Published online before print May 4, 2009, doi: 10.1161/CIRCULATIONAHA.108.827691
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(Circulation. 2009;119:2587-2596.)
© 2009 American Heart Association, Inc.
Molecular Cardiology |
Sca-1+ Stem Cell Survival and Engraftment in the Infarcted Heart
Dual Role for Preconditioning-Induced Connexin-43
From the Department of Pathology and Laboratory Medicine, 231 Albert Sabin Way, University of Cincinnati, Cincinnati, Ohio.
Correspondence to Professor Muhammad Ashraf, Department of Pathology and Laboratory Medicine, 231 Albert Sabin Way, University of Cincinnati, Cincinnati, OH 45267-0529. E-mail ashrafm{at}ucmail.uc.edu
Received October 14, 2008; accepted March 20, 2009.
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Abstract |
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Background— We report that elevated connexin-43 (Cx-43) in stem cells preconditioned with insulin-like growth factor-1 (IGF-1) is cytoprotective and reprograms the cells for cardiomyogenic differentiation.
Methods and Results— Sca-1+ cells were preconditioned with 100 nmol/L IGF-1 for 30 minutes followed by 8 hours of oxygen glucose deprivation to assess the cytoprotective effects of preconditioning. LDH release assay, cytochrome c release, and mitochondrial membrane potential assay showed improved survival of preconditioned Sca-1+ cells under oxygen glucose deprivation compared with nonpreconditioned Sca-1+ cells via PI3K/Akt-dependent caspase-3 downregulation. We observed PI3K/Akt-dependent upregulation of cardiac-specific markers including MEF-2c (2.5-fold), GATA4 (3.1-fold), and Cx-43 (3.5-fold). Cx-43 inhibition with specific RNA interference reduced cell survival under oxygen glucose deprivation and after transplantation. In vivo studies were performed in a female rat model of acute myocardial infarction (n=78). Animals were grouped to receive intramyocardially 70 µL Dulbecco modified Eagles medium without cells (group 1) or containing male 1x106 nonpreconditioned Sca-1+ cells (group 2) or preconditioned Sca-1+ (group 3) cells labeled with PKH26. Survival of the preconditioned Sca-1+ cells was 5.5-fold higher in group 3 compared with group 2 at 7 days after transplantation. Confocal imaging after actinin and Cx-43 specific immunostaining showed extensive engraftment and myogenic differentiation of preconditioned Sca-1+ cells. Compared with group 2, group 3 showed increased blood vessel density (22.3±1.7 per microscopic field; P<0.0001) and attenuated infarction size (23.3±3.6%; P=0.002). Heart function indices including ejection fraction (56.2±3.5; P=0.029) and fractional shortening (24.3±2.1; P=0.03) were improved in group 3 compared with group 2.
Conclusions— Preconditioning with IGF-1 reprograms Sca-1+ for prosurvival signaling and cardiomyogenic differentiation with an important role for Cx-43 in this process.
Key Words: angiogenesis • apoptosis • connexin 43 • insulin-like growth factor-1 • stem cells
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Introduction |
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The electric and mechanical integrity of the heart is compromised after myocardial infarction because of massive loss of functioning myocytes. Heart cell therapy provides an unconventional corrective measure to compensate for myocyte loss in the infarcted heart.1–4 Nevertheless, poor survival of donor cells is one of the major concerns that hampers a better prognosis.5 Additionally, poor engraftment and lack of functional coupling of donor cells with the viable host tissue greatly impede cell-to-cell signaling and electric communication. Most previous strategies have addressed the issue of cell survival alone, although with limited success.6–8 Because functional improvement of the heart is proportional to the number of injected cells, we therefore proposed that a strategy that concurrently addresses both of these issues would increase the effectiveness of the procedure. Connexin-43 (Cx-43), with its dual role as an antiapoptotic and as a gap-junctional protein, can effectively resolve both of these issues.9–11
Clinical Perspective on p 2596
The connexin family of genes encodes for >20 proteins, of which Cx-30, Cx-37, Cx-40, Cx-43, and Cx-45 have been studied extensively for their role in the heart.12 Cx-43 is predominantly synthesized in the plasma membrane of cardiomyocytes and forms intercellular channels to link cytoplasmic compartments of the adjacent myocytes. As an alternative to the paracrine mechanism of intercellular communication, Cx-43 ensures a direct transfer of ions and signaling molecules that regulates intracellular calcium and cell survival via releasing ATP, NAD+, or glutamate and propagation of electric impulses.13,14 Gap-junctional intercellular communication is also important for cellular proliferation and differentiation.15 Under physiological conditions, Cx-43 participates in cellular response to ischemia.16,17 Moreover, localization of Cx-43 in intracellular structures such as the mitochondria seems to be cardioprotective.18,19 Any reduction in Cx-43 renders the heart more susceptible to electric instability. Cell-based delivery of the Cx-43 transgene prevented ventricular arrhythmia after infarction.19 The functional versatility of Cx-43 supports our study rationale that pharmacological targeting of Cx-43 in stem cells may improve their survival and integration in the infarcted heart. Different growth factors potentiate Cx-43 expression.20–22 Insulin-like growth factor-1 (IGF-1) increases intracellular Cx-43.22 Our results emphasize the importance of IGF-1/IGF-1 receptor (IGF-1R) interaction to initiate downstream survival signaling involving Cx-43, which primarily curtailed stem cell apoptosis and promoted their survival, factors crucial for subsequent engraftment of donor cells in the infarcted heart. These are novel findings that underscore the need to exploit a dual role for the preconditioning-induced Cx-43 to promote stem cell survival and their electromechanical coupling after transplantation for better prognosis.
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Methods |
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The experimental protocols are described in the expanded Methods in the online-only Data Supplement. The lists of antibodies and primer sequences used are given in Tables I and II in the online-only Data Supplement.
Statistical Analysis
All experiments were performed at least 3 times to assess reproducibility of the results. The data were expressed as mean±SEM. Student t test or 1-way or 2-way ANOVA was performed to analyze statistical differences in each response variable. Prespecified comparisons between groups were made, and Bonferroni or Tukey adjustment for multiple comparisons was done when appropriate. A value of P
0.05 was considered statistically significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors read and agreed to the manuscript as written.
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Results |
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Flow cytometry showed that 92.9% of cells expressed Sca-1 and were very low in c-kit (0.7%) and CD45 (4.3%) expression (Figure 1A1 to 1A4). The homogeneity of the purified cells was confirmed by fluorescent immunostaining for Sca-1 antigen (Figure 1B). These cells were later used in all of the in vitro and in vivo experiments.
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Cytoprotection by IGF-1 Pretreatment
The percent cell viability under oxygen glucose deprivation (OGD) was significantly higher in preconditioned Sca-1+ cells (PCSca-1+) compared with nonpreconditioned Sca-1+ cells (non-PCSca-1+). Preconditioning reduced the nonviable cells from 5.9% to 1.7% under 4-hour OGD and from 31.4% to 13.7% under 8-hour OGD (P=0.001 by 2-way ANOVA; Figure 1C). These results were confirmed by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL), which showed that 8-hour OGD caused higher TUNEL positivity in non-PCSca-1+ compared with PCSca-1+ (Figure 1C2). JC-1, a cationic dye that exhibits membrane potential–dependent accumulation in the mitochondria, was used to detect the early stage of apoptosis (Figure 1D1 to 1D3). IGF-1 pretreatment significantly reduced the percentage of early apoptotic cells (green) under OGD (Figure 1D3). Moreover, cytochrome c–specific immunostaining revealed a typical punctuate distribution of fluorescence in the mitochondria of PCSca-1+ (Figure 1E1), whereas release of cytochrome c from the mitochondria after OGD was indicated by diffused fluorescence in the cytoplasm (Figure 1E2). Phase contrast microscopy revealed well-preserved cell morphology in PCSca-1+ (Figure 1E3) compared with non-PCSca-1+ (Figure 1E4). Annexin V–fluorescein isothiocyanate/propidium iodide staining coupled with flow cytometry showed that preconditioning reduced apoptotic cells from 69.7% in non-PCSca-1+ to 50.5% in PCSca-1+ (Figure 1F). Taken together, IGF-1 preconditioning offered cytoprotection against OGD in vitro.
Cell Signaling in IGF-1 Initiated Cytoprotection
Sca-1+ cells expressed IGF-1 receptors that were phosphorylated by 30-minute pretreatment with 100 nmol/L IGF-1 (Figure 2A). PI3K/Akt signaling plays a significant role downstream of IGF-1/IGF-1R ligand/receptor interaction.23 We observed higher pAkt in PCSca-1+ compared with non-PCSca-1+ (Figure 2B). OGD for 8 hours significantly reduced pAkt in non-PCSca-1+, whereas it still remained high in PCSca-1+ (Figure 2B). Pretreatment with PI3K inhibitor (LY294002; 40 µmol/L) abolished pAkt in PCSca-1+. The effect of LY294002 was more pronounced in the cells subjected to 8-hour OGD compared with normoxia. LDH release assay showed higher PCSca-1+ survival under 8-hour OGD compared with non-PCSca-1+ (Figure 2C). However, cytoprotection was abolished in PCSca-1+ pretreated with LY294002 (17.9% in PCSca-1+ versus 31.2% in IGF-1+LY294002–treated cells). Western blotting showed that 8-hour OGD increased caspase-3 cleavage (activation of caspase-3) in non-PCSca-1+ compared with PCSca-1+. Contrarily, pretreatment with LY294002 showed a higher level of caspase-3 cleavage in PCSca-1+, thus showing an inverse relation between the dynamics of caspase-3 cleavage and Akt phosphorylation (Figure 2D1). Treatment with caspase-3 inhibitor and subsequent exposure to 8-hour OGD showed enhanced cell survival under OGD (31.1%) compared with inhibitor untreated cells (64.9%) (Figure 2D2).
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Preconditioning With IGF-1 Favored Cardiomyogenesis
Although IGF-1 treatment for 1 day and 7 days significantly induced mRNA expression of cardiac marker proteins, 7-day IGF-1 treatment was more effective in upregulating GATA-4 (3.1-fold), Cx-43 (3.5-fold), and MEF-2c (2.5-fold), whereas troponin-I upregulation remained insignificant (Figure 3A). Western blotting confirmed these findings except for GATA-4, which remained undetectable (Figure 3B). LY294002 pretreatment reversed the effect of IGF-1 on cells grown in normoxia (Figure 3C). We observed that cardiac-specific gene expression was sensitive to anoxia. Cells cultured with 0.5% bovine serum albumin for 12 hours under OGD showed abrogation of Cx-43 and MEF2c, whereas the presence of IGF-1 during OGD significantly enhanced their gene expression (Figure 3D). The presence of IGF-1 during OGD also offered cytoprotection in addition to promoting cardiac-specific proteins (Figure 3E). These data suggested that OGD not only imposed a survival challenge for cells but also impeded their differentiation. Even though downregulation of cardiac markers under OGD was not seen to be fully reversed from our 12-hour study, the presence of IGF-1 supported Sca-1+ cells toward cardiac differentiation. Hence, it is reasonable to project a more pronounced effect of IGF-1 in vivo when one considers the kinetics of its expression in the infarcted heart, which significantly affects the transplanted cells. In accordance with Western blotting results (Figure 3F), PCSca-1+ transplanted hearts showed higher IGF-1 on day 1 and day 7 (Figure 3G).
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Cx-43 Confers Cytoprotection
Because we observed that IGF-1 preconditioning increased Cx-43 in Sca-1+, we established its role in cytoprotection. Cx-43 was abolished in Cx-43 small interfering RNA (siRNA) transfected cells grown under normoxia or OGD compared with scramble siRNA (Sc siRNA) transfected cells (Figure I in the online-only Data Supplement). These results were confirmed by Western blotting (Figure 4A1). LDH assay showed higher cell death in Cx-43 siRNA transfected cells under 4-hour and 8-hour OGD compared with Sc siRNA transfected cells (P=0.0075 by 2-way ANOVA; Figure 4A2). Additionally, Sca-1+ transfected with Cx-43 siRNA showed shrunken and rounded morphology (Figure 4A3) compared with Sc siRNA transfected cells (Figure 4A4). Elucidating the mechanism for increased death in Cx-43 siRNA transfected cells under 8-hour OGD, we observed that caspase-3 cleavage was significantly increased (Figure 4A5). Double fluorescence immunostaining revealed punctate and colocalized distribution of Cx-43 (red) and cytochrome c (green) in the mitochondria (Figure 4A6). Figure 4A7 shows a magnified image of an Sca-1+ cell from Figure 4A6 (white box) to show colocalization of Cx-43 (red) and cytochrome c (green). Western blotting on subcellular fractions confirmed a higher presence of Cx-43 in the mitochondrial fraction in PcSca-1+ (Figure 4B). Voltage-dependent anion channel protein was used as an indicator of purity of the mitochondrial fraction. Real-time polymerase chain reaction (PCR)–based gene array showed that 10 genes from the apoptotic cascade had >2-fold higher expression in Cx-43 siRNA transfected cells (Figure II in the online-only Data Supplement). The expression of caspase recruitment domain family member-10 (Card-10), which increased 6.02-fold in Cx-43 siRNA transfected cells under normoxia, led to higher sensitivity to anoxia and increased to >10-fold (Figure III in the online-only Data Supplement). We observed that elevation of Cx-43 in response to IGF-1 treatment abrogated Card-10 with a concomitant increase in cell survival under OGD (Figure 4C).
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Sca-1+ cells from male mice transfected with Cx-43 siRNA (n=4) or Sc siRNA cells (n=4) were transplanted in a female rat heart model of acute myocardial infarction. Real-time PCR for sry gene on day-7 myocardial samples showed that survival of Cx-43 siRNA transfected cells was significantly lower compared with Sc siRNA transfected cells (Figure 4D).
In Vivo Proliferation and Survival of PCSca-1+
Immunostaining showed pronounced pAkt at the site of cell graft in PCSca-1+ transplanted animals (group 3) (Figure 5A1 to 5A3) compared with non-PCSca-1+ transplanted animals (group 2) (Figure 5A4 to 5A6). The number of TUNEL+ cells was significantly higher in group 2 compared with group 3 on day 7 after engraftment (Figure 5B1 to 5B5). We also observed a higher presence of Ki67+ cells in group 3 (P<0.001 versus group 2), especially in the cell-engrafted regions (Figure 5C1 to 5C2). For an overall estimation of donor cell survival, the entire left ventricle (LV) was used for mice sry gene estimation in female recipient rat heart at 1 day, 4 days, and 7 days after engraftment of male donor cells. Significantly higher cell survival was observed in group 3 compared with group 2 at all the studied time points (Table III in the online-only Data Supplement and Figure 5D). Dulbecco modified Eagles medium–injected female animal hearts (group 1) were used as a control.
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Angiomyogenic Fate of PCSca-1+
Confocal imaging after immunostaining for 
-sarcomeric actinin 7 days after cell engraftment depicted extensive neomyogenesis in the PCSca-1+ transplanted area (Figure 6A1 to 6A4). Colocalization of PKH26-labeled cells (red) with cardiac actinin (green) was observed in the infarct and peri-infarct regions, indicating myogenic differentiation of the engrafted cells. However, the propensity of differentiating non-PCSca-1+ was obviously low compared with PCSca-1+ (Figure 6B1 to 6B4). These results suggest that Sca-1+ has inherent differentiation potential that is accentuated by preconditioning. Immunostaining of histological sections from group 3 for myosin heavy chain (slow isoform) and Cx-43 showed that neofibers also expressed Cx-43 and were well engrafted in the host tissue (Figure 6C1 to 6C4). Electron microscopy confirmed the presence of tight junctions and myofilaments in the newly differentiating Sca-1+ (Figure 6D1 to 6D4).
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To evaluate angiogenesis and maturation index at the 6-week time point, histological sections were immunostained for von Willebrand factor VIII alone (Figure 7A1 to 7A7) or with smooth muscle actin (Figure 7B1 to 7B4). The highest angiogenic activity was observed in group 3 (Figure 7A7). By 2-way ANOVA, blood vessel density in infarct and peri-infarct regions (22.3±1.7 and 32±2.2, respectively) was higher compared with group 2 (16.9±1.5, P<0.0001; 24.3±1.4, P<0.0001) and group 1 (11.3±1.6, P<0.0001; 19.3±2.1, P<0.0001). No significant difference in maturation index was found among the 3 groups in the infarct region, but group 3 showed higher maturation index in the peri-infarct region compared with group 2 (P=0.11 by 2-way ANOVA; Figure 7B1 to 7B4). However, the total number of mature blood vessels was higher in group 3 compared with the other groups.
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Infarction Size and Heart Function
Cross sections at the midpapillary muscle level showed transmural infarction in all the animals. Marked LV wall thinning was observed at 6 weeks in group 1 with 51.9±5.6% infarction of the LV (Figure 8A1). Comparatively, infarction size at 6 weeks was attenuated in group 2 (38.8±1.2%) and group 3 (23.3±3.6%) (P=0.002 versus group 2) (Figure 8A2).
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The indices of systolic function including LV ejection fraction and LV fractional shortening were higher in group 2 (43.3±2.8%, P=0.024 and 17.3±1.4%, P=0.024) and group 3 (56.2±3.5%, P=0.001 and 24.3±2.1%, P=0.001) compared with group 1 (28.3±4.7%; 10.5±2%) (Figure 8B and 8C). LV ejection fraction (P=0.029) and LV fractional shortening (P=0.03) between group 3 and group 2 also showed a significant difference. LV end-systolic dimension (in centimeters) was smaller in group 3 (0.57±0.05; P=0.0003 versus all other groups by 2-way ANOVA) compared with group 1 (0.71±0.03) and group 2 (0.7±0.01) (Figure 8D). LV wall thickness improved from 0.07 cm in group 1 to 0.08±0.01 cm in group 2 (group 2 versus group 1, P=0.59) and 0.11±0.01 cm in group 3 (group 3 versus group 1, P=0.02) (Figure 8E).
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Discussion |
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IGF-1 acts through the IGF-1/IGF-1R ligand/receptor system and significantly affects LV remodeling.24 The IGF-1 autocrine and paracrine systems in the viable cardiomyocytes are altered transiently in response to myocardial infarction.25 Therefore, strategies were designed to manipulate intrinsic IGF-1 for its continuous participation in myocardial repair. To this end, IGF-1 protein and transgene delivery either by direct DNA injection or by ex vivo cell-based delivery has been studied.26,27 We have recently shown that IGF-1 transgene overexpression in the mesenchymal stem cells promoted their survival after transplantation.28 Similar beneficial effects were achieved by preconditioning to activate IGF-1–mediated signaling in the stem cells. Our preconditioning approach with IGF-1 protein is simpler and much safer to follow from a clinical perspective compared with IGF-1 transgene overexpression. The results of preconditioning also implied that IGF-1 was not required to be carried by stem cells. Preconditioning improved cell survival through classic survival signaling involving Akt activation, which preserved mitochondrial integrity and prevented cytochrome c release, thus preventing caspase-3 cleavage with a possible role for Cx-43. Multiple cellular and physiological functions of IGF-1 have been reported through activation of PI3K-Akt.27,29,30 Interestingly, 30-minute preconditioning in this study generated pAkt, which remained elevated after 8-hour OGD in vitro and for 24 hours after transplantation. We infer that better engraftment of PcSca-1+ after transplantation in this study was due to improved cell survival, reduced cell apoptosis, and increased cell proliferation.
Another interesting observation in this study was elevation of IGF-1 in the infarcted heart after PCSca-1+ engraftment. Besides adoption of cardiac phenotype, the beneficial effect of heart cell therapy is attributed to release of paracrine factors that are accentuated by their genetic and pharmacological manipulation.6,31 Western blotting showed copious expression of IGF-1 from Sca-1+ under OGD in vitro and in the infarcted heart until day 7 of observation. Additionally, we observed that short-term IGF-1 treatment for preconditioning and the continuous presence of IGF-1 during OGD had distinct effects on stem cells in terms of cardiomyogenic gene expression. First, the timing of IGF-1 paracrine release was optimal to provide a second window of preconditioning when the effects of initial in vitro preconditioning vanished. Second, in agreement with our in vitro results from supplementation of recombinant IGF-1 for 7 days in culture under normoxia as well as anoxia, the duration of IGF-1 paracrine release in vivo spanning 7 days prompted expression of cardiac-specific marker proteins including Cx-43. Therefore, PCSca-1+ manifested enhanced cardiomyogenesis compared with their non-PCSca-1+ counterparts. This was evident from immunohistological studies in which -sarcomeric actinin– and myosin heavy chain (slow isoform)–positive structures were colocalized with Cx-43 expression, thus signifying stem cell engraftment and coupling with existing fibers. We therefore inferred that the cardiomyogenesis observed in this study was due to extensive transdifferentiation of PCSca-1+. Although the neofibers were connected by gap junctions, their real contribution to contractility was not determined. Additional studies are therefore needed to demonstrate that neofibers were functionally competent and contributed to improved heart function. On the basis of the present data, it is safe to say that improvement in global heart function was due to implantation of mesenchymal stem cells forming neofibers and release of paracrine factors.1,2
We also discovered that Cx-43 expression indeed played an important role in the cytoprotective effects of preconditioning. As discussed earlier, Cx-43 is responsible for electric coupling and maintenance of homeostasis between the adjacent cardiomyocytes. In other cells such as astrocytes, gap junctions composed of Cx-43 reduced apoptotic neuronal damage in cerebral ischemia.32 Moreover, Cx-43 hemichannels are capable of responding to extracellular cues and induce survival signals via extracellular signal–regulated kinase activation.33 Cx-43 is also located in the mitochondrial membrane of cardiomyocytes and is upregulated in response to ischemic preconditioning.34 Additionally, Cx-43 translocates into the mitochondrial membrane in response to ischemic stress.35 In both cases, Cx-43 becomes a modulator of mitochondrial functions during cell apoptosis. We observed that abrogation of Cx-43 in Sca-1+ caused poor cell survival under OGD and in the infarcted heart. Although the mechanism of Cx-43 in conferring cytoprotection after preconditioning requires more in-depth studies, our data showing punctate and colocalized distribution of Cx-43 and cytochrome c in Sca-1+ cells confirm its mitochondrial distribution. Supporting an antiapoptotic role for Cx-43, further studies will be required to delineate the mechanism by which Cx-43 prevents cytochrome c translocation from the mitochondria into the cytoplasm. We are currently working on our hypothesis that Cx-43 either integrates into the membrane transition pores or develops a complex with cytochrome c through hydrophobic interaction, thus preventing its release into the cytoplasm. Furthermore, expression of Card-10, with its well-documented role in caspase activation in the context of apoptosis, showed sensitivity to Cx-43 overexpression as a result of IGF-1 treatment.36 Taken together, preconditioning with IGF-1 concurrently incurred cytoprotection leading to cardiomyogenesis; these were not mutually exclusive molecular events but instead intertwined with each other.
These are important findings although with limitations that warrant additional studies. One limitation is the lack of functional evidence for electric coupling of the newly formed myofibers. Selection of optimal cell type remains a major challenge for transplantation in the heart. Bone marrow–derived cells in this regard were the focus of interest2 and have been studied extensively for their ability to adopt a cardiac phenotype in the infarcted heart.6,7 More recent studies have shown that they share electric characteristics similar to those of native cardiomyocytes.37 We were particularly interested in the Sca-1+ cell population because of the association of Sca-1 antigen with cell growth activity and differentiation potential.38 Sca-1+ cells have wide distribution in the body tissues including skeletal muscle, heart, and bone marrow and have been manipulated to adopt cardiac phenotypes.39 High-level Sca-1 cells expressing mesenchymal multipotent stem cells differentiate into cardiomyocytes.40 Further studies should focus on defining Sca-1+ with multiple surface markers to select sublineages with greater cardiomyogenic capacity and on assessing functional coupling. The cytoprotective role of IGF-1–induced Cx-43 could implicate Bcl10 and nuclear factor-
B signaling. Despite these study limitations, the overwhelmingly beneficial effects of IGF-1 preconditioning, as well as the simplicity, reproducibility, easy adoptability, and lack of safety issues, make this approach highly appealing for clinical applications.
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Acknowledgments |
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Sources of Funding
This work was supported by National Institutes of Health grants R37-HL074272, HL-080686, HL087246 (Dr Ashraf), and HL087288 and HL089535 (Dr Haider).
Disclosures
None.
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.827691/DC1.
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