ABSTRACT

In addition to multi-nucleated muscle fibres, numerous resident and infiltrating mononuclear cells populate the muscle compartment. As most epigenetic assays in skeletal muscle are conducted on whole tissue homogenates, essentially nothing is known about regulatory processes exclusively within muscle fibres in vivo. Utilizing a novel genetically modified mouse model developed by our laboratory, we (1) outline a simple and rapid workflow for isolating pure myonuclei from small tissue samples via fluorescent activated cell sorting and extracting high-quality large-fragment DNA for downstream analyses, and (2) provide information on myonuclear and interstitial cell nuclear CpG DNA methylation via reduced representation bisulphite sequencing (RRBS) using mice that were subjected to an acute mechanical overload of the plantaris muscle. In 3-month-old mice, myonuclei are ~50% of total nuclei in sham and ~30% in 3-d overloaded muscle, the difference being attributable to mononuclear cell infiltration and proliferation with overload. In purified myonuclei, pathway analysis of hypomethylated promoter regions following overload was distinct from interstitial nuclei and revealed marked regulation of factors that converge on the master regulator of muscle growth mTOR, and on autophagy. Specifically, acute hypomethylation of Rheb, Rictor, Hdac1, and Hdac2, in addition to a major driver of ribosome biogenesis Myc, reveals the epigenetic regulation of hypertrophic signalling within muscle fibres that may underpin the long-term growth response to loading. This study provides foundational information on global myonuclear epigenetics in vivo using RRBS, and demonstrates the importance of isolating specific nuclear populations to study the epigenetic regulation of skeletal muscle fibre adaptation.

 抽象的

除了多核肌纤维外，肌肉室中还存在大量驻留和浸润单核细胞。由于骨骼肌中的大多数表观遗传学测定都是在整个组织匀浆上进行的，因此基本上对体内肌肉纤维内的调节过程一无所知。利用我们实验室开发的新型转基因小鼠模型，我们 (1) 概述了一个简单快速的工作流程，通过荧光激活细胞分选从小组织样本中分离纯肌核，并提取高质量的大片段 DNA 用于下游分析，以及（ 2) 使用遭受跖肌急性机械超负荷的小鼠，通过还原代表性亚硫酸氢盐测序 (RRBS) 提供有关肌核和间质细胞核 CpG DNA 甲基化的信息。在 3 个月大的小鼠中，假手术中肌核占总细胞核的约 50%，而 3 d 超负荷肌肉中肌核占总核的约 30%，差异归因于单核细胞浸润和超负荷增殖。在纯化的肌核中，过载后低甲基化启动子区域的通路分析与间质核不同，并揭示了对肌肉生长主调节器 mTOR 和自噬的集中因子的显着调节。具体来说， Rheb、Rictor、Hdac1和Hdac2的急性低甲基化，除了核糖体生物发生Myc的主要驱动因素之外，揭示了肌纤维内肥大信号的表观遗传调控，可能支撑对负荷的长期生长反应。 这项研究使用 RRBS 提供了体内整体肌核表观遗传学的基础信息，并证明了分离特定核群体对于研究骨骼肌纤维适应的表观遗传调控的重要性。