detection of cytokine expression in transplanted cells, we cannot exclude the possibility that transplantation of cardiomyocytes and skeletal muscle cells but not of fibroblasts triggered cytokine expression in endogenous cardiomyocytes.

Table 2. Secretion of growth factors and cytokines by skeletal myoblasts and ESC-derived cardiomyocytes (semiquantitative assay)

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.
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Cytokines Improve the Viability of Isolated Cardiomyocytes In Vitro
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To further delineate the impact of individual growth factors and cytokines released by ESC-derived cardiomyocytes and myoblasts on cardiomyocytes, we incubated isolated adult rat cardiomyocytes for 5 and 10 days with selected growth factors that were either released by ESC-derived cardiomyocytes (IL-6), myoblasts (IGF-I), or both cell types (OSM, TNF-ß, and IL-1). All growth factors clearly increased the number of surviving cardiomyocytes in culture by approximately 30% after 10 days in culture (Fig. 7; data not shown) compared with nontreated controls. In addition, we observed a considerable induction of the rate of protein synthesis within cardiomyocytes after 5 days, which probably reflected adaptive patterns of protein expression inflicted by the different cytokines used. The strongest effect was elicited by IGF-I (2.1-fold) and OSM (1.8-fold), followed by slightly lower rates (1.5-fold) in TNF-ß-, IL-1-, and Il-6-treated cultures (Fig. 7). Remodeling of myocytes and synthesis of new sarcomers was monitored by staining of cultures for F-actin and sarcomeric -actinin (Fig. 7). In comparison with control cultures, IGF-I showed the highest density of myofibrils, ranging from the center of the cell down to the end of newly built-up extensions. In contrast, OSM-treated cardiomyocytes were characterized by a substantial cell lengthening and a limited appearance of new cross-striations, which probably reflects reduced myofibrillogenesis. IL-1 and IL-6 induced a phenotype that was similar to but less pronounced than that of OSM-treated myocytes.

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¨CL) and in nontreated controls (A, D) at day 10. All cultures contained 2% fetal calf serum. The insets in (D¨CF) and (J¨CL) show higher magnifications to reveal the cross-striations in cultured cells. Total 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.- K6 m. t/ U; a( F) }
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DISCUSSION

Transplantation of stem cells seems a promising concept for the treatment of myocardial infarction and heart failure. However, despite encouraging results from first clinical trials that demonstrated beneficial effects of different transplantation procedures , the mechanisms that underlie the improvement of cardiac function are still enigmatic. In particular, it has been questioned whether and to what extent transplanted cells might acquire characteristics of functional cardiac muscle cells and improve the contractile force of damaged hearts.& g6 Q7 }/ z) _' N
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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.
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Skeletal myoblasts have been proposed to improve heart function after myocardial infarction in several animal models and in humans .

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.- C" x% X" B  P3 P) X
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Several groups have reported an improvement of cardiac function after transplantation of ESC-derived cardiomyocytes (a recent review is given in ). 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  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./ ~1 p( G( }0 D9 X6 Z6 E7 o% J
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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  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' V! e' O, H- A. n  b' [
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ACKNOWLEDGMENTS% R4 e' n8 G* u$ n4 @+ E, U4 u

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.# t; b4 k- U: }1 M/ N/ f: a; R
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Rumyantsev PP. Interrelations of the proliferation and differentiation processes during cardiac myogenesis and regeneration. Int Rev Cytol 1977;51:186¨C273.3 p% V) W/ n- U! ]; s* g; r

Laugwitz KL, Moretti A, Lam J et al. Postnatal isl1  cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 2005;433:647¨C653.
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