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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 promotes enhanced myocyte fusion. (A) C2C12 myoblasts stably expressing HA-βgal or HA-FHL1 were differentiated and costained with anti-MHC (MF20, green), myogenin antibodies (blue), and propidium iodide (red). Cells were imaged using confocal microscopy. Shown are representative images after 96 h of differentiation. Myoblast differentiation was quantified from 0–96 h. Arrows indicate examples of transfected myotubes. Bar, 70 μm. (B) The percentage of total nuclei positive for myogenin. (C) The differentiation index, determined by scoring the proportion of total nuclei within MHC-positive cells (myocytes and myotubes). (D) The fusion index, the mean number of nuclei per MHC-positive cell. Black and gray lines represent HA-βgal control and HA-FHL1–expressing C2C12 cells, respectively. Data represent the mean from three independent experiments (n = 3) ± SEM in which ≥100 cells were counted for each replicate. (*, P < 0.05; **, P < 0.01). (E) Lysates (25 μg) from differentiating C2C12 cells stably expressing HA-βgal or HA-FHL1 were immunoblotted with MyoD, Myf5, myogenin, MHC, and β-tubulin antibodies (loading control). (F) MHC expression was analyzed by densitometry and quantified relative to β-tubulin. Data represent the mean from three independent experiments ± SEM (n = 3; **, P < 0.01). (G) The DNA/protein ratio was determined. Data represent the mean from four independent experiments ± SEM (n = 4; **, P < 0.01).
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fig2: FHL1 promotes enhanced myocyte fusion. (A) C2C12 myoblasts stably expressing HA-βgal or HA-FHL1 were differentiated and costained with anti-MHC (MF20, green), myogenin antibodies (blue), and propidium iodide (red). Cells were imaged using confocal microscopy. Shown are representative images after 96 h of differentiation. Myoblast differentiation was quantified from 0–96 h. Arrows indicate examples of transfected myotubes. Bar, 70 μm. (B) The percentage of total nuclei positive for myogenin. (C) The differentiation index, determined by scoring the proportion of total nuclei within MHC-positive cells (myocytes and myotubes). (D) The fusion index, the mean number of nuclei per MHC-positive cell. Black and gray lines represent HA-βgal control and HA-FHL1–expressing C2C12 cells, respectively. Data represent the mean from three independent experiments (n = 3) ± SEM in which ≥100 cells were counted for each replicate. (*, P < 0.05; **, P < 0.01). (E) Lysates (25 μg) from differentiating C2C12 cells stably expressing HA-βgal or HA-FHL1 were immunoblotted with MyoD, Myf5, myogenin, MHC, and β-tubulin antibodies (loading control). (F) MHC expression was analyzed by densitometry and quantified relative to β-tubulin. Data represent the mean from three independent experiments ± SEM (n = 3; **, P < 0.01). (G) The DNA/protein ratio was determined. Data represent the mean from four independent experiments ± SEM (n = 4; **, P < 0.01).

Mentions: Skeletal muscle hypertrophy is characterized by enhanced transcription/translation leading to increased protein synthesis, predominantly of myofibrillar proteins, which may be coupled with reduced protein breakdown (Furmanczyk and Quinn, 2003; Boonyarom and Inui, 2006). Concomitant with increased protein synthesis, and perhaps a feature of more advanced hypertrophy (Sandri, 2008), is activation and fusion of quiescent myoblasts called satellite cells with each other or with existing myofibers, resulting in myonuclei addition (Adams, 2006; O'Connor and Pavlath, 2007). We next evaluated whether FHL1-induced myotube hypertrophy was a consequence of enhanced myoblast fusion and/or differentiation and if this was accompanied by increased protein synthesis. Myoblasts stably expressing HA-FHL1 or HA-βgal control generated as described previously (McGrath et al., 2006) were induced to differentiate for 0–96 h and stained with markers of myoblast differentiation, myogenin and sarcomeric myosin heavy chain (MHC; Fig. 2 A; Cottle et al., 2007). Differentiation was assessed by scoring the percentage of myogenin-positive nuclei, which revealed a twofold increase in HA-FHL1–expressing cells, but only after 96 h differentiation (Fig. 2 B). The differentiation index represents the proportion of nuclei that are localized within MHC-positive myotubes and is used as a measure of myoblast differentiation (Sabourin et al., 1999; Erbay et al., 2003). Normally, this does not exceed ∼50% (Yoshida et al., 1998). HA-FHL1 myotubes exhibited a similar differentiation index to HA-βgal myotubes up until 72 h, but then increased approximately twofold at 96 h (Fig. 2 C). Myoblast fusion was scored as total nuclei per MHC-positive myotube (Sabourin et al., 1999). After a 96-h differentiation, HA-FHL1 cell lines showed enhanced cell fusion (Fig. 2 D). Therefore, FHL1 may promote myoblast fusion. We detected no changes in the levels or temporal appearance of the muscle regulatory factors MyoD, Myf5, or myogenin in FHL1-expressing cells during differentiation (Fig. 2 E). However, a fivefold increase in MHC expression was detected in FHL1-expressing cells at 48 h of differentiation (Fig. 2, E and F). Expression of MHC is induced during the latter stages of myoblast differentiation (Cole et al., 1993); however, accretion of this major myofibrillar protein is also indicative of muscle anabolism and hypertrophy (Furmanczyk and Quinn, 2003). Analysis of the protein/DNA ratios in FHL1-expressing myotubes compared with controls revealed a twofold increase in protein synthesis relative to DNA, a feature of skeletal muscle hypertrophy (Fig. 2 G; De Arcangelis et al., 2005). These studies reveal that FHL1 increases the fusion of myoblasts. The increased protein/DNA ratio coupled with evidence of MHC accretion may be indicative of myotube hypertrophy. An imbalance in the nuclear/cytoplasmic ratio caused by hyperfusion may lead to an increase in protein translation, which increases the myotube cytoplasmic volume (Adams, 2006; O'Connor and Pavlath, 2007).


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 promotes enhanced myocyte fusion. (A) C2C12 myoblasts stably expressing HA-βgal or HA-FHL1 were differentiated and costained with anti-MHC (MF20, green), myogenin antibodies (blue), and propidium iodide (red). Cells were imaged using confocal microscopy. Shown are representative images after 96 h of differentiation. Myoblast differentiation was quantified from 0–96 h. Arrows indicate examples of transfected myotubes. Bar, 70 μm. (B) The percentage of total nuclei positive for myogenin. (C) The differentiation index, determined by scoring the proportion of total nuclei within MHC-positive cells (myocytes and myotubes). (D) The fusion index, the mean number of nuclei per MHC-positive cell. Black and gray lines represent HA-βgal control and HA-FHL1–expressing C2C12 cells, respectively. Data represent the mean from three independent experiments (n = 3) ± SEM in which ≥100 cells were counted for each replicate. (*, P < 0.05; **, P < 0.01). (E) Lysates (25 μg) from differentiating C2C12 cells stably expressing HA-βgal or HA-FHL1 were immunoblotted with MyoD, Myf5, myogenin, MHC, and β-tubulin antibodies (loading control). (F) MHC expression was analyzed by densitometry and quantified relative to β-tubulin. Data represent the mean from three independent experiments ± SEM (n = 3; **, P < 0.01). (G) The DNA/protein ratio was determined. Data represent the mean from four independent experiments ± SEM (n = 4; **, P < 0.01).
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fig2: FHL1 promotes enhanced myocyte fusion. (A) C2C12 myoblasts stably expressing HA-βgal or HA-FHL1 were differentiated and costained with anti-MHC (MF20, green), myogenin antibodies (blue), and propidium iodide (red). Cells were imaged using confocal microscopy. Shown are representative images after 96 h of differentiation. Myoblast differentiation was quantified from 0–96 h. Arrows indicate examples of transfected myotubes. Bar, 70 μm. (B) The percentage of total nuclei positive for myogenin. (C) The differentiation index, determined by scoring the proportion of total nuclei within MHC-positive cells (myocytes and myotubes). (D) The fusion index, the mean number of nuclei per MHC-positive cell. Black and gray lines represent HA-βgal control and HA-FHL1–expressing C2C12 cells, respectively. Data represent the mean from three independent experiments (n = 3) ± SEM in which ≥100 cells were counted for each replicate. (*, P < 0.05; **, P < 0.01). (E) Lysates (25 μg) from differentiating C2C12 cells stably expressing HA-βgal or HA-FHL1 were immunoblotted with MyoD, Myf5, myogenin, MHC, and β-tubulin antibodies (loading control). (F) MHC expression was analyzed by densitometry and quantified relative to β-tubulin. Data represent the mean from three independent experiments ± SEM (n = 3; **, P < 0.01). (G) The DNA/protein ratio was determined. Data represent the mean from four independent experiments ± SEM (n = 4; **, P < 0.01).
Mentions: Skeletal muscle hypertrophy is characterized by enhanced transcription/translation leading to increased protein synthesis, predominantly of myofibrillar proteins, which may be coupled with reduced protein breakdown (Furmanczyk and Quinn, 2003; Boonyarom and Inui, 2006). Concomitant with increased protein synthesis, and perhaps a feature of more advanced hypertrophy (Sandri, 2008), is activation and fusion of quiescent myoblasts called satellite cells with each other or with existing myofibers, resulting in myonuclei addition (Adams, 2006; O'Connor and Pavlath, 2007). We next evaluated whether FHL1-induced myotube hypertrophy was a consequence of enhanced myoblast fusion and/or differentiation and if this was accompanied by increased protein synthesis. Myoblasts stably expressing HA-FHL1 or HA-βgal control generated as described previously (McGrath et al., 2006) were induced to differentiate for 0–96 h and stained with markers of myoblast differentiation, myogenin and sarcomeric myosin heavy chain (MHC; Fig. 2 A; Cottle et al., 2007). Differentiation was assessed by scoring the percentage of myogenin-positive nuclei, which revealed a twofold increase in HA-FHL1–expressing cells, but only after 96 h differentiation (Fig. 2 B). The differentiation index represents the proportion of nuclei that are localized within MHC-positive myotubes and is used as a measure of myoblast differentiation (Sabourin et al., 1999; Erbay et al., 2003). Normally, this does not exceed ∼50% (Yoshida et al., 1998). HA-FHL1 myotubes exhibited a similar differentiation index to HA-βgal myotubes up until 72 h, but then increased approximately twofold at 96 h (Fig. 2 C). Myoblast fusion was scored as total nuclei per MHC-positive myotube (Sabourin et al., 1999). After a 96-h differentiation, HA-FHL1 cell lines showed enhanced cell fusion (Fig. 2 D). Therefore, FHL1 may promote myoblast fusion. We detected no changes in the levels or temporal appearance of the muscle regulatory factors MyoD, Myf5, or myogenin in FHL1-expressing cells during differentiation (Fig. 2 E). However, a fivefold increase in MHC expression was detected in FHL1-expressing cells at 48 h of differentiation (Fig. 2, E and F). Expression of MHC is induced during the latter stages of myoblast differentiation (Cole et al., 1993); however, accretion of this major myofibrillar protein is also indicative of muscle anabolism and hypertrophy (Furmanczyk and Quinn, 2003). Analysis of the protein/DNA ratios in FHL1-expressing myotubes compared with controls revealed a twofold increase in protein synthesis relative to DNA, a feature of skeletal muscle hypertrophy (Fig. 2 G; De Arcangelis et al., 2005). These studies reveal that FHL1 increases the fusion of myoblasts. The increased protein/DNA ratio coupled with evidence of MHC accretion may be indicative of myotube hypertrophy. An imbalance in the nuclear/cytoplasmic ratio caused by hyperfusion may lead to an increase in protein translation, which increases the myotube cytoplasmic volume (Adams, 2006; O'Connor and Pavlath, 2007).

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