Zhang Yujie, Hao Jingran, Feng Yueyao, Qiu Tongtong, Wu Jinjin, Zhou Xuenan, Fan Heng, Chang Yongsheng
Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Key Laboratory of Cellular Homeostasis and Disease, Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, China.
Department of Reproductive Medicine, General Hospital of Ningxia Medical University (The First Clinical Medical College of Ningxia Medical University), Yinchuan, China.
J Cachexia Sarcopenia Muscle. 2025 Aug;16(4):e70020. doi: 10.1002/jcsm.70020.
Glucocorticoids (GCs) are the most important and frequently used class of anti-inflammatory drugs. However, the mechanisms underlying excessive glucocorticoid-mediated induction of muscle atrophy remain incompletely understood.
We generated skeletal muscle-specific Klf9 transgenic mice (mKlf9TG) and skeletal muscle-specific Klf9 knockout mice (Klf9). The body weight, tissue weight, body composition, grip strength, running distance and muscle fibre cross section of mKlf9TG, Klf9 mice and their littermate controls were examined. Expression of genes related to muscle protein synthesis and degradation pathways were also tested in the mKlf9TG mice, Klf9 mice and their littermate controls. We performed Klf9 gain- or loss-of-function studies in differentiated C2C12 myotubes using lentiviruses encoding Klf9 or the shRNA specific to Klf9 in vitro. Luciferase reporter gene assay and ChIP assay were performed to explore the molecular mechanism of Klf9 action. Klf9 and Klf9 mice were treated with dexamethasone (Dex). Multiple genetic and pharmacological approaches were also used to investigate the intracellular signalling cascades underlying the Dex/Klf9-ediated skeletal muscle wasting.
Skeletal muscle Klf9 gene expression was significantly upregulated by Dex (p < 0.05 or p < 0.01 vs. vehicle group). Compared with littermate control mice (R-loxP), mKlf9TG mice exhibited decreased skeletal muscle mass (TA 0.101 ± 0.018 vs. 0.040 ± 0.007 g, p < 0.001) and impaired grip strength (forelimb 157.4 ± 3.7 vs. 93.45 ± 9.8 and four limbs 255.3 ± 23.1 vs. 170.1 ± 36.2, p < 0.001). Conversely, compared with Klf9, Klf9 mice exhibited increased skeletal muscle mass (TA 0.103 ± 0.012 vs. 0.123 ± 0.005 g, p < 0.001) and enhanced grip strength (forelimb 110.3 ± 5.8 vs. 156.8 ± 10.0 and four limbs 155.5 ± 6.3 vs. 226.5 ± 19.7, p < 0.001). Skeletal muscle Klf9 deficiency alleviated muscle atrophy induced by acute high-dose Dex treatment (p < 0.001). Mechanistically, Klf9 induces the expression of myostatin (Mstn) and muscle atrophy F-box (MAFbx) by directly binding to and activating the transcription of their promoters. Treatment of AAV-MSTN reduced the increased grip strength of Klf9 mice (forelimb 143.5 ± 22.3 vs. 118.8 ± 3.1 and four limbs 249.8 ± 24.7 vs. 208.7 ± 9.0, p < 0.001).
In summary, our study provides novel insights into the mechanisms underlying GC-induced muscular atrophy and reveals that skeletal muscle induction of Klf9 expression is a mechanism underlying GC therapy-induced muscle loss. Thus, targeting Klf9 may offer novel approaches to the treatment of skeletal muscle wasting diseases.
糖皮质激素（GCs）是最重要且最常用的一类抗炎药物。然而，糖皮质激素介导的肌肉萎缩过度诱导的潜在机制仍未完全明确。
我们构建了骨骼肌特异性Klf9转基因小鼠（mKlf9TG）和骨骼肌特异性Klf9基因敲除小鼠（Klf9）。检测了mKlf9TG小鼠、Klf9小鼠及其同窝对照小鼠的体重、组织重量、身体组成、握力、跑步距离和肌纤维横截面积。还在mKlf9TG小鼠、Klf9小鼠及其同窝对照小鼠中检测了与肌肉蛋白质合成和降解途径相关的基因表达。我们在体外使用编码Klf9的慢病毒或Klf9特异性短发夹RNA（shRNA）在分化的C2C12肌管中进行了Klf9功能获得或缺失研究。进行了荧光素酶报告基因测定和染色质免疫沉淀（ChIP）测定以探索Klf9作用的分子机制。用 dexamethasone（Dex）处理Klf9和Klf9小鼠。还使用了多种遗传和药理学方法来研究Dex/Klf9介导的骨骼肌消瘦的细胞内信号级联反应。
Dex显著上调骨骼肌Klf9基因表达（与溶剂组相比，p < 0.05或p < 0.01）。与同窝对照小鼠（R-loxP）相比，mKlf9TG小鼠的骨骼肌质量降低（胫前肌0.101 ± 0.018 vs. 0.040 ± 0.007 g，p < 0.001）且握力受损（前肢157.4 ± 3.7 vs. 93.45 ± 9.8，四肢255.3 ± 23.1 vs. 170.1 ± 36.2，p < 0.001）。相反，与Klf9相比，Klf9小鼠的骨骼肌质量增加（胫前肌0.103 ± 0.012 vs. 0.123 ± 0.005 g，p < 0.001）且握力增强（前肢110.3 ± 5.8 vs. 156.8 ± 10.0，四肢155.5 ± 6.3 vs. 226.5 ± 19.7，p < 0.001）。骨骼肌Klf9缺乏减轻了急性高剂量Dex治疗诱导的肌肉萎缩（p < 0.001）。机制上，Klf9通过直接结合并激活其启动子转录来诱导肌肉生长抑制素（Mstn）和肌肉萎缩F盒（MAFbx）的表达。腺相关病毒-MSTN（AAV-MSTN）治疗降低了Klf9小鼠增加的握力（前肢143.5 ± 22.3 vs. 118.8 ± 3.1，四肢249.8 ± 24.7 vs. 208.7 ± 9.0，p < 0.001）。
总之，我们的研究为GC诱导的肌肉萎缩的潜在机制提供了新见解，并揭示骨骼肌中Klf9表达的诱导是GC治疗诱导肌肉丢失的一种机制。因此，靶向Klf9可能为治疗骨骼肌消瘦疾病提供新方法。
