Relation between skeletal muscle strength and ubiquitin ligase expression in muscle atrophy models in zebrash larvae (Danio Rerio)

Background Skeletal muscle atrophy is often seen in patients with chronic diseases or muscle disuse. Upregulated expression of ubiquitin ligases such as Muscle Ring Finger 1 (MuRF-1) and Muscle Atrophy F-box (MAFbx) has been shown in different immobilization-induced atrophy models, which is believed to be responsible for the enhanced muscle protein proteolysis and thereafter results in muscle weakness. However, currently used immobilization animal models can include artefacts due to diculty of food intake and stress. Zebrash larvae 5-6 days after hatching do not require active movement for food intake, and it is possible to assess the muscle function of their trunk muscle. Therefore, we established two muscle atrophy models using zebrash larvae and aimed to investigate the role of the MuRFs and MAFbx in relation with muscle function. Methods Maximal active force of examined and the between adjacent laments in sarcomeric structure was measured using small angle x-ray diffraction. MuRF and MAFbx expression was determined using real time PCR. Two-way ANOVA was used to analyze the difference between groups. Results We found a signicant up-regulation of MuRF-1 and a lower active force generation in dexamethasone treated larvae. However, although the BTS immobilization induced muscle weakness, it was associated with decreased of MuRF-1 to 3 and MAFbx. After 3 days of immobilization, sarcomere became more compressed compared to the

MuRF-1 is considered to target proteins associated with thick myosin laments for further degradation during muscle atrophy [10,11], whereas MAFbx attacks several intermediate lament proteins including desmin and vimentin [12]. General hormonal changes can affect the gene expression. For example, glucocorticoids up regulate the expression of MuRFs and MAFbx in myotubes [10,13,14], by directly affecting transcriptional factors such as KLF15 and FoxO [15]. However, the factors affecting the expression of ubiquitin ligases and their role in inactivity-induced muscle atrophy are still unclear.
Although studies have shown upregulation of MuRF-1 and MAFbx in different atrophy animal models, using this as an indicator of muscle protein breakdown could be problematic since they are also involved in muscle remodelling activity and metabolism [16].
To study mechanisms involved in immobilization-induced atrophy, animal models (mainly rodents) involving denervation, cast immobilization, or limb suspension have been applied [17]. However, artefacts may be introduced due to the di culty of food and water intake when physical movement is restrained or other systemic changes induced by the intervention [18]. Denervation may induce other physiological consequences due to stress or wound recovery. Since these factors can upregulate the ubiquitin ligases expression through different pathways [16], it is di cult to ascertain the independent relationship between muscle inactivity and MuRF-1 and expression level under such circumstances. Zebra sh (Danio Rerio) has become an important model organism in developmental biology since most organ systems, including muscles, are fully developed in a few days after hatching and since several disease models are available [19]. The active force produced by the sarcomeres of the contractile system can be addressed using single larvae technique [20,21]. It is thus possible to determine effects of different interventions on muscle function in larvae up to 5-6 days after hatching. Recently, it has also been shown that early immobilization [22] affects sarcomere assembly and that a chemical blocker of actin-myosin interaction BTS [23] can reversibly inhibit swimming activity for longer time and affect disease development in muscle dystrophy [24]. An important advantage of the zebra sh models is that the early larvae, up to 5-6 days after fertilization (dpf), live on the yolk sack and do not require active movement for food intake.
BTS treatment inhibits the striated muscle and larval muscle movements, but does not affect the heart or blood circulation. The BTS immobilisation model can thus provide an ethically more attractive animal model compared to larger animal models, enabling full muscle immobilization for several days without effects on general physiology still enabling analysis of muscle function.
The aim of the present study was to investigate the role of the regulatory factors MuRFs and MAFbx in muscle atrophy. Two models were applied: glucocorticoid treatment and pharmaceutical immobilization using zebra sh larvae. Maximal active force of the trunk muscles was examined and correlated with studies of sarcomeric structure using small angle x-ray diffraction and with expression analysis of the ubiquitin kinases using real time PCR. We demonstrate that whereas growth/development and glucorticoid-induced atrophy was associated with increased ubiquitin ligase expression, the immobilization-induced atrophy was associated with decreased of ubiquitin kinases, showing that atrophy can be associated with both increased and decreased expression of ubiquitin kinases.

Zebra sh larvae
Zebra sh larvae (Danio Rerio, AB strain) were obtained from the Zebra sh Facility at Karolinska Institutet and kept in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , and 0.33 mM MgSO 4 , pH 6.8-6.9, with 200 µl of 0.05% methylene blue in 1 L) at 28 ºC to 6 dpf in the laboratory. The larvae were anesthetized in E3 medium containing 0.0017% MS-222 tricaine (3-amino benzoic acid ethylester) prior to mechanical analysis, small angle x-ray (SAXS) analysis and xation for mRNA extraction. The maintenance and mating of adult sh at the zebra sh facility are approved by the local authorities and follow required routines.
2. Pharmacological immobilization and glucocorticoid treatment BTS (N-benzyl-p-toluene sulphonamide), a small-molecule inhibitor of actin-myosin interaction in muscle [23,25], was added to E3 medium from 3 to 6 dpf to keep the larvae completely immobilized [24]. In one series, BTS was added at 48 hours post fertilization after dechorionation to ensure that these larvae would never move. The lowest BTS dose required for full immobilization (when larvae did not react to gentle touch) of 10 larvae in 5 ml of E3 medium was titrated to 40-50 µM, and the medium was replaced every day. Control larvae that were actively swimming from 3-6 dpf, were treated with solvent control (0.1% DMSO) in E3 medium. To study the recovery process of larvae that were immobilized 1-2 days from 3 dpf, these larvae were transferred to BTS-free E3 medium for recovery of 1 or 2 days. Prior to analysis, DMSO and BTS treated larvae were kept in regular E3 medium for 1 h to wash out BTS and DMSO. In a series where acute effects of immobilization were studied, BTS was only introduced to larvae at 5 dpf. Zebra sh larvae were thereafter immobilized for 1, 2 and 4 h and immediately sampled for further mRNA extraction. In a separate series where the effect of glucocorticoids was investigated, 300 µM dexamethasone was added to 10 larvae in 5 ml of E3 medium from 3 to 5 dpf.

Mechanical analysis
The maximal active force generated by zebra sh larvae was analysed at each day following the protocol by Dou et al, 2008 [20]. Anesthetized larvae were transferred and held in a physiological buffer solution at 22 ºC (composition in mM: 118 NaCl, 24 MOPS, 5 KCl, 1.2 MgCl 2 , 1.2 Na 2 HPO 4 , 1.6 CaCl 2 and 10 glucose at pH 7.4). The larva was anesthetised with 0.017% tricaine (3-amino benzoic acid ethylester) and then the head was crushed. A segment of 1-1.5 mm including the mid portion of the trunk muscles was prepared and attached with aluminium clips. The preparation was thereafter mounted between a force transducer and a xed hook attached to a micro manipulator, enabling the muscle length to be varied. The preparation was held in the physiological buffer solution at 22 ºC and stimulated with 0.5 ms electrical pulses at supra-maximal voltage at 2 min intervals. The muscles were mounted at slack length (when passive force is just noticeable) and stimulated 2-3 times to ensure reproducible responses. Thereafter the preparations were stretched in steps of 10% of the initial length between contractions. At each length step the passive and active force were recorded to determine the length-force relationships.
The maximal active force at optimal stretch (at muscle length about 1.3 to the slack length) was taken as a measure of the muscle function of the larvae.

Small angle x-ray scattering
Lattice spacing of contractile laments at 6 dpf was determined by small angle x-ray scattering (SAXS) using synchrotron radiation. as previously described [20,26,27] at beamline PO3 at Petra III, Deutsches Elektronen-Synchrotron (DESY) synchrotron facility in Hamburg Germany [28]. Larvae muscle preparations with aluminum clips were mounted between two hooks in a Kapton cuvette in the physiological solution as described (Sect. 3 above) at 22 ºC. The preparations were stretched to optimal length. The equatorial diffraction patterns were recorded with 0.1 to 1 s exposures using a PILATUS detector (DECTRIS Ltd.). A beam size of 16 × 22 µm 2 , a camera length of 5.1 m and a wavelength of 0.95 Å were used. Slightly stretched, air-dried rat tail tendon was used to calibrate the diffraction signal in both directions using the collagen re ections (periodicity 65 nm).

Quantitative PCR analysis
Quantitative real time PCR (qPCR) was used to follow the expression of Muscle Ring Finger protein 1-3 and MAFbx/atrogin-1 at different time points during zebra sh larvae development, immobilization and glucocorticoid treatment. For each analysis at different time points, 10 larvae were anesthetized and stored in 1 ml of RNAlater solution (Qiagen) at 4 ºC for further preparation. For mRNA extraction, 10 larvae from the RNAlater solution were homogenized with a pestle homogenizer and mRNA was extracted using the RNeasy mini kit (Qiagen). The purity and concentration of the obtained mRNA was controlled (NanoDrop 1000, 3.6.0 program). Following reverse transcription (QuantiTect Reverse Transcription kit; Qiagen), cDNA was used for ampli cations with primers for the candidate genes (listed in Table 1).
Before quantitative PCR analysis, the PCR products of each pair of primers were analysed with 3% agarose gel electrophoresis and con rmed to have a single band by using GelRed Nucleic Acid Gel Stain (Biotium) and visualized on a Bio-Rad system (Bio-Rad Laboratories). cDNA was thereafter mixed with Fast SyBR Green Master (Qiagen) mix according to the protocol provided by the company and qPCR was performed using Step One Plus™ Real-Time PCR System (Thermo Fischer).

Results
The rst step was to examine the dynamics of MuRF expression in the zebra sh larvae in relation to contractile function, using dexamethasone treatment known to up-regulate MuRF expression in skeletal muscle [10]. We treated larvae with 300 µM dexamethasone from 3 to 5 dpf, and found a signi cant upregulation of MuRF-1 and a lower active force generation as shown in Fig. 1. To explore if pharmacological immobilization affected MuRF-1 expression and active force we immobilized zebra sh larvae from 3 to 6 dpf using an actomyosin inhibitor (BTS) to inhibit contractile function and active swimming completely. Although the larvae stopped swimming and become unresponsive to gentle touch, normal heart beating and blood circulation were observed by microscopy. After removal of BTS prior to mechanical analysis, larvae swimming recovered in all the groups within 40 min, and therefore a 60 min BTS-wash out time period was used to ensure that no residual BTS could in uence subsequent mechanical analysis. Maximal active force at optimal length was determined at different developmental and BTS immobilisation time. Immobilization had signi cant effect on active force (Table 2). As shown in Fig. 2, active force increased gradually with development from 4 to 6 dpf in DMSO-treated controls. In the immobilized group, active force was slightly lower but not signi cantly different compared to the controls after 1 day of immobilization (at 4 dpf). However, the difference became bigger after 2 days of immobilization (at 5 dpf). Immobilized larvae were signi cantly weaker compared to the controls at 5-6 dpf as illustrated in Fig. 2. To see if the effect of immobilization on active force is reversible, larvae treated with BTS for 1 day were transferred to BTS-free medium for recovery. After 2-day recovery (from day 4 to 6 dpf), active force was similar to that in the controls at 6 dpf (hatched bar compared to open bar at 6 dpf, Fig. 2). In contrast, larvae immobilized for 2-days and recovered only for 1-day did not recover (grey bar compared to open bar at 6 dpf, Fig. 2).
The expression of MuRF-1 to 3, and MAFbx/atrogin-1 were altered due to immobilization during 4-6 dpf as summarized in Table 2. As shown in Fig. 3A, MuRF-1 had the highest expression among these four genes, whereas MuRF-3 expression was the lowest in general. In the controls, the expression of the MuRF-1 and MAFbx increased gradually during muscle development from 4-6 dpf. After 1-day immobilization, no difference was noticeable in any of the examined ligases expression. There is a tendency that immobilization reduced ubiquitin ligase expression as illustrated in Fig. 2A. After 1-day immobilization (at 5 dpf), compared to the controls, MuRF-1 and − 2 expression became signi cantly lower. After 3-day immobilization, MuRF-2, -3 and MAFbx expression was signi cantly lower in treated groups compared to the controls. To examine if immobilization would induce more rapid (within hours) changes in MuRFs and MAFbx, control larvae at 5 dpf were immobilized with BTS for only 1, 2 and 4 hours. As seen in Fig. 3B, no changes were observed in these genes in this shorter time perspective. In a separate series we dechorionated the larvae at 18 h post fertilization, and thereafter immobilized them until 4-6 dpf. In this circumstance where larvae never moved until being analysed, the changes of ligase expression were similar compared to the ones immobilized from 3 dpf. In the controls, MAFbx expression increased signi cantly from 4-6 dpf ( Table 2), which correlated with muscle development.
To explore that if the immobilization affected the contractile lament structure, we performed x-ray scattering experiments. Strong scattering patterns were recorded from 6 dpf larvae with clear 1.1 and 1.0 spacings of about 25 and 43 nm respectively as shown in Fig. 4. Panel A and B show the equatorial x-ray pattern from 6 dpf controls and 6 dpf larvae immobilized from 3 dpf. A clear outward movement of 1.0 and 1.1 re ections was observed after 3-day immobilization, demonstrating the shrinkage of the lament lattice. The average spacing of the 1.0 re ection (d1.0) from multiple analysis was signi cantly smaller in the BTS treated group (Fig. 4C).

Discussion
Zebra sh muscle developed their size and active force during the rst days after hatching, most likely re ecting an interplay between mechanical activity (swimming) and the growth of muscles. It has been previously shown that immobilization affects muscle contractile function in muscle injury and dystrophic zebra sh larvae models [21,22]. Our study builds on these observations. We provided evidence that freely-swimming zebra sh larvae development during 4-6 days after fertilization is associated with an increase of active force of the trunk muscles, but immobilization could inhibit this effect. In the immobilization group, we showed that the inhibitory effect of BTS in muscle contraction is fully reversible and did not signi cantly affect the cardiac function. However, immobilization led to a signi cant loss of active force, demonstrating that muscle contraction is required for muscle strength development. After 1day immobilization and 2-day swimming active force could be fully restored, but after 2-day immobilization, a full recovery of contractile force was not achieved within 1 day although the larvae were actively swimming and alive. This model could be of interest for further studies for movement recovery after immobilization-induced muscle weakness. Dexamethasone treatment for 2 days successfully lowered the active force of zebra sh larvae, suggesting muscle atrophy probably had occurred although active swimming was observed all the time. Glucocorticoids stimulate MuRF-1 upregulation, accelerating proteolysis degradation of myosin heavy chain [10]. The effect of glucocorticoid is mediated by direct targeting on transcriptional factors KLF15 and FoxO [15]. We observed almost twofold of increase in MuRF-1 in the dexamethasone-treated larvae. This nding con rmed with previous studies that proteolysis due to ubiquitin ligase activation may be involved in glucocorticoid-induced muscle atrophy [10,29,30].
The present study shows that ubiquitination ligase signalling pathway is involved in the zebra sh larvae muscle developmental stage and can be altered by muscle immobilization. The mechanistic process could be that the active contraction of sarcomeres stimulates the signalling pathway via the mechanical load sensing elements such as titin, which acts like a link between thick laments and z-disk [31]. MAFbx expression correlated positively with muscle development in the controls. A tendency of MuRF-1and − 3 up-regulation was also observed although was not signi cant. This nding most likely re ects the essential role of ubiquitin ligases in muscle development. As reported previously, MuRF-1 and − 3 double knockout mice exhibited skeletal myopathy due to abnormal MHC accumulation and subsequent bre degradation during growth [32]. Despite the role in muscle atophy, MAFbx was increasingly expressed during the postnatal stages after hatching in chicken [33], indicating its important role in muscle growth.
MAFbx activates the degradation of transcriptional factors MyoD and myogenic that controls myogenic stem cell function [34][35][36]. In addition, ubiquitin ligase plays an important role in microtubules stabilization thus affects sarcomere assembly [37]. Therefore, the current study is consistent with the view that protein degradation via the activation of the ubiquitin-proteasome system is required for sarcomeric protein turnover and maintenance of skeletal muscle function during muscle development. In contrast, the expression of all the investigated ubiquitin ligases reduced signi cantly after immobilization for 2 days accompanied with a loss of active force. Opposite to studies nding un up-regulation of MuRF-1 and MAFbx in muscle disuse animal models including rat [38] and mouse [9,39] as well as in humans [40], our ndings suggest that up-regulation of ubiquitin ligases are not mandatory for observing a loss of active force in all the circumstances. This is in a line with previous observations showing non-consistent responses of MuRFs and MAFbx to muscle disuse in different animal models (see review [17]).
As revealed by our SAXS data, the lack of active contraction not only negatively in uence the contraction force, but also resulted in smaller distance between thick and thin laments. The loss of cytoskeletal components affects sarcomeric structure which correlates with contractile dysfunction. Several muscular atrophies have been shown associated with alterations in lament lattice as demonstrated in zebra sh models [26,27]. However, the extent of lattice compression itself was suggested not be a major component explaining the loss of active force. Most likely it is a marker for the loss of sarcomeric components that in turns in uence the force production [22]. Ubiquitin ligases located within sarcomere and targets on important skeletal proteins. MuRF-1 associates with the Z-disks and M-line whereby MuRF-3 is found at the Z-disks [41][42][43]. MuRF-1 targets proteins associated in thick laments in muscle atrophy such as titin and myosin heavy chain [10,11,44]. It has been shown that MuRF-3 binds to, and has a stabilizing effect, on microtubules [41]. Intermediate lament proteins including desmin and vimentin are substrate for MAFbx [12]. As shown by us, the down-regulation of ubiquitin ligases expression appears to be related to the shrinkage of inter lamental space, it implicates that ubiquitination process may affect proteins responsible for inter lamental space enlargement.

Conclusion
In summary we demonstrate that it was possible to establish muscle atrophy model by BTS-induced immobilization and glucocorticoids treatment in zebra sh larvae. We have observed signi cantly reduced active force of trunk muscle in both models during 3-6 dpf. Ubiquitin ligase MuRF-1 expression was elevated in glucocorticoid treated larvae whereas MuRF 1-3 and MAbx expression was lowered in immobilized larvae compared to the controls, suggesting that ubiquitin ligase expression play a complex role in different muscle atrophy expression. MAFbx expression increases with muscle strengthening during larvae early development stage in the controls. Availability of data and materials

List Of Abbreviations
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval
The experiments were performed according to European guidelines for animal research, complied with national regulations for the care of experimental animals. Experiments on zebra sh larvae up to 6 dpf are not required to be approved by ethics committee.

Consent for publication
Not applicable Figure 1 Effects of dexamethasone (300 µM, introduced at 3 dpf) on MuRF-1 expression (Panel A) and maximal active force (Panel B) at 5 dpf. Open bars: DMSO treated controls; lled bars: dexamethasone treated.