Received 8 January 2015; accepted 5 April 2015; published 14 April 2015

ABSTRACT

Fyn kinase-dependent cellular events were related with skeletal muscle denervation. In the present study, we used a combination of techniques to measure ER stability and the related Gb1g2 trafficking following muscle mobilization, and demonstrated a temporally and Fyn-dependent up-regulation of Ca²⁺ level in the mobilized muscle. In parallel, Fyn activity in ER was gradually decreased, which was accompanied by enhanced PTP1B activity and expressions of ER proteins (calnexin, Grp94, cyclophilin, and Hsp70). Moreover, during muscle mobilization, there was more membrane protein breakdown than protein synthesis, which probably featured robust Gb1g2 internalization, and Rab1-dependent transport into ER compartment; the signaling was related to disruption of PI3K-Akt signaling and decrement of muscular functions. Then, Gb1g2 trafficking is a key component necessary for the early recovery processes regarding muscle atrophy, which would be the therapeutic consideration for muscle repair and regeneration.

Keywords:

Muscle Mobilization, Fyn, Rab1, Gb1g2, Endoplasmic Reticulum

1. Introduction

Skeletal muscle comprises up to 40% of human body mass, which is a highly ordered, structurally stable tissue. Muscular function is dependent on intact nerve supply; for example, sciatic nerve injury causes profound structural and functional changes including rapid loss of muscle mass and contractile force [1] [2] . As reported, growth factors usually result in a quick and adaptive response, and mediate changes allowing an animal to effectively face mobilization [3] -[7] . Coincidently, we have characterized that Fyn, one of Src kinases, represented the predominant signaling in VEGFR-2 activation and VEGFR-1 shuttling between membrane and ER, which initiated stereotypic inflammation and muscle-T cell interaction during muscle denervation [8] - [10] . Therefore, it was proposed that subcellular communication might form a viable link to functional muscular stabilization.

As reported, Fyn-related signaling served as integral components for remodeling of G-protein-coupled receptors (GPCR) [11] [12] . As reported, heterotrimeric G protein signaling to the plasma membrane could be activated by GPCR, which promoted the exchange of GDP (guanosine diphosphate) for GTP in the Ga subunit and its dissociation from Gbg. GTP-loaded Gbg served as part of the mechanism of PI3-kinase activation [13] - [16] ; such examples included internalization of Gbg in response to the action of b-adrenergic receptors. Additionally, this pool of free Gbg is associated with endosomes, and capable to cause remodeling of Golgi apparatus [17] - [25] .

Intracellular trafficking of diverse signal transduction proteins is coordinated by a variety of Rab GTPases that regulate the budding and fission of vesicles from compartments [23] - [25] ; among them, Rab1 is a molecule for transport from ER to Golgi [14] - [16] . Then, it raised possibility that cross-link of Gbg-Rab1 might involve in muscle adaptation during muscle mobilization. A deeper understanding of phenotype-dependent stability of ER compartment would certainly provide insight into novel preventive or therapeutic regimens to alleviate or delay muscular damage.

2. Methods

2.1. Leg Casting Disuse Atrophy Model and Ethics Statement

Mice were anesthetized with isoflurane, and the lower right leg was casted from the knee to the toes with heat-activated casting material (Vet Lite, Kruuse, Marslev, Denmark ). The test materials were administered by continuous infusion via implantation of an osmotic minipump. After casting, animals were euthanized by carbon dioxide asphyxiation followed by cervical dislocation. Gastrocnemius was dissected rapidly from both legs when the cast was removed, and the muscles were cleaned of tendons and connective tissue and weighed.

All experiments using animals were conducted in accordance with the relevant codes of practice for the care and use of animals for scientific purposes (National Institutes of Health, 1985). All experimental protocols were approved by Ethics Committee of Fudan University, all efforts were made to minimize the numbers of animal used and their suffering.

2.2. Immunohistochemistry

Serial sections of gastrocnemius were carried out on a cryostat at 15 μm. Sections were mounted on glass slides (Superfrost plus) and labeled with antibody against Gb1g2 (1:1000, Abcam, Cambridge , MA ) and Alexa Fluor594 conjugated secondary antibody. Antibodies were diluted in 0.01M PB with 3% normal goat serum, 1% BSA, 0.5% Triton X-100, and 0.05% sodium azide, pH7.4. Sections were viewed under microscope (Leica L 2000A ), images were acquired and processed by Leica Quin software. In separate experiments, we tested different antigen retrieval techniques including microwave heating and SDS-pretreatment.

2.3. Satellite Cell Culture

Gastrocnemius was weighted after being cleaned of tendons and fat tissue, cut in fragments, rinsed in PBS, incubated in 15 ml of dissociation medium and incubated at 37˚C in a water bath for 1 h. Every 10 min tube was shaken gently. Afterwards, muscle fragments were dispersed using a 10 ml pipette with a large extremity in the presence of the dissociation medium. Supernatant was decanted and filtered through a 200 μm nylon mesh into a tube containing an equal volume of culture medium to isolate free cells. The remaining muscle tissue was further dissociated in fresh dissociation medium before being filtered in a tube to isolate additional free cells. Free cells were centrifuged (300 g, 10 min) and resuspended in 5 ml of DMEM with 10% (v/v) fetal bovine serum, then were seeded at a density of 6 - 10 × 10⁴ cells per 25 cm ² flask (Nunc) in a total volume of 4 ml culture medium. The cultures were grown at 37˚C with humidified atmosphere containing 5% CO₂. 4 days later, cells were assayed.

For macrophage or T cell induction, lipopolysaccharide (LPS, 100 ng/mL) and lymphocytes (1 × 10⁶) were added to cultures for 48 hours. Conditioned medium was collected and concentrated 100-fold using Amicon ultra centrifuge filters (Millipore, Billerica, MA, USA).

2.4. Assays for Protein Metabolism

Gastrocnemius was rinsed and homogenized, the precipitated proteins were collected by centrifugation. The supernatant was used for measurement of L-[3,4,5-³H] phenylalanine specific activities. The pellet was washed three times in 2% (v/v) perchloric acid and then hydrolyzed in 2 M NaOH. The fractional protein synthesis rates were calculated using the equation: fr PS (% day) = Sb/Si×t, where Sb and Si are the specific activities of the protein-bound and free acid-soluble tissue phenylalanine pools respectively; t is the time after the isotope injection in days.

To assess protein breakdown rates, gastrocnemius was tied by the tendons at resting length to stainless steel supports and preincubated in a shaking water bath for 30 min at 37˚C in individual stoppered 25 ml flasks containing 3 ml oxygenated (95% O₂ - 5% CO₂) Krebs-Henseleit bicarbonate buffer (pH 7.4) with 10 mM glucose. Muscles were then incubated in fresh buffer containing 0.5 mM cycloheximide for 2 h. The net production of tyrosine and 1-methyl-histidine (1-Me-His) during incubation was assayed using HPLC. Incubation studies were carried out using 6 - 8 muscles per group, each gastrocnemius obtained from a separate rat.

2.5. Intracellular Ca²⁺ Measurement

One day before the assay, CORNING^® black with clear flat bottom 96-well-assay plate was coated with poly-L- Lysine. Gastrocnemius was homogenized, suspended and plated in 150 μl medium and incubated in a humidified atmosphere of 5% CO₂ at 37˚C overnight. At the day of assay, 100 μl medium was removed from plate, 50 μl FLIPR^® calcium assay reagent (Molecular Devices Corp) dissolved in 1× reagent buffer (1× HANKs buffer with 20 mM HEPES), pH 7.4, with 5 mM probenecid was added and the plate was incubated at 37˚C for 1 h. Treatment reagents were dissolved in the assay buffer (HBSS: KCl 5 mM, KH₂PO₄ 0.3 mM, NaCl 138 mM, NaHCO₃ 4 mM, Na₂HPO₄ 0.3 mM, D-glucose 5.6 mM, with additional 20 mM HEPES, 2.5 mM probenecid and 13 mM CaCl₂). Using a FlexStation (Molecular Devices Corp.), the [Ca²⁺]i fluorescence were monitored every 1.52 s intervals with excitation wavelength at 485 nm and emission wavelength at 525 nm. The [Ca²⁺]i fluorescence was measured up to 90 s, the fluorescence intensity from 3 wells were averaged and the relative amount of [Ca²⁺]i release was determined by integrating the area under the peak of the [Ca²⁺]i fluorescence averages.

2.6. Recombinant Adenovirus Construction

Recombinant adenovirus expressing mouse Gb1g2, Rab1 siRNA were constructed by inserting into the adenoviral shuttle vector pDE1sp 1A (Microbix Biosystems, Inc., Canada), and the insert was then switched to the adenoviral vector through LR recombination. After homologous recombination with the backbone vector PJM17, plaques resulting from viral cytopathic effects were selected and expanded in 293 cells. Positive plaques were further purified and large-scale production of adenovirus was carried out by two sequential CsCl gradients and PD-10 Sephadex chromatography.

2.7. Subcellular Fractionation

For nuclear extracts, gastrocnemius was washed twice in ice-cold PBS, resuspended in buffer A (10 mM HEPES, 10 mM NaCl, 3 mM MgCl₂, 1 mM EGTA, 0.1% Triton X-100, pH 7.5) supplemented with 50 mM NaF, 1 mM Na₃VO₄, 1 mM DTT, 1 mM PMSF, and 1 μg/ml protease inhibitor cocktail and incubated on ice for 40 min. The nuclei were pelleted by centrifugation at 2400 g for 10 min at 4˚C and resuspended in buffer B (25 mM HEPES, 300 mM NaCl, 5 mM MgCl₂, 1 mM EGTA, 20% glycerol, pH 7.4) supplemented with 50 mM NaF, 1 mM Na₃VO₄, 1 mM DTT, 1 mM PMSF, and 1 μg/ml protease inhibitor cocktail. After incubation on ice for 60 min, the lysates were centrifuged at 12,000 g for 20 min at 4˚C. The supernatants (nuclear extracts) were collected and assayed.

For ER separation, gastrocnemius was homogenized in 2 ml of 10 mM Tris-HCl (pH 7.5) and 150 mM sucrose, the resulting cell lysate was mixed with 6.5 ml of 1 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 63.5% sucrose and layered under a sucrose density gradient (1.5/1.7/2.1 M sucrose). Gradients were centrifuged at 100,000 ×g for 3 h and ER fractions were collected manually. ER fractions were washed with 2 vol. of 10 mM Tris-HCl (pH 7.5) with centrifugation, and resuspended in 10 mM Tris-HCl (pH 7.5) with 150 mM sucrose, then stored for later analysis.

2.8. Immunoprecipitation and Western Blotting

Gastrocnemius was homogenized and centrifuged, the supernatants were incubated with anti-Rab1, AchRα antibody (1:200; BD Transduction Laboratories, Lexington, KY) at 4˚C overnight with slow rotation. 60 μl of protein G-agarose beads (Invitrogen, Carlsbad, CA) were added and further incubated for 3 h. Afterwards, the beads were washed and protein sample were eluted from with 1x SDS sample buffer.

For Western blot analysis, protein samples were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ). Membranes were probed with anti-cSrc, anti-Gb1g2 (1:1000; Abcam, Cambridge, MA), anti-AchRβ (1:500; Abcam), anti-calnexin, anti-Grp94, anti-cyclophilin, anti-Hsp70 (1:500; BD Transduction Laboratories) respectively. Protein bands were detected using alkaline phosphatase conjugated secondary antibodies (1:5000) and ECF substrate, and then scanned using a Storm 860 imaging system. Band intensities were quantified and analyzed with ImageQuant software (GE Healthcare).

2.9. In Vitro Fyn and MuSK Kinase Assay

Proteins in anti-Fyn or anti-MuSK antibody immunoprecipitated pool (1:200; Abcam) were precipitated with 5% (w/v) TCA. The resulting pellets were washed with acetone and incubated at 30˚C with 5 μg of SRC substrate peptide (KVEKIGEGTYGVVYK, corresponding to amino acids 6 - 20 of p34cdc2; Upstate Biotechnology, Lake Placid, New York) in kinase buffer containing 5 μCi of [γ-³²P]-adenosine triphosphate ([γ-³²P]-ATP; PerkinElmer Life Sciences, Waltham, Massachusetts), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM MnCl₂, 25 μM ATPase, 1 mM dithiothreitol, and 100 μM Na₃VO₄. After 30 min, the reaction was terminated by the addition of 10 μl of 40% (w/v) TCA, and samples were spotted on P81 cellulose phosphate paper (Upstate Biotech). The paper was washed three times with 1% (w/v) phosphoric acid and once with acetone. Radioactivity retained on the P81 paper was quantified by liquid scintillation counting. Blank counts (without tissue lysate) were subtracted from each result, and radioactivity (cpm) was converted to picomoles per minute (pmol/min).

2.10. PTP 1B Assay

Gastrocnemius was collected and homogenized in RIPA buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 2 mM EDTA, 1.0% Triton X-100 and complete protease inhibitor mixture). Equal amounts of protein were incubated at 4˚C for 2 h with anti-PTP1B antibody and then, 20 μl of protein G sepharose was added and incubated for additional 2 h with mixing. Immunoprecipitated complexes were washed twice in RIPA buffer, once with the assay buffer (25 mM imidazole [pH 7.2], 0.1 mg/ml BSA, 10 mM DTT) and finally they were resuspended in 25 μl of assay buffer. The phosphatase substrate raytide was labeled at its unique tyrosine residue in the presence of [γ-³²P] ATP. Assay mixtures (30 μl) containing the immunoprecipitated pellet and [γ-³²P]-labeled raytide (1 × 10⁵ cpm) were incubated at 30˚C for 2 h and the reaction was terminated by adding 750 μl of a charcoal mixture (0.9 M HCl, 90 mM sodium pyrophosphate, 2 mM NaH₂PO₄, 4% vol/vol Norit A). After centrifugation, the radioactivity in 400 μl of the supernatant was measured by scintillation counting. Blanks were determined by measuring the free γ-³²P in reactions where the immunoprecipitates were either boiled or omitted, and these values were subtracted from the reaction values.

2.11. Whole Muscle Mechanical Properties

The experiments were performed in vitro at 30˚C in a vertical muscle apparatus (300B, Aurora Scientific) containing a Ringer solution (120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 3.15 mM MgCl₂, 1.3 mM NaH₂PO₄, 25 mM NaHCO₃, 11 mM glucose, 30 μM d-tubocurarine, pH 7.2 - 7.4) continuously bubbled with 95%O₂ - 5%CO₂. The muscles were stretched to the optimal length, i.e., the length that allowed maximal tension development in response to a single pulse. The muscles were then electrically stimulated, through two parallel electrodes, with supramaximal pulses (0.5 ms duration) delivered by a Grass S44 electronic stimulator through a stimulus isolation unit (Grass SIU5). Muscle response was recorded by isometric force transducer (Grass FT03). Data were analyzed by LabView software. Tetanic stimulation was obtained by applying trains of supramaximal stimuli at 80- and 150-Hz frequency for the muscles. Tetanic tension was normalized to the muscle wet weight (specific tension, N/g). Muscles were weighed at the end of each experiment.

2.12. Statistics

Data were represented as mean ± SEM and analyzed with Prism 5 software. For all data sets, normality and homocedasticity assumptions were reached, validating the application of the one-way ANOVA, followed by Dunnett test as post hoc test to do comparisons. Differences were considered significant for p < 0.05.

3. Results

3.1. Temporal Alteration of ER in Response to Muscle Mobilization

In the present study, muscle mobilization was established by leg casting, 1, 7, 14 and 21 days later, extracellular Ca²⁺ levels in gastrocnemius were measured using FlexStation. As shown in Figure 1(a), Ca²⁺ levels rose rapidly at day 1 - 7, which were approximately 2.4 and 2.1 folds over control in Fyn^(+/−) mice, and 1.3 folds over control in Fyn^(−/−) mice. Intriguingly, the elevation could be partly inhibited by thapsigargin (1 μM, 16 h), indicating that there might have functional alteration of ER during muscle mobilization. As a confirmation, it was demonstrated that following leg casting, Fyn activity was increased with compartment dependent manner, in ER, the elevation of [γ-³²P] incorporation was transient, which rose approximately 3.4 folds over control values within 7 days, instead in nucleus, Fyn activation happened persistently, which increased around 3.3 folds over control (Figure 1(b)). PTP1B activity was also investigated by [γ-³²P] incorporation, as demonstrated, PTP1B

activity remained at control level at day 1 - 7 following muscle mobilization, however, by day 14 - 21, which was considerably strengthened to 4.4 folds over control (Figure 1(c)). Furthermore, ER proteins (calnexin, Grp94, cyclophilin and HSP70) were measured by Western Blot analysis, it was demonstrated that whose expression was remarkably and timely increased by day 14 - 21, the levels were increased to 2.4 - 2.9 fold over control respectively (Figure 1(d), Figure 1(e)).

3.2. Decrease in Membrane Protein Synthesis during Muscle Mobilization

It was previously reported that ER is associated with protein synthesis [26] [27] , the functional alteration was proposed to implicate in the cellular responses during prolonged muscle denervation [8] [9] . In the present study, Fyn^(+/−) mice were undergone leg casting, gastrocnemius was separated, Figure 2(a) revealed that protein synthesis in membrane was markedly decreased by day 14 following muscle mobilization, however, protein breakdown was increased in the same time (Figure 2(b), Figure 2(c)).

3.3. Membrane Gb1g2 Expression during Muscle Mobilization

Gastrocnemius was separated from Fyn^(+/−) and Fyn^(−/−) mice, by Western Blot analysis, signal quantification revealed that Gb1g2 membrane expression was greatly disrupted in Fyn^−/− mice (Figure 3(a)). When Fyn^(+/−) mice were undergone leg casting, it was revealed that Gb1g2 membrane expression in gastrocnemius was attenuated by day 14 following muscle mobilization (Figure 3(b), Figure 3(c)). In parallel, PI3K-Akt signaling was considerably down-regulated (Figure 3(d), Figure 3(e)).

3.4. Membrane Gb1g2 Expression Was Dependent on ER Functional Alteration

Satellite cells were cultured to detect the modulation of Gb1g2 membrane expression, as illustrated in Figure 4(a), Gb1g2 membrane expression was remarkably inhibited when exposed to brefeldin A (Golgi transport block, 10 μg/ml, 16 h), turnicamycin (N glycosylation block, 2 μg/ml, 18 h), or leptomycin B (nuclear export block, 20 nM, 30 min). PI3K-Akt signaling was attenuated in the same condition (Figure 4(b), Figure 4(c)), then the data indicated that ER functional alteration might lead to Gb1g2 internalization and the resultant disruption of G-protein coupled receptor signaling.

3.5. Gb1g2 Internalization Was Modulated by Rab1

By immunofluorescent analysis, it was illustrated that there was no detectable alteration in total Gb1g2 fluorescent densities, intriguingly, the fluorescence was considerably translocated into cytosol at day 14 following muscle mobilization (Figure 5(a)). Subsequently, molecular connection of Rab1 and Gb1g2 was detected by immunoprecipitation, in which anti-Rab1 was used as immunoprecipitated antibody and anti-Gb1g2 as immunoblot antibody, it was demonstrated that the expression levels were strengthened around 3.0 folds over control at 14 days following muscle mobilization (Figure 5(b)). Additionally, in satellite cells, by Western Blot analysis, it was revealed that there was a significant decrease in membrane Gb1g2 expression upon challenged with macrophage or T cell conditioned medium, the effect could be blocked by Rab1 knockdown (Figure 5(c), Figure 5(d)). In the same time, satellite cell proliferation was disrupted in the presence of macrophage or T cell conditioned medium, the effect was dependent on Rab1 (Figure 5(e)).

3.6. Gb1g2 Internalization Was Associated with Motor Endplate Alteration

Gb1g2 and Rab1 were over- or down-expressed in gastrocnemius, then mice were undergone leg casting, it was revealed that cell survival was decreased considerably at day 14 - 21 following muscle mobilization: the levels reduced to 54.1 ± 6.1 and 53.1% ± 5.9% control respectively, which could be partly rescued in the presence of adeno-Gb1g2 or Rab1 siRNA (Figure 6(a)). Similarly, MuSK activity and AchR cluster, as well as muscle mechanical power was disrupted at day 14 following muscle mobilization, which could also be partly ameliorated by infection of adeno-Gb1g2 or Rab1 siRNA (Figure 6(b) and Figure 6(c)).

4. Discussion

Previously, we reported that timely Fyn activation triggered a particular microenvironment in the denervated gastrocnemius, which evoked early muscular inflammation and later local immunological changes via specific subcellular communication [8] [9] . Herein, gastrocnemius mobilization was established and it was observed that extracellular calcium level was elevated, which happened rapidly and sustained within one week of operation.

Intriguingly, the elevation was sensitive to Fyn and could be partly inhibited by thapsigargin, a ER Ca²⁺ ATPase. Coincidently, we showed that Fyn activity was timely inhibited during muscle mobilization, which accompanied by enhanced PTP1B activity and expression of ER proteins (calnexin, Grp94, cyclophilin, and Hsp70). Thereby the present observations supported previous observation that muscle atrophy was related to PTP1B evoked organelle interconnection [10] [28] -[34] , and provoked the idea that signaling in the ER compartment was proposed to feature and trigger certain distinctive functional outcomes during muscle mobilization.

Moreover, we demonstrated that prolonged muscle mobilization resulted in a remarkable increase in membrane protein breakdown and decrease in protein synthesis. In parallel, Gb1g2 appeared to be targeted and led to disruption in PI3K-Akt signaling. Recently, substantial evidence implicated that G-protein coupled receptors play an important role on skeletal muscle growth and differentiation, whose cross-talk with growth factor receptors make the host less vulnerable to the insults [35] [36] . Regarding current observation, Gb1g2 was presumed to be sensitized by muscle mobilization and might perpetuate and lower the threshold for musular dysfunction.

Coincidently, we observed that in later stage (14 - 21 day) of leg casting, Gb1g2 underwent vigorous internalization and cross-link with Rab1, a molecule for transport from ER to Golgi, this cellular event was primarily initiated in the presence of macrophage or T cells. Then, the data indicated that along with prolonged muscle mobilization, Gb1g2 was translocated into ER, which seemed to result in marked impairment in the related G-protein couple receptor signaling. Additionally, it was illustrated that cross-link of Gb1g2 and Rab1 caused an increased decay of PI3K-Akt signaling and decrement of muscle cell survival. Therefore, a teleological argument is Gb1g2 translocation might trigger complex alterations in ER compartment, by which result in dynamic and variable damage to muscular function [37] -[41] .

Skeletal muscle had the capacity to change its phenotype in response to altered functional demands, muscle atrophy was usually in parallel to the decline in protein synthesis and metabolism in motor endplate, and harbored different endogenous levels of muscle-specific tyrosine kinase (MuSK) and AchR cluster [42] -[46] . In the present study, we demonstrated that inhibition of Gb1g2-Rab1 cross-link appeared to be able to rescue muscular function, including reorganization of muscle cell viability, agrin initiated MuSK activity and AchR cluster, as well as muscle mechanical power. Therefore, we realized that stability of ER compartment was proposed to be important for the maintenance of muscular function, which could be deteriorated along with muscle mobilization.

5. Conclusion

Collectively, we demonstrated a dramatic and temporal up-regulation of extracellular calcium level during muscle mobilization, which was sensitive to Fyn and partly dependent on ER function. Coincidently, Fyn activity in ER was decreased with prolonged muscle mobilization, which was accompanied by enhanced PTP1B activity and expressions of ER proteins including calnexin, Grp94, cyclophilin, and Hsp70; in parallel, there was more protein breakdown than protein synthesis in membrane, which probably featured robust Gb1g2 internalization, and Rab1-related transport into ER compartment. Most importantly, muscle mobilization resulted in disruption of PI3K-Akt signaling and decrement of muscular functions, including muscle cell survival, agrin initiated MuSK activity as well as AchR cluster, and muscle mechanical power, which could be rescued by inhibition of Gb1g2-Rab1 cross-link. Thus, stability of ER compartment might overlap with stability of motor end-plate, which is a key component necessary for the early recovery processes regarding muscle atrophy, and would be the therapeutic considerations for muscle repair and regeneration.

Acknowledgements

This work is supported by the Natural scientific program in Minhang District of Shanghai (2011MHZ21), National nature sciences of China (81471370).

References

NOTES

^*Corresponding author.