Wei Chengjie, Wang Dandan, Ma Yitian, Wang Shengnan, Pan Dengke, Ma Yuehui, Jiang Lin
Laboratory of Animal (Poultry) Genetics Breeding and Reproduction, Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.
National Germplasm Center of Domestic Animal Resources, Ministry of Science and Technology of the People's Republic of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.
J Cachexia Sarcopenia Muscle. 2025 Jun;16(3):e13829. doi: 10.1002/jcsm.13829.
Effective treatments for skeletal muscle atrophy, a debilitating condition linked to ageing and glucocorticoid therapy, remain lacking. Zinc finger BED-type containing 6 (ZBED6), a transcriptional repressor, enhances muscle growth and protects against sepsis-induced atrophy, but its role in ageing- and dexamethasone (Dex)-induced muscle atrophy remains unknown. This study investigated the protective role of ZBED6 knockout (KO) against muscle atrophy through the Dkk3-Fbxo32 pathway.
The muscle mass, ratio and myofibrillar morphology of 5-day-old (wild-type (WT): KO, n = 5:3), 5-month-old (n = 8:9) and 8-month-old (n = 3:3) ZBED6-KO pigs and 18-month-old mice (n = 3:3) were analysed. A model of Dex-induced muscle atrophy was established using 3-month-old mice (n = 6:6) via intraperitoneal injections (15 mg/kg/day for 10 days). C2C12 myotubes were treated with 100 μM Dex for 24 h. Muscle morphology was analysed through H&E and immunofluorescence staining. Gene expression was assessed through RNA-seq, qRT-PCR and western blotting. The downstream targets were identified through ChIP-seq using anti-ZBED6 antibodies and RNA-seq analysis of the gastrocnemius muscle from ZBED6-KO and WT pigs. Dkk3 was overexpressed by injecting AAV9-myo2A-Dkk3 (2 × 10) into the tibialis anterior muscle of 3-month-old ZBED6-KO mice (n = 4), which were harvested 1 month postinjection. ZBED6-KO C2C12 cells were generated via CRISPR/Cas9 and treated with Dex to assess the effects on myotube diameter and gene expression.
The muscle mass and muscle-to-carcass ratio in ZBED6-KO pigs increased by 27% and 12%, respectively (p < 0.05), while the Dkk3-Fbxo32 pathway was suppressed by 50% (p < 0.01). ChIP-seq/RNA-seq identified Dkk3 as the most significant ZBED6 target (log2FC = -3.38, p < 0.01). The myofibrillar cross-sectional areas (CSAs) increased twofold in aged ZBED6-KO mice, while fibrosis and the Dkk3-Fbxo32 pathway were suppressed by 76% and 50%, respectively (all p < 0.01). Dkk3 overexpression reduced the tibialis anterior muscle weight and CSA in ZBED6-KO mice by 31% and 61%, respectively (p < 0.01). Dex reduced the CSA in WT mice (45%, p < 0.01), but ZBED6-KO mice resisted atrophy (CSA similar to untreated WT). ZBED6-KO increased myotube diameter by twofold (p < 0.01) and inhibited the activation of the Dkk3-Fbxo32 pathway (p < 0.01). Conversely, Zbed6 overexpression reduced the CSA and myotube diameter by 32% and 64%, respectively (p < 0.01) and rescued by Dkk3 silencing (50% recovery, p < 0.01).
ZBED6 depletion mitigates ageing- and Dex-induced muscle atrophy via the Dkk3-Fbxo32 axis, highlighting its therapeutic potential.
骨骼肌萎缩是一种与衰老和糖皮质激素治疗相关的衰弱性疾病，目前仍缺乏有效的治疗方法。含锌指BED结构域6（ZBED6）是一种转录抑制因子，可促进肌肉生长并预防脓毒症诱导的萎缩，但其在衰老和地塞米松（Dex）诱导的肌肉萎缩中的作用尚不清楚。本研究通过Dkk3-Fbxo32途径研究了ZBED6基因敲除（KO）对肌肉萎缩的保护作用。
分析了5日龄（野生型（WT）：KO，n = 5:3）、5月龄（n = 8:9）和8月龄（n = 3:3）ZBED6-KO猪以及18月龄小鼠（n = 3:3）的肌肉质量、比例和肌原纤维形态。通过对3月龄小鼠（n = 6:6）腹腔注射（15 mg/kg/天，共10天）建立Dex诱导的肌肉萎缩模型。用100 μM Dex处理C2C12肌管24小时。通过苏木精-伊红（H&E）染色和免疫荧光染色分析肌肉形态。通过RNA测序、定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法评估基因表达。使用抗ZBED6抗体通过染色质免疫沉淀测序（ChIP-seq）和对ZBED6-KO猪和WT猪腓肠肌的RNA测序分析来鉴定下游靶点。通过向3月龄ZBED6-KO小鼠（n = 4）的胫前肌注射腺相关病毒9-肌动蛋白2A-Dkk3（2×10）来过表达Dkk3，注射后1个月收获小鼠。通过CRISPR/Cas9技术构建ZBED6-KO C2C12细胞，并用地塞米松处理以评估对肌管直径和基因表达的影响。
ZBED6-KO猪的肌肉质量和肌肉与胴体比例分别增加了27%和12%（p < 0.05），而Dkk3-Fbxo32途径被抑制了50%（p < 0.01）。ChIP-seq/RNA-seq鉴定出Dkk3是最显著的ZBED6靶点（log2FC = -3.38，p < 0.01）。老年ZBED6-KO小鼠的肌原纤维横截面积（CSA）增加了两倍，而纤维化和Dkk3-Fbxo32途径分别被抑制了76%和50%（均p < 0.01）。过表达Dkk3使ZBED6-KO小鼠胫前肌重量和CSA分别降低了31%和61%（p < 0.01）。Dex使WT小鼠的CSA降低（45%，p < 0.01），但ZBED6-KO小鼠抵抗了萎缩（CSA与未处理的WT小鼠相似）。ZBED6-KO使肌管直径增加了两倍（p < 0.01），并抑制了Dkk3-Fbxo,32途径的激活（p < 0.01）。相反，过表达Zbed6使CSA和肌管直径分别降低了32%和64%（p < 0.01），而Dkk3沉默可使其恢复（恢复50%，p < 0.01）。
ZBED6缺失通过Dkk3-Fbxo32轴减轻衰老和Dex诱导的肌肉萎缩，凸显了其治疗潜力。
