State Key Laboratory of Pharmaceutical Biotechnology, Medical School, Nanjing University & Division of Sports Medicine and Adult Reconstructive Surgery, Department of Orthopedic Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China.
Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing, Jiangsu, China.
J Cachexia Sarcopenia Muscle. 2022 Dec;13(6):3163-3180. doi: 10.1002/jcsm.13106. Epub 2022 Oct 13.
Sarcopenia is a common and progressive skeletal muscle disorder characterized by atrophic muscle fibres and contractile dysfunction. Accumulating evidence shows that the number and function of satellite cells (SCs) decline and become impaired during ageing, which may contribute to impaired regenerative capacity. A series of myokines/small extracellular vesicles (sEVs) released from muscle fibres regulate metabolism in muscle and extramuscular tissues in an autocrine/paracrine/endocrine manner during muscle atrophy. It is still unclear whether myokines/sEVs derived from muscle fibres can affect satellite cell function during ageing.
Aged mice were used to investigate changes in the myogenic capacity of SCs during ageing-induced muscle atrophy. The effects of atrophic myotube-derived sEVs on satellite cell differentiation were investigated by biochemical methods and immunofluorescence staining. Small RNA sequencing was performed to identify differentially expressed sEV microRNAs (miRNAs) between the control myotubes and atrophic myotubes. The target genes of the miRNA were predicted by bioinformatics analysis and verified by luciferase activity assays. The effects of identified miRNA on the myogenic capacity of SCs in vivo were investigated by intramuscular injection of adeno-associated virus (AAV) to overexpress or silence miRNA in skeletal muscle.
Our study showed that the myogenic capacity of SCs was significantly decreased (50%, n = 6, P < 0.001) in the tibialis anterior muscle of aged mice. We showed that atrophic myotube-derived sEVs inhibited satellite cell differentiation in vitro (n = 3, P < 0.001) and in vivo (35%, n = 6, P < 0.05). We also found that miR-690 was the most highly enriched miRNA among all the screened sEV miRNAs in atrophic myotubes [Log (Fold Change) = 7, P < 0.001], which was verified in the atrophic muscle of aged mice (threefold, n = 6, P < 0.001) and aged men with mean age of 71 ± 5.27 years (2.8-fold, n = 10, P < 0.001). MiR-690 can inhibit myogenic capacity of SCs by targeting myocyte enhancer factor 2, including Mef2a, Mef2c and Mef2d, in vitro (n = 3, P < 0.05) and in vivo (n = 6, P < 0.05). Specific silencing of miR-690 in the muscle can promote satellite cell differentiation (n = 6, P < 0.001) and alleviate muscle atrophy in aged mice (n = 6, P < 0.001).
Our study demonstrated that atrophic muscle fibre-derived sEV miR-690 may inhibit satellite cell differentiation by targeting myocyte enhancer factor 2 during ageing.
肌少症是一种常见的进行性骨骼肌疾病，其特征为纤维萎缩和收缩功能障碍。越来越多的证据表明，卫星细胞（SCs）的数量和功能在衰老过程中下降并受损，这可能导致再生能力受损。肌肉纤维释放的一系列肌因子/小细胞外囊泡（sEVs）以自分泌/旁分泌/内分泌的方式调节肌肉和肌肉外组织的代谢，在肌肉萎缩过程中。目前尚不清楚肌肉纤维来源的肌因子/sEV 是否会影响衰老过程中卫星细胞的功能。
使用老年小鼠研究衰老诱导的肌肉萎缩过程中 SC 成肌能力的变化。通过生化方法和免疫荧光染色研究萎缩肌管衍生的 sEV 对卫星细胞分化的影响。通过小 RNA 测序鉴定对照肌管和萎缩肌管之间差异表达的 sEV microRNAs（miRNAs）。通过生物信息学分析预测 miRNA 的靶基因，并通过荧光素酶活性测定进行验证。通过肌内注射腺相关病毒（AAV）过表达或沉默 miRNA 来研究鉴定的 miRNA 对体内 SC 成肌能力的影响。
我们的研究表明，老年小鼠的比目鱼肌中 SC 的成肌能力显著下降（50%，n=6，P<0.001）。我们表明，萎缩肌管衍生的 sEV 抑制体外（n=3，P<0.001）和体内（35%，n=6，P<0.05）卫星细胞分化。我们还发现，miR-690 是所有筛选的 sEV miRNAs 中在萎缩肌管中含量最丰富的 miRNA[Log（倍数变化）=7，P<0.001]，在衰老小鼠的萎缩肌肉中得到验证（三倍，n=6，P<0.001）和平均年龄为 71±5.27 岁的老年男性（2.8 倍，n=10，P<0.001）。MiR-690 可以通过靶向肌细胞增强因子 2（包括 Mef2a、Mef2c 和 Mef2d）来抑制 SC 的成肌能力，包括体外（n=3，P<0.05）和体内（n=6，P<0.05）。肌肉中 miR-690 的特异性沉默可以促进卫星细胞分化（n=6，P<0.001）并缓解老年小鼠的肌肉萎缩（n=6，P<0.001）。
我们的研究表明，衰老过程中，萎缩肌纤维来源的 sEV miR-690 可能通过靶向肌细胞增强因子 2 抑制卫星细胞分化。
