Proceedings of The Physiological Society

University of Manchester (2010) Proc Physiol Soc 19, C61

Oral Communications

Quantification of skeletal muscle microvascular endothelial nitric oxide synthase (eNOS) content using immunofluorescence microscopy

M. Cocks1, C. S. Shaw1, A. M. Wagenmakers1

1. School of Sport and Exercise Science, Univesity of Birmingham, Birmingham, United Kingdom.

Impaired skeletal muscle microvascular function is an early event in the pathogenesis leading to hypertension, insulin resistance and cardiovascular disease. A reduction in either nitric oxide (NO) synthesis or bioavailability has been implicated in the development of microvascular dysfunction. The rate limiting enzyme of NO synthesis is endothelial nitric oxide synthase (eNOS) and the content and activation of eNOS will influence microvascular function. Reports suggest a decrease in eNOS content in obesity. Analysis of eNOS protein content within skeletal muscle has been performed previously using whole muscle homogenates but such methods can not account for differences in skeletal muscle microvascular density associated with obesity and exercise training. Therefore we aimed to develop a technique to visualise and quantify eNOS content within skeletal muscle microvascular endothelium using immunofluorescence microscopy. Muscle biopsies were taken from the vastus lateralis of two male subjects (22 years, BMI 22.6 kg/m2 and 57 years, BMI 36.4 kg/m2) using the Bergstrom needle technique. Cryosections were stained using anti-eNOS coupled to appropriately targeted secondary antibodies in combination with the endothelial marker lectin (UEI-I FITC). Analysis was performed in duplicate and repeated 5 times. Images were viewed and captured using widefield fluorescence microscopy. eNOS staining was present in microvessels in skeletal muscle and colocalised with the endothelial marker in both capillaries and larger vessels. Quantification of eNOS content within the microvascular endothelium was achieved by analysing eNOS fluorescence intensity within the area stained positive for the endothelium. An average of (mean ± SD) 177± 54 microvessels were analysed per cryosection. The coefficient of variation for eNOS content was 7.7% (fluorescence intensity, mean ± SD 50.8± 3.9) for participant 1 and 6.3% (fluorescence intensity, mean ± SD 36.2± 2.3) for participant 2. We have developed a novel immunofluorescence staining method to investigate the eNOS content in the microvascular endothelium in skeletal muscle. This new technique allows for reproducible measurements of microvascular eNOS content in human muscle biopsies. This method allows the analysis of eNOS content independent of changes in microvascular density which may confound the use of techniques using whole muscle homogenates. This novel approach may improve our understanding of the mechanisms responsible for alterations in skeletal muscle microvascular function associated with obesity, insulin resistance and physical activity.

Where applicable, experiments conform with Society ethical requirements