Partial resistance to HDAC inhibitors in FAPs of dystrophic muscles at late stages of disease is associated to epigenetic and transcriptional features of cellular senescence [RNA-seq]. Partial resistance to HDAC inhibitors in FAPs of dystrophic muscles at late stages of disease is associated to epigenetic and transcriptional features of cellular senescence [RNA-seq]

Pharmacological treatment of Duchenne Muscular Dystrophy (DMD) with histone deacetylase inhibitors (HDACi) is currently being tested in clinical trials. Pre-clinical studies performed in mdx mice - the mouse model of DMD - have shown that HDACi promote compensatory muscle regeneration, while inhibiting fibro-adipogenic degeneration, by targeting fibro-adipogenic progenitors (FAPs); however, these beneficial effects are restricted to early stages of disease progression. We show here that FAPs from late stage mdx mice exhibit epigenetic and transcriptional features of senescence that could not be fully reversed by HDACi. In particular, genome-wide increase in H3K9/14 acetylation at gene promoters of Senescence Associated Secretory Phenotype (SASP) genes was associated with their upregulation in late stage mdx FAPs. Treatment with the HDACi Trichostatin A (TSA) could inhibit SASP gene activation in FAPs, by decreasing H3K9/14 acetylation. Conversely, combinatorial decrease of H3K27 and/or H3K9/14 acetylation at promoters of genes required for cycle activation and progression was associated with their downregulation in FAPs from late stage mdx mice. However, these epigenetic and transcriptional alterations could not be reversed by TSA, due to a general resistance exhibited by FAPs from late stage mdx mice to HDACi-induced H3K9/14 hyperacetylation. Overall, this data reveal that disease-associated features of senescence develop in FAPs of DMD muscle through epigenetically distinct and pharmacologically dissociable events, and suggests that HDACi might at least retain anti- fibrotic and inflammatory activity at late stages of DMD, by repressing FAP-derived SASP. Overall design: Integrative analysis of 2 different histone modifications (H3K9/14ac and H3K27ac ChIP-seq) and expression profiling (RNAseq) in FAPs derived from mdx mice at early and late stages of disease progression in response to TSA treatment. Comparative analysis of FAP transcriptome to other muscle-derived cells (MUSCs and Macrophages) to evaluate cell-specific features.

当前针对杜氏肌营养不良症（Duchenne Muscular Dystrophy, DMD）的组蛋白去乙酰化酶抑制剂（HDACi）疗法，目前正处于临床试验阶段。在DMD的经典小鼠模型mdx小鼠中开展的临床前研究显示，HDACi可通过靶向脂肪生成祖细胞（FAPs），促进代偿性肌肉再生并抑制脂肪纤维化变性，但这类有益效应仅局限于疾病进程的早期阶段。
本研究证实，晚期mdx小鼠的FAPs呈现出细胞衰老的表观遗传与转录特征，且此类特征无法被HDACi完全逆转。具体而言，衰老相关分泌表型（SASP）基因的启动子区域H3K9/14乙酰化水平的全基因组升高，与晚期mdx小鼠FAPs中该类基因的上调表达显著相关。经HDACi曲古抑菌素A（TSA）处理后，可通过降低H3K9/14乙酰化水平，抑制FAPs中的SASP基因激活。
与之相反，细胞周期激活与进程所需基因的启动子区域H3K27及/或H3K9/14乙酰化水平的协同降低，与晚期mdx小鼠FAPs中该类基因的下调表达密切相关。但由于晚期mdx小鼠的FAPs普遍对HDACi诱导的H3K9/14高乙酰化产生抗性，TSA无法逆转这类表观遗传与转录改变。
综上，本研究数据表明，DMD肌肉FAPs中出现的疾病相关衰老特征，是通过表观遗传层面独立且药理学上可分离的事件介导的；同时研究提示，HDACi或可通过抑制FAPs分泌的SASP，在DMD疾病晚期仍保留抗纤维化与抗炎活性。
整体实验设计：对不同疾病进程阶段（早期与晚期）的mdx小鼠来源的FAPs，经TSA处理后，开展两种组蛋白修饰（H3K9/14ac与H3K27ac染色质免疫共沉淀测序, ChIP-seq）及表达谱分析（RNA测序, RNA-seq）的整合分析。同时将FAPs的转录组与其他肌肉来源细胞（肌干细胞, MUSCs与巨噬细胞, Macrophages）进行比较分析，以评估细胞的特异性特征。