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Regulation of the growth of multinucleated muscle cells by an NFATC2-dependent pathway.

Horsley V, Friday BB, Matteson S, Kegley KM, Gephart J, Pavlath GK - J. Cell Biol. (2001)

Bottom Line: The growth defect is intrinsic to muscle cells, since the lack of NFATC2 in primary muscle cultures results in reduced cell size and myonuclear number in myotubes.Taken together, these results implicate a novel role for NFATC2 in skeletal muscle growth.We demonstrate that during growth of multinucleated muscle cells, myoblasts initially fuse to form myotubes with a limited number of nuclei and that subsequent nuclear addition and increases in myotube size are controlled by a molecular pathway regulated by NFATC2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University, Atlanta, Georgia 30322, USA.

ABSTRACT
The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the development and differentiation of several tissue types. Here, we examine the role of NFATC2 in skeletal muscle by analyzing adult NFATC2(-/)- mice. These mice exhibit reduced muscle size due to a decrease in myofiber cross-sectional area, suggesting that growth is blunted. Muscle growth was examined during regeneration after injury, wherein NFATC2- myofibers form normally but display impaired growth. The growth defect is intrinsic to muscle cells, since the lack of NFATC2 in primary muscle cultures results in reduced cell size and myonuclear number in myotubes. Retroviral-mediated expression of NFATC2 in the mutant cells rescues this cellular phenotype. Myonuclear number is similarly decreased in NFATC2(-/)- mice. Taken together, these results implicate a novel role for NFATC2 in skeletal muscle growth. We demonstrate that during growth of multinucleated muscle cells, myoblasts initially fuse to form myotubes with a limited number of nuclei and that subsequent nuclear addition and increases in myotube size are controlled by a molecular pathway regulated by NFATC2.

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NFATC2−/− myoblasts form small myotubes with decreased nuclear number in vitro. (A) Cellular proteins from wild-type and NFATC2−/− myoblasts were collected at various time points during differentiation and analyzed by immunoblotting for myogenin and EMyHC. The expression patterns of myogenin and EMyHC are similar between differentiating wild-type and NFATC2−/− muscle cells. A portion of a Coomassie-stained gel demonstrates relative protein loading. (B) Wild-type and NFATC2−/− myoblasts were induced to differentiate in DM for 48 h and then immunostained for EMyHC. (C) The number of nuclei within individual myotubes (at least two nuclei) was counted. Myotubes were grouped into two categories, and the percentage of myotubes in each category was determined. Each bar represents mean ± standard error of three independent cell isolates each performed in duplicate (*P < 0.05). Bar, 60 μm.
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Figure 4: NFATC2−/− myoblasts form small myotubes with decreased nuclear number in vitro. (A) Cellular proteins from wild-type and NFATC2−/− myoblasts were collected at various time points during differentiation and analyzed by immunoblotting for myogenin and EMyHC. The expression patterns of myogenin and EMyHC are similar between differentiating wild-type and NFATC2−/− muscle cells. A portion of a Coomassie-stained gel demonstrates relative protein loading. (B) Wild-type and NFATC2−/− myoblasts were induced to differentiate in DM for 48 h and then immunostained for EMyHC. (C) The number of nuclei within individual myotubes (at least two nuclei) was counted. Myotubes were grouped into two categories, and the percentage of myotubes in each category was determined. Each bar represents mean ± standard error of three independent cell isolates each performed in duplicate (*P < 0.05). Bar, 60 μm.

Mentions: Myofiber growth is dependent on both nonmuscle and muscle cells. To determine if the defects observed in myofiber growth in NFATC2−/− mice are intrinsic to muscle cells, myoblasts were isolated from wild-type and NFATC2−/− mice and studied in vitro. To determine if NFATC2−/− myoblasts can differentiate normally, immunoblot analyses were performed on muscle cells in DM using antibodies against myogenin and EMyHC, markers of myogenic commitment and differentiation, respectively (Merlie and Gros 1976; Andres and Walsh 1996). Myogenin levels increase at 24 h and are downregulated at 48 h in both wild-type and NFATC2−/− cells (Fig. 4 A). A small amount of myogenin is detected in both types of myoblasts, presumably due to spontaneous differentiation in the cultures. EMyHC is expressed at similar levels in both cell types after 24 and 48 h in DM. Thus, the expression patterns of myogenin and EMyHC in wild-type and NFATC2−/− muscle cells are similar, indicating that the early stages of muscle differentiation are not regulated by NFATC2. Consistent with these results, after 48 h in DM wild-type and NFATC2−/− cultures appear morphologically differentiated, containing many multinucleated cells with an elongated appearance. However, NFATC2−/− cells form myotubes that are smaller than wild-type myotubes (Fig. 4 B), indicating a muscle-intrinsic role for NFATC2 in regulating myotube size.


Regulation of the growth of multinucleated muscle cells by an NFATC2-dependent pathway.

Horsley V, Friday BB, Matteson S, Kegley KM, Gephart J, Pavlath GK - J. Cell Biol. (2001)

NFATC2−/− myoblasts form small myotubes with decreased nuclear number in vitro. (A) Cellular proteins from wild-type and NFATC2−/− myoblasts were collected at various time points during differentiation and analyzed by immunoblotting for myogenin and EMyHC. The expression patterns of myogenin and EMyHC are similar between differentiating wild-type and NFATC2−/− muscle cells. A portion of a Coomassie-stained gel demonstrates relative protein loading. (B) Wild-type and NFATC2−/− myoblasts were induced to differentiate in DM for 48 h and then immunostained for EMyHC. (C) The number of nuclei within individual myotubes (at least two nuclei) was counted. Myotubes were grouped into two categories, and the percentage of myotubes in each category was determined. Each bar represents mean ± standard error of three independent cell isolates each performed in duplicate (*P < 0.05). Bar, 60 μm.
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Related In: Results  -  Collection

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Figure 4: NFATC2−/− myoblasts form small myotubes with decreased nuclear number in vitro. (A) Cellular proteins from wild-type and NFATC2−/− myoblasts were collected at various time points during differentiation and analyzed by immunoblotting for myogenin and EMyHC. The expression patterns of myogenin and EMyHC are similar between differentiating wild-type and NFATC2−/− muscle cells. A portion of a Coomassie-stained gel demonstrates relative protein loading. (B) Wild-type and NFATC2−/− myoblasts were induced to differentiate in DM for 48 h and then immunostained for EMyHC. (C) The number of nuclei within individual myotubes (at least two nuclei) was counted. Myotubes were grouped into two categories, and the percentage of myotubes in each category was determined. Each bar represents mean ± standard error of three independent cell isolates each performed in duplicate (*P < 0.05). Bar, 60 μm.
Mentions: Myofiber growth is dependent on both nonmuscle and muscle cells. To determine if the defects observed in myofiber growth in NFATC2−/− mice are intrinsic to muscle cells, myoblasts were isolated from wild-type and NFATC2−/− mice and studied in vitro. To determine if NFATC2−/− myoblasts can differentiate normally, immunoblot analyses were performed on muscle cells in DM using antibodies against myogenin and EMyHC, markers of myogenic commitment and differentiation, respectively (Merlie and Gros 1976; Andres and Walsh 1996). Myogenin levels increase at 24 h and are downregulated at 48 h in both wild-type and NFATC2−/− cells (Fig. 4 A). A small amount of myogenin is detected in both types of myoblasts, presumably due to spontaneous differentiation in the cultures. EMyHC is expressed at similar levels in both cell types after 24 and 48 h in DM. Thus, the expression patterns of myogenin and EMyHC in wild-type and NFATC2−/− muscle cells are similar, indicating that the early stages of muscle differentiation are not regulated by NFATC2. Consistent with these results, after 48 h in DM wild-type and NFATC2−/− cultures appear morphologically differentiated, containing many multinucleated cells with an elongated appearance. However, NFATC2−/− cells form myotubes that are smaller than wild-type myotubes (Fig. 4 B), indicating a muscle-intrinsic role for NFATC2 in regulating myotube size.

Bottom Line: The growth defect is intrinsic to muscle cells, since the lack of NFATC2 in primary muscle cultures results in reduced cell size and myonuclear number in myotubes.Taken together, these results implicate a novel role for NFATC2 in skeletal muscle growth.We demonstrate that during growth of multinucleated muscle cells, myoblasts initially fuse to form myotubes with a limited number of nuclei and that subsequent nuclear addition and increases in myotube size are controlled by a molecular pathway regulated by NFATC2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Emory University, Atlanta, Georgia 30322, USA.

ABSTRACT
The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the development and differentiation of several tissue types. Here, we examine the role of NFATC2 in skeletal muscle by analyzing adult NFATC2(-/)- mice. These mice exhibit reduced muscle size due to a decrease in myofiber cross-sectional area, suggesting that growth is blunted. Muscle growth was examined during regeneration after injury, wherein NFATC2- myofibers form normally but display impaired growth. The growth defect is intrinsic to muscle cells, since the lack of NFATC2 in primary muscle cultures results in reduced cell size and myonuclear number in myotubes. Retroviral-mediated expression of NFATC2 in the mutant cells rescues this cellular phenotype. Myonuclear number is similarly decreased in NFATC2(-/)- mice. Taken together, these results implicate a novel role for NFATC2 in skeletal muscle growth. We demonstrate that during growth of multinucleated muscle cells, myoblasts initially fuse to form myotubes with a limited number of nuclei and that subsequent nuclear addition and increases in myotube size are controlled by a molecular pathway regulated by NFATC2.

Show MeSH
Related in: MedlinePlus