Bidirectional effect of uric acid on C2C12 myotubes and its partial mechanism

To explore the effects and mechanisms of different concentrations of uric acid on skeletal muscle cells.


Introduction
Sarcopenia was first identified by Rosenberg in the 1980s and categorized as an independent condition in the International Classification of Diseases in 2016.It is a progressive and generalized skeletal muscle disorder that involves the accelerated loss of muscle mass and function, with adverse outcomes such as falls, functional decline, frailty, and mortality. 1,2The causes of sarcopenia are very complex, including systemic chronic low-grade inflammation, insulin resistance, hormone disorder, oxidative stress, and mitochondrial dysfunction. 3,4Exploring the pathogenesis of sarcopenia is necessary to prevent sarcopenia, treat patients with the disease, and improve the quality of life of elderly individuals.
Uric acid is the final product of purine in the human body.As a strong antioxidant, it can remove more than half of the free radicals in the body. 5Several studies have shown that skeletal muscle mass and strength are positively correlated with serum uric acid levels.Middle-aged and elderly individuals with higher serum uric acid levels show better grip strength, which is generally believed to be related to the antioxidant effect of uric acid. 6,7However, a study conducted in the United States involving 7544 subjects found that participants in the highest grouping (>8 mg/dL) of serum uric acid concentration had 2.0 times the odds of manifesting sarcopenia compared with participants in the lowest grouping (<6 mg/dL) (P < 0.01). 8Recently, two cross-sectional studies in Japan and western China also suggested that hyperuricaemia was associated with poor muscle strength. 9,10here thus seems to be some controversy over the effect of uric acid on skeletal muscle.Therefore, we treated C2C12 myotubes with a uric acid concentration gradient to explore the effect of uric acid on C2C12 myotubes as well as the possible mechanism.

C2C12 cell culture and differentiation
The C2C12 cell line was purchased from Procell (#CL-0044, Wuhan, China) and cultured in complete medium containing Dulbecco's modified Eagle medium (DMEM), 10% foetal bovine serum (FBS), and 1% penicillin/streptomycin (P/S).Upon reaching 70% confluency, the medium was changed to differentiation medium (DMEM with 2% horse serum and 1% P/S) for 1 week to induce differentiation, and changed at least every 3 days.

Dosage regimen
Referring to the principle of drug concentration gradient design, myotubes were treated with 0 μM, 200 μM, 400 μM, 600 μM, 800 μM, 1000 μM and 1200 μM uric acid (MedChemExpress, Shanghai, China) for 72 h to obtain the most suitable concentration for cell damage.In the following experiments, myotubes were treated at this concentration with or without the cGAS inhibitor RU.521 (MedChemExpress), dissolved in Dimethyl sulfoxide (DMSO), and diluted to 1 μM.The final concentration of DMSO was <0.1%.

Cell viability
Cell viability was determined by Cell Counting Kit-8 assay (Dojindo, Kumamoto, Japan).C2C12 cells were seeded in 96-well plates at a density of 1 Â 10 4 cells per well.After culture in complete medium for 24 h, C2C12 were treated with different concentrations of UA for 72 h.Then cell viability was determined by incubation with DMEM containing CCK-8 for 40 min.The absorbance at 450 nm was measured with a microplate reader.

Measurement of myotube diameter
The myotubes were photographed with an optical microscope, and the diameter of the myotubes was measured using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).Ten random culture fields were taken for each sample, and at least 100 myotubes were counted.

MyHC staining
C2C12 myotubes cultured in 12-well plates were fixed with paraformaldehyde for 15 min after the indicated treatment.First, the cells were soaked with Triton X-100 (Servicebio, Wuhan, China) for 10 min.Goat serum (200 μL) was added and incubated at 37 C for 1 h.Then, 200 μL of MyHC primary antibody (1:100, Santa Cruz, Dallas, TX, USA) was added and incubated overnight at 4 C.A fluorescent secondary antibody conjugated with Alexa Fluor 488 (1:500, Fine Biotech, Wuhan, China) was added and incubated for 1 h at room temperature.Finally, the nuclei were stained with 4-6 diamidino-2-phenylindole (DAPI) for 5 min, and an anti-fluorescence quencher was added.The cells were washed with Phosphate buffered saline (PBS) three times between each step.Images were captured, and fluorescence intensity was measured with ImageJ.

Intracellular ROS level
The intracellular ROS levels were measured with a Reactive oxygen species (ROS) assay kit (Beyotime, Shanghai, China).After the indicated treatment, cells cultured in 12-well plates were incubated with 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) diluted in serum-free medium at 37 C for 20 min.Images were captured, and fluorescence intensity was measured with ImageJ.

Mitochondrial membrane potential
Mitochondrial membrane potential was measured using a JC-1 probe (Beyotime Biotechnology, a probe for detecting mitochondrial membrane potential).Cells cultured in 6-well plates after the indicated treatment were incubated with 100-μL JC-1 staining solution (5 μg/mL) at 37 C for 20 min and rinsed twice with JC-1 staining buffer.The mitochondrial membrane potential was monitored using a fluorescence microscope to measure the relative fluorescence intensity from mitochondrial JC-1 monomers or aggregates under 488-nm and 594-nm laser excitation.

Western blotting
Myotubes were lysed with Radio immunoprecipitation assay (RIPA) buffer containing Phenylmethanesulfonyl fluoride (PMSF) on ice for 30 min, lysates were centrifuged at 12 000 Â g for 15 min, and the supernatants were transferred into new tubes.The protein concentration was quantified using a Bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology).Proteins (30 μg) were separated by 8%, 10%, or 12% Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), transferred, blocked, washed, and incubated with specific primary antibodies and secondary antibodies.Signals were detected using an enhanced chemiluminescence substrate (Zenbio, Chengdu, China).Then, specific protein bands were visualized using Fusion F.X.EDGE Imaging System (Vilber Bio Imaging, France) Biotechnology, a probe for detecting mitochondrial membrane potential.The intensity of individual bands in Western blots was quantitated using Evolution-Capt software.The measures of protein relative abundance in the different samples were expressed relative to the reference protein signal.

Statistical analysis
All data were analysed using GraphPad Prism software 8.0 to generate charts and perform statistical tests.Multiple comparisons were analyzed with one-way analysis of variance (ANOVA).P < 0.05 was considered statistically significant.Data are represented as the mean AE SD or standard error of the mean (SEM).

Effects of different concentrations of uric acid on C2C12 myotubes and autophagy
C2C12 myotubes were treated with different concentrations of uric acid.With increasing concentration, the cell viability showed an inverted "J" curve: C2C12 cell viability was the highest in 400-μM UA, while it was inhibited significantly in 1000-μM, 1200-μM, and 1400-μM UA compared with 0 μM (Fig. 1a).Moreover, myotubes showed phenotypic changes: the diameter treated with 400-μM UA was enlarged compared with that in 0-μM UA, while it was significantly reduced for 1000-μM, 1200-μM, and 1400-μM UA (Fig. 1b,c).In addition, Western blot and immunofluorescence results showed that the expression level of MyHC in cells treated with 400-μM UA was significantly increased, while those for 1000-μM, 1200-μM and 1400-μM UA progressively decreased in turn (Fig. 1d-g).Autophagy was assessed using the autophagy markers LC3B and P62; the results show that the ratio of LC3BII/LC3BI was increased in myotubes treated with 800-μM UA, but the difference was not statistically significant, and it was significantly increased for treatment with 1000-μM, 1200-μM, and 1400-μM UA.Meanwhile, P62 protein level progressively decreased in turn at 800-μM, 1000-μM, 1200-μM, and 1400-μM UA (Fig. 1h-j).These results indicate that uric acid has a bidirectional effect on C2C12 myotubes because of different concentrations.Hyperuricaemia enhances autophagy in myotubes.

Hyperuricemia induced redox balance disorder and activated cGAS-Sting signaling pathways in C2C12 myotubes
The ROS fluorescence staining showed that ROS was decreased in 400-μM UA, while it was significantly enhanced in 1000-μM, 1200-μM, and 1400-μM UA (Fig. 2a,b).Concurrently, mtDNA transcript levels increased as the uric acid concentration increased (Fig. 2c).Green fluorescence of JC-1 staining increased and red fluorescence decreased as uric acid concentration increased, indicating that the mitochondrial membrane potential was abnormal (Fig. 2d,e).The above results suggest that hyperuricaemia induced myotube injury and autophagy accompanied by enhanced oxidative stress and impaired mitochondrial function.The expression of cGAS protein in myotubes was upregulated, and the p-Sting/Sting ratio was gradually increased in 1000-μM, 1200-μM, and 1400-μM UA compared with 0-μM UA (Fig. 2f-h).Hyperuricaemia activates the cGAS-Sting signaling pathway.

cGAS inhibitor reversed hyperuricemia-induced redox balance disorder in C2C12 myotubes
Uric acid could induce diameter changes, MyHC protein expression, autophagy, and cGAS, p-Sting/Sting expression changes in myotubes.We speculated that the cGAS-Sting pathway may be involved in the effect of uric acid on myotubes.To test this hypothesis, the cGAS inhibitor RU.521 was used for the following experiments.The results show that compared with the 1000-μM UA group, ROS fluorescence intensity and mtDNA levels decreased (Fig. 3a-c), and green fluorescence of JC-1 staining decreased while red fluorescence increased (Fig. 3d,e) in 1000-μM UA with RU.521 group.Hyperuricaemia promotes oxidative stress and mitochondrial damage in myotubes by activating the cGAS-Sting pathway.We further observed the effect of cGAS inhibitors on autophagy and phenotype in myotubes.The results show that compared with the 1000-μM UA group, the RU.521 treatment group exhibited increased P62 protein levels (Fig. 4a,b) and a decreased LC3BII/LC3BI ratio (Fig. 4a,c).RU.521 ameliorated the hyperuricaemia-induced myotube diameter decrease (Fig. 4d,e).Western blot and immunofluorescence results show MyHC protein expression recovery (Fig. 4f-i).These findings suggest that uric acid induces autophagy and atrophy in myotubes by activating the cGAS-Sting pathway.

Discussion
In this experiment, we studied the effect of different uric acid concentrations on C2C12 myotubes and excessive concentrations of uric acid stimulate myotube atrophy possible mechanism.
Our study showed that for the uric acid concentration gradient, myotubes cultured in 400-μM UA medium exhibited the largest fiber size, and MyHC protein expression was upregulated.Cell viability, myotube diameter, and MyHC protein expression levels were decreased in myotubes cultured in 200-μM and 600-μM UA medium compared with myotubes cultured in 400-μM UA medium but increased compared with those for standard medium.The results suggest that an appropriate concentration of uric acid is beneficial for myotubes.Uric acid is a strong antioxidant, and its antioxidant capacity is much higher than that of other nonenzymatic antioxidants, uric acid can react with peroxynitrite and free radicals. 11It has been shown that higher serum uric acid levels may be independently associated with greater skeletal muscle function, and a certain concentration of uric acid can protect skeletal muscle from ROS-induced damage because of the strong antioxidant capacity of uric acid. 12,13However, our experimental results showed that myotubes were slightly thinner in 800-μM UA medium than in plain medium, and MyHC protein expression decreased, but with no statistical significance.At 1000-μM UA, the myotube diameter significantly decreased, and MyHC protein expression was greatly downregulated.Myotube diameter and MyHC protein expression levels were further decreased for 1200-μM and 1400-μM UA.Thus, excessive concentrations of uric acid play a role in skeletal muscle injury.A study showed that 750-μM UA medium increased oxidative stress by 32% and reduced endogenous routine respiration, complex II-  | 437 linked oxidative phosphorylation, and electron transfer system capacities, which increased triglyceride levels by 237% and promoted lipogenesis in myotubes. 14The hydrophobic environment formed by intracellular lipids is not conducive to the antioxidant effect of uric acid but rather promotes the pro-oxidative properties of uric acid. 15We further measured the oxidative stress indicators of myotubes in different concentrations of uric acid, showing that cells cultured with 1000-μM, 1200-μM, and 1400-μM UA had higher ROS and mtDNA levels, and abnormal mitochondrial membrane potential.The CCK8 assay results and myotube diameter and MyHC protein expression results suggest that the relationship between uric acid concentration and myotube generation is bidirectional, and the statistical analysis showed an inverted J-shaped curve.Appropriate uric acid concentrations may protect skeletal muscle through antioxidant effects, while hyperuricaemia has pro-oxidative capacity, and skeletal muscle atrophy is closely related to hyperuricaemia-induced oxidative stress.
With changes in myotube diameter and MyHC expression, we found that autophagy-related parameters also changed.LC3 and P62 both are marker proteins for autophagosome membrane formation.An increased LC3BII/LCB3I ratio indicates enhanced autophagic function, 16 while decreasing P62 indicates the activation of autophagy. 17In this experiment, the LC3BII/LC3BI ratio and P62 protein level of myotubes cultured with uric acid at concentrations of 600 μM and below were not significantly changed.At 800-μM UA, the LC3BII/LC3BI ratio increased, but with no statistical significance, while the P62 protein level was significantly reduced.With myotubes cultured in uric acid at concentrations of 1000 μM and above, the LC3BII/LC3BI ratio was significantly increased and the P62 protein level was decreased.That indicates that autophagic function in myotubes is enhanced with high concentrations of uric acid.In recent studies, Hu and Choi et al. also found that autophagy activation leads to skeletal muscle atrophy, and the expression of LC3II and Atg7 was upregulated in skeletal muscle, while P62 was inhibited.Moreover, the mass and strength of skeletal muscle were significantly decreased in mice, and the expression of skeletal muscle-related proteins (myogenin, atrogin-1, MuRF1) was altered. 18,19In response to hyperuricaemia, the LC3BII/LC3BI ratio increased, the P62 protein level decreased, and myotube parameters (diameter, MyHC) decreased.Therefore, we can speculate that the damage to skeletal muscle by hyperuricaemia is achieved at least in part through the excessive activation of autophagy.
The cGAS-Sting pathway is an important signaling pathway for autoimmunity, sterile inflammatory responses, cellular senescence, and the induction of autophagy. 20,21Recent studies have found that oxidative stress damages mitochondria, and released mtDNA can activate the cGAS-Sting pathway. 22In our study, the expression of cGAS and p-Sting/Sting in myotubes was significantly upregulated for cells cultured in high concentrations of uric acid compared with cells cultured in standard medium.To further investigate how uric acid activates autophagy and damages skeletal muscle, a specific inhibitor of cGAS protein (RU.521) was used in this study.The results showed that myotubes cultured with high concentrations of uric acid and RU.521 had decreased ROS fluorescence intensity, mtDNA level and JC-1 green fluorescence, and increased red fluorescence compared with cells treated with high concentrations of uric acid alone, suggesting that inhibition of the cGAS protein can attenuate hyperuricaemia-induced oxidative stress and mitochondrial damage.In addition, the LC3BII/LC3BI ratio decreased and P62 protein level increased in the inhibitor group compared with myotubes in the hyperuricaemia group, indicating that inhibition of cGAS protein can attenuate the hyperactivation of autophagy in myotubes induced by hyperuricaemia.Moreover, the myotube diameter decreased and MyHC protein expression level decreased in the hyperuricaemia group compared with the blank control group, but the myotube diameter increased and MyHC protein expression level increased in the myotubes treated with the inhibitor, clarifying that inhibiting the cGAS-Sting pathway can reduce the adverse effects of hyperuricaemia on skeletal myocytes.In this study, we examined the cGAS-Sting pathway, hyperuricaemia, and autophagy in the context of sarcopenia for the first time, and found that hyperuricaemia could induce oxidative stress, damage mitochondria, and release mtDNA to activate the cGAS-Sting pathway.The cGAS-Sting pathway can positively regulate cellular oxidative stress, promote excessive autophagy, and lead to myotube atrophy in myotubes.
At present, clinical studies have investigated the effect of serum uric acid on skeletal muscle.Appropriate concentrations of uric acid exerted antioxidant effects and improved skeletal muscle function.High concentrations of uric acid damaged skeletal muscle owing to pro-oxidant effects, and subjects had reduced grip strength and walking speed.However, the concentration of uric acid that is damaging to skeletal muscle cells at the cellular level may not apply to animals in vivo.Therefore, our findings depend on further in vivo experiments or clinical trials for validation.

Conclusion
In this study, we observed the effects of different uric acid concentrations on skeletal muscle cells in the laboratory for the first time and explored excessive concentrations of uric acid stimulate myotube atrophy mechanisms.Our research confirms that uric acid has a bidirectional effect on C2C12 myotubes: suitable concentrations of uric acid facilitate myotube growth, and excessive concentrations of uric acid stimulate myotube atrophy, accompanied by the activation of autophagy function.In addition, high concentrations of uric acid were observed to induce oxidative stress in C2C12 myotubes and damage mitochondria, thereby activating the cGAS-Sting signaling pathway and overactivating cellular autophagy, leading to myotube atrophy.

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