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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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Generation of FHL1 transgenic mice. (A) The linearized transgene contains the 2.2-kb HSA promoter, fused in frame to the human FHL1 coding sequence with an N-terminal HA tag and a C-terminal bovine growth hormone polyA tail (BGHpA). FHL1-RBM mutants C132F and H123Y are indicated. (B) Skeletal muscle lysates were immunoblotted for HA-FHL1 or β-tubulin in wild-type (WT) or FHL1 transgenic (TG) muscle (GAS, gastrocnemius; EDL, extensor digitorum longus; SOL, soleus plantaris; PLA, plantaris; ECU, extensor carpi ulnaris; FDP, flexor digitorum profundus). A lysate prepared from C2C12 cells transfected with HA-FHL1 was used as a positive control (+). (C) The relative expression of HA-FHL1 was determined by densitometry of HA-immunoreactive polypeptides, standardized relative to β-tubulin loading. HA-FHL1 expression is represented as a fold difference, relative to GAS, arbitrarily defined as one. Graph depicts mean ± SEM (GAS, EDL, SOL, and PLA: n = 3 mice; ECU and FDP: ***, P < 0.005; n = 1 mouse). (D) Tissue lysates from WT or TG mice were immunoblotted for HA-FHL1 using anti-HA antibodies or β-tubulin. Lysates prepared from C2C12 cells transfected with HA-FHL1 were used as a positive control (+). (E) Longitudinal gastrocnemius muscle sections from WT or FHL-TG mice were stained with anti-FHL1 (green) or anti-HA (red) antibodies and imaged by confocal microscopy, then images were further resolved by deconvolution. Bars, 5 μm.
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fig3: Generation of FHL1 transgenic mice. (A) The linearized transgene contains the 2.2-kb HSA promoter, fused in frame to the human FHL1 coding sequence with an N-terminal HA tag and a C-terminal bovine growth hormone polyA tail (BGHpA). FHL1-RBM mutants C132F and H123Y are indicated. (B) Skeletal muscle lysates were immunoblotted for HA-FHL1 or β-tubulin in wild-type (WT) or FHL1 transgenic (TG) muscle (GAS, gastrocnemius; EDL, extensor digitorum longus; SOL, soleus plantaris; PLA, plantaris; ECU, extensor carpi ulnaris; FDP, flexor digitorum profundus). A lysate prepared from C2C12 cells transfected with HA-FHL1 was used as a positive control (+). (C) The relative expression of HA-FHL1 was determined by densitometry of HA-immunoreactive polypeptides, standardized relative to β-tubulin loading. HA-FHL1 expression is represented as a fold difference, relative to GAS, arbitrarily defined as one. Graph depicts mean ± SEM (GAS, EDL, SOL, and PLA: n = 3 mice; ECU and FDP: ***, P < 0.005; n = 1 mouse). (D) Tissue lysates from WT or TG mice were immunoblotted for HA-FHL1 using anti-HA antibodies or β-tubulin. Lysates prepared from C2C12 cells transfected with HA-FHL1 were used as a positive control (+). (E) Longitudinal gastrocnemius muscle sections from WT or FHL-TG mice were stained with anti-FHL1 (green) or anti-HA (red) antibodies and imaged by confocal microscopy, then images were further resolved by deconvolution. Bars, 5 μm.

Mentions: We next investigated whether FHL1 expression in postmitotic myofibers induced skeletal muscle hypertrophy in vivo. The FHL1 cDNA was fused to an HA tag (HA-FHL1) and linked to the human skeletal muscle α-actin (HSA) promoter (Fig. 3 A), which drives skeletal muscle–specific expression (Brennan and Hardeman, 1993). Skeletal α-actin is expressed only after myoblast fusion to form myotubes, and expression persists in adult muscle (Cox et al., 1990) in the elongated postmitotic cells of the somites, as well as in adult mature myofibers, but not satellite cells (Gunning et al., 1987; Miniou et al., 1999; Shen et al., 2003). FHL1 transgenic mice were generated and two lines were characterized. Transgenic HA-FHL1 expression was restricted to skeletal muscle, most prominent in fast-twitch enriched muscles including gastrocnemius (Fig. 3, B and C, GAS), extensor digitorum longus (EDL), plantaris, extensor carpi ulnaris (ECU), and flexor digitorum profundus (FDP), which is consistent with HSA promoter activity (Tinsley et al., 1996), with no expression in other tissues (Fig. 3 D). FHL1 transgenic sarcomere structure appeared undisturbed, with normal α-actinin localization at the Z-line (Fig. S1, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200804077/DC1). HA-FHL1 colocalized with endogenous FHL1 at the I-band (Fig. 3 E; McGrath et al., 2006).


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)

Generation of FHL1 transgenic mice. (A) The linearized transgene contains the 2.2-kb HSA promoter, fused in frame to the human FHL1 coding sequence with an N-terminal HA tag and a C-terminal bovine growth hormone polyA tail (BGHpA). FHL1-RBM mutants C132F and H123Y are indicated. (B) Skeletal muscle lysates were immunoblotted for HA-FHL1 or β-tubulin in wild-type (WT) or FHL1 transgenic (TG) muscle (GAS, gastrocnemius; EDL, extensor digitorum longus; SOL, soleus plantaris; PLA, plantaris; ECU, extensor carpi ulnaris; FDP, flexor digitorum profundus). A lysate prepared from C2C12 cells transfected with HA-FHL1 was used as a positive control (+). (C) The relative expression of HA-FHL1 was determined by densitometry of HA-immunoreactive polypeptides, standardized relative to β-tubulin loading. HA-FHL1 expression is represented as a fold difference, relative to GAS, arbitrarily defined as one. Graph depicts mean ± SEM (GAS, EDL, SOL, and PLA: n = 3 mice; ECU and FDP: ***, P < 0.005; n = 1 mouse). (D) Tissue lysates from WT or TG mice were immunoblotted for HA-FHL1 using anti-HA antibodies or β-tubulin. Lysates prepared from C2C12 cells transfected with HA-FHL1 were used as a positive control (+). (E) Longitudinal gastrocnemius muscle sections from WT or FHL-TG mice were stained with anti-FHL1 (green) or anti-HA (red) antibodies and imaged by confocal microscopy, then images were further resolved by deconvolution. Bars, 5 μm.
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Related In: Results  -  Collection

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fig3: Generation of FHL1 transgenic mice. (A) The linearized transgene contains the 2.2-kb HSA promoter, fused in frame to the human FHL1 coding sequence with an N-terminal HA tag and a C-terminal bovine growth hormone polyA tail (BGHpA). FHL1-RBM mutants C132F and H123Y are indicated. (B) Skeletal muscle lysates were immunoblotted for HA-FHL1 or β-tubulin in wild-type (WT) or FHL1 transgenic (TG) muscle (GAS, gastrocnemius; EDL, extensor digitorum longus; SOL, soleus plantaris; PLA, plantaris; ECU, extensor carpi ulnaris; FDP, flexor digitorum profundus). A lysate prepared from C2C12 cells transfected with HA-FHL1 was used as a positive control (+). (C) The relative expression of HA-FHL1 was determined by densitometry of HA-immunoreactive polypeptides, standardized relative to β-tubulin loading. HA-FHL1 expression is represented as a fold difference, relative to GAS, arbitrarily defined as one. Graph depicts mean ± SEM (GAS, EDL, SOL, and PLA: n = 3 mice; ECU and FDP: ***, P < 0.005; n = 1 mouse). (D) Tissue lysates from WT or TG mice were immunoblotted for HA-FHL1 using anti-HA antibodies or β-tubulin. Lysates prepared from C2C12 cells transfected with HA-FHL1 were used as a positive control (+). (E) Longitudinal gastrocnemius muscle sections from WT or FHL-TG mice were stained with anti-FHL1 (green) or anti-HA (red) antibodies and imaged by confocal microscopy, then images were further resolved by deconvolution. Bars, 5 μm.
Mentions: We next investigated whether FHL1 expression in postmitotic myofibers induced skeletal muscle hypertrophy in vivo. The FHL1 cDNA was fused to an HA tag (HA-FHL1) and linked to the human skeletal muscle α-actin (HSA) promoter (Fig. 3 A), which drives skeletal muscle–specific expression (Brennan and Hardeman, 1993). Skeletal α-actin is expressed only after myoblast fusion to form myotubes, and expression persists in adult muscle (Cox et al., 1990) in the elongated postmitotic cells of the somites, as well as in adult mature myofibers, but not satellite cells (Gunning et al., 1987; Miniou et al., 1999; Shen et al., 2003). FHL1 transgenic mice were generated and two lines were characterized. Transgenic HA-FHL1 expression was restricted to skeletal muscle, most prominent in fast-twitch enriched muscles including gastrocnemius (Fig. 3, B and C, GAS), extensor digitorum longus (EDL), plantaris, extensor carpi ulnaris (ECU), and flexor digitorum profundus (FDP), which is consistent with HSA promoter activity (Tinsley et al., 1996), with no expression in other tissues (Fig. 3 D). FHL1 transgenic sarcomere structure appeared undisturbed, with normal α-actinin localization at the Z-line (Fig. S1, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200804077/DC1). HA-FHL1 colocalized with endogenous FHL1 at the I-band (Fig. 3 E; McGrath et al., 2006).

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