A novel in vitro model for the assessment of postnatal myonuclear accretion.
A novel in vitro model for the assessment of postnatal myonuclear accretion.
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DOI：
10.1186/s13395-018-0151-4
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发表时间：
2018-02-14
期刊：
影响因子：
4.9
通讯作者：
Langen R
中科院分区：
文献类型：
--
作者：
Kneppers A;Verdijk L;de Theije C;Corten M;Gielen E;van Loon L;Schols A;Langen R
由于肌细胞核的有丝分裂后性质，出生后肌发生对于骨骼肌的生长、修复和再生至关重要。这一过程由卫星细胞通过增殖、分化和随后与预先存在的肌纤维融合（即，肌层增生）。目前对肌发生的认识主要基于成肌细胞体外形成合胞体，其代表发育性肌发生的方面，但可能不完全描绘出生后肌发生。因此，我们的目的是建立一个体外模型，更好地反映出生后的肌肉发生，研究细胞的内在和外在的过程和信号参与的调控出生后的肌肉发生。将增殖的C2C12成肌细胞用胰蛋白酶消化并与分化5天的C2C12肌管共培养3天。通过活细胞延时成像和通过用Vybrant® DiD和DiO进行细胞标记的细胞追踪来视觉评估出生后肌纤维增生。此外，开发了基于Cre/LoxP的细胞系统，通过成肌细胞-肌管融合后荧光素酶的条件性表达来半定量地评估体外出生后肌纤维增生。荧光素酶活性通过光度法评估并针对总蛋白质含量进行校正。活细胞延时成像、基于染色的细胞示踪和重组依赖性荧光素酶活性显示了出生后体外肌层增生的发生。用生肌因子IGF-I（p <0.001）和细胞因子IL-13（p <0.05）和IL-4（p <0.001）处理的共培养物增加了出生后的肌生长，而生肌抑制剂细胞松弛素D（p <0.001）、肌生长抑制素（p <0.05）和TNF α（p <0.001）减少了出生后的肌生长。此外，在从电脉冲刺激诱导的纤维损伤（p <0.001）和LY29004诱导的萎缩（p <0.001）恢复后，出生后肌纤维增生增加。此外，细胞类型特异性siRNA介导的成肌细胞（p <0.001）而非肌管中肌生成因子的敲低，降低了出生后肌生成。我们开发了一种生理相关的，敏感的，高通量的细胞系统，用于半定量评估体外出生后肌纤维增生，它可以用来模拟生理肌生成触发器，并可以区分细胞类型的特定作用的信号和反应的调节出生后肌生成。因此，这种方法适用于对出生后肌发生调节的基础和转化研究，并将提高我们对卫星细胞数量或功能受损导致的肌肉病理的理解。本文的在线版本（10.1186/s13395 - 018 - 0151 - 4）包含补充材料，可供授权用户使用。
Due to the post-mitotic nature of myonuclei, postnatal myogenesis is essential for skeletal muscle growth, repair, and regeneration. This process is facilitated by satellite cells through proliferation, differentiation, and subsequent fusion with a pre-existing muscle fiber (i.e., myonuclear accretion). Current knowledge of myogenesis is primarily based on the in vitro formation of syncytia from myoblasts, which represents aspects of developmental myogenesis, but may incompletely portray postnatal myogenesis. Therefore, we aimed to develop an in vitro model that better reflects postnatal myogenesis, to study the cell intrinsic and extrinsic processes and signaling involved in the regulation of postnatal myogenesis. Proliferating C2C12 myoblasts were trypsinized and co-cultured for 3 days with 5 days differentiated C2C12 myotubes. Postnatal myonuclear accretion was visually assessed by live cell time-lapse imaging and cell tracing by cell labeling with Vybrant® DiD and DiO. Furthermore, a Cre/LoxP-based cell system was developed to semi-quantitatively assess in vitro postnatal myonuclear accretion by the conditional expression of luciferase upon myoblast–myotube fusion. Luciferase activity was assessed luminometrically and corrected for total protein content. Live cell time-lapse imaging, staining-based cell tracing, and recombination-dependent luciferase activity, showed the occurrence of postnatal myonuclear accretion in vitro. Treatment of co-cultures with the myogenic factor IGF-I (p < 0.001) and the cytokines IL-13 (p < 0.05) and IL-4 (p < 0.001) increased postnatal myonuclear accretion, while the myogenic inhibitors cytochalasin D (p < 0.001), myostatin (p < 0.05), and TNFα (p < 0.001) decreased postnatal myonuclear accretion. Furthermore, postnatal myonuclear accretion was increased upon recovery from electrical pulse stimulation-induced fiber damage (p < 0.001) and LY29004-induced atrophy (p < 0.001). Moreover, cell type-specific siRNA-mediated knockdown of myomaker in myoblasts (p < 0.001), but not in myotubes, decreased postnatal myonuclear accretion. We developed a physiologically relevant, sensitive, high-throughput cell system for semi-quantitative assessment of in vitro postnatal myonuclear accretion, which can be used to mimic physiological myogenesis triggers, and can distinguish the cell type-specific roles of signals and responses in the regulation of postnatal myogenesis. As such, this method is suitable for both basal and translational research on the regulation of postnatal myogenesis, and will improve our understanding of muscle pathologies that result from impaired satellite cell number or function. The online version of this article (10.1186/s13395-018-0151-4) contains supplementary material, which is available to authorized users.
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影响因子：
10.5
作者：
Kassar-Duchossoy, L;Giacone, E;Tajbakhsh, S
通讯作者：
Tajbakhsh, S
影响因子：
5.4
作者：
Dull, T;Zufferey, R;Naldini, L
通讯作者：
Naldini, L
影响因子：
4.8
作者：
Joanisse, Sophie;Gillen, Jenna B.;Parise, Gianni
通讯作者：
Parise, Gianni
DOI：
10.1073/pnas.80.15.4856
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BLAU, HM;WEBSTER, C;PAVLATH, GK
通讯作者：
PAVLATH, GK
影响因子：
4
作者：
Jacquemin, Virginie;Butler-Browne, Gillian Sandra;Mouly, Vincent
通讯作者：
Mouly, Vincent