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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 transgenic mice exhibit skeletal muscle hypertrophy, increased strength, and fatigue resistance. (A) Transverse gastrocnemius sections were stained with a dystrophin antibody and viewed by confocal microscopy. Higher-magnification images are shown in the insets, with one fiber outlined. Bars, 50 μm. Images were analyzed for fiber area (B) and diameter (C; n = 3–4 mice; *, P < 0.05). GAS, gastrocnemius. (D) Transverse FDP sections were stained with MHC isoform-specific antibodies, and the percentage of 2A, 2X, and 2B fibers was calculated. The bar graph displays the frequency of each fiber type in wild-type (WT) versus transgenic (TG) mice (n = 3–5 mice; ***, P < 0.005). (E) Transverse FDP sections were stained for 2B MHC fibers; one fiber is outlined per image. Bars, 40 μm. (F) The diameters of FDP-2B fibers from WT versus TG mice are shown (n = 3–4 mice; *, P < 0.05). (G) The frequency of Pax-7–positive nuclei in the gastrocnemius was scored, relative to total nuclei. The bar graph represents the frequency of Pax-7–positive nuclei for WT and TG mice (n = 3–5 mice; **, P < 0.01). (H) Transverse FDP sections were hematoxylin and eosin stained. Arrows indicate centralized nuclei. Bars, 40 μm. (I) The frequency of centralized nuclei were scored, relative to total myofiber nuclei, in transverse gastrocnemius and FDP sections (n = 4–6 mice; *, P < 0.05; ***, P < 0.001). (J) A whole animal strength test measured overall limb and abdominal strength as a percentage pass rate over 15 trials for WT versus TG 12-mo-old mice (n = 4–8; **, P < 0.01). All graphs represent mean ± SEM; WT, white; TG, gray. (K) Muscle fatigability was assessed as a drop in force with repeated tetanic contractions in EDL muscles from WT (black) versus TG (red) mice, expressed as a percentage of initial contraction over time, mean ± SEM (n = 7, P < 0.02).
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fig4: FHL1 transgenic mice exhibit skeletal muscle hypertrophy, increased strength, and fatigue resistance. (A) Transverse gastrocnemius sections were stained with a dystrophin antibody and viewed by confocal microscopy. Higher-magnification images are shown in the insets, with one fiber outlined. Bars, 50 μm. Images were analyzed for fiber area (B) and diameter (C; n = 3–4 mice; *, P < 0.05). GAS, gastrocnemius. (D) Transverse FDP sections were stained with MHC isoform-specific antibodies, and the percentage of 2A, 2X, and 2B fibers was calculated. The bar graph displays the frequency of each fiber type in wild-type (WT) versus transgenic (TG) mice (n = 3–5 mice; ***, P < 0.005). (E) Transverse FDP sections were stained for 2B MHC fibers; one fiber is outlined per image. Bars, 40 μm. (F) The diameters of FDP-2B fibers from WT versus TG mice are shown (n = 3–4 mice; *, P < 0.05). (G) The frequency of Pax-7–positive nuclei in the gastrocnemius was scored, relative to total nuclei. The bar graph represents the frequency of Pax-7–positive nuclei for WT and TG mice (n = 3–5 mice; **, P < 0.01). (H) Transverse FDP sections were hematoxylin and eosin stained. Arrows indicate centralized nuclei. Bars, 40 μm. (I) The frequency of centralized nuclei were scored, relative to total myofiber nuclei, in transverse gastrocnemius and FDP sections (n = 4–6 mice; *, P < 0.05; ***, P < 0.001). (J) A whole animal strength test measured overall limb and abdominal strength as a percentage pass rate over 15 trials for WT versus TG 12-mo-old mice (n = 4–8; **, P < 0.01). All graphs represent mean ± SEM; WT, white; TG, gray. (K) Muscle fatigability was assessed as a drop in force with repeated tetanic contractions in EDL muscles from WT (black) versus TG (red) mice, expressed as a percentage of initial contraction over time, mean ± SEM (n = 7, P < 0.02).

Mentions: FHL1 transgenic gastrocnemius muscle exhibited a significant increase in the total fiber cross-sectional area (CSA) at both 6 (1.2-fold; Fig. 4, A and B) and 12 weeks of age (not depicted). Analysis of gastrocnemius fiber diameters revealed an increase in the frequency of larger fibers (≥50 μm) in transgenic mice (Fig. 4 C). Myofibrils from FHL1 transgenic mice were significantly wider (1.3-fold) than the wild type (Fig. S1 C; Rosenblatt and Woods, 1992). Therefore, FHL1 induces skeletal muscle hypertrophy in vivo. Skeletal muscle hypertrophy can be accompanied by a fiber type conversion (Dunn et al., 1999), identified by scoring MHC isoform expression. A 10% increase in oxidative type 2A, MHC-positive fibers was detected in FHL1 transgenic FDP muscle, with a corresponding decrease in 2X fibers; however, this latter trend was not statistically significant (Fig. 4 D). No difference was detected in the frequency of type 2B (Fig. 4 D) or type 1 fibers in FHL1 transgenic FDP muscle (not depicted); however, the FDP is composed of <1% of type 1 fibers. Therefore, FHL1 promotes skeletal muscle hypertrophy associated with a switch toward oxidative fiber type expression.


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 transgenic mice exhibit skeletal muscle hypertrophy, increased strength, and fatigue resistance. (A) Transverse gastrocnemius sections were stained with a dystrophin antibody and viewed by confocal microscopy. Higher-magnification images are shown in the insets, with one fiber outlined. Bars, 50 μm. Images were analyzed for fiber area (B) and diameter (C; n = 3–4 mice; *, P < 0.05). GAS, gastrocnemius. (D) Transverse FDP sections were stained with MHC isoform-specific antibodies, and the percentage of 2A, 2X, and 2B fibers was calculated. The bar graph displays the frequency of each fiber type in wild-type (WT) versus transgenic (TG) mice (n = 3–5 mice; ***, P < 0.005). (E) Transverse FDP sections were stained for 2B MHC fibers; one fiber is outlined per image. Bars, 40 μm. (F) The diameters of FDP-2B fibers from WT versus TG mice are shown (n = 3–4 mice; *, P < 0.05). (G) The frequency of Pax-7–positive nuclei in the gastrocnemius was scored, relative to total nuclei. The bar graph represents the frequency of Pax-7–positive nuclei for WT and TG mice (n = 3–5 mice; **, P < 0.01). (H) Transverse FDP sections were hematoxylin and eosin stained. Arrows indicate centralized nuclei. Bars, 40 μm. (I) The frequency of centralized nuclei were scored, relative to total myofiber nuclei, in transverse gastrocnemius and FDP sections (n = 4–6 mice; *, P < 0.05; ***, P < 0.001). (J) A whole animal strength test measured overall limb and abdominal strength as a percentage pass rate over 15 trials for WT versus TG 12-mo-old mice (n = 4–8; **, P < 0.01). All graphs represent mean ± SEM; WT, white; TG, gray. (K) Muscle fatigability was assessed as a drop in force with repeated tetanic contractions in EDL muscles from WT (black) versus TG (red) mice, expressed as a percentage of initial contraction over time, mean ± SEM (n = 7, P < 0.02).
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fig4: FHL1 transgenic mice exhibit skeletal muscle hypertrophy, increased strength, and fatigue resistance. (A) Transverse gastrocnemius sections were stained with a dystrophin antibody and viewed by confocal microscopy. Higher-magnification images are shown in the insets, with one fiber outlined. Bars, 50 μm. Images were analyzed for fiber area (B) and diameter (C; n = 3–4 mice; *, P < 0.05). GAS, gastrocnemius. (D) Transverse FDP sections were stained with MHC isoform-specific antibodies, and the percentage of 2A, 2X, and 2B fibers was calculated. The bar graph displays the frequency of each fiber type in wild-type (WT) versus transgenic (TG) mice (n = 3–5 mice; ***, P < 0.005). (E) Transverse FDP sections were stained for 2B MHC fibers; one fiber is outlined per image. Bars, 40 μm. (F) The diameters of FDP-2B fibers from WT versus TG mice are shown (n = 3–4 mice; *, P < 0.05). (G) The frequency of Pax-7–positive nuclei in the gastrocnemius was scored, relative to total nuclei. The bar graph represents the frequency of Pax-7–positive nuclei for WT and TG mice (n = 3–5 mice; **, P < 0.01). (H) Transverse FDP sections were hematoxylin and eosin stained. Arrows indicate centralized nuclei. Bars, 40 μm. (I) The frequency of centralized nuclei were scored, relative to total myofiber nuclei, in transverse gastrocnemius and FDP sections (n = 4–6 mice; *, P < 0.05; ***, P < 0.001). (J) A whole animal strength test measured overall limb and abdominal strength as a percentage pass rate over 15 trials for WT versus TG 12-mo-old mice (n = 4–8; **, P < 0.01). All graphs represent mean ± SEM; WT, white; TG, gray. (K) Muscle fatigability was assessed as a drop in force with repeated tetanic contractions in EDL muscles from WT (black) versus TG (red) mice, expressed as a percentage of initial contraction over time, mean ± SEM (n = 7, P < 0.02).
Mentions: FHL1 transgenic gastrocnemius muscle exhibited a significant increase in the total fiber cross-sectional area (CSA) at both 6 (1.2-fold; Fig. 4, A and B) and 12 weeks of age (not depicted). Analysis of gastrocnemius fiber diameters revealed an increase in the frequency of larger fibers (≥50 μm) in transgenic mice (Fig. 4 C). Myofibrils from FHL1 transgenic mice were significantly wider (1.3-fold) than the wild type (Fig. S1 C; Rosenblatt and Woods, 1992). Therefore, FHL1 induces skeletal muscle hypertrophy in vivo. Skeletal muscle hypertrophy can be accompanied by a fiber type conversion (Dunn et al., 1999), identified by scoring MHC isoform expression. A 10% increase in oxidative type 2A, MHC-positive fibers was detected in FHL1 transgenic FDP muscle, with a corresponding decrease in 2X fibers; however, this latter trend was not statistically significant (Fig. 4 D). No difference was detected in the frequency of type 2B (Fig. 4 D) or type 1 fibers in FHL1 transgenic FDP muscle (not depicted); however, the FDP is composed of <1% of type 1 fibers. Therefore, FHL1 promotes skeletal muscle hypertrophy associated with a switch toward oxidative fiber type expression.

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