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TRANSLATIONAL AND CLINICAL RESEARCH |
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 on June 20, 2006;
accepted for publication on September 8, 2006.
First published online in STEM CELLS EXPRESS September 14, 2006.
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ABSTRACT |
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, 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.
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INTRODUCTION |
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TopAbstractIntroductionMethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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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 [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.
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METHODS |
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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).
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RESULTS |
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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.
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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.
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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.
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, 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.
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). 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.
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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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.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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TopAbstractIntroductionMethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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REFERENCES |
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, p < .05 PBS versus myoblasts; #, p < .05 PBS versus cardiomyocytes. See text and 
