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Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle.

Matsuura K, Wada H, Nagai T, Iijima Y, Minamino T, Sano M, Akazawa H, Molkentin JD, Kasanuki H, Komuro I - J. Cell Biol. (2004)

Bottom Line: Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes.Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo.In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan.

ABSTRACT
The concept of the plasticity or transdifferentiation of adult stem cells has been challenged by the phenomenon of cell fusion. In this work, we examined whether neonatal cardiomyocytes fuse with various somatic cells including endothelial cells, cardiac fibroblasts, bone marrow cells, and endothelial progenitor cells spontaneously in vitro. When cardiomyocytes were cocultured with endothelial cells or cardiac fibroblasts, they fused and showed phenotypes of cardiomyocytes. Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes. Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo. In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins. These results suggest that cardiomyocytes fuse with other cells and enter the cell cycle by maintaining their phenotypes.

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Cre/lox recombination assay for detection of cell fusion in vivo. (A and C) Schematic representation of the transgenes expressed by the mouse line and the adenovirus used in HUVEC transplantation model (A) and in skeletal muscle–derived cell transplantation model (C). (B) Cre-expressing HUVEC were transplanted to the heart of CAG-CAT-LacZ mice. Two adjacent sections of 6 μm were prepared. One section treated with X-gal staining demonstrated a β-gal+ cell in the myocardium (a and b, arrow). The adjacent section, which was double stained with mouse monoclonal anti-Cre and goat polyclonal anti-cTnT antibodies, showed the expression of Cre (c, green, arrow) and cTnT (c, red, arrow) with the fine-striated pattern in the same cell in a and b. Bars: a, 50 μm; b and c, 100 μm. (D) Skeletal muscle–derived cells isolated from CAG-CAT-LacZ mice were transplanted to the heart of MerCreMer mice treated with tamoxifen. Sections were analyzed by X-gal staining or by triple staining with rabbit polyclonal anti-β-gal, goat polyclonal anti-cTnT, and mouse monoclonal anti-Cre antibodies. X-gal staining revealed a β-gal+ cell in the myocardium (a). The immunofluorescent confocal images demonstrated that β-gal+ (b and d, green) cells also expressed cTnT (c and d, red) and Cre (c and d, blue). The merged images of the same view of d were taken by fluorescent microscope (e). Nuclei were stained with Hoechst 33258 (e, blue). Arrows indicate the fused cells. Bars, 100 μm.
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fig8: Cre/lox recombination assay for detection of cell fusion in vivo. (A and C) Schematic representation of the transgenes expressed by the mouse line and the adenovirus used in HUVEC transplantation model (A) and in skeletal muscle–derived cell transplantation model (C). (B) Cre-expressing HUVEC were transplanted to the heart of CAG-CAT-LacZ mice. Two adjacent sections of 6 μm were prepared. One section treated with X-gal staining demonstrated a β-gal+ cell in the myocardium (a and b, arrow). The adjacent section, which was double stained with mouse monoclonal anti-Cre and goat polyclonal anti-cTnT antibodies, showed the expression of Cre (c, green, arrow) and cTnT (c, red, arrow) with the fine-striated pattern in the same cell in a and b. Bars: a, 50 μm; b and c, 100 μm. (D) Skeletal muscle–derived cells isolated from CAG-CAT-LacZ mice were transplanted to the heart of MerCreMer mice treated with tamoxifen. Sections were analyzed by X-gal staining or by triple staining with rabbit polyclonal anti-β-gal, goat polyclonal anti-cTnT, and mouse monoclonal anti-Cre antibodies. X-gal staining revealed a β-gal+ cell in the myocardium (a). The immunofluorescent confocal images demonstrated that β-gal+ (b and d, green) cells also expressed cTnT (c and d, red) and Cre (c and d, blue). The merged images of the same view of d were taken by fluorescent microscope (e). Nuclei were stained with Hoechst 33258 (e, blue). Arrows indicate the fused cells. Bars, 100 μm.

Mentions: Next, we examined whether cardiomyocytes fuse with other mature somatic cells in vivo as well as in vitro. RFP+ HUVEC or RFP+ skeletal muscle–derived cells isolated from neonatal Sprague-Dawley rats were first injected into the hearts of adult GFP transgenic Sprague-Dawley rats. At 7 d after injection, we observed GFP and RFP double-positive cells, which also expressed cTnT, in the heart (Fig. 7 A). The expression of two different kinds of dyes in the single cell suggests that spontaneous cell fusion could also occur in vivo in the heart. Furthermore, we examined the cell fusion in vivo by using the Cre/lox recombination assay. HUVEC infected with adenovirus containing the nuclear-localized Cre recombinase gene (Kanegae et al., 1995) were transplanted directly to the heart of mice that carry the loxP-flanked chloramphenicol acetyltransferase (CAT) gene located between the CAG promoter and the LacZ gene (Fig. 8 A, CAG-CAT-LacZ). At 4 d after transplantation, some β-gal+ cells were observed in the myocardium, and the same cells in the adjacent sections showed the expression of Cre and cTnT with the fine striated pattern. A typical image was presented in Fig. 8 B. Skeletal muscle–derived cells isolated from CAG-CAT-LacZ mice were transplanted to the heart of MerCreMer mice (Sohal et al., 2001) in which the expression of Cre was restricted to the cardiomyocytes under the control of the αMHC promoter after treatment with tamoxifen (Fig. 8 C). At 4 d after transplantation, we observed some β-gal+ cells that coexpressed Cre and cTnT in the myocardium (Fig. 8 D). These genetic results strongly suggest that HUVEC and skeletal muscle–derived cells could fuse spontaneously with cardiomyocytes in vivo. Next, we examined whether cardiomyocytes spontaneously fuse with endogenous surrounding cells in injured heart tissue. When adult rat hearts were cryoinjured there were cells expressing both cTnT and vWF at the border zone, but not at the normal and injured areas (Fig. 7 B). Staining of adjacent sections revealed that cells that expressed both cTnT and vWF also expressed desmin and Ki67 (Fig. 7 B), whereas there were no Ki67-expressing cardiomyocytes in the normal adult heart. These findings suggest that cardiomyocytes fuse with surrounding endothelial lineage cells and reenter the cell cycle also in vivo.


Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle.

Matsuura K, Wada H, Nagai T, Iijima Y, Minamino T, Sano M, Akazawa H, Molkentin JD, Kasanuki H, Komuro I - J. Cell Biol. (2004)

Cre/lox recombination assay for detection of cell fusion in vivo. (A and C) Schematic representation of the transgenes expressed by the mouse line and the adenovirus used in HUVEC transplantation model (A) and in skeletal muscle–derived cell transplantation model (C). (B) Cre-expressing HUVEC were transplanted to the heart of CAG-CAT-LacZ mice. Two adjacent sections of 6 μm were prepared. One section treated with X-gal staining demonstrated a β-gal+ cell in the myocardium (a and b, arrow). The adjacent section, which was double stained with mouse monoclonal anti-Cre and goat polyclonal anti-cTnT antibodies, showed the expression of Cre (c, green, arrow) and cTnT (c, red, arrow) with the fine-striated pattern in the same cell in a and b. Bars: a, 50 μm; b and c, 100 μm. (D) Skeletal muscle–derived cells isolated from CAG-CAT-LacZ mice were transplanted to the heart of MerCreMer mice treated with tamoxifen. Sections were analyzed by X-gal staining or by triple staining with rabbit polyclonal anti-β-gal, goat polyclonal anti-cTnT, and mouse monoclonal anti-Cre antibodies. X-gal staining revealed a β-gal+ cell in the myocardium (a). The immunofluorescent confocal images demonstrated that β-gal+ (b and d, green) cells also expressed cTnT (c and d, red) and Cre (c and d, blue). The merged images of the same view of d were taken by fluorescent microscope (e). Nuclei were stained with Hoechst 33258 (e, blue). Arrows indicate the fused cells. Bars, 100 μm.
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Related In: Results  -  Collection

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fig8: Cre/lox recombination assay for detection of cell fusion in vivo. (A and C) Schematic representation of the transgenes expressed by the mouse line and the adenovirus used in HUVEC transplantation model (A) and in skeletal muscle–derived cell transplantation model (C). (B) Cre-expressing HUVEC were transplanted to the heart of CAG-CAT-LacZ mice. Two adjacent sections of 6 μm were prepared. One section treated with X-gal staining demonstrated a β-gal+ cell in the myocardium (a and b, arrow). The adjacent section, which was double stained with mouse monoclonal anti-Cre and goat polyclonal anti-cTnT antibodies, showed the expression of Cre (c, green, arrow) and cTnT (c, red, arrow) with the fine-striated pattern in the same cell in a and b. Bars: a, 50 μm; b and c, 100 μm. (D) Skeletal muscle–derived cells isolated from CAG-CAT-LacZ mice were transplanted to the heart of MerCreMer mice treated with tamoxifen. Sections were analyzed by X-gal staining or by triple staining with rabbit polyclonal anti-β-gal, goat polyclonal anti-cTnT, and mouse monoclonal anti-Cre antibodies. X-gal staining revealed a β-gal+ cell in the myocardium (a). The immunofluorescent confocal images demonstrated that β-gal+ (b and d, green) cells also expressed cTnT (c and d, red) and Cre (c and d, blue). The merged images of the same view of d were taken by fluorescent microscope (e). Nuclei were stained with Hoechst 33258 (e, blue). Arrows indicate the fused cells. Bars, 100 μm.
Mentions: Next, we examined whether cardiomyocytes fuse with other mature somatic cells in vivo as well as in vitro. RFP+ HUVEC or RFP+ skeletal muscle–derived cells isolated from neonatal Sprague-Dawley rats were first injected into the hearts of adult GFP transgenic Sprague-Dawley rats. At 7 d after injection, we observed GFP and RFP double-positive cells, which also expressed cTnT, in the heart (Fig. 7 A). The expression of two different kinds of dyes in the single cell suggests that spontaneous cell fusion could also occur in vivo in the heart. Furthermore, we examined the cell fusion in vivo by using the Cre/lox recombination assay. HUVEC infected with adenovirus containing the nuclear-localized Cre recombinase gene (Kanegae et al., 1995) were transplanted directly to the heart of mice that carry the loxP-flanked chloramphenicol acetyltransferase (CAT) gene located between the CAG promoter and the LacZ gene (Fig. 8 A, CAG-CAT-LacZ). At 4 d after transplantation, some β-gal+ cells were observed in the myocardium, and the same cells in the adjacent sections showed the expression of Cre and cTnT with the fine striated pattern. A typical image was presented in Fig. 8 B. Skeletal muscle–derived cells isolated from CAG-CAT-LacZ mice were transplanted to the heart of MerCreMer mice (Sohal et al., 2001) in which the expression of Cre was restricted to the cardiomyocytes under the control of the αMHC promoter after treatment with tamoxifen (Fig. 8 C). At 4 d after transplantation, we observed some β-gal+ cells that coexpressed Cre and cTnT in the myocardium (Fig. 8 D). These genetic results strongly suggest that HUVEC and skeletal muscle–derived cells could fuse spontaneously with cardiomyocytes in vivo. Next, we examined whether cardiomyocytes spontaneously fuse with endogenous surrounding cells in injured heart tissue. When adult rat hearts were cryoinjured there were cells expressing both cTnT and vWF at the border zone, but not at the normal and injured areas (Fig. 7 B). Staining of adjacent sections revealed that cells that expressed both cTnT and vWF also expressed desmin and Ki67 (Fig. 7 B), whereas there were no Ki67-expressing cardiomyocytes in the normal adult heart. These findings suggest that cardiomyocytes fuse with surrounding endothelial lineage cells and reenter the cell cycle also in vivo.

Bottom Line: Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes.Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo.In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan.

ABSTRACT
The concept of the plasticity or transdifferentiation of adult stem cells has been challenged by the phenomenon of cell fusion. In this work, we examined whether neonatal cardiomyocytes fuse with various somatic cells including endothelial cells, cardiac fibroblasts, bone marrow cells, and endothelial progenitor cells spontaneously in vitro. When cardiomyocytes were cocultured with endothelial cells or cardiac fibroblasts, they fused and showed phenotypes of cardiomyocytes. Furthermore, cardiomyocytes reentered the G2-M phase in the cell cycle after fusing with proliferative noncardiomyocytes. Transplanted endothelial cells or skeletal muscle-derived cells fused with adult cardiomyocytes in vivo. In the cryoinjured heart, there were Ki67-positive cells that expressed both cardiac and endothelial lineage marker proteins. These results suggest that cardiomyocytes fuse with other cells and enter the cell cycle by maintaining their phenotypes.

Show MeSH
Related in: MedlinePlus