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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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Regenerating myofibers form, but their growth is impaired in NFATC2−/− mice. (A) Northern blots of TA muscles from wild-type and NFATC2−/− mice 4 d after injury are shown. A portion of an ethidium bromide–stained gel containing 28S and 18S rRNAs demonstrates relative RNA loading. (B) 7 d after injury, TA muscles were collected from wild-type and NFATC2−/− mice, and the number of regenerated myofibers was counted in sections from the central region of the lesion. (C) A comparison of hematoxylin and eosin–stained sections of regenerating TA muscles at day 25 after injury demonstrates the smaller size of the mutant myofibers (top). The CSA of regenerated myofibers in the central region of the lesion was determined for various time points after injury (bottom). Data are mean ± standard error; n = 3–7 for each genotype (*P < 0.05). Bar, 60 μm.
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Figure 3: Regenerating myofibers form, but their growth is impaired in NFATC2−/− mice. (A) Northern blots of TA muscles from wild-type and NFATC2−/− mice 4 d after injury are shown. A portion of an ethidium bromide–stained gel containing 28S and 18S rRNAs demonstrates relative RNA loading. (B) 7 d after injury, TA muscles were collected from wild-type and NFATC2−/− mice, and the number of regenerated myofibers was counted in sections from the central region of the lesion. (C) A comparison of hematoxylin and eosin–stained sections of regenerating TA muscles at day 25 after injury demonstrates the smaller size of the mutant myofibers (top). The CSA of regenerated myofibers in the central region of the lesion was determined for various time points after injury (bottom). Data are mean ± standard error; n = 3–7 for each genotype (*P < 0.05). Bar, 60 μm.

Mentions: During postnatal development and during regeneration after injury, myofibers grow in size, a process involving addition of nuclei to the myofiber. Since myonuclei are postmitotic, myofiber growth involves the fusion of muscle precursor cells with myofibers. These muscle precursor cells, called satellite cells, lie underneath the basal lamina surrounding each myofiber and are in close juxtaposition to the myofiber itself. Satellite cells are normally quiescent but become activated and start proliferating in response to growth factors. Since satellite cells are required for skeletal muscle regeneration (Robertson et al. 1992; Quinlan et al. 1997) and muscle growth (Darr and Schultz 1989; Rosenblatt and Parry 1993; Mozdziak et al. 2000), we used regenerating muscle as a model to examine satellite cell function in NFATC2−/− mice. Northern analysis was performed on regenerating TA muscles of wild-type and NFATC2−/− mice on day 4 after injury using myoD and myogenin as markers of activation/proliferation and differentiation, respectively. At this time point, satellite cells have become activated and are proliferating and beginning to differentiate. No differences are observed in the levels of either MyoD or myogenin mRNA (Fig. 3 A). In addition, the number of myoblasts isolated from wild-type and NFATC2−/− muscles 2 d after regeneration does not differ (data not shown), further suggesting that NFATC2−/− satellite cells are able to activate and proliferate normally. We next analyzed the ability of satellite cells to fuse and form new myofibers. When myofibers form after injury, their nuclei are centrally localized, facilitating identification of regenerating myofibers. The number of centrally nucleated myofibers in the core of the injury 7 d after injury was counted as described in Materials and Methods. At this time point, the area of injury is completely filled with these regenerating myofibers. As seen in Fig. 3 B, no significant difference exists in the number of regenerated myofibers between wild-type and NFATC2−/− mice. Therefore, satellite cell function appears normal in the early stages of muscle regeneration in the absence of NFATC2.


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)

Regenerating myofibers form, but their growth is impaired in NFATC2−/− mice. (A) Northern blots of TA muscles from wild-type and NFATC2−/− mice 4 d after injury are shown. A portion of an ethidium bromide–stained gel containing 28S and 18S rRNAs demonstrates relative RNA loading. (B) 7 d after injury, TA muscles were collected from wild-type and NFATC2−/− mice, and the number of regenerated myofibers was counted in sections from the central region of the lesion. (C) A comparison of hematoxylin and eosin–stained sections of regenerating TA muscles at day 25 after injury demonstrates the smaller size of the mutant myofibers (top). The CSA of regenerated myofibers in the central region of the lesion was determined for various time points after injury (bottom). Data are mean ± standard error; n = 3–7 for each genotype (*P < 0.05). Bar, 60 μm.
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Related In: Results  -  Collection

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Figure 3: Regenerating myofibers form, but their growth is impaired in NFATC2−/− mice. (A) Northern blots of TA muscles from wild-type and NFATC2−/− mice 4 d after injury are shown. A portion of an ethidium bromide–stained gel containing 28S and 18S rRNAs demonstrates relative RNA loading. (B) 7 d after injury, TA muscles were collected from wild-type and NFATC2−/− mice, and the number of regenerated myofibers was counted in sections from the central region of the lesion. (C) A comparison of hematoxylin and eosin–stained sections of regenerating TA muscles at day 25 after injury demonstrates the smaller size of the mutant myofibers (top). The CSA of regenerated myofibers in the central region of the lesion was determined for various time points after injury (bottom). Data are mean ± standard error; n = 3–7 for each genotype (*P < 0.05). Bar, 60 μm.
Mentions: During postnatal development and during regeneration after injury, myofibers grow in size, a process involving addition of nuclei to the myofiber. Since myonuclei are postmitotic, myofiber growth involves the fusion of muscle precursor cells with myofibers. These muscle precursor cells, called satellite cells, lie underneath the basal lamina surrounding each myofiber and are in close juxtaposition to the myofiber itself. Satellite cells are normally quiescent but become activated and start proliferating in response to growth factors. Since satellite cells are required for skeletal muscle regeneration (Robertson et al. 1992; Quinlan et al. 1997) and muscle growth (Darr and Schultz 1989; Rosenblatt and Parry 1993; Mozdziak et al. 2000), we used regenerating muscle as a model to examine satellite cell function in NFATC2−/− mice. Northern analysis was performed on regenerating TA muscles of wild-type and NFATC2−/− mice on day 4 after injury using myoD and myogenin as markers of activation/proliferation and differentiation, respectively. At this time point, satellite cells have become activated and are proliferating and beginning to differentiate. No differences are observed in the levels of either MyoD or myogenin mRNA (Fig. 3 A). In addition, the number of myoblasts isolated from wild-type and NFATC2−/− muscles 2 d after regeneration does not differ (data not shown), further suggesting that NFATC2−/− satellite cells are able to activate and proliferate normally. We next analyzed the ability of satellite cells to fuse and form new myofibers. When myofibers form after injury, their nuclei are centrally localized, facilitating identification of regenerating myofibers. The number of centrally nucleated myofibers in the core of the injury 7 d after injury was counted as described in Materials and Methods. At this time point, the area of injury is completely filled with these regenerating myofibers. As seen in Fig. 3 B, no significant difference exists in the number of regenerated myofibers between wild-type and NFATC2−/− mice. Therefore, satellite cell function appears normal in the early stages of muscle regeneration in the absence of NFATC2.

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