Ganassi Massimo, Zammit Peter S, Hughes Simon M
Randall Centre for Cell and Molecular Biophysics, King's College London, London, UK.
Methods Mol Biol. 2023;2640:21-43. doi: 10.1007/978-1-0716-3036-5_3.
Adult skeletal musculature experiences continuous physical stress, and hence requires maintenance and repair to ensure its continued efficient functioning. The population of resident muscle stem cells (MuSCs), termed satellite cells, resides beneath the basal lamina of adult myofibers, contributing to both muscle hypertrophy and regeneration. Upon exposure to activating stimuli, MuSCs proliferate to generate new myoblasts that differentiate and fuse to regenerate or grow myofibers. Moreover, many teleost fish undergo continuous growth throughout life, requiring continual nuclear recruitment from MuSCs to initiate and grow new fibers, a process that contrasts with the determinate growth observed in most amniotes. In this chapter, we describe a method for the isolation, culture, and immunolabeling of adult zebrafish myofibers that permits examination of both myofiber characteristics ex vivo and the MuSC myogenic program in vitro. Morphometric analysis of isolated myofibers is suitable to assess differences among slow and fast muscles or to investigate cellular features such as sarcomeres and neuromuscular junctions. Immunostaining for Pax7, a canonical stemness marker, identifies MuSCs on isolated myofibers for study. Furthermore, the plating of viable myofibers allows MuSC activation and expansion and downstream analysis of their proliferative and differentiative dynamics, thus providing a suitable, parallel alternative to amniote models for the study of vertebrate myogenesis.
成年骨骼肌组织不断受到物理应激，因此需要维持和修复以确保其持续高效运作。驻留肌肉干细胞（MuSCs）群体，即卫星细胞，位于成年肌纤维的基膜下方，对肌肉肥大和再生均有贡献。在受到激活刺激时，MuSCs增殖以产生新的成肌细胞，这些成肌细胞分化并融合以再生或生长肌纤维。此外，许多硬骨鱼终生持续生长，需要从MuSCs持续招募细胞核以启动和生长新的纤维，这一过程与大多数羊膜动物的确定性生长形成对比。在本章中，我们描述了一种分离、培养和免疫标记成年斑马鱼肌纤维的方法，该方法允许在体外检查肌纤维特征以及MuSC的生肌程序。对分离的肌纤维进行形态测量分析适用于评估慢肌和快肌之间的差异，或研究诸如肌节和神经肌肉接头等细胞特征。对典型干性标记物Pax7进行免疫染色可识别分离肌纤维上的MuSCs以供研究。此外， viable myofibers的接种可使MuSCs激活和扩增，并对其增殖和分化动态进行下游分析，从而为研究脊椎动物肌生成提供了一种合适的、与羊膜动物模型并行的替代方法。 （注：原文中“viable myofibers”表述有误，可能是“isolated myofibers”，已按正确理解翻译）
