In this experiment, we studied the effect of different concentrations of uric acid medium on C2C12 myotubes and its possible mechanism.
Our study showed that among C2C12 myotubes cultured with a uric acid concentration gradient, C2C12 myotubes cultured in 400 µM uric acid medium exhibited the largest fibre sizes, and MyHC protein expression was significantly upregulated. Cell viability, myotube diameter, and MyHC protein expression levels were decreased in myotubes cultured with 200 µM and 600 µM uric acid medium compared to myotubes cultured with 400 µM uric acid medium but increased compared to myotubes cultured with standard medium. The results suggest that an appropriate concentration of uric acid is beneficial for the development of myotubes. Uric acid is a strong antioxidant, and its antioxidant capacity is much higher than that of other nonenzymatic antioxidants, which can react with peroxynitrite and free radicals produced by its decomposition. It has been shown that higher serum uric acid levels may be independently associated with greater skeletal muscle function. This greater skeletal muscle function may be related to the strong antioxidant capacity of uric acid, and a certain concentration of uric acid can protect skeletal muscle from ROS-induced damage[11]. However, our experimental results showed that when the uric acid concentration reached 800 µM, myotubes were slightly smaller in diameter than myotubes cultured in plain medium, and MyHC protein expression levels decreased, but there was no statistically significant difference. At 1000 µM uric acid, myotube diameter significantly decreased, and MyHC protein expression was significantly downregulated. Myotube diameter and MyHC protein expression levels were decreased in myotubes cultured with 1200 µM and 1400 µM uric acid compared to myotubes cultured with 1000 µM uric acid. Thus, excessive concentrations of uric acid play an important role in skeletal muscle injury. A study showed that 750 µM uric acid medium increased oxidative stress by 32% and reduced endogenous routine respiration, complex II-linked oxidative phosphorylation, and electron transfer system capacities, which simultaneously increased triglyceride levels by 237% and promoted lipogenesis[12]. The 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[13]. We further measured the ROS levels and mitochondrial membrane potential of C2C12 myotubes in different concentrations of uric acid medium, and the results showed that cells cultured with 1000 µM, 1200 µM, and 1400 µM uric acid medium had higher ROS levels, mtDNA levels and abnormal mitochondrial membrane potential. The CCK8 assay results, the light microscopy results of myotube diameter and MyHC protein expression results suggest that the relationship between uric acid concentration and C2C12 myotube generation is bidirectional, and the statistical analysis showed an inverted J-shaped curve. Appropriate concentrations of uric acid 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 protein expression, we found that autophagy-related parameters (LC3BII/LC3BI, P62) also changed. LC3 is a marker protein for autophagosome membrane formation. When autophagy occurs, the LC3BI protein binds phosphatidylethanolamine to form the LC3BII protein, inducing the formation of autophagosomes. An increased LC3BII/LCB3I ratio indicates enhanced autophagic function[14]. The p62 protein is a degradative substrate of cellular autophagy, and when autophagy occurs, it binds to LC3 and then translocates to autolysosomes for degradation. A decrease in P62 protein levels indicates the activation of autophagy[15]. In this experiment, the LC3BII/LC3BI ratio and P62 protein level of C2C12 myotubes cultured with uric acid at concentrations of 600 µM and below were not significantly changed. At 800 µM uric acid, the LC3BII/LC3BI ratio increased, but there was no statistically significant difference, and the P62 protein level was significantly reduced. The LC3BII/LC3BI ratio was significantly increased and the P62 protein level was significantly decreased in C2C12 myotubes cultured with uric acid at concentrations of 1000 µM and above. These results indicate enhanced autophagic function in C2C12 myotubes cultured with high concentrations of uric acid. In recent studies, Hu and Choi et al. also found that activation of autophagy leads to skeletal muscle atrophy, and the expression of LC3II and Atg7 was upregulated in skeletal muscle, while the expression of 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[16, 17]. In response to hyperuricaemia, the LC3BII/LC3BI ratio increased, the P62 protein level decreased, and C2C12 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 signalling pathway is an important signalling pathway for autoimmunity, sterile inflammatory responses, cellular senescence, and the induction of autophagy[18, 19]. Recent studies have found that oxidative stress damages mitochondria, and released mitochondrial DNA (mtDNA) can activate the cGAS-sting signalling pathway[20]. In this study, we also found that the expression of cGAS and p-sting/sting in C2C12 myotubes was significantly upregulated in cells cultured with 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 C2C12 myotubes cultured with high concentrations of uric acid and RU.521 had decreased ROS fluorescence intensity, mtDNA level, JC-1 green fluorescence and increased JC-1 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 the P62 protein level increased in C2C12 myotubes in the inhibitor group compared with C2C12 myotubes in the hyperuricaemia group, indicating that inhibition of cGAS protein can attenuate the hyperactivation of autophagy in C2C12 myotubes induced by hyperuricaemia. Moreover, the C2C12 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 signalling pathway can reduce the adverse effects of hyperuricaemia on skeletal myocytes. In this study, we examined the cGAS-sting signalling pathway, hyperuricaemia, and autophagy in the context of sarcopenia for the first time, and 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 C2C12 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 due 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.