Sant'ana Pereira J A, Wessels A, Nijtmans L, Moorman A F, Sargeant A J
Department of Muscle and Exercise Physiology, Vrije University Amsterdam, The Netherlands.
J Muscle Res Cell Motil. 1995 Feb;16(1):21-34. doi: 10.1007/BF00125307.
In the present study we have developed a method which, by combining histochemical, immunohistochemical, electrophoretic and immunoblotting analyses on a single fibre, enables a sensitive characterization of human skeletal muscle fibres dissected from freeze-dried biopsy samples. For histochemical (and immunohistochemical) analysis fibre fragments (500 microns) of individual fibres were mounted in an embedding medium to allow cryostat sections of normalized thickness to be reproducibly obtained. The specificity of the myofibrillar Ca2+ ATPase (mATPase) staining profiles in gelatin-embedded single fibre sections was tested by immunohistochemical reactions with anti-myosin heavy chain (MyHC) monoclonal antibodies specific to human MyHC I, IIA, IIB and IIA + IIB and by gel electrophoresis. The combined methodologies demonstrated the specificity of the mATPase staining patterns which correlated to the expression of distinct MyHC isoforms. In addition the results provide evidence that many fibres co-expressed different MyHC isoforms in variable relative amounts, forming a continuum. Staining intensities for mATPase, converted into optical density values by image analysis revealed that a relationship between mATPase and MyHC expression holds for hybrid fibres even when displaying one MyHC type with overwhelming dominance. The results also revealed that three MyHC isoforms I, IIA and IIB can be co-expressed on a single muscle fibre. In such a case mATPase alone, with the current protocols, does not allow an accurate characterization of the specific MyHC-based fibre type(s). Although some hybrid fibres may have displayed a non-uniform expression of myosins along their lengths, most fibres from the IIA/B group (type) remained very stable with respect to the relative amounts of the MyHCs expressed. Finally, a second slow MyHC isoform was recognized on immunoblots of a mixed muscle sample.
在本研究中，我们开发了一种方法，通过对单根纤维进行组织化学、免疫组织化学、电泳和免疫印迹分析，能够对从冻干活检样本中分离出的人类骨骼肌纤维进行灵敏的表征。对于组织化学（和免疫组织化学）分析，将单根纤维的纤维片段（500微米）嵌入包埋介质中，以便可重复地获得标准化厚度的冰冻切片。通过与针对人类肌球蛋白重链（MyHC）I、IIA、IIB以及IIA + IIB的抗MyHC单克隆抗体进行免疫组织化学反应，并通过凝胶电泳，测试了明胶包埋的单根纤维切片中肌原纤维Ca2+ ATP酶（mATPase）染色图谱的特异性。联合方法证明了mATPase染色模式的特异性，其与不同MyHC亚型的表达相关。此外，结果提供了证据表明许多纤维以可变的相对量共表达不同的MyHC亚型，形成一个连续体。通过图像分析将mATPase的染色强度转换为光密度值，结果显示即使在一种MyHC类型占主导的杂种纤维中，mATPase与MyHC表达之间也存在关系。结果还表明，三种MyHC亚型I、IIA和IIB可以在单根肌纤维上共表达。在这种情况下，仅使用当前方案的mATPase不能准确表征基于特定MyHC的纤维类型。尽管一些杂种纤维可能在其长度上显示出肌球蛋白的不均匀表达，但IIA/B组（类型）的大多数纤维在表达的MyHC相对量方面保持非常稳定。最后，在混合肌肉样本的免疫印迹中识别出第二种慢MyHC亚型。
