Immunofluorescent visualisation of focal adhesion kinase in human skeletal muscle and its associated microvasculature

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.