Rumsey John W, Das Mainak, Kang Jung-Fong, Wagner Robert, Molnar Peter, Hickman James J
NanoScience Technology Center, 12424 Research Parkway, Suite 400, University of Central Florida, Orlando, FL 32826, USA.
Biomaterials. 2008 Mar;29(8):994-1004. doi: 10.1016/j.biomaterials.2007.10.042.
While much is known about muscle spindle structure, innervation and function, relatively few factors have been identified that regulate intrafusal fiber differentiation and spindle development. Identification of these factors will be a crucial step in tissue engineering functional muscle systems. In this study, we investigated the role of the growth factor, neuregulin 1-beta-1 (Nrg 1-beta-1) EGF, for its ability to influence myotube fate specification in a defined culture system utilizing the non-biological substrate N-1[3-(trimethoxysilyl)propyl]-diethylenetriamine (DETA). Based on morphological and immunocytochemical criteria, Nrg 1-beta-1 treatment of developing myotubes increases the ratio of nuclear bag fibers to total myotubes from 0.019 to 0.100, approximately a five-fold increase. The myotube cultures were evaluated for expression of the intrafusal fiber-specific alpha cardiac-like myosin heavy chain and for the expression of the non-specific slow myosin heavy chain. Additionally, the expression of ErbB2 receptors on all myotubes was observed, while phosphorylated ErbB2 receptors were only observed in Nrg 1-beta-1-treated intrafusal fibers. After Nrg 1-beta-1 treatment, we were able to observe the expression of the intrafusal fiber-specific transcription factor Egr3 only in fibers exhibiting the nuclear bag phenotype. Finally, nuclear bag fibers were characterized electrophysiologically for the first time in vitro. This data shows conclusively, in a serum-free system, that Nrg 1-beta-1 is necessary to drive specification of forming myotubes to the nuclear bag phenotype.
尽管人们对肌梭的结构、神经支配和功能了解颇多，但已确定的调节肌梭内纤维分化和梭发育的因素相对较少。识别这些因素将是组织工程功能性肌肉系统的关键一步。在本研究中，我们利用非生物底物N-1[3-(三甲氧基甲硅烷基)丙基]-二亚乙基三胺（DETA），在特定培养系统中研究了生长因子神经调节蛋白1-β-1（Nrg 1-β-1）表皮生长因子（EGF）影响肌管命运特化的能力。基于形态学和免疫细胞化学标准，用Nrg 1-β-1处理发育中的肌管，可使核袋纤维与总肌管的比例从0.019增加到0.100，约增加了五倍。对肌管培养物进行了肌梭内纤维特异性α心脏样肌球蛋白重链表达和非特异性慢肌球蛋白重链表达的评估。此外，观察了所有肌管上ErbB2受体的表达，而仅在经Nrg 1-β-1处理的肌梭内纤维中观察到磷酸化的ErbB2受体。Nrg 1-β-1处理后，我们仅在表现出核袋表型的纤维中观察到肌梭内纤维特异性转录因子Egr3的表达。最后，首次在体外对核袋纤维进行了电生理特征分析。该数据确凿地表明，在无血清系统中，Nrg 1-β-1是驱动形成的肌管特化为核袋表型所必需的。
