FHL1 activates myostatin signalling in skeletal muscle and promotes atrophy

Highlights • Myostatin signals via SMADs to promote muscle wasting.• FHL1 normally promotes hypertrophy but can activate SMAD signalling.• FHL1 promoted myostatin signalling in vitro.• FHL1 promoted hypertrophy in the absence of myostatin but atrophy in its presence.


Introduction
Voluntary movement is essential for a normal healthy life and the performance of daily activities. Such movement requires a sufficient quantity of skeletal muscle especially in the locomotor muscles and the appropriate proportions of the different fibre types. Different fibre-types have distinct rates of contraction and abilities to endure activity. The overall phenotype of a muscle is related to the relative proportions of the different fibres it contains. Muscle phenotype is plastic and the size and proportion of the individual fibres can change dependent on a number of factors including physical activity.
Changes in muscle mass and phenotype are important aspects of a number of chronic diseases such as chronic obstructive pulmonary disease (COPD), heart failure and cancer and have prognostic ability. Indeed exercise capacity and strength are better predictors of survival in patients with COPD than standard measures of pulmonary function [1]. Muscle mass is also lost in ageing and there is a marked change in phenotype again with prognostic implications [2,3]. Consequently the factors that affect muscle mass are being intensively studied.
Not all fibres atrophy at the same rate and a number of studies have shown that type II fibres are more likely to atrophy than type I fibres in diseases as varied as COPD [4], heart failure [5] and osteoarthritis [6] as well as in normal human ageing [7]. As there is a shift towards type II fibres in the quadriceps muscles in chronic disease, this increased sensitivity of type II fibres to atrophy is likely to contribute to accelerated wasting. Under some conditions (e.g. starvation) where muscle acts as an emergency fuel store, this response may be important; however, in chronic disease it is likely to be detrimental.
Myostatin is one factor likely to be involved in the increased susceptibility of type II fibres to atrophy. This growth regulator is a member of the transforming growth factor-b (TGF-b) family that was identified from natural mutations in animals with a double muscled phenotype [8,9]. Germ-line deletion of the myostatin gene from mice resulted in a similar hypermuscular phenotype suggesting that myostatin is an inhibitor of muscle growth. Deletion of myostatin also increased the proportion of the fastest type IIB fibres suggesting that the major effects of myostatin were on this fibre type [10,11]. Furthermore myostatin expression is highest in type IIB fibres [12], is elevated in response to hind limb suspension and is a target for the type I fibre restricted microRNA, miR-499. Indeed it has been shown that increased myostatin mRNA and protein are associated with type II muscle atrophy [13].
Myostatin signals by binding to an activin IIB/alk4/5 receptor complex promoting the phosphorylation of SMAD-2/3. Consequently factors that modify SMAD-2/3 phosphorylation are likely to alter myostatin signalling. One protein that activates SMAD proteins is the four and a half LIM domain protein FHL1 which binds to CKd and promotes SMAD phosphorylation [14]. In muscle cells such an activity would promote muscle atrophy but under normal conditions FHL1 appears to promote hypertrophy. For example, over-expression of FHL1 leads to muscle hypertrophy [15] and patients with mutations in FHL1 have a range of myopathic conditions including X-linked myopathy with postural muscle atrophy (XMPMA) [16]. However, a number of studies have shown that FHL1 can associate with atrophy. For example, denervation in mice increases FHLI [17], long-term training in humans reduces FHL1 expression [18] and we observed that FHL1 was associated with weakness in COPD patients [19]. These observations raise the possibility FHL1 potentiates myostatin signalling in muscle cells so contributes to muscle wasting under a subset of conditions. Consequently in this study we determined the effect of FHL1 on SMAD reporter gene expression in response to myostatin, as well as the effect of FHL1 on myostatin induced myotube wasting. Finally we used electroporation of the tibialis anterior (TA) in mice to determine the effect of FHL1 on myostatin induced muscle wasting in vivo.

Cloning
Full-length murine myostatin and FHL1 were sub-cloned into pGEMT by PCR from image clones (Source Bioscience) then shuttled into empty pCAGGS expression vector (containing a CMVenhancer and chicken b-actin promoter) [20] and sequenced. Large-scale plasmid preparations were carried out using the Endo-Free Mega kit (Qiagen) according to manufacturer's instructions and plasmids were eluted in sterile dH 2 0.

Cell culture, luciferase reporter and myotube diameter assays
For luciferase experiments, C2C12 myoblasts were cultured and transfected using lipofectamine as described in [21] scaled for culture in 24 well plates. A total of 0.4 lg DNA was transfected per well in total: 0.2 lg of (CAGA) 12 -luciferase plasmid [22], 0.1 lg of pRLTK plasmid, 0.1 lg of either FHL1-pcDNA or 0.1 lg pcDNA as the control. After transfection the cells were cultured in DMEM supplemented with 10% (v/v) FBS. Twenty-four hours later, cells were serum starved for 7 h then washed and treated with serumfree DMEM containing either TGF-b1 (Insight Biotechnology) or Myostatin (PromoKine) for 16 h. Luciferase activity was assayed using the Dual-Luciferase Ò Reporter Assay System (Promega) according to the manufacturer's instructions. Firefly luciferase activity was normalised to Renilla luciferase activity to account for transfection efficiency. All data were normalised to the mean of the pcDNA untreated (0 ng/mL TGF-b) group for that set of transfections.
For myotube diameter measurements, C2C12 myoblasts were cultured and transfected as described in [23] with a total of 2 lg DNA consisting of 0.5 lg pCAGGS-GFP [24]and either 1.5 lg FHL1-pcDNA or 1.5 lg pcDNA. Thus, myoblasts that were successfully transfected with pCAGGS-GFP would also be transfected with the FHL1 expression vector and allow identification of fluorescent 'transfected' myotubes. After 4.5 h incubation, media was replaced with fresh DMEM + 10% FBS and the cells returned to the incubator. The medium was replaced every 2 days and allowed to become confluent (approximately 3 days 470 nm/40 nm. Average myotube diameter was ascertained by measuring the shortest distance across the myotube at five points along the length of fluorescent myotubes using Image J.

Electroporation
Mouse experiments were approved by the Royal Veterinary College Ethical Review Process (ERP-A-2010-WS01) and were licensed by the UK Secretary of State for the Home Office under Project License PPL 70/6797. Twelve female CD1 mice (7.5 weeks old) were anaesthetised with Hypnorm (VetaPharma) and Hypnovel (Roche), both lower legs were shaved and 10U (25 ll) of bovine hyaluronidase (Sigma) was injected percutaneously into each TA to increase transfection efficiency [27,28]. Mice were allowed to partially recover at 37°C and after 1.5 h they were reanaesthetised using 5% isofluorane and maintained at 2% isofluorane. The TA muscles were injected with 25 ll of the appropriate plasmid at 1 mg/mL. Immediately following the plasmid injection electro-conductive cream was applied to electrodes which were placed either side of TA, separated by approximately 5 mm. Electroporation was performed using 10 pulses of 85 V each for 20 ms, at a frequency of 1 Hz.
Following electroporation, mice recovered and were left for 2 weeks, after which the mice were sacrificed and TA muscles were harvested and placed upright onto small pieces of cork with a small amount of OCT at the bottom to fix the bottom of the TAs onto cork and flash frozen in liquid nitrogen cooled iso-pentane.
Muscle samples were sectioned as previously described [27] to obtain tissue for histology and RNA analysis from defined levels within the muscle. Muscle sections were stained with haematoxylin and eosin, and by immunofluorescence for fibre type as previously described [29]. Random fields were captured at 20Â magnification using an Olympus CKX41 camera and Cell^D software (Olympus Europe).

RNA extraction from tissue
Muscle sections from regions adjacent to the histology samples were placed into CK-14 ceramic beaded tubes containing 500 ll of TRIzol Ò (Invitrogen) and homogenised with the Precellys 24 (Stretton Scientific) for 2 Â 15 s cycles at 5500 rpm. The samples were centrifuged at 8000 rpm for 3 min at 4°C and the supernatant transferred to fresh micro-centrifuge tubes and RNA extracted according to manufacturer's instructions (Qiagen). The RNA was resuspended in 30 ll RNase-free dH 2 O and stored at À80°C. RNA concentration was quantified using a Nanodrop TM .

Quantitative real-time PCR (QPCR)
cDNA was synthesised from 150 ng RNA and amplified by qPCR as previously described [30]. The PCR primers used have been described previously [31]. Data was normalised to a geometric mean of ribosomal protein large P0 (RPLPO) and b2 microglobulin using the DDCt method. FHL1 enhances myostatin induced wasting in C2C12 myotubes. C2C12 myoblasts were co-transfected with an EGFP expression vector (pCAGGS-EGFP) and either pCDNA or pCAGGS-FHL1 and differentiated into myotubes before treatment with myostatin for 4 days. FHL1 expression induced myotube hypertrophy. At 20 ng/mL, myostatin alone had no effect on myotube diameter, however, in the presence of FHL1, myostatin reduced myotube diameter. Representative images of each group at day 2 are shown in (A) with fluorescent images alone on the top row and merged with the brightfield images below. Quantitation of myotube diameters after 2 (B) and 4 (C) days of treatment. Images captured at Â10 magnification, scale bar represents 120 lm. Graphs represent median ± IQR of pooled data from 3 independent experiments ( ⁄ p < 0.05 and ⁄⁄⁄ p < 0.001 Mann Whitney).

Statistical analysis
Data are presented as mean ± SEM for data with a parametric distribution and median (interquartile range) for non-parametric data. Differences were determined by t-test for parametric data and by Mann-Whitney test for non-parametric data. To establish differences in muscle fibre profiles data were compared by oneway ANOVA for each point. Significance was set at a 2 tailed p value = 0.05).

Effect of FHL1 on SMAD reporter gene expression in response to TGFb ligands
Treatment of the cells with 10 ng/mL TGFb alone increased CAGA 12 luciferase reporter gene expression by $4-fold and this was increased further to $7-fold by expression of FHL1 (Fig. 1a) consistent with an effect of FHL1 on TGF-b signalling. FHL1 expression had no effect on luciferase activity in the absence of added TGF-b. In the absence of transfection with an FHL1 expression plasmid (i.e. in the cells transfected with pCDNA), myostatin had no effect on luciferase activity at concentrations below 100 ng/mL. At 100 ng/mL myostatin alone increased luciferase activity $1.4fold (p = 0.025). However, in the presence of FHL1, myostatin caused a detectable increase in luciferase activity at all concentrations above 20 ng/mL (20 ng/mL 1.4-fold, p = 0.003, 50 ng/mL, 1.8fold p < 0.001, 100 ng/mL 1.7-fold p = 0.001). Furthermore, at all doses myostatin caused a larger increase in mean luciferase activity in the presence of FHL1 than in its absence (Fig. 1b) which reached statistical significance for 50 ng/mL (20 ng/mL 1.3-fold, p = 0.065, 50 ng/mL, 1.5-fold p = 0.001, 100 ng/mL 1.2-fold p = 0.221). These data show that FHL1 increases the activity of myostatin signalling in myoblasts. The lack of a significant effect of FHL1 on luciferase activity at 100 ng/mL myostatin raises the possibility that FHL1 increases the sensitivity of the cell to myostatin rather than the size of the response. Alternatively it may reflect the weakness of the response of the reporter system to myostatin compared to TGF-b.

Effect of FHL1 on myotube diameter in response to myostatin
To determine whether FHL1 enhanced myostatin dependent myotube wasting, we determined the effect of myostatin on myotube size in the presence or absence of FHL1 expression. Treatment of the cells with 20 ng/mL myostatin alone did not alter the size of the myotubes 2 or 4 days after treatment (2 days-myostatin

Effect of FHL1 on myostatin induced muscle wasting in vivo
To determine whether FHL1 also increased myostatin dependent atrophy in vivo, we over-expressed myostatin and FHL1, alone or in combination in the tibialis anterior (TA) muscles of mice by electroporation. To determine the effect of myostatin or FHL1 alone, mice were injected in the right TA with pCAGGSmyostatin (M1) or pCAGGS-FHL1 (F) and in the left TA with empty pCAGGS as a control (C1 and C2 respectively). To determine the effect of FHL1 on myostatin activity, mice were electroporated in the right TA with both pCAGGS-myostatin and pCAGGS-FHL1 (M +F) and in the left TA with pCAGGS-myostatin (M2). Electroporation with pCAGGS-myostatin increased myostatin mRNA expression in M1 compared to the contralateral C1 (Fig. 3A). Similarly, electroporation of pCAGGS-FHL1 increased FHL1 expression in F and F+M muscles compared to their respective controls (C2 and M2) but this only reached statistical significance in F+M vs M2 (Fig. 3B). However, there was no effect of myostatin on FHL1 expression and FHL1 mRNA was significantly higher in the F and F+M groups combined than in all other groups combined (2-fold, p < 0.01, Fig. 3B). Myostatin expression in the muscle electroporated with both plasmids (F+M) did not differ from that in the contralateral muscle electroporated with pCAGGS-myostatin alone (M2) ( Table 3).
Sections from each electroporated muscle were stained with H&E to analyse the effect of electroporation on muscle fibre diameter (Fig 4). Consistent with previous studies, myostatin expression alone caused a $10% decrease in fibre diameter [32] (Fig. 4A and F, from 37.8 ± 0.9 lm (n = 8) to 33.1 ± 0.9 lm (n = 8) p < 0.001) whereas FHL1 expression increased fibre diameter by 10% compared with the control TA ( Fig. 4B and F to 42.7 ± 0.5 lm (n = 4) p < 0.001). Co-expression of FHL1 with myostatin in the same TA caused a larger decrease in fibre diameter than expression of myostatin alone (8.7% further reduction, to 30 ± 1.2 lm (n = 4), Fig. 4C and F, p = 0.033). Comparing the diameter of myofibres overexpressing FHL1 in the presence or absence of myostatin showed that myostatin decreased fibre diameter from 42.7 ± 5 lm to 30 ± 1.2 lm a reduction of approximately 25% (p < 0.001).
To analyse further the effects on muscle fibre size, we determined the proportion of fibres within 5 lm bins and the proportion of fibres below a given fibre diameter again in 5 lm steps (Fig. 5). Comparison of the two sets of control muscles with each other and the two sets of myostatin muscles with each other, showed no significant difference at any point, validating the technique and showing that expression of myostatin or FHL1 in the contralateral TA did not affect fibre size ( Fig. 5E and F). This approach also allowed us to pool the data from the control and the data from the myostatin treated muscles. Myostatin caused a significant shift to smaller fibres compared to control electroporated muscles (Fig. 5A and G). FHL1 caused a significant increase in the proportion of larger fibres compared to control fibres. (Fig. 5B, E and G). In FHL1 expressing muscles, myostatin caused a greater increase in the smallest fibres than myostatin alone with a significant increase in the fibres below 25 lm (Fig 5C, F and G).
Fibre proportions were analysed by immunofluorescence (Figs. 6 and 7). Comparison of the proportion of fibres in each muscle showed that myostatin expression reduced the proportion of type IIB fibres and increased the proportion of IIA fibres compared to the controls. FHL1 alone did not affect the fibre proportion compared to controls and in combination with myostatin appeared to cause an increase in type IIB/type IIX fibres (p = 0.037, Fig. 6).
Gene expression within the groups was then examined to identify changes in pathways associated with muscle wasting. Given that myostatin over-expression alone caused wasting, downstream TGF-b signalling was analysed, revealing a significant increase in PAI-1 (1.49-fold, M1 compared to C1, p < 0.05, Table 1), suggesting activation of the TGF-b signalling pathway. To identify the atrophy pathway induced by the myostatin signalling, expression of components of proteasomal degradation, autophagy and apoptosis were measured. No significant differences were observed in the expression of any of these genes but there was a trend to a decrease in expression of ATG4B (0.73-fold, p = 0.059) and ATG12 l (0.58-fold, p = 0.076), genes that are associated with autophagy. Changes in the expression of the myosin heavy chains were identified; with an increase in MHCI (13.1-fold, p < 0.01) and a decrease in expression of MHCIIB (0.34, p < 0.05, consistent with the observed reduction in type IIB fibres) and a trend towards a reduction in MHCIIX (0.64, p = 0.06). There was no apparent increase in the expression of MHCIIA even though there was an increase in type IIA fibre proportion possibly due to the small number of samples analysed. There was also a significant increase in myogenin expression (1.82, p < 0.05).
Expression of FHL1 alone did not significantly alter the expression of any of the genes tested (F compared to C2, Table 2). However, there was a trend to an increase in VEGF-C (1.2-fold, p = 0.085). Additionally there was a trend to a decrease in BAX expression (0.81-fold, p = 0.075). (n = 4) showed that myostatin reduced fibre size compared to control (p < 0.001), FHL1 increased fibre size compared to control (p < 0.001) but FHL1 + myostatin caused a larger reduction in fibre diameter than myostatin alone (p < 0.001 FHL1 vs FHL1 + myostatin, p = 0.033 myostatin vs FHL1 + myostatin). ⁄⁄⁄ p < 0.001, ⁄ p < 0.05.
Expression of FHL1 and myostatin in the same muscle (F + M compared to M2, Table 3) caused a significant increase in the expression of genes associated with autophagy (ATG12 l; 1.46fold, p < 0.05 with a trend to an increase in ATG4B; 1.41-fold, p = 0.052) and apoptosis (BAD; 1.36-fold, p < 0.05 and BAX; 1.29fold, p < 0.01) as well as a trend to an increase in MuRF1 (1.27fold, p = 0.056, Table 3) compared to the expression of myostatin alone. There were no significant differences in the expression of myosin heavy chains in between muscles expressing myostatin alone and myostatin and FHL1 but there was a trend towards an increase in the expression of MHCIIX (1.88-fold, p = 0.058) and MHCI (2.99-fold, p = 0.075).

Discussion
Our data indicate that exogenous expression of FHL1 increases myostatin activity in skeletal muscle cells in vitro and exacerbates muscle wasting in the presence of elevated myostatin in vivo. The accepted role for FHL1 is as a promoter of hypertrophy, as shown by the effects of overexpression of FHL1 in mice and postural muscle atrophy in patients with mutations in FHL1 [15,16]. In the absence of myostatin we observed an increase in both myotube and myofibre diameter in response to FHL1 consistent with the prior data. However, in the presence of myostatin we found that FHL1 enhanced the effects of myostatin both in vitro and in vivo.
These data are consistent with FHL1 potentiating the effects of TGF-b as identified in hepatic carcinoma cells [14]. However, in muscle cells FHL1 did not increase SMAD signalling in the absence of a TGF-b ligand as observed by Ding et al. in tumor cells [14]. The reason for this difference is not clear but differences in cell type with different relative levels of expression or localisation of CK1d or SMAD proteins may provide an explanation.
The activation of myostatin by FHL1 may help to explain the increase in FHL1 following sciatic nerve section [17] and the association of FHL1 with weakness in COPD patients [19] as myostatin expression increases in both situations [33,34]. Furthermore, FHL1 is expressed at higher levels in type II fibres than in type I fibres raising the possibility that it contributes to the greater sensitivity of type II fibres to myostatin.
Myostatin has previously been shown to increase the expression of the atrogenes MuRF-1 and atrogin-1 and we have previously shown that myostatin increases the expression of autophagy-associated genes in vitro [31,35]. Whilst there was no detectable increase in the expression of genes associated with these pathways in the muscles treated with myostatin alone in the presence of both myostatin and FHL1, the expression of genes associated with autophagy and apoptosis were increased consistent with muscle atrophy. The lack of increase in the presence of myostatin alone may have been the result of the time point studied thus any earlier increase in the expression of these genes may have been missed. Such an explanation is consistent with the small increases observed in the presence of myostatin and FHL1 where the atrophy was greater.

Critique of the experimental approach
The data present a consistent argument that FHL1 increases the functional effects of myostatin in vitro and in vivo. This observation is also consistent with activation of SMAD signalling by FHL1 in other cells. However, there are a number of potential confounding factors in the data that need to be considered. First it should be noted that the experimental approach relies on over-expression and it is possible that this causes an artefact. Against this suggestion is the consistency of the responses in myoblasts, myotubes and muscle in vivo as FHL1 increases myostatin activity measured by 3 different assays (luciferase based SMAD reporter assays, myotube wasting in vitro and myofibre wasting in vivo. The second potential confounding issue is that the in vivo response occurs on the background of a regeneration response to electroporation injury complicating any picture. However, our observations on the effects of the single agents (myostatin and FHL1) are consistent with previous studies using transgenic over-expression, which does not cause injury and regeneration [15,36]; with myostatin alone causing atrophy and FHL1 alone causing hypertrophy. Furthermore, the effects we observe in vitro are the same as those we observe in vivo (i.e. FHL1 alone increases myotube size whereas in combination with myostatin it causes a greater reduction in myotube size than myostatin alone). Together these observations suggest that the combined response in vivo is not caused by the experimental approach. The third confounding factor is that we did not observe large changes in gene expression in response to either FHL1 or myostatin. Indeed only the increase in myostatin (which we over expressed) and MCH-I exceeded a 2-fold change although a number of other changes reached statistical significance. These small changes are likely to result from the timepoint chosen for the analysis which is 2 weeks after the electroporation and the likely reduction in the expression of the transfected genes with time. Such small changes and the fact that many trends did not reach statistical significance may account for the apparent opposite direction of change observed for a number of the genes between myostatin compared to control (Table 1) and myostatin + FHL1 compared to myostatin (Table 3). However, as FHL1 also    interacts with a number of transcription factors including NFATC1 [15] it is also possible that these differences in direction of change reflect a different time course that results from the interaction between FHL1 and myostatin or some other factor within the experimental system.

Conclusion
In conclusion we demonstrate that exogenous FHL1 expression exacerbates the atrophic effects of myostatin in vitro and in vivo. These observations together with the relative restriction of FHL1 to type II fibres observed in the literature [16], suggest that FHL1 may contribute to type II fibre atrophy under the appropriate conditions. However, further experiments are required to confirm that FHL1 contributes to the increased sensitivity of type II fibres to myostatin dependent atrophy.