Evidence for Fusion Between Cardiac and Skeletal Muscle Cells

Abstract

Abstract-Cardiomyoplasty with skeletal myoblasts may benefit cardiac function after infarction. Recent reports indicate that adult stem cells can fuse with other cell types. Because myoblasts are "fusigenic" cells by nature, we hypothesized they might be particularly likely to fuse with cardiomyocytes. To test this, neonatal rat cardiomyocytes labeled with LacZ and green fluorescent protein (GFP) were cocultured with unlabeled C2C12 myoblasts. After 3 days, we observed a small population of skeletal myotubes that expressed LacZ and GFP, indicating cell fusion. To test whether such fusion occurred in vivo, LacZ-expressing C2C12 myoblasts were grafted into normal nude mouse hearts. At 2 weeks after grafting, cells at the graft-host interface expressed both LacZ and cardiac-specific myosin light chain 2v (MLC2v). To test more definitively whether fusion between skeletal and cardiac muscle could occur, we used a Cre/lox reporter system that activated LacZ only upon cell fusion. When neonatal cardiomyocytes from ␣-myosin heavy chain promoter (␣-MHC)-Cre mice were cocultured with myoblasts from floxed-lacZ reporter mice, LacZ was activated in a subset of cells, indicating cell fusion occurred in vitro. Finally, we grafted the floxed-lacZ myoblasts into normal hearts of ␣-MHC-Cre ϩ and ␣-MHC-Cre Ϫ mice (nϭ5 each). Hearts analyzed at 4 days and 1 week after transplantation demonstrated activation of LacZ when the skeletal muscle cells were implanted into hearts of ␣-MHC-Cre ϩ mice, but not after implantation into ␣-MHC-Cre Ϫ mice. These data indicate that skeletal muscle cell grafting gives rise to a subpopulation of skeletal-cardiac hybrid cells with a currently unknown phenotype. The full text of this article is available online at http://circres.ahajournals.org. Key Words: cardiomyoplasty Ⅲ skeletal myoblast Ⅲ cell transplantation Ⅲ cell fusion Ⅲ Cre/lox C ellular cardiomyoplasty has emerged as a promising therapy for myocardial infarct repair. Skeletal myoblasts and bone marrow-derived stem cells have been tried in various animal models and are now undergoing clinical trials. [1][2][3][4] A recent study has shown that circulating cells derived from transplanted bone marrow can fuse with cardiomyocytes, Purkinje cells and hepatocytes. 5 Because skeletal myoblasts are "fusigenic" cells by nature, we tested if myoblasts can fuse with cardiomyocytes. Our study shows that skeletal myoblast grafting into the heart results in fusion with host cardiomyocytes, giving rise to a subpopulation of hybrid cells. Materials and Methods Cell CultureMouse neonatal cardiomyocytes and primary neonatal skeletal myoblasts were isolated as described previously. 6,7 Spontaneous cell fusion (coculture) experiments were set up in gelatin-coated 6-well plates at a total density of 2ϫ10 4 cells/cm 2 (1:1 ratio) in DMEM/ M199 (4:1) supplemented with 10% horse serum, 5% FBS, and 6 g/mL insulin. These conditions were optimized for cardiomyocytes while allowing terminal differentiation of skeletal myoblasts into multinucleated myotubes.Forced fusi...

Progress and prospects: Cell based regenerative therapy for cardiovascular disease

“…Studies using reporter genes such as EGFP and b-galactosidase, or Y chromosome fluorescence in situ hybridization (FISH) 16 allow for easier recognition of the transplanted cells. The application of the Cre/lox reporter system to this problem 17 is an example of progress in this direction.…”

“…The existence of cell fusion has been clearly demonstrated in several organs [17][18][19] although its functional significance has not been determined. The combination of reporter genes and the Cre/lox system has also been used to study cell fusion in vivo.…”

“…The combination of reporter genes and the Cre/lox system has also been used to study cell fusion in vivo. 17 More recently, cells have been shown to establish connections via nanotubules that facilitate intercellular transport of macromolecules, and this could be an alternative explanation for phenotype switches of injected multipotent cells. 20 Cell integration, another plausible mechanism of cell transfer benefit, refers to the ability of certain cell types such as skeletal myoblasts, to integrate into the cardiac syncytium without trans-differentiating into the cardiac muscle in the process improving cardiac performance.…”

“…20 Cell integration, another plausible mechanism of cell transfer benefit, refers to the ability of certain cell types such as skeletal myoblasts, to integrate into the cardiac syncytium without trans-differentiating into the cardiac muscle in the process improving cardiac performance. Electromechanical coupling between the transplanted skeletal myoblasts and the myocardium 17,21 would contribute to this functional integration, although its existence has been challenged. 22 Another potential mechanism of the observed beneficial effects is the possibility that cell transfer can stimulate proliferation and/or differentiation of endogenous progenitors, based on a paracrine effect of transplanted cells (i.e.…”

“…FloxedLacZ myoblasts injected into hearts of mice that had cardiac restricted expression of Cre (a-MHC-Cre) were shown to fuse with cardiomyocytes. 17 The allure of a nonimmunogenic, autologous cell integrating into injured myocardium with possible trans-differentiation into rapidly contracting units has stimulated the initiation of clinical trials, which will be discussed below. To date, the evidence for electro-mechanical coupling between myoblasts and cardiomyocytes is indirect with no in vivo demonstration of synchronously contracting cardiomyocytes and myoblasts.…”