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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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The lack of NFATC2 in mutant myotubes is responsible for the cellular phenotype. (A) RT-PCR of RNA from wild-type and NFATC2−/− myoblasts was performed for various NFAT isoforms and normalized to the expression of 18S rRNA. NFATC4 is not expressed in myoblasts of either genotype (data not shown). Data are mean ± standard error; n = 3 for each genotype. (B) Primary myoblasts were infected with an NFAT-responsive reporter and either control (Ctrl) or NFATC2 retroviruses. Cells were induced to differentiate, and luciferase assays were subsequently performed. Data are reported as fold increase in luciferase activity over control cells. Each bar represents the mean ± standard error of four independent experiments each performed in triplicate (*P < 0.05). (C) Wild-type and NFATC2−/− myoblasts were infected with either control or NFATC2 retroviruses (RV) and induced to differentiate for 48 h. NFATC2−/− cultures infected with NFATC2 retrovirus display increased myotube size with an increased number of nuclei. (D) The number of nuclei within individual myotubes (at least two nuclei) was counted for each of the retrovirally infected cultures. Myotubes were grouped into two categories as in the legend to Fig. 4 C, and the percentage of myotubes in each category was calculated. The expression of NFATC2 in NFATC2−/− cultures returns the nuclear number proportions back to that of wild-type distributions. Each bar represents mean ± standard error of three experiments each performed in duplicate (**significantly different from +/+ Ctrl P < 0.05; *significantly different from −/− Ctrl P < 0.05). Bar, 60 μm.
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Figure 5: The lack of NFATC2 in mutant myotubes is responsible for the cellular phenotype. (A) RT-PCR of RNA from wild-type and NFATC2−/− myoblasts was performed for various NFAT isoforms and normalized to the expression of 18S rRNA. NFATC4 is not expressed in myoblasts of either genotype (data not shown). Data are mean ± standard error; n = 3 for each genotype. (B) Primary myoblasts were infected with an NFAT-responsive reporter and either control (Ctrl) or NFATC2 retroviruses. Cells were induced to differentiate, and luciferase assays were subsequently performed. Data are reported as fold increase in luciferase activity over control cells. Each bar represents the mean ± standard error of four independent experiments each performed in triplicate (*P < 0.05). (C) Wild-type and NFATC2−/− myoblasts were infected with either control or NFATC2 retroviruses (RV) and induced to differentiate for 48 h. NFATC2−/− cultures infected with NFATC2 retrovirus display increased myotube size with an increased number of nuclei. (D) The number of nuclei within individual myotubes (at least two nuclei) was counted for each of the retrovirally infected cultures. Myotubes were grouped into two categories as in the legend to Fig. 4 C, and the percentage of myotubes in each category was calculated. The expression of NFATC2 in NFATC2−/− cultures returns the nuclear number proportions back to that of wild-type distributions. Each bar represents mean ± standard error of three experiments each performed in duplicate (**significantly different from +/+ Ctrl P < 0.05; *significantly different from −/− Ctrl P < 0.05). Bar, 60 μm.

Mentions: To confirm that the absence of NFATC2 is responsible for the observed defects in myotube size and nuclear number in vitro, two types of experiments were performed. First, the expression levels of other NFAT isoforms in the mutant cells were analyzed to rule out compensatory changes that could contribute to defects in myotube size. We analyzed the expression of the three other known calcium-responsive NFAT isoforms, NFATC1, NFATC3, and NFATC4, in wild-type and NFATC2−/− cells. RT-PCR was performed on RNA isolated from high-density wild-type and NFATC2−/− myoblasts using gene-specific primers and normalized to the expression of 18S RNA. NFATC4 was not detected in either genotype (data not shown). The expression of NFATC1 and NFATC3 is not significantly different between the two types of muscle cells (Fig. 5 A). Thus, the absence of NFATC2 does not alter the expression of NFATC1 or NFATC3 in skeletal muscle cells. These results are consistent with the results of Ranger et al. 2000, indicating that no compensatory changes occur in the expression of individual NFAT isoforms in chondrocytes lacking 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)

The lack of NFATC2 in mutant myotubes is responsible for the cellular phenotype. (A) RT-PCR of RNA from wild-type and NFATC2−/− myoblasts was performed for various NFAT isoforms and normalized to the expression of 18S rRNA. NFATC4 is not expressed in myoblasts of either genotype (data not shown). Data are mean ± standard error; n = 3 for each genotype. (B) Primary myoblasts were infected with an NFAT-responsive reporter and either control (Ctrl) or NFATC2 retroviruses. Cells were induced to differentiate, and luciferase assays were subsequently performed. Data are reported as fold increase in luciferase activity over control cells. Each bar represents the mean ± standard error of four independent experiments each performed in triplicate (*P < 0.05). (C) Wild-type and NFATC2−/− myoblasts were infected with either control or NFATC2 retroviruses (RV) and induced to differentiate for 48 h. NFATC2−/− cultures infected with NFATC2 retrovirus display increased myotube size with an increased number of nuclei. (D) The number of nuclei within individual myotubes (at least two nuclei) was counted for each of the retrovirally infected cultures. Myotubes were grouped into two categories as in the legend to Fig. 4 C, and the percentage of myotubes in each category was calculated. The expression of NFATC2 in NFATC2−/− cultures returns the nuclear number proportions back to that of wild-type distributions. Each bar represents mean ± standard error of three experiments each performed in duplicate (**significantly different from +/+ Ctrl P < 0.05; *significantly different from −/− Ctrl P < 0.05). Bar, 60 μm.
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Figure 5: The lack of NFATC2 in mutant myotubes is responsible for the cellular phenotype. (A) RT-PCR of RNA from wild-type and NFATC2−/− myoblasts was performed for various NFAT isoforms and normalized to the expression of 18S rRNA. NFATC4 is not expressed in myoblasts of either genotype (data not shown). Data are mean ± standard error; n = 3 for each genotype. (B) Primary myoblasts were infected with an NFAT-responsive reporter and either control (Ctrl) or NFATC2 retroviruses. Cells were induced to differentiate, and luciferase assays were subsequently performed. Data are reported as fold increase in luciferase activity over control cells. Each bar represents the mean ± standard error of four independent experiments each performed in triplicate (*P < 0.05). (C) Wild-type and NFATC2−/− myoblasts were infected with either control or NFATC2 retroviruses (RV) and induced to differentiate for 48 h. NFATC2−/− cultures infected with NFATC2 retrovirus display increased myotube size with an increased number of nuclei. (D) The number of nuclei within individual myotubes (at least two nuclei) was counted for each of the retrovirally infected cultures. Myotubes were grouped into two categories as in the legend to Fig. 4 C, and the percentage of myotubes in each category was calculated. The expression of NFATC2 in NFATC2−/− cultures returns the nuclear number proportions back to that of wild-type distributions. Each bar represents mean ± standard error of three experiments each performed in duplicate (**significantly different from +/+ Ctrl P < 0.05; *significantly different from −/− Ctrl P < 0.05). Bar, 60 μm.
Mentions: To confirm that the absence of NFATC2 is responsible for the observed defects in myotube size and nuclear number in vitro, two types of experiments were performed. First, the expression levels of other NFAT isoforms in the mutant cells were analyzed to rule out compensatory changes that could contribute to defects in myotube size. We analyzed the expression of the three other known calcium-responsive NFAT isoforms, NFATC1, NFATC3, and NFATC4, in wild-type and NFATC2−/− cells. RT-PCR was performed on RNA isolated from high-density wild-type and NFATC2−/− myoblasts using gene-specific primers and normalized to the expression of 18S RNA. NFATC4 was not detected in either genotype (data not shown). The expression of NFATC1 and NFATC3 is not significantly different between the two types of muscle cells (Fig. 5 A). Thus, the absence of NFATC2 does not alter the expression of NFATC1 or NFATC3 in skeletal muscle cells. These results are consistent with the results of Ranger et al. 2000, indicating that no compensatory changes occur in the expression of individual NFAT isoforms in chondrocytes lacking 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