Exercise Metabolism Research Group, School of Sport and Exercise Sciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Histochem Cell Biol. 2012 Oct;138(4):617-26. doi: 10.1007/s00418-012-0980-x. Epub 2012 Jun 30.
Within animal skeletal muscle, focal adhesion kinase (FAK) has been associated with load-dependent molecular and metabolic adaptation including the regulation of insulin sensitivity. This study aimed to generate the first visual images of the localisation of FAK within human skeletal muscle fibres and its associated microvasculature using widefield and confocal immunofluorescence microscopy. Percutaneous muscle biopsies, taken from five lean, active males, were frozen and 5-μm cryosections were incubated with FAK antibodies for visualisation in muscle fibres and the microvasculature. Anti-myosin heavy chain type I was used for fibre-type differentiation. Muscle sections were also incubated with anti-dihydropyridine receptor (DHPR) to investigate co-localisation of FAK with the t-tubules. FITC-conjugated Ulex europaeus Agglutinin I stained the endothelium of the capillaries, whilst anti-smooth muscle actin stained the vascular smooth muscle of arterioles. Fibre-type differences in the intensity of FAK immunofluorescence were determined with image analysis software. In transversely and longitudinally orientated fibres, FAK was localised at the sarcolemmal regions. In longitudinally orientated fibres, FAK staining also showed uniform striations across the fibre and co-staining with DHPR suggests FAK associates with the t-tubules. There was no fibre-type difference in sarcoplasmic FAK content. Within the capillary endothelium and arteriolar smooth muscle, FAK was distributed heterogeneously as clusters. This is the first study to visualise FAK in human skeletal muscle microvasculature and within the (sub)sarcolemmal and t-tubule regions using immunofluorescence microscopy. This technique will be an important tool for investigating the role of FAK in the intracellular signalling of human skeletal muscle and the endothelium of its associated microvasculature.
在动物骨骼肌中，粘着斑激酶（FAK）与依赖于负荷的分子和代谢适应有关，包括胰岛素敏感性的调节。本研究旨在使用宽场和共聚焦免疫荧光显微镜首次可视化人骨骼肌纤维内 FAK 的定位及其相关的微血管。从 5 名瘦、活跃的男性中采集经皮肌肉活检标本，冷冻后用 5μm 冷冻切片进行 FAK 抗体孵育，以在肌肉纤维和微血管中可视化。抗肌球蛋白重链 I 型用于纤维型分化。肌肉切片还与抗二氢吡啶受体（DHPR）孵育，以研究 FAK 与 t 小管的共定位。FITC 缀合的欧洲菘蓝凝集素 I 染色毛细血管的内皮，而抗平滑肌肌动蛋白染色小动脉的血管平滑肌。使用图像分析软件确定 FAK 免疫荧光的强度在纤维型之间的差异。在横向和纵向取向的纤维中，FAK 定位于肌膜区域。在纵向取向的纤维中，FAK 染色也显示纤维上均匀的条纹，与 DHPR 的共染色表明 FAK 与 t 小管相关。肌浆内 FAK 含量在纤维型之间没有差异。在毛细血管内皮细胞和小动脉平滑肌中，FAK 呈不均匀分布的簇状。这是首次使用免疫荧光显微镜在人骨骼肌微血管中和（亚）肌膜和 t 小管区域可视化 FAK 的研究。这项技术将成为研究 FAK 在人骨骼肌细胞内信号转导及其相关微血管内皮细胞中的作用的重要工具。
