Sothers Hanna, Hu Xianzhen, Crossman David K, Si Ying, Alexander Matthew S, McDonald Merry-Lynn N, King Peter H, Lopez Michael A
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham (UAB), Birmingham, Alabama, USA.
Genetics, Genomics and Bioinformatics, Graduate School of Biomedical Sciences, University of Alabama at Birmingham (UAB), Birmingham, Alabama, USA.
J Cachexia Sarcopenia Muscle. 2025 Aug;16(4):e70005. doi: 10.1002/jcsm.70005.
Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease due to loss-of-function variants in the DYSTROPHIN gene. DMD-related skeletal muscle wasting is typified by an aberrant immune response involving upregulation of the TGFβ family of cytokines, like TGFβ1 and BMP4. We previously demonstrated that bone morphogenetic protein 4 (BMP4) is increased in DMD and BMP4 stimulation induces a 20-fold upregulation of Smad8 transcription in muscle cells. However, the role of BMP4 in late-stage DMD skeletal muscle is unknown. We hypothesized that BMP4 signalling is a driver of aberrant gene expression in late-stage human DMD skeletal muscle detectable by a transcriptomic signature.
Transcriptomes from skeletal muscle biopsies of late-stage DMD versus non-DMD controls and C2C12 muscle cells with or without BMP4 stimulation were generated using RNA-Seq. We tested transcriptional differences at the single transcript level in skeletal muscle biopsy samples from three patients with DMD and compared them to three non-DMD. They were then analyzed by Ingenuity Pathway Analysis, weighted gene coexpression network analyses (WGCNA) and Gene Set Enrichment Analysis (GSEA). Key hub and high-fold change genes overlapping in the DMD and BMP4 muscle transcriptomes were validated in additional primary and bulk skeletal muscle samples.
A total of 3048 transcripts in the human muscle and 5291 transcripts in C2C12 muscle cells were differentially expressed. WGCNA identified an overlapping molecular signature of 1027 genes dysregulated in DMD muscle that were induced in BMP4-stimulated C2C12 muscle cells. SERPING1 and Aff3 were identified as the top hub genes. Highly upregulated DMD muscle transcripts that overlapped with BMP4-stimulated C2C12 muscle cells included ADAM12, SERPING1, SMAD8 and SFRP4. DMD skeletal muscle analysis showed aberrant upregulation of TGFβ signalling, extracellular matrix remodelling and collagen biosynthesis pathways, in contrast to inhibited mitochondrial and metabolic pathways.
In summary, the DMD transcriptome was characterized by dysregulation of immune function, ECM remodelling and muscle bioenergetic metabolism. We additionally define a late-stage DMD skeletal muscle transcriptome that overlaps with a BMP4-induced molecular signature in C2C12 muscle cells. This supports BMP4/Smad8 pathway as a disease-driving regulator of transcriptomic changes in late-stage DMD skeletal muscle. Further exploration of this cross-species transcriptomic signature may expand our understanding of the evolution of dystrophic signalling pathways and the associated gene networks, which could be evaluated for therapeutic development.
杜氏肌营养不良症（DMD）是一种致命的X连锁隐性疾病，由肌营养不良蛋白基因的功能丧失变异引起。与DMD相关的骨骼肌萎缩的典型特征是异常的免疫反应，涉及细胞因子TGFβ家族（如TGFβ1和BMP4）的上调。我们之前证明，骨形态发生蛋白4（BMP4）在DMD中增加，并且BMP4刺激可诱导肌肉细胞中Smad8转录上调20倍。然而，BMP4在晚期DMD骨骼肌中的作用尚不清楚。我们假设BMP4信号传导是晚期人类DMD骨骼肌中异常基因表达的驱动因素，可通过转录组特征检测到。
使用RNA测序生成晚期DMD与非DMD对照以及有或无BMP4刺激的C2C12肌肉细胞的骨骼肌活检转录组。我们在三名DMD患者的骨骼肌活检样本中测试了单个转录本水平的转录差异，并将其与三名非DMD患者进行比较。然后通过 Ingenuity Pathway Analysis、加权基因共表达网络分析（WGCNA）和基因集富集分析（GSEA）对它们进行分析。在DMD和BMP4肌肉转录组中重叠的关键枢纽基因和高倍数变化基因在额外的原代和整块骨骼肌样本中得到验证。
人类肌肉中共有3048个转录本和C2C12肌肉细胞中有5291个转录本差异表达。WGCNA确定了在DMD肌肉中失调的1027个基因的重叠分子特征，这些基因在BMP4刺激的C2C12肌肉细胞中被诱导。SERPING1和Aff3被确定为顶级枢纽基因。与BMP4刺激的C2C12肌肉细胞重叠的高度上调的DMD肌肉转录本包括ADAM12、SERPING1、SMAD8和SFRP4。DMD骨骼肌分析显示TGFβ信号传导、细胞外基质重塑和胶原蛋白生物合成途径异常上调，而线粒体和代谢途径则受到抑制。
总之，DMD转录组的特征是免疫功能失调、细胞外基质重塑和肌肉生物能量代谢异常。我们还定义了晚期DMD骨骼肌转录组，它与C2C12肌肉细胞中BMP4诱导的分子特征重叠。这支持BMP4/Smad8途径作为晚期DMD骨骼肌转录组变化的疾病驱动调节因子。对这种跨物种转录组特征的进一步探索可能会扩展我们对营养不良信号通路和相关基因网络进化的理解，这可用于评估治疗开发。
