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Identification of FHL1 as a regulator of skeletal muscle mass: implications for human myopathy.

Cowling BS, McGrath MJ, Nguyen MA, Cottle DL, Kee AJ, Brown S, Schessl J, Zou Y, Joya J, Bönnemann CG, Hardeman EC, Mitchell CA - J. Cell Biol. (2008)

Bottom Line: In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes.Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity.NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Victoria, Australia.

ABSTRACT
Regulators of skeletal muscle mass are of interest, given the morbidity and mortality of muscle atrophy and myopathy. Four-and-a-half LIM protein 1 (FHL1) is mutated in several human myopathies, including reducing-body myopathy (RBM). The normal function of FHL1 in muscle and how it causes myopathy remains unknown. We find that FHL1 transgenic expression in mouse skeletal muscle promotes hypertrophy and an oxidative fiber-type switch, leading to increased whole-body strength and fatigue resistance. Additionally, FHL1 overexpression enhances myoblast fusion, resulting in hypertrophic myotubes in C2C12 cells, (a phenotype rescued by calcineurin inhibition). In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes. FHL1 binds with the calcineurin-regulated transcription factor NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1), enhancing NFATc1 transcriptional activity. Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity. NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle. Therefore, via NFATc1 signaling regulation, FHL1 appears to modulate muscle mass and strength enhancement.

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FHL1 regulates the transcriptional activity of NFATc1 and expression of the calcineurin-responsive gene GATA-2. (A) C2C12 myoblasts were transiently transfected with HA-tagged wild-type (WT) FHL1, FHL1C132F, or FHL1H123Y and allowed to differentiate for 72 h, then costained with anti-HA antibody (green) and To-Pro-3 (blue) to detect nuclei. Cells were imaged using confocal microscopy. Arrows indicate hypertrophic myotubes. Bars, 100 μm. (B) Maximum myotube diameter was measured in myotubes transiently transfected with HA-tagged wild-type (WT) FHL1, FHL1C132F, FHL1H123Y, or HA-βgal control, then differentiated for 96 h and stained as described in Materials and methods. The bar graph represents the mean frequency of myotubes with diameters of 0–20 μm, >20–40 μm, and >40 μm, ±SEM (n > 300 myotubes from three experiments; n = 3; *, P < 0.05). (C) C2C12 myoblasts were cotransfected with various combinations of expression vectors as indicated, and with NFATc1-dependent (pGL2-IL-2) and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 48 h and assayed for luciferase activity. Gal4DBD activity is represented by white bars and Gal4DBD-NFATc1 by gray bars. Error bars represent ± SEM (n ≥ 5 experiments; *, P < 0.05). (D) Transfected myoblasts were differentiated for 48 h and assayed for luciferase activity (48 h; n = 4 experiments). (E) Gastrocnemius (GAS) lysates from wild-type (WT) or HA-FHL1 transgenic (TG) mice were immunoblotted for GATA-2 or β-tubulin (loading control). (F) GATA-2 protein expression levels in WT (white) versus TG (gray) gastrocnemius muscle was quantified using densitometry and standardized to the loading control. The bar graph represents the mean ± SEM (n = 5 mice per genotype; *, P < 0.05). (G) Transverse gastrocnemius muscle sections were costained with anti–GATA-2 (green), anti-vinculin (red; sarcolemma) antibodies, and To-Pro-3 iodide nuclear stain (blue), then imaged by confocal microscopy. Images were taken at the same intensity. Bars, 40 μm. (H) C2C12 cells were cotransfected with various combinations of expression vectors for Gal4DBD (white) or Gal4DBD-NFATc1 (gray) and either scrambled (control) or FHL1 siRNA oligonucleotides, as indicated, with NFATc1-dependent (pGL2-IL-2) and Renilla luciferase reporters. Assays were performed under growth conditions. Error bars represent ±SEM (n = 5 experiments; *, P < 0.05). (I) Luciferase assays performed in H were repeated under differentiation (48 h) conditions (n = 3 experiments; *, P < 0.05).
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fig7: FHL1 regulates the transcriptional activity of NFATc1 and expression of the calcineurin-responsive gene GATA-2. (A) C2C12 myoblasts were transiently transfected with HA-tagged wild-type (WT) FHL1, FHL1C132F, or FHL1H123Y and allowed to differentiate for 72 h, then costained with anti-HA antibody (green) and To-Pro-3 (blue) to detect nuclei. Cells were imaged using confocal microscopy. Arrows indicate hypertrophic myotubes. Bars, 100 μm. (B) Maximum myotube diameter was measured in myotubes transiently transfected with HA-tagged wild-type (WT) FHL1, FHL1C132F, FHL1H123Y, or HA-βgal control, then differentiated for 96 h and stained as described in Materials and methods. The bar graph represents the mean frequency of myotubes with diameters of 0–20 μm, >20–40 μm, and >40 μm, ±SEM (n > 300 myotubes from three experiments; n = 3; *, P < 0.05). (C) C2C12 myoblasts were cotransfected with various combinations of expression vectors as indicated, and with NFATc1-dependent (pGL2-IL-2) and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 48 h and assayed for luciferase activity. Gal4DBD activity is represented by white bars and Gal4DBD-NFATc1 by gray bars. Error bars represent ± SEM (n ≥ 5 experiments; *, P < 0.05). (D) Transfected myoblasts were differentiated for 48 h and assayed for luciferase activity (48 h; n = 4 experiments). (E) Gastrocnemius (GAS) lysates from wild-type (WT) or HA-FHL1 transgenic (TG) mice were immunoblotted for GATA-2 or β-tubulin (loading control). (F) GATA-2 protein expression levels in WT (white) versus TG (gray) gastrocnemius muscle was quantified using densitometry and standardized to the loading control. The bar graph represents the mean ± SEM (n = 5 mice per genotype; *, P < 0.05). (G) Transverse gastrocnemius muscle sections were costained with anti–GATA-2 (green), anti-vinculin (red; sarcolemma) antibodies, and To-Pro-3 iodide nuclear stain (blue), then imaged by confocal microscopy. Images were taken at the same intensity. Bars, 40 μm. (H) C2C12 cells were cotransfected with various combinations of expression vectors for Gal4DBD (white) or Gal4DBD-NFATc1 (gray) and either scrambled (control) or FHL1 siRNA oligonucleotides, as indicated, with NFATc1-dependent (pGL2-IL-2) and Renilla luciferase reporters. Assays were performed under growth conditions. Error bars represent ±SEM (n = 5 experiments; *, P < 0.05). (I) Luciferase assays performed in H were repeated under differentiation (48 h) conditions (n = 3 experiments; *, P < 0.05).

Mentions: Differentiation of skeletal myoblasts (myogenesis) is defined by an ordered series of events (Abmayr et al., 2003). After induction of differentiation, myoblasts withdraw from the cell cycle, then elongate, align, and fuse to form primary multinucleated myotubes. A second wave of myoblast fusion results in further myonuclei addition to nascent myotubes, followed by the assembly of myofibrillar proteins into the costamere and sarcomere (Sanger et al., 2002). We have previously demonstrated that FHL1 promotes myoblast elongation in Sol8 myoblast cell lines (McGrath et al., 2003) and is important for sarcomere formation (McGrath et al., 2006). To investigate the role FHL1 plays in myoblast fusion and differentiation, C2C12 skeletal myoblasts were transiently transfected with HA-FHL1 or HA-βgal control and induced to differentiate to myotubes. Overexpression of FHL1 resulted in the formation of large “sac-like” myotubes containing dense clusters of nuclei (Fig. 1, C and D, arrows), in contrast to the thin, elongated myotubes containing a linear arrangement of myonuclei observed in HA-βgal control cells (Fig. 1, A and B). Analysis of myotube diameters revealed a shift toward larger myotubes, with FHL1 overexpression resulting in ∼20% of myotubes being >40 μm in diameter, compared with only 5% of control myotubes (see Fig. 7 B).


Identification of FHL1 as a regulator of skeletal muscle mass: implications for human myopathy.

Cowling BS, McGrath MJ, Nguyen MA, Cottle DL, Kee AJ, Brown S, Schessl J, Zou Y, Joya J, Bönnemann CG, Hardeman EC, Mitchell CA - J. Cell Biol. (2008)

FHL1 regulates the transcriptional activity of NFATc1 and expression of the calcineurin-responsive gene GATA-2. (A) C2C12 myoblasts were transiently transfected with HA-tagged wild-type (WT) FHL1, FHL1C132F, or FHL1H123Y and allowed to differentiate for 72 h, then costained with anti-HA antibody (green) and To-Pro-3 (blue) to detect nuclei. Cells were imaged using confocal microscopy. Arrows indicate hypertrophic myotubes. Bars, 100 μm. (B) Maximum myotube diameter was measured in myotubes transiently transfected with HA-tagged wild-type (WT) FHL1, FHL1C132F, FHL1H123Y, or HA-βgal control, then differentiated for 96 h and stained as described in Materials and methods. The bar graph represents the mean frequency of myotubes with diameters of 0–20 μm, >20–40 μm, and >40 μm, ±SEM (n > 300 myotubes from three experiments; n = 3; *, P < 0.05). (C) C2C12 myoblasts were cotransfected with various combinations of expression vectors as indicated, and with NFATc1-dependent (pGL2-IL-2) and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 48 h and assayed for luciferase activity. Gal4DBD activity is represented by white bars and Gal4DBD-NFATc1 by gray bars. Error bars represent ± SEM (n ≥ 5 experiments; *, P < 0.05). (D) Transfected myoblasts were differentiated for 48 h and assayed for luciferase activity (48 h; n = 4 experiments). (E) Gastrocnemius (GAS) lysates from wild-type (WT) or HA-FHL1 transgenic (TG) mice were immunoblotted for GATA-2 or β-tubulin (loading control). (F) GATA-2 protein expression levels in WT (white) versus TG (gray) gastrocnemius muscle was quantified using densitometry and standardized to the loading control. The bar graph represents the mean ± SEM (n = 5 mice per genotype; *, P < 0.05). (G) Transverse gastrocnemius muscle sections were costained with anti–GATA-2 (green), anti-vinculin (red; sarcolemma) antibodies, and To-Pro-3 iodide nuclear stain (blue), then imaged by confocal microscopy. Images were taken at the same intensity. Bars, 40 μm. (H) C2C12 cells were cotransfected with various combinations of expression vectors for Gal4DBD (white) or Gal4DBD-NFATc1 (gray) and either scrambled (control) or FHL1 siRNA oligonucleotides, as indicated, with NFATc1-dependent (pGL2-IL-2) and Renilla luciferase reporters. Assays were performed under growth conditions. Error bars represent ±SEM (n = 5 experiments; *, P < 0.05). (I) Luciferase assays performed in H were repeated under differentiation (48 h) conditions (n = 3 experiments; *, P < 0.05).
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fig7: FHL1 regulates the transcriptional activity of NFATc1 and expression of the calcineurin-responsive gene GATA-2. (A) C2C12 myoblasts were transiently transfected with HA-tagged wild-type (WT) FHL1, FHL1C132F, or FHL1H123Y and allowed to differentiate for 72 h, then costained with anti-HA antibody (green) and To-Pro-3 (blue) to detect nuclei. Cells were imaged using confocal microscopy. Arrows indicate hypertrophic myotubes. Bars, 100 μm. (B) Maximum myotube diameter was measured in myotubes transiently transfected with HA-tagged wild-type (WT) FHL1, FHL1C132F, FHL1H123Y, or HA-βgal control, then differentiated for 96 h and stained as described in Materials and methods. The bar graph represents the mean frequency of myotubes with diameters of 0–20 μm, >20–40 μm, and >40 μm, ±SEM (n > 300 myotubes from three experiments; n = 3; *, P < 0.05). (C) C2C12 myoblasts were cotransfected with various combinations of expression vectors as indicated, and with NFATc1-dependent (pGL2-IL-2) and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 48 h and assayed for luciferase activity. Gal4DBD activity is represented by white bars and Gal4DBD-NFATc1 by gray bars. Error bars represent ± SEM (n ≥ 5 experiments; *, P < 0.05). (D) Transfected myoblasts were differentiated for 48 h and assayed for luciferase activity (48 h; n = 4 experiments). (E) Gastrocnemius (GAS) lysates from wild-type (WT) or HA-FHL1 transgenic (TG) mice were immunoblotted for GATA-2 or β-tubulin (loading control). (F) GATA-2 protein expression levels in WT (white) versus TG (gray) gastrocnemius muscle was quantified using densitometry and standardized to the loading control. The bar graph represents the mean ± SEM (n = 5 mice per genotype; *, P < 0.05). (G) Transverse gastrocnemius muscle sections were costained with anti–GATA-2 (green), anti-vinculin (red; sarcolemma) antibodies, and To-Pro-3 iodide nuclear stain (blue), then imaged by confocal microscopy. Images were taken at the same intensity. Bars, 40 μm. (H) C2C12 cells were cotransfected with various combinations of expression vectors for Gal4DBD (white) or Gal4DBD-NFATc1 (gray) and either scrambled (control) or FHL1 siRNA oligonucleotides, as indicated, with NFATc1-dependent (pGL2-IL-2) and Renilla luciferase reporters. Assays were performed under growth conditions. Error bars represent ±SEM (n = 5 experiments; *, P < 0.05). (I) Luciferase assays performed in H were repeated under differentiation (48 h) conditions (n = 3 experiments; *, P < 0.05).
Mentions: Differentiation of skeletal myoblasts (myogenesis) is defined by an ordered series of events (Abmayr et al., 2003). After induction of differentiation, myoblasts withdraw from the cell cycle, then elongate, align, and fuse to form primary multinucleated myotubes. A second wave of myoblast fusion results in further myonuclei addition to nascent myotubes, followed by the assembly of myofibrillar proteins into the costamere and sarcomere (Sanger et al., 2002). We have previously demonstrated that FHL1 promotes myoblast elongation in Sol8 myoblast cell lines (McGrath et al., 2003) and is important for sarcomere formation (McGrath et al., 2006). To investigate the role FHL1 plays in myoblast fusion and differentiation, C2C12 skeletal myoblasts were transiently transfected with HA-FHL1 or HA-βgal control and induced to differentiate to myotubes. Overexpression of FHL1 resulted in the formation of large “sac-like” myotubes containing dense clusters of nuclei (Fig. 1, C and D, arrows), in contrast to the thin, elongated myotubes containing a linear arrangement of myonuclei observed in HA-βgal control cells (Fig. 1, A and B). Analysis of myotube diameters revealed a shift toward larger myotubes, with FHL1 overexpression resulting in ∼20% of myotubes being >40 μm in diameter, compared with only 5% of control myotubes (see Fig. 7 B).

Bottom Line: In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes.Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity.NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Victoria, Australia.

ABSTRACT
Regulators of skeletal muscle mass are of interest, given the morbidity and mortality of muscle atrophy and myopathy. Four-and-a-half LIM protein 1 (FHL1) is mutated in several human myopathies, including reducing-body myopathy (RBM). The normal function of FHL1 in muscle and how it causes myopathy remains unknown. We find that FHL1 transgenic expression in mouse skeletal muscle promotes hypertrophy and an oxidative fiber-type switch, leading to increased whole-body strength and fatigue resistance. Additionally, FHL1 overexpression enhances myoblast fusion, resulting in hypertrophic myotubes in C2C12 cells, (a phenotype rescued by calcineurin inhibition). In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes. FHL1 binds with the calcineurin-regulated transcription factor NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1), enhancing NFATc1 transcriptional activity. Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity. NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle. Therefore, via NFATc1 signaling regulation, FHL1 appears to modulate muscle mass and strength enhancement.

Show MeSH
Related in: MedlinePlus