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IGF-I increases bone marrow contribution to adult skeletal muscle and enhances the fusion of myelomonocytic precursors.

Sacco A, Doyonnas R, LaBarge MA, Hammer MM, Kraft P, Blau HM - J. Cell Biol. (2005)

Bottom Line: One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC).However, the molecular components responsible for this injury-related response remain largely unknown.These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Muscle damage has been shown to enhance the contribution of bone marrow-derived cells (BMDCs) to regenerating skeletal muscle. One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC). However, the molecular components responsible for this injury-related response remain largely unknown. In this paper, we show that delivery of insulin-like growth factor I (IGF-I) to adult skeletal muscle by three different methods-plasmid electroporation, injection of genetically engineered myoblasts, and recombinant protein injection-increases the integration of BMDCs up to fourfold. To investigate the underlying mechanism, we developed an in vitro fusion assay in which co-cultures of MMCs and myotubes were exposed to IGF-I. The number of fusion events was substantially augmented by IGF-I, independent of its effect on cell survival. These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

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IGF-I promotes fusion of MMCs with myotubes. (a) MMCs were isolated by FACS from fresh bone marrow from GFP transgenic mice, maintained in myoblast GM for 3 d, and co-cultured with 3-d-old primary wild-type myotubes, in the presence or absence of 100 ng/ml IGF-I, according to Fig. 4 a (3). Asterisk indicates total numbers of GFP+myogenin+ multinucleated myotubes observed after 4 d, shown with a 95% confidence interval based on the Poisson distribution. (b) For analyses of cell survival, MMCs were isolated and immediately cultured in myotube-conditioned DM in the presence or absence of 100 ng/ml IGF-I. Cell counts were performed on days 0 and 3. Final cell numbers are expressed as percentages of the initial number of cells plated on day 0 (± SEM). P value was determined with a t test. P > 0.5. (c) Laser-scanning confocal image of an example of GFP+myogenin+ multinucleated myotube; immunofluorescence shows nuclear muscle myogenin (red) and MMCs GFP (green).
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fig7: IGF-I promotes fusion of MMCs with myotubes. (a) MMCs were isolated by FACS from fresh bone marrow from GFP transgenic mice, maintained in myoblast GM for 3 d, and co-cultured with 3-d-old primary wild-type myotubes, in the presence or absence of 100 ng/ml IGF-I, according to Fig. 4 a (3). Asterisk indicates total numbers of GFP+myogenin+ multinucleated myotubes observed after 4 d, shown with a 95% confidence interval based on the Poisson distribution. (b) For analyses of cell survival, MMCs were isolated and immediately cultured in myotube-conditioned DM in the presence or absence of 100 ng/ml IGF-I. Cell counts were performed on days 0 and 3. Final cell numbers are expressed as percentages of the initial number of cells plated on day 0 (± SEM). P value was determined with a t test. P > 0.5. (c) Laser-scanning confocal image of an example of GFP+myogenin+ multinucleated myotube; immunofluorescence shows nuclear muscle myogenin (red) and MMCs GFP (green).

Mentions: Experiments were designed to test the effect of IGF-I on the fusion of MMCs with myotubes. A dose–response curve for IGF-I effect on fusion was performed (50, 100, 200, and 400 ng/ml; unpublished data), and 100 ng/ml was the optimal IGF-I concentration used for further experiments. GFP+ MMCs that had been exposed in culture to myoblast GM for 3 d were co-cultured with 3-d-old myotubes that had been exposed to the DNA synthesis inhibitor Ara-C for 3 d to eliminate proliferating myoblasts. Co-cultures were maintained for the subsequent 4 d in the presence or absence of IGF-I (Fig. 7 a). As shown in Fig. 7 a, we observed a twofold increase in the number of GFP+ myotubes containing myogenin-expressing nuclei when IGF-I was added to co-cultures (95% confidence interval based on the Poisson distribution). Under these conditions, as shown in Fig. 5, GFP+ myotubes were visible as early as 5 h after co-culture and increased for a period of 4 d thereafter. This effect was not attributable to an increased survival of these cells during co-culture because the number of MMCs does not change in the presence or absence of IGF-I in DM conditioned by myotubes (P > 0.5; Fig. 7 b). An example of a multinucleated GFP+ myotube with all nuclei positive for myogenin is shown in Fig. 7 c. We conclude that IGF-I promotes fusion of MMCs with mature myotubes and that this effect is not attributable to an increase in cell survival under the experimental conditions used.


IGF-I increases bone marrow contribution to adult skeletal muscle and enhances the fusion of myelomonocytic precursors.

Sacco A, Doyonnas R, LaBarge MA, Hammer MM, Kraft P, Blau HM - J. Cell Biol. (2005)

IGF-I promotes fusion of MMCs with myotubes. (a) MMCs were isolated by FACS from fresh bone marrow from GFP transgenic mice, maintained in myoblast GM for 3 d, and co-cultured with 3-d-old primary wild-type myotubes, in the presence or absence of 100 ng/ml IGF-I, according to Fig. 4 a (3). Asterisk indicates total numbers of GFP+myogenin+ multinucleated myotubes observed after 4 d, shown with a 95% confidence interval based on the Poisson distribution. (b) For analyses of cell survival, MMCs were isolated and immediately cultured in myotube-conditioned DM in the presence or absence of 100 ng/ml IGF-I. Cell counts were performed on days 0 and 3. Final cell numbers are expressed as percentages of the initial number of cells plated on day 0 (± SEM). P value was determined with a t test. P > 0.5. (c) Laser-scanning confocal image of an example of GFP+myogenin+ multinucleated myotube; immunofluorescence shows nuclear muscle myogenin (red) and MMCs GFP (green).
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Related In: Results  -  Collection

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fig7: IGF-I promotes fusion of MMCs with myotubes. (a) MMCs were isolated by FACS from fresh bone marrow from GFP transgenic mice, maintained in myoblast GM for 3 d, and co-cultured with 3-d-old primary wild-type myotubes, in the presence or absence of 100 ng/ml IGF-I, according to Fig. 4 a (3). Asterisk indicates total numbers of GFP+myogenin+ multinucleated myotubes observed after 4 d, shown with a 95% confidence interval based on the Poisson distribution. (b) For analyses of cell survival, MMCs were isolated and immediately cultured in myotube-conditioned DM in the presence or absence of 100 ng/ml IGF-I. Cell counts were performed on days 0 and 3. Final cell numbers are expressed as percentages of the initial number of cells plated on day 0 (± SEM). P value was determined with a t test. P > 0.5. (c) Laser-scanning confocal image of an example of GFP+myogenin+ multinucleated myotube; immunofluorescence shows nuclear muscle myogenin (red) and MMCs GFP (green).
Mentions: Experiments were designed to test the effect of IGF-I on the fusion of MMCs with myotubes. A dose–response curve for IGF-I effect on fusion was performed (50, 100, 200, and 400 ng/ml; unpublished data), and 100 ng/ml was the optimal IGF-I concentration used for further experiments. GFP+ MMCs that had been exposed in culture to myoblast GM for 3 d were co-cultured with 3-d-old myotubes that had been exposed to the DNA synthesis inhibitor Ara-C for 3 d to eliminate proliferating myoblasts. Co-cultures were maintained for the subsequent 4 d in the presence or absence of IGF-I (Fig. 7 a). As shown in Fig. 7 a, we observed a twofold increase in the number of GFP+ myotubes containing myogenin-expressing nuclei when IGF-I was added to co-cultures (95% confidence interval based on the Poisson distribution). Under these conditions, as shown in Fig. 5, GFP+ myotubes were visible as early as 5 h after co-culture and increased for a period of 4 d thereafter. This effect was not attributable to an increased survival of these cells during co-culture because the number of MMCs does not change in the presence or absence of IGF-I in DM conditioned by myotubes (P > 0.5; Fig. 7 b). An example of a multinucleated GFP+ myotube with all nuclei positive for myogenin is shown in Fig. 7 c. We conclude that IGF-I promotes fusion of MMCs with mature myotubes and that this effect is not attributable to an increase in cell survival under the experimental conditions used.

Bottom Line: One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC).However, the molecular components responsible for this injury-related response remain largely unknown.These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Muscle damage has been shown to enhance the contribution of bone marrow-derived cells (BMDCs) to regenerating skeletal muscle. One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC). However, the molecular components responsible for this injury-related response remain largely unknown. In this paper, we show that delivery of insulin-like growth factor I (IGF-I) to adult skeletal muscle by three different methods-plasmid electroporation, injection of genetically engineered myoblasts, and recombinant protein injection-increases the integration of BMDCs up to fourfold. To investigate the underlying mechanism, we developed an in vitro fusion assay in which co-cultures of MMCs and myotubes were exposed to IGF-I. The number of fusion events was substantially augmented by IGF-I, independent of its effect on cell survival. These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

Show MeSH
Related in: MedlinePlus