Differential DNA Methylation and Transcriptional Signatures Characterize Impairment of Progenitor Cells in Pediatric Human Muscle Contractures Following Brain Injury [RNA-seq]. Differential DNA Methylation and Transcriptional Signatures Characterize Impairment of Progenitor Cells in Pediatric Human Muscle Contractures Following Brain Injury [RNA-seq]

Limb contractures are a debilitating and progressive consequence of a wide range of pediatric conditions that affect skeletal muscles, including perinatal brain injury causing cerebral palsy (CP). While several rehabilitation therapies are currently used in the clinical setting, their long-term effectiveness in treating contractures is marginal since they do not change underlying muscle biological properties. Therefore, new therapies based on a biological understanding of contracture development are needed. Here we show that myoblast progenitor cells from contractured muscle in children with CP had higher rates of proliferation than control cells from typically developing children. This phenotype was associated with upregulation of DNMT3a and patterns of DNA hypermethylation and gene expression that favored cell expansion over quiescence. Treatment of CP progenitors with 5-azacytidine, a DNMT inhibitor and hypomethylating agent, normalized this epigenetic imprint and promoted exit from mitosis. Together with previous studies demonstrating reduction in myoblast differentiation capacity, these data suggest that mechanisms of early myofiber growth and establishment of an adult population of quiescent stem cells could be compromised in CP. Hypomethylating agents like 5-azacytidine could be used to rescue myogenesis and promote muscle growth in contractured muscle and thus may represent a new approach to treating this devastating condition Overall design: We performed RNA sequencing analysis of dividing satellite cell-derived myoblast precursors from contractured cerebral palsy (CP) muscles and typically developing (TD) control muscles, treated and non-treated with hypomethylating agent 5-azacytidine (AZA)

肢体挛缩（Limb contractures）是一类衰弱且呈进行性发展的后遗症，可由多种累及骨骼肌的儿科病症引发，其中包括导致脑瘫（Cerebral Palsy, CP）的围产期脑损伤。目前临床已应用多种康复治疗手段，但此类疗法对挛缩的长期疗效有限，因其无法改变肌肉的潜在生物学特性，故而亟需基于挛缩发病生物学机制的新型治疗方案。

本研究证实，脑瘫患儿挛缩肌肉来源的成肌祖细胞（myoblast progenitor cells）的增殖速率显著高于典型发育儿童的对照细胞。该表型与DNA甲基转移酶3a（DNMT3a）的上调，以及偏向细胞扩增而非静息状态的DNA高甲基化模式和基因表达谱密切相关。采用5-氮胞苷（5-azacytidine）——一种DNA甲基转移酶抑制剂兼低甲基化试剂——处理脑瘫来源的成肌祖细胞，可使其表观遗传印记恢复正常，并促进细胞退出有丝分裂。结合此前关于成肌细胞分化能力受损的相关研究，上述数据表明，脑瘫患者的肌纤维早期生长以及静息干细胞成年群体的建立过程可能存在缺陷。

诸如5-氮胞苷这类低甲基化试剂，可用于修复挛缩肌肉的肌发生过程并促进肌肉生长，因此有望成为治疗这一毁灭性病症的全新策略。

实验设计概述：我们对挛缩型脑瘫（CP）肌肉及典型发育（TD）对照肌肉来源的增殖性卫星细胞衍生成肌前体细胞开展了RNA测序分析，所有样本均经或未经低甲基化试剂5-氮胞苷（AZA）处理。