Yang Jia-He, Gao Jun, E Ya-Qi, Jiao Li-Jie, Wu Ren, Yan Qiu-Yi, Wei Zi-Yi, Yan Guo-Liang, Liang Jin-Long, Li Hong-Zhu
Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China.
Department of Pathophysiology, School of Medicine, Xiamen University, Xiamen, Fujian, China.
J Cachexia Sarcopenia Muscle. 2024 Oct;15(5):2118-2133. doi: 10.1002/jcsm.13560. Epub 2024 Aug 27.
Hydrogen sulfide (HS), the third gasotransmitter discovered, regulates a variety of physiological functions. Whether HS alleviates skeletal muscle ageing by regulating autophagy has not been reported.
Mice were administered 150 mg/kg/day of D-galactose (D-gal), and C2C12 myotubes were cultured in 20 g/L D-gal to induce ageing. Sodium hydrosulfide (NaHS) was employed as an exogenous donor in the treatment group. The intracellular concentration of HS was quantified by the 7-azido-4-methylcoumarin fluorescence probe. The proteins involved in the ubiquitin-mediated degradation of AMPKα1 were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation (Co-IP). S-sulfhydration of USP5 was tested by a biotin-switch assay. Associated proteins were analysed by western blot.
NaHS was found to effectively restore the HS content in both ageing gastrocnemius (+91.89%, P < 0.001) and C2C12 myotubes (+27.55%, P < 0.001). In comparison to the D-gal group, NaHS was observed to increase the mean cross-sectional area of muscle fibres (+44.91%, P < 0.001), to decrease the collagen volume fraction of gastrocnemius (-81.32%, P = 0.001) and to reduce the β-galactosidase-positive area of C2C12 myotubes (-28.74%, P < 0.001). NaHS was also found to reverse the expression of muscle atrophy F box protein (MAFbx), muscle-specific RING finger protein 1 (MuRF1), Cyclin D1 and p21 in the ageing gastrocnemius tissue (MAFbx: -31.73%, P = 0.008; MuRF1: -32.37%, P = 0.003; Cyclin D1: +45.34%, P = 0.010; p21: -25.53%, P = 0.022) and C2C12 myotubes (MAFbx: -16.38%, P < 0.001; MuRF1: -16.45%, P = 0.003; Cyclin D1: +40.23%, P < 0.001; p21: -35.85%, P = 0.026). The AMPKα1-ULK1 pathway was activated and autophagy was up-regulated in NaHS-treated gastrocnemius tissue (p-AMPKα1: +61.61%, P = 0.018; AMPKα1: +30.64%, P = 0.010; p-ULK1/ULK1: +85.87%, P = 0.005; p62: -29.07%, P < 0.001; Beclin1: +24.75%, P = 0.007; light chain 3 II/I [LC3 II/I]: +55.78%, P = 0.004) and C2C12 myotubes (p-AMPKα1: +77.49%, P = 0.018; AMPKα1: +26.18%, P = 0.022; p-ULK1/ULK1: +38.34%, P = 0.012; p62: -9.02%, P = 0.014; Beclin1: +13.36%, P < 0.001; LC3 II/I: +79.38%, P = 0.017; autophagy flux: +24.88%, P = 0.034) compared with the D-gal group. The effects of NaHS on autophagy were comparable to those of acadesine and LYN-1604, and chloroquine could reverse its effects on ageing. LC-MS/MS and Co-IP experiments demonstrated that USP5 is a deubiquitinating enzyme of AMPKα1. Following the knockdown of USP5, the activation of AMPKα1 was decreased (p-AMPKα1: -42.10%, P < 0.001; AMPKα1: -43.93%, P < 0.001), autophagy was inhibited (p-ULK1/ULK1: -27.51, P = 0.001; p62: +36.00, P < 0.001; Beclin1: -22.15%, P < 0.001) and NaHS lost its ability to up-regulate autophagy. NaHS was observed to restore the expression (gastrocnemius: +62.17%, P < 0.001; C2C12 myotubes: +37.51%, P = 0.003) and S-sulfhydration (+53.07%, P = 0.009) of USP5 and reduce the ubiquitination of AMPKα1.
HS promotes the deubiquitination of AMPKα1 by increasing the expression and S-sulfhydration of USP5, thereby up-regulating autophagy and alleviating skeletal muscle ageing.
硫化氢（HS）是发现的第三种气体信号分子，可调节多种生理功能。HS是否通过调节自噬来减轻骨骼肌衰老尚未见报道。
给小鼠腹腔注射150 mg/kg/天的D-半乳糖（D-gal），并在20 g/L D-半乳糖中培养C2C12肌管以诱导衰老。治疗组使用硫氢化钠（NaHS）作为外源性供体。采用7-叠氮基-4-甲基香豆素荧光探针定量细胞内HS浓度。通过液相色谱串联质谱（LC-MS/MS）和免疫共沉淀（Co-IP）检测参与泛素介导的AMPKα1降解的蛋白质。采用生物素转换法检测USP5的S-硫醇化修饰。通过蛋白质免疫印迹法分析相关蛋白。
发现NaHS能有效恢复衰老腓肠肌（+91.89%，P<0.001）和C2C12肌管（+27.55%，P<0.001）中的HS含量。与D-gal组相比，NaHS可增加肌纤维的平均横截面积（+44.91%，P<0.001），降低腓肠肌的胶原体积分数（-81.32%，P=0.001），减少C2C12肌管中β-半乳糖苷酶阳性面积（-28.74%，P<0.001）。还发现NaHS可逆转衰老腓肠肌组织中肌肉萎缩F盒蛋白（MAFbx）、肌肉特异性RING指蛋白1（MuRF1）、细胞周期蛋白D1和p21的表达（MAFbx：-31.73%，P=0.008；MuRF1：-32.37%，P=0.003；细胞周期蛋白D1：+45.34%，P=0.010；p21：-25.53%，P=0.022）以及C2C12肌管中的表达（MAFbx：-16.38%，P<0.001；MuRF1：-16.45%，P=0.003；细胞周期蛋白D1：+40.23%，P<0.001；p21：-35.85%，P=0.026）。在NaHS处理的腓肠肌组织（p-AMPKα1：+61.61%，P=0.018；AMPKα1：+30.64%，P=0.010；p-ULK1/ULK1：+85.87%，P=0.005；p62：-29.07%，P<0.001；Beclin1：+24.75%，P=0.007；轻链3 II/I [LC3 II/I]：+55.78%，P=0.004）和C2C12肌管（p-AMPKα1：+77.49%，P=0.018；AMPKα1：+26.18%，P=0.022；p-ULK1/ULK1：+38.34%，P=0.012；p62：-9.02%，P=0.014；Beclin1：+13.36%，P<0.001；LC3 II/I：+79.38%，P=0.017；自噬通量：+24.88%，P=0.034）中，与D-gal组相比，AMPKα1-ULK1通路被激活，自噬上调。NaHS对自噬的影响与AICAR和LYN-1604相当，氯喹可逆转其对衰老的影响。LC-MS/MS和Co-IP实验表明，USP5是AMPKα1的去泛素化酶。敲低USP5后，AMPKα1的激活降低（p-AMPKα1：-42.10%，P<0.001；AMPKα1：-43.93%，P<0.001），自噬受到抑制（p-ULK1/ULK1：-27.51，P=0.001；p62：+36.00，P<0.001；Beclin1：-22.15%，P<0.001），且NaHS失去上调自噬的能力。观察到NaHS可恢复USP5的表达（腓肠肌：+62.17%，P<0.001；C2C12肌管：+37.51%，P=0.003）和S-硫醇化修饰（+53.07%，P=0.009），并减少AMPKα1的泛素化。
HS通过增加USP5的表达和S-硫醇化修饰促进AMPKα1的去泛素化，从而上调自噬并减轻骨骼肌衰老。
