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• W2087935545 abstract "The cardiomyogenic potential of adult bone marrow (BM) cells after being directly transplanted into the ischemically injured heart remains a controversial issue. In this study, we investigated the ability of transplanted BM cells to develop intracellular calcium ([Ca2+]i) transients in response to membrane depolarization in situ. Low-density mononuclear (LDM) BM cells, c-kit-enriched (c-kitenr) BM cells, and highly enriched lin– c-kit+ BM cells were obtained from adult transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP), and injected into peri-infarct myocardiums of nontransgenic mice. After 9–10 days the mice were killed, and the hearts were removed, perfused in Langendorff mode, loaded with the calcium-sensitive fluorophore rhod-2, and subjected to two-photon laser scanning fluorescence microscopy (TPLSM) to monitor action potential–induced [Ca2+]i transients in EGFP-expressing donor-derived cells and non-expressing host cardiomyocytes. Whereas spontaneous and electrically evoked [Ca2+]i transients were found to occur synchronously in host cardiomyocytes along the graft–host border and in areas remote from the infarct, they were absent in all of the >3,000 imaged BM-derived cells that were located in clusters throughout the infarct scar or peri-infarct zone. We conclude that engrafted BM-derived cells lack attributes of functioning cardiomyocytes, calling into question the concept that adult BM cells can give rise to substantive cardiomyocyte regeneration within the infarcted heart. The cardiomyogenic potential of adult bone marrow (BM) cells after being directly transplanted into the ischemically injured heart remains a controversial issue. In this study, we investigated the ability of transplanted BM cells to develop intracellular calcium ([Ca2+]i) transients in response to membrane depolarization in situ. Low-density mononuclear (LDM) BM cells, c-kit-enriched (c-kitenr) BM cells, and highly enriched lin– c-kit+ BM cells were obtained from adult transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP), and injected into peri-infarct myocardiums of nontransgenic mice. After 9–10 days the mice were killed, and the hearts were removed, perfused in Langendorff mode, loaded with the calcium-sensitive fluorophore rhod-2, and subjected to two-photon laser scanning fluorescence microscopy (TPLSM) to monitor action potential–induced [Ca2+]i transients in EGFP-expressing donor-derived cells and non-expressing host cardiomyocytes. Whereas spontaneous and electrically evoked [Ca2+]i transients were found to occur synchronously in host cardiomyocytes along the graft–host border and in areas remote from the infarct, they were absent in all of the >3,000 imaged BM-derived cells that were located in clusters throughout the infarct scar or peri-infarct zone. We conclude that engrafted BM-derived cells lack attributes of functioning cardiomyocytes, calling into question the concept that adult BM cells can give rise to substantive cardiomyocyte regeneration within the infarcted heart. IntroductionConflicting data exist as to the ability of adult bone marrow (BM) cells to give rise to cardiomyocytes within the injured heart.1Rubart M Field LJ Cardiac regeneration: repopulating the heart.Annu Rev Physiol. 2006; 68: 29-49Crossref PubMed Scopus (198) Google Scholar The possibility of cardiomyocyte formation by adult BM-derived stem cells was initially demonstrated in a study by Bittner et al.,2Bittner RE Schöfer C Weipoltshammer K Ivanova S Streubel B Hauser E et al.Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice.Anat Embryol (Berl). 1999; 199: 391-396Crossref PubMed Scopus (384) Google Scholar who observed dystrophin-expressing cardiomyocytes in mdx mice after BM reconstitution with wild-type donor cells. Subsequently, Jackson et al.3Jackson KA Majka SM Wang H Pocius J Hartley CJ Majesky MW et al.Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells.J Clin Invest. 2001; 107: 1395-1402Crossref PubMed Scopus (1764) Google Scholar induced myocardial ischemia/reperfusion injury in mice after BM reconstitution with so-called side population cells that were genetically engineered to express β-galactosidase. Donor cell–derived cardiomyocytes were detected in the peri-infarct zone, albeit at a very low prevalence (0.02%). A study by Xaymardan et al.4Xaymardan M Tang L Zagreda L Pallante B Zheng J Chazen JL et al.Platelet-derived growth factor-AB promotes the generation of adult bone marrow-derived cardiac myocytes.Circ Res. 2004; 94: E39-E45Crossref PubMed Scopus (72) Google Scholar showed that a small percentage of fluorescently labeled unfractionated BM cells obtained from adult rats can acquire a cardiomyocyte phenotype in an infarct transplantation model. Similarly, fluorescently tagged BM mononuclear cells from patients with previous myocardial infarction were shown to transdifferentiate into cardiomyocytes when seeded on cryoinjured mouse ventricles in culture.5Fernández-Avilés F San Román JA García-Frade J Fernández ME Peñarrubia MJ de la Fuente L et al.Experimental and clinical regenerative capability of human bone marrow cells after myocardial infarction.Circ Res. 2004; 95: 742-748Crossref PubMed Scopus (411) Google ScholarA limited number of studies have reported extensive myocardial regeneration from BM-derived cells within the infarcted heart.6Orlic D Kajstura J Chimenti S Jakoniuk I Anderson SM Li B et al.Bone marrow cells regenerate infarcted myocardium.Nature. 2001; 410: 701-705Crossref PubMed Scopus (4672) Google Scholar,7Kajstura J Rota M Whang B Cascapera S Hosoda T Bearzi C Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fusion.Circ Res. 2005; 96: 127-137Crossref PubMed Scopus (432) Google Scholar In these latter studies, female mice were subjected to permanent coronary artery ligation, and c-kit-positive BM cells from adult male transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP) were directly injected into the viable peri-infarct region. De novo formation of donor cell–derived cardiomyocytes was assessed by co-staining for cardiomyocyte-specific markers plus Y chromosome or EGFP. By this approach, it was demonstrated that transplanted BM cells could give rise to millions of new cardiomyocytes by 9 days after intracardiac injection, resulting in partial replacement of the scar with functioning muscle. The concomitant improvement of left ventricular contractility led the authors to suggest that de novo cardiomyocytes became electromechanically integrated and were thus capable of directly contributing to the overall pump function of the injured heart.Numerous other studies have failed to observe cardiomyogenic differentiation from adult BM-derived donor cells. For example, Balsam et al.8Balsam LB Wagers AJ Christensen JL Kofidis T Weissman IL Robbins RC Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.Nature. 2004; 428: 668-673Crossref PubMed Scopus (1530) Google Scholar demonstrated that purified populations of adult hematopoetic stem cells obtained from transgenic mice widely expressing EGFP transiently engrafted within the infarcted myocardium, but did not express cardiac tissue–specific markers. Rather, most of the donor-derived cells expressed the pan-hematopoetic marker CD45 and the myeloid marker Gr-1. Nygren et al.9Nygren JM Jovinge S Breitbach M Säwén P Röll W Hescheler J et al.Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.Nat Med. 2004; 10: 494-501Crossref PubMed Scopus (906) Google Scholar did not observe cardiomyogenic transformation of unfractionated BM cells or c-kit+-enriched hematopoetic stem cells after engraftment within the infarcted mouse myocardium, as evidenced by the absence of cardiomyocyte-specific immune reactivity in the donor cells. Murry et al.10Murry CE Soonpaa MH Reinecke H Nakajima H Nakajima HO Rubart M et al.Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.Nature. 2004; 428: 664-668Crossref PubMed Scopus (1893) Google Scholar utilized cardiomyocyte-restricted transgenes (expressing either β-galactosidase or EGFP under the regulation of the α-cardiac myosin heavy chain promoter) as well as a ubiquitously expressed EGFP reporter transgene (in conjunction with histochemical analyses) to track the fate of two purified populations of BM-derived hematopoetic stem cells (lin– c-kit+ and lin– c-kit+ sca-1+ cells) after their transplantation into injured hearts. Although donor cells did engraft in the damaged muscle, no transdifferentiation events were detected, as evidenced by the lack of expression of a cardiomyocyte-restricted reporter transgene, as well as the absence of cardiomyocyte-specific immune reactivity in the donor cells.10Murry CE Soonpaa MH Reinecke H Nakajima H Nakajima HO Rubart M et al.Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.Nature. 2004; 428: 664-668Crossref PubMed Scopus (1893) Google ScholarWhen all these findings are considered collectively, the question of whether regeneration of cardiomyocytes can be achieved by engrafting adult BM-derived cells within the injured heart remains highly controversial, despite the use of seemingly identical donor cells and experimental conditions in various studies. In this study, we elected to use an assay that relied on examination of function rather than of potentially subjective immune histologic endpoints, to examine the regenerative potential of adult BM-derived cells in the infarcted heart. Specifically, we systematically probed the ability of engrafted BM-derived cells to develop intracellular calcium ([Ca2+]i) transients in response to membrane depolarization within the intact heart, using a previously developed two-photon laser scanning microscopy (TPLSM)-based imaging technique.11Rubart M Wang E Dunn KW Field LJ Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts.Am J Physiol Cell Physiol. 2003; 284: C1654-C1668Crossref PubMed Scopus (69) Google Scholar,12Rubart M Pasumarthi KB Nakajima H Soonpaa MH Nakajima HO Field LJ Physiological coupling of donor and host cardiomyocytes after cellular transplantation.Circ Res. 2003; 92: 1217-1224Crossref PubMed Scopus (186) Google Scholar A side-by-side comparison was performed, of low-density mononuclear (LDM) BM cells, c-kit-enriched (c-kitenr) BM cells, and highly enriched hematopoetic stem cells (lin– c-kit+ BM cells), obtained from adult transgenic mice ubiquitously expressing EGFP. We demonstrate that all donor cell types were efficiently engrafted in the infarcted tissue of nontransgenic recipient hearts at day 9 after the cell injection. However, while spontaneous and electrical stimulation–induced [Ca2+]i transients were observed to occur synchronously in host cardiomyocytes along the graft–host border, they were absent in all of the >3,000 imaged BM-derived cells that were located in clusters within the infarct scar or border zone. These results indicate that engrafted BM-derived cells lack fundamental attributes of functioning cardiomyocytes, and further call into question the concept that adult BM cells can give rise to substantive cardiomyocyte regeneration within the infarcted heart.ResultsDonor cell preparationACT-EGFP mice were used for isolating donor BM cells. These mice express EGFP under the control of the chicken β-actin promoter.13Okabe M Ikawa M Kominami K Nakanishi T Nishimune Y “Green mice” as a source of ubiquitous green cells.FEBS Lett. 1997; 407: 313-319Abstract Full Text Full Text PDF PubMed Scopus (2248) Google Scholar In control experiments, a comparison of 10-μm sections obtained from adult ACT-EGFP and wild-type mouse hearts under epifluorescence illumination demonstrated that all cardiomyocytes in the transgenic hearts expressed EGFP throughout the cytoplasm and nuclei (Figure 1). The kinetics of electrically evoked [Ca2+]i transients in adult ACT-EGFP hearts were indistinguishable from those in wild-type hearts (data not shown). From these observations, we expect any BM-derived cardiomyocytes from ACT-EGFP mice to express EGFP stably, and to function normally.Three different donor BM cell populations were studied, namely, LDM BM cells, c-kit-enriched (c-kitenr) BM cells, and highly enriched lineage negative (lin–) c-kit+ BM cells. LDM BM cells were isolated using a density centrifugation protocol as published earlier.9Nygren JM Jovinge S Breitbach M Säwén P Röll W Hescheler J et al.Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.Nat Med. 2004; 10: 494-501Crossref PubMed Scopus (906) Google Scholar c-kitenr BM cells were isolated using magnet-activated cell sorting. Flow cytometry analyses of the c-kitenr BM cells demonstrated that the percentage of c-kit-positive cells in the eluted (i.e., labeled) fraction from the magnetic column was approximately fivefold to sixfold larger than that in the eluent (i.e., unlabeled) fraction (Figure 2a). The c-kitenr BM cell population was further assayed for the expression of EGFP and a variety of surface antigens. Before enrichment, 35% (dotted area in Figure 2b) of the cells in the initial LDM BM preparation were EGFP positive (Figure 2c). We confirmed the percentage of EGFP-expressing cells using additional epifluorescence analyses. Our number is in good agreement with earlier studies that used EGFP reporter transgenes to track the cardiomyogenic potential of marrow-derived cells.7Kajstura J Rota M Whang B Cascapera S Hosoda T Bearzi C Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fusion.Circ Res. 2005; 96: 127-137Crossref PubMed Scopus (432) Google Scholar,14Orlic D Kajstura J Chimenti S Bodine DM Leri A Anversa P Transplanted adult bone marrow cells repair myocardial infarcts in mice.Ann NY Acad Sci. 2001; 938 (discussion 229–230): 221-229Crossref PubMed Scopus (390) Google Scholar c-kitenr BM cells were 45% negative for a cocktail of antibodies that recognize mature hematopoetic cells, including lymphocytes, monocytes, granulocytes, neutrophils, and erythrocytes (Figure 2d). Markers for hematopoetic stem and progenitor cells (CD34, sca-1, flk-1) were present in 4% (flk-1) to 21% (sca-1) of the c-kitenr BM cell population (Figure 2e–g). Restricting the flow cytometry analysis to the subfraction of EGFP-positive cells within the c-kitenr BM cell population did not alter the expression profile of surface antigens (see Supplementary Figure S1), thereby suggesting that the presence of the fluorescent protein does not impart negative or positive selections on BM subpopulations. Highly enriched lin– c-kit+ BM cells were prepared by first removing cells expressing hematopoetic lineage markers through magnetic immunobead subtraction.15Orlic D Fischer R Nishikawa S Nienhuis AW Bodine DM Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor.Blood. 1993; 82: 762-770PubMed Google Scholar This lin– fraction was then reacted with an anti-c-kit monoclonal antibody and separated using fluorescence-activated cell sorting into c-kit+ and c-kit– cells (Figure 2h). Lin– cells that showed c-kit expression levels 2-fold to 100-fold higher than EGFP–/c-kit– cells were used for intracardiac transplantation (only EGFP-expressing cells were used).Figure 2Analysis of c-kitenr (panels a–g) and highly enriched lin– c-kit+ (panel h) bone marrow (BM) cells. (a) Representative flow cytometry dot plots of the eluted (positively labeled; right panel) and eluent (unlabeled; middle panel) fractions of c-kitenr BM cells based on levels of enhanced green fluorescent protein (EGFP) (x-axis) and c-kit (y-axis) expressions. The left panel shows isotype control. The axes are log-scaled. (b–g) Flow cytometry analyses of surface antigen expression in ACT-EGFP c-kitenr BM cells. c-kitenr BM cells were selected for c-kit+ (CD117+) cells using magnet-activated cell sorting as described in Materials and Methods. (b) Light scatter gating of eluted cells identified ∼70% of the cells within the low-density mononuclear gate (FSC, forward scatter). (c) Analysis of GFP expression versus side scatter (SSC) identified 35% of eluted cells as GFP+. (d) Expression of lineage markers (x-axis) versus c-kit expression (y-axis) among cells falling within the mononuclear gate as defined in b. (e) Expression of CD34 (x-axis) versus c-kit expression (y-axis) among cells falling within the lymphocyte gate. (f) Expression of Sca-1 (x-axis) versus c-kit expression (y-axis) among cells falling within the mononuclear gate. (g) Expression of Flk-1 (x-axis) versus c-kit expression (y-axis) among cells falling within the mononuclear gate. (h) Fluorescence-activated cell sorting (FACS) of ACT-EGFP, lineage-depleted (lin–) mouse BM cells based on c-kit and EGFP expression. Representative FACS dot plot of lin– BM cells stained with ACK-4/Biotin/streptavidin–phycoerythrin (streptavidin–PE). Antibody-labeled cells were separated using FACS into four subsets based on the number of c-kit receptors (y-axis) and levels of EGFP (x-axis) expression. The box in upper right rectangle indicates the fraction of c-kit+/EGFP+ cells that was used for cell transplantation (lin– c-kit+ group). The mean expression levels of both c-kit and EGFP in c-kit+/EGFP+ cells were ∼2-fold to ∼100-fold higher than those in c-kit–/EGFP– cells (lower left rectangle).View Large Image Figure ViewerDownload Hi-res image Download (PPT)ACT-EGFP BM-derived cells stably engraft infarcted myocardiumThe peri-infarct regions of nontransgenic hearts were injected with 100,000 LDM, c-kitenr, or lin– c-kit+ BM cells. The mice were subsequently killed and the hearts were removed and processed to monitor scar formation and donor cell viability, using sirius red/fast green histochemical staining and a chromogenic anti-EGFP immune reactivity assay, respectively. The analysis was performed at 9 to 10 days after the engraftment, because earlier studies had suggested that extensive cardiomyocyte regeneration from lin– c-kit+ BM cells takes place within this period.6Orlic D Kajstura J Chimenti S Jakoniuk I Anderson SM Li B et al.Bone marrow cells regenerate infarcted myocardium.Nature. 2001; 410: 701-705Crossref PubMed Scopus (4672) Google Scholar,7Kajstura J Rota M Whang B Cascapera S Hosoda T Bearzi C Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fusion.Circ Res. 2005; 96: 127-137Crossref PubMed Scopus (432) Google Scholar,16Rota M Kajstura J Hosoda T Bearzi C Vitale S Esposito G et al.Bone marrow cells adopt the cardiomyogenic fate in vivo.Proc Natl Acad Sci USA. 2007; 104: 17783-17788Crossref PubMed Scopus (257) Google Scholar Figure 3 shows representative examples of grafts generated with each cell type. In accordance with earlier observations,6Orlic D Kajstura J Chimenti S Jakoniuk I Anderson SM Li B et al.Bone marrow cells regenerate infarcted myocardium.Nature. 2001; 410: 701-705Crossref PubMed Scopus (4672) Google Scholar,7Kajstura J Rota M Whang B Cascapera S Hosoda T Bearzi C Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fusion.Circ Res. 2005; 96: 127-137Crossref PubMed Scopus (432) Google Scholar,8Balsam LB Wagers AJ Christensen JL Kofidis T Weissman IL Robbins RC Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.Nature. 2004; 428: 668-673Crossref PubMed Scopus (1530) Google Scholar,9Nygren JM Jovinge S Breitbach M Säwén P Röll W Hescheler J et al.Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.Nat Med. 2004; 10: 494-501Crossref PubMed Scopus (906) Google Scholar,10Murry CE Soonpaa MH Reinecke H Nakajima H Nakajima HO Rubart M et al.Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.Nature. 2004; 428: 664-668Crossref PubMed Scopus (1893) Google Scholar,16Rota M Kajstura J Hosoda T Bearzi C Vitale S Esposito G et al.Bone marrow cells adopt the cardiomyogenic fate in vivo.Proc Natl Acad Sci USA. 2007; 104: 17783-17788Crossref PubMed Scopus (257) Google Scholar we found high levels of EGFP+ cells that were primarily located in clusters throughout the infarcted tissue and in the bordering peri-infarct zone rather than in the viable myocardium. The analysis showed that all three BM-derived donor cell types survived and efficiently engrafted the infarcted myocardium.Figure 3Direct transplantation and engraftment of ACT-EGFP bone marrow (BM) cells into infarcted myocardium of nontransgenic recipients. Left panels: Sirius red– and fast green–stained histological sections from infarcted hearts at 9 days after left coronary artery ligation and transplantation of 100,000 ACT-EGFP BM cells. Right panels: adjacent sections from the same hearts immunostained for EGFP (brown signal, horseradish peroxidase–conjugated secondary antibody, signal developed with diaminobenzidine reaction). Scale bar: 30 μm. EGFP, enhanced green fluorescent protein; LDM, low-density mononuclear cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It had been reported earlier that green autofluorescence can be mistaken for EGFP fluorescence in the ischemically injured heart under epifluorescence illumination.17Laflamme MA Murry CE Regenerating the heart.Nat Biotechnol. 2005; 23: 845-856Crossref PubMed Scopus (798) Google Scholar TPLSM imaging of peri-infarct regions in Langendorff-perfused mouse hearts similarly revealed strongly autofluorescent structures. They could however be reliably distinguished from EGFP fluorescence on the basis of their distinct emission profiles under the vital imaging conditions used in this study (for details, see Supplementary Data S1 and Supplementary Figure S2).Engrafted donor-derived cells lack electrically evoked [Ca2+]i transientsWe next investigated the functional fate of ACT-EGFP BM-derived cells after transplantation into peri-infarct regions. The hearts that had received LDM BM cells were examined first. The mice were killed 9 or 10 days after the BM cell injection, and the hearts were removed, loaded with rhod-2 (a calcium-sensitive fluorescent dye), and subjected to TPLSM imaging. Imaging was initially performed along the graft–host border, with the presence of [Ca2+]i transients in host cardiomyocytes within the same microscopic field providing a good positive control. A representative frame-mode image obtained during remote point stimulation at 4 Hz is shown in Figure 4a. A cluster of small (<10 μm diameter), round, donor-derived cells, readily identifiable by virtue of their EGFP fluorescence, were present adjacent to the peri-infarct border zone of the host myocardium. The host cardiomyocytes located on either side of the BM cell graft exhibited periodic increases in rhod-2 fluorescence (denoted by asterisks), reflecting cyclic increases in intracellular free calcium triggered by propagated action potentials. The calcium responses in these cells appear to be in synchrony and at the same frequency as remote stimulation, indicating that they are functionally coupled to the remote myocardium outside the peri-infarct region. In contrast, no [Ca2+]i transients were detectable in the cluster of EGFP-expressing donor-derived cells.Figure 4Simultaneous imaging of rhod-2 and enhanced green fluorescent protein (EGFP) fluorescence in a nontransgenic heart at 9 days after coronary artery ligation and injection of ACT-EGFP low-density mononuclear bone marrow cells into the peri-infarct zone. (a) Full-frame two-photon laser scanning fluorescence microscopy (TPLSM) image of the graft–host myocardium border zone. The hearts were loaded with rhod-2. Red (rhod-2) and green (EGFP) fluorescence signals were superimposed. Host cardiomyocytes and donor-derived cells (green/yellow) are apparent. The preparation was paced by point stimulation at a remote site at 4 Hz. The white bar demarcates the position of line-scan mode data acquisition. Asterisks denote host cardiomyocytes with [Ca2+]i transients. Scale bar: 20 μm. (b) Stacked line-scan images of the regions in a demarcated by the blue dotted lines. The line-scans traverse one non-EGFP-expressing (host) cardiomyocyte and one EGFP-expressing (donor derived) cell. Scale bars: 20 μm horizontally, 125 ms vertically. (c) Spatially integrated changes in rhod-2 and EGFP fluorescence for one host cardiomyocyte and one juxtaposed donor-derived cell. The fluorescence signal across the entire cell was averaged. (d) Full-frame TPLSM images obtained from the heart depicted in a at increasing depths. The heart was paced by point stimulation at a remote site at 4 Hz. The numbers indicate nominal distance of the focal plane from the epicardial surface. Scale bar: 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also obtained line-scan images from the graft–host border by repeatedly scanning at a high rate (500 Hz) along the white line in Figure 4a during remote electrical stimulation at 4 Hz. The scan line traversed a host cardiomyocyte, a juxtaposed capillary, and a small donor-derived cell. The line scans were then stacked such that the y-axis represents time and the x-axis represents distance (Figure 4b). Spatially averaged traces for the red and green fluorescence signals were then generated from the line-scan data (Figure 4c). These traces confirm the presence of action potential–evoked calcium responses in the host cardiomyocytes, and the absence of calcium responses in the neighboring LDM BM-derived cells. A series of X, Y scans taken at 4-μm z-steps along the graft–host border during continuous electrical point stimulation at 4 Hz similarly did not reveal calcium responses in the cluster of donor-derived cells (Figure 4d).In order to exclude the possibility that the absence of spontaneous and remote stimulation-evoked [Ca2+]i transients in donor-derived cells results from a lack of electrical coupling between donor and host cells (rather than from an intrinsic inability to raise cytosolic calcium in response to membrane depolarization), we also monitored changes in [Ca2+]i during electrical field stimulation (100 V, 2 ms, 3 Hz). Under these conditions, the development of [Ca2+]i transients is no longer dependent on intercellular action potential propagation. Electrical field stimulation readily evoked [Ca2+]i transients in host cardiomyocytes at sites remote from the graft as well as in areas bordering donor cell clusters, but not in donor-derived cells within the clusters. Collectively, these data indicate that engrafted LDM BM-derived cells within the infarcted tissue lack the ability to raise cytosolic calcium transiently in response to electrical membrane excitation. Similarly, no [Ca2+]i transients were inducible in BM-derived cell clusters in the center of the scar during spontaneous sinus rhythm, remote electrical point stimulation, or electrical field stimulation. A total of >300 LDM BM-derived cells, imaged and distributed among eight animals, were shown to lack spontaneous or electrically evoked [Ca2+]i transients.We next examined the functional fate of c-kitenr and lin– c-kit+ BM cells following their direct injection into peri-infarct regions. Representative TPLSM images obtained from hearts 9 days after the transplantation of either donor cell type are shown Figures 5 and 6. Frame-mode and line-scan mode images revealed synchronous and periodic increases in rhod-2 fluorescence in host cardiomyocytes at the graft–host border during electrical point stimulation at 4 Hz, but not in donor-derived cell clusters which, in many cases, appeared to be in physical contact with functioning host myocytes (see in Figures 5a and 6a). Line scan data (Figures 5b and 6b) were used for generating spatially averaged traces for the green and red fluorescent signals (Figures 5c and 6c), which confirmed the presence of action potential-evoked [Ca2+]i transients in host cardiomyocytes, and the absence of such transients in donor-derived cells, respectively. Electrical field stimulation also failed to evoke cytosolic calcium transients in these donor-derived cell populations. Moreover, we did not detect spontaneous or electrically evoked calcium responses in donor-derived cells located within the infarct scar remote from functioning host cardiomyocytes. Identical results were obtained in a total of >1,700 and >1,300 donor-derived cells after transplantation of c-kitenr and lin– kit+ BM cells, respectively. These cells were distributed among 10 animals per group. Therefore, our results indicate that the clustered EGFP-expressing cells, whether they are derived from LDM, c-kitenr, or lin– c-kit+ BM cells, lack the ability to develop cytosolic calcium transients in response to membrane depolarization, and consequently do not function as cardiomyocytes following direct transplantation into ischemically injured heart muscle.Figure 5Simultaneous imaging of rhod-2 and enhanced green fluorescent protein (EGFP) fluorescence in a nontran" @default.
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• W2087935545 title "Adult Bone Marrow–derived Cells Do Not Acquire Functional Attributes of Cardiomyocytes When Transplanted into Peri-infarct Myocardium" @default.
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