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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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RBM-FHL1 mutations alter localization of NFATc1. (A) C2C12 myoblasts were cotransfected with HA-FHL1 wild-type (top), HA-FHL1C132F (middle), or HA-FHL1H123Y (bottom), and myc-NFATc1, then differentiated for 72 h. Myotubes were fixed and costained with HA (green) and myc (red) antibodies, costained with menadione-NBT, and imaged by fluorescence microscopy. White arrowheads indicate perinuclear aggregates colocalizing with the reducing-body marker menadione-NBT in the absence of substrate (black arrowheads). Yellow arrowheads reveal the region shown in the high-magnification images showing colocalization between FHL1, NFATc1, and menadione-NBT in the insets. (B) Transverse skeletal muscle sections were prepared from human RBM muscle biopsies, with FHLC132F (top) or FHL1H123Y (bottom) mutations. Sections were stained with antibodies recognizing FHL1 (green) and NFATc1 (red), then costained with DAPI (nuclei). Arrowheads identify reducing bodies. (C) Vector control, FHL1 wild-type (FHL1-WT), or indicated FHL1 RBM mutants were cotransfected with myc-NFATc1 into C2C12 myoblasts. Myoblasts were differentiated for 72 h, then ionomycin-stimulated for 30 min, and costained with anti-myc (NFATc1, glow-over images are shown) and anti-HA (to identify cotransfected cells, not depicted here). White arrows identify NFATc1 nuclear staining, yellow arrows indicate NFATc1-negative nuclei. High-magnification images are shown in the insets. (D) The percentage of transfected cells containing nuclear NFATc1 was scored after ionomycin stimulation. The bar graph equals mean ± SEM (n = 4 experiments; *, P < 0.05). Bars: (A and B) 20 μm; (C) 50 μm.
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fig6: RBM-FHL1 mutations alter localization of NFATc1. (A) C2C12 myoblasts were cotransfected with HA-FHL1 wild-type (top), HA-FHL1C132F (middle), or HA-FHL1H123Y (bottom), and myc-NFATc1, then differentiated for 72 h. Myotubes were fixed and costained with HA (green) and myc (red) antibodies, costained with menadione-NBT, and imaged by fluorescence microscopy. White arrowheads indicate perinuclear aggregates colocalizing with the reducing-body marker menadione-NBT in the absence of substrate (black arrowheads). Yellow arrowheads reveal the region shown in the high-magnification images showing colocalization between FHL1, NFATc1, and menadione-NBT in the insets. (B) Transverse skeletal muscle sections were prepared from human RBM muscle biopsies, with FHLC132F (top) or FHL1H123Y (bottom) mutations. Sections were stained with antibodies recognizing FHL1 (green) and NFATc1 (red), then costained with DAPI (nuclei). Arrowheads identify reducing bodies. (C) Vector control, FHL1 wild-type (FHL1-WT), or indicated FHL1 RBM mutants were cotransfected with myc-NFATc1 into C2C12 myoblasts. Myoblasts were differentiated for 72 h, then ionomycin-stimulated for 30 min, and costained with anti-myc (NFATc1, glow-over images are shown) and anti-HA (to identify cotransfected cells, not depicted here). White arrows identify NFATc1 nuclear staining, yellow arrows indicate NFATc1-negative nuclei. High-magnification images are shown in the insets. (D) The percentage of transfected cells containing nuclear NFATc1 was scored after ionomycin stimulation. The bar graph equals mean ± SEM (n = 4 experiments; *, P < 0.05). Bars: (A and B) 20 μm; (C) 50 μm.

Mentions: The FHL proteins play critical roles as transcriptional coactivators or repressors (Johannessen et al., 2006; Cottle et al., 2007). Calcineurin signaling is implicated in regulating myopathy-related muscle degeneration, positively in some myopathies and negatively in others (Chakkalakal et al., 2004; St-Pierre et al., 2004; Parsons et al., 2007). Calcineurin is a calmodulin-dependent, calcium-activated protein phosphatase. Once activated, calcineurin dephosphorylates cytosolic NFAT transcription factors, exposing a nuclear localization sequence and thereby promoting their nuclear translocation and transcriptional events. NFATc1-c3 and NFAT5 are expressed in skeletal muscle (Abbott et al., 1998; O'Connor et al., 2007). NFATc1 promotes skeletal muscle hypertrophy and oxidative myofiber-type identity (Chin et al., 1998; Semsarian et al., 1999b), NFATc2 governs myofiber number (Horsley et al., 2001), and NFATc3 regulates primary myotube formation during myogenesis (Kegley et al., 2001). NFAT5 is a constitutively nuclear, calcineurin-independent transcription factor, which regulates skeletal muscle cell migration and differentiation (O'Connor et al., 2007). As FHL1 promoted hypertrophy and fiber type switching in vivo, which is reminiscent of NFATc1 function, we evaluated whether FHL1 interacted with NFATc1. GST-FHL1 bound His-NFATc1 in purified component binding studies (Fig. 5 A). In control studies, GST–GATA-2 complexed with His-NFATc1 as described previously (Musarò et al., 1999). Also, GST-FHL1, but not GST-coupled glutathione-Sepharose, incubated with skeletal muscle lysate pulled down an ∼100-kD polypeptide that corresponds to endogenous NFATc1 (Fig. 5 B). We next asked whether FHL1-RBM mutants could also complex with NFATc1. C2C12 myoblasts were cotransfected with myc-NFATc1 and HA-FHL1 (wild type), HA-FHL1C132F, or HA-FHL1H123Y. HA-FHL1 was detected in myc-NFATc1 immunoprecipitates (Fig. 5 C). In contrast, binding of RBM-FHL1 mutants to myc-NFATc1, although evident (Fig. 5 C), was reduced by ∼80% (Fig. 5 D). Mutant FHL1 is sequestered to reducing body aggregates in the skeletal muscle of RBM-affected individuals (Schessl et al., 2008). Therefore, the RBM mutant FHL1–NFATc1 complex may be less accessible for immunoprecipitation because of its sequestration in reducing body aggregates. To investigate this possibility, we examined the effect of wild-type or RBM mutant FHL1 on the subcellular localization of NFATc1; the latter has been found to localize to the myotube cytosol, and in response to calcineurin activation, in some but not all nuclei (Abbott et al., 1998). In differentiated C2C12 cells, wild-type HA-FHL1 and myc-NFATc1 colocalized in the cytosol (Fig. 6 A). FHL1C132F and FHL1H123Y localized diffusely in the cytosol but also in perinuclear cytoplasmic aggregates (Fig. 6 A, white arrowheads), which stained with the reducing body marker menadione–nitro blue tetrazolium chloride (NBT) in the absence of substrate (Fig. 6 A, black arrowheads), consistent with reducing-body aggregates in RBM skeletal muscle (Schessl et al., 2008). Interestingly, NFATc1, when coexpressed with HA-FHL1C132F or HA-FHL1H123Y but not wild-type FHL1, also localized to reducing body aggregates (Fig. 6 A, insets). We obtained muscle biopsies from individuals with RBM and examined NFATc1 localization, revealing its reducing body aggregate colocalization with FHL1 (Fig. 6 B, arrowheads). We also determined the affect of FHL1-RBM mutants on NFATc1 nuclear translocation in response to the Ca2+ ionophore, ionomycin, which activates calcineurin signaling (Abbott et al., 1998). NFATc1 nuclear translocation was detected in 35% of myotube nuclei scored after ionomycin pretreatment, and this localization was not affected by FHL1 coexpression (Fig. 6, C and D). In contrast, NFATc1 nuclear localization was significantly reduced by HA-FHL1C132F or HA-FHL1H123Y coexpression, revealing that RBM-FHL1 mutants may sequester NFATc1 to reducing bodies and therefore reduce NFATc1 activity in the nucleus.


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)

RBM-FHL1 mutations alter localization of NFATc1. (A) C2C12 myoblasts were cotransfected with HA-FHL1 wild-type (top), HA-FHL1C132F (middle), or HA-FHL1H123Y (bottom), and myc-NFATc1, then differentiated for 72 h. Myotubes were fixed and costained with HA (green) and myc (red) antibodies, costained with menadione-NBT, and imaged by fluorescence microscopy. White arrowheads indicate perinuclear aggregates colocalizing with the reducing-body marker menadione-NBT in the absence of substrate (black arrowheads). Yellow arrowheads reveal the region shown in the high-magnification images showing colocalization between FHL1, NFATc1, and menadione-NBT in the insets. (B) Transverse skeletal muscle sections were prepared from human RBM muscle biopsies, with FHLC132F (top) or FHL1H123Y (bottom) mutations. Sections were stained with antibodies recognizing FHL1 (green) and NFATc1 (red), then costained with DAPI (nuclei). Arrowheads identify reducing bodies. (C) Vector control, FHL1 wild-type (FHL1-WT), or indicated FHL1 RBM mutants were cotransfected with myc-NFATc1 into C2C12 myoblasts. Myoblasts were differentiated for 72 h, then ionomycin-stimulated for 30 min, and costained with anti-myc (NFATc1, glow-over images are shown) and anti-HA (to identify cotransfected cells, not depicted here). White arrows identify NFATc1 nuclear staining, yellow arrows indicate NFATc1-negative nuclei. High-magnification images are shown in the insets. (D) The percentage of transfected cells containing nuclear NFATc1 was scored after ionomycin stimulation. The bar graph equals mean ± SEM (n = 4 experiments; *, P < 0.05). Bars: (A and B) 20 μm; (C) 50 μm.
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fig6: RBM-FHL1 mutations alter localization of NFATc1. (A) C2C12 myoblasts were cotransfected with HA-FHL1 wild-type (top), HA-FHL1C132F (middle), or HA-FHL1H123Y (bottom), and myc-NFATc1, then differentiated for 72 h. Myotubes were fixed and costained with HA (green) and myc (red) antibodies, costained with menadione-NBT, and imaged by fluorescence microscopy. White arrowheads indicate perinuclear aggregates colocalizing with the reducing-body marker menadione-NBT in the absence of substrate (black arrowheads). Yellow arrowheads reveal the region shown in the high-magnification images showing colocalization between FHL1, NFATc1, and menadione-NBT in the insets. (B) Transverse skeletal muscle sections were prepared from human RBM muscle biopsies, with FHLC132F (top) or FHL1H123Y (bottom) mutations. Sections were stained with antibodies recognizing FHL1 (green) and NFATc1 (red), then costained with DAPI (nuclei). Arrowheads identify reducing bodies. (C) Vector control, FHL1 wild-type (FHL1-WT), or indicated FHL1 RBM mutants were cotransfected with myc-NFATc1 into C2C12 myoblasts. Myoblasts were differentiated for 72 h, then ionomycin-stimulated for 30 min, and costained with anti-myc (NFATc1, glow-over images are shown) and anti-HA (to identify cotransfected cells, not depicted here). White arrows identify NFATc1 nuclear staining, yellow arrows indicate NFATc1-negative nuclei. High-magnification images are shown in the insets. (D) The percentage of transfected cells containing nuclear NFATc1 was scored after ionomycin stimulation. The bar graph equals mean ± SEM (n = 4 experiments; *, P < 0.05). Bars: (A and B) 20 μm; (C) 50 μm.
Mentions: The FHL proteins play critical roles as transcriptional coactivators or repressors (Johannessen et al., 2006; Cottle et al., 2007). Calcineurin signaling is implicated in regulating myopathy-related muscle degeneration, positively in some myopathies and negatively in others (Chakkalakal et al., 2004; St-Pierre et al., 2004; Parsons et al., 2007). Calcineurin is a calmodulin-dependent, calcium-activated protein phosphatase. Once activated, calcineurin dephosphorylates cytosolic NFAT transcription factors, exposing a nuclear localization sequence and thereby promoting their nuclear translocation and transcriptional events. NFATc1-c3 and NFAT5 are expressed in skeletal muscle (Abbott et al., 1998; O'Connor et al., 2007). NFATc1 promotes skeletal muscle hypertrophy and oxidative myofiber-type identity (Chin et al., 1998; Semsarian et al., 1999b), NFATc2 governs myofiber number (Horsley et al., 2001), and NFATc3 regulates primary myotube formation during myogenesis (Kegley et al., 2001). NFAT5 is a constitutively nuclear, calcineurin-independent transcription factor, which regulates skeletal muscle cell migration and differentiation (O'Connor et al., 2007). As FHL1 promoted hypertrophy and fiber type switching in vivo, which is reminiscent of NFATc1 function, we evaluated whether FHL1 interacted with NFATc1. GST-FHL1 bound His-NFATc1 in purified component binding studies (Fig. 5 A). In control studies, GST–GATA-2 complexed with His-NFATc1 as described previously (Musarò et al., 1999). Also, GST-FHL1, but not GST-coupled glutathione-Sepharose, incubated with skeletal muscle lysate pulled down an ∼100-kD polypeptide that corresponds to endogenous NFATc1 (Fig. 5 B). We next asked whether FHL1-RBM mutants could also complex with NFATc1. C2C12 myoblasts were cotransfected with myc-NFATc1 and HA-FHL1 (wild type), HA-FHL1C132F, or HA-FHL1H123Y. HA-FHL1 was detected in myc-NFATc1 immunoprecipitates (Fig. 5 C). In contrast, binding of RBM-FHL1 mutants to myc-NFATc1, although evident (Fig. 5 C), was reduced by ∼80% (Fig. 5 D). Mutant FHL1 is sequestered to reducing body aggregates in the skeletal muscle of RBM-affected individuals (Schessl et al., 2008). Therefore, the RBM mutant FHL1–NFATc1 complex may be less accessible for immunoprecipitation because of its sequestration in reducing body aggregates. To investigate this possibility, we examined the effect of wild-type or RBM mutant FHL1 on the subcellular localization of NFATc1; the latter has been found to localize to the myotube cytosol, and in response to calcineurin activation, in some but not all nuclei (Abbott et al., 1998). In differentiated C2C12 cells, wild-type HA-FHL1 and myc-NFATc1 colocalized in the cytosol (Fig. 6 A). FHL1C132F and FHL1H123Y localized diffusely in the cytosol but also in perinuclear cytoplasmic aggregates (Fig. 6 A, white arrowheads), which stained with the reducing body marker menadione–nitro blue tetrazolium chloride (NBT) in the absence of substrate (Fig. 6 A, black arrowheads), consistent with reducing-body aggregates in RBM skeletal muscle (Schessl et al., 2008). Interestingly, NFATc1, when coexpressed with HA-FHL1C132F or HA-FHL1H123Y but not wild-type FHL1, also localized to reducing body aggregates (Fig. 6 A, insets). We obtained muscle biopsies from individuals with RBM and examined NFATc1 localization, revealing its reducing body aggregate colocalization with FHL1 (Fig. 6 B, arrowheads). We also determined the affect of FHL1-RBM mutants on NFATc1 nuclear translocation in response to the Ca2+ ionophore, ionomycin, which activates calcineurin signaling (Abbott et al., 1998). NFATc1 nuclear translocation was detected in 35% of myotube nuclei scored after ionomycin pretreatment, and this localization was not affected by FHL1 coexpression (Fig. 6, C and D). In contrast, NFATc1 nuclear localization was significantly reduced by HA-FHL1C132F or HA-FHL1H123Y coexpression, revealing that RBM-FHL1 mutants may sequester NFATc1 to reducing bodies and therefore reduce NFATc1 activity in the nucleus.

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