The effect of different training modes on skeletal muscle microvascular density and endothelial enzymes controlling NO availability

Abstract

It is becoming increasingly apparent that a high vasodilator response of the skeletal muscle microvasculature to insulin and exercise is of critical importance for adequate muscle perfusion and long-term microvascular and muscle metabolic health. Previous research has shown that a sedentary lifestyle, obesity and ageing lead to impairments in the vasodilator response, while a physically active lifestyle keeps both microvascular density and vasodilator response high. To investigate the molecular mechanisms behind these impairments and the benefits of exercise training interventions, our laboratory has recently developed quantitative immunofluorescence microscopy methods to measure protein content of eNOS and NAD(P)Hoxidase specifically in the endothelial layer of capillaries and arterioles of human skeletal muscle. As eNOS produces nitric oxide (NO) and NAD(P)Hoxidase produces superoxide anions (O2 (-) , quenching NO) we propose that the eNOS/NAD(P)Hoxidase protein ratio is a marker of vasodilator capacity. The novel methods show that endurance training (ET) and high intensity interval training (HIT), generally regarded as a time-efficient alternative to ET, increase eNOS protein content and the eNOS/NADP(H)oxidase protein ratio in previously sedentary lean and obese young men. Resistance exercise training had smaller but qualitatively similar effects. Western blot data of other laboratories suggest that endurance exercise training leads to similar changes in sedentary elderly men. Future research will be required to investigate the relative importance of other sources and tissues in the balance between NO and O2 (-) production seen by the vascular smooth muscle layer of terminal arterioles.

Figure 1. The balance between nitric oxide (NO) production and NO scavenging in the skeletal muscle microvasculature impacts human metabolic health

Highly trained individuals have an optimal balance between eNOS and NAD(P)Hoxidase protein content and activity in the skeletal muscle microvasculature, and combine high physical activity levels with a high exercise tolerance, high anabolic response to resistance exercise, and the highest reported levels of insulin sensitivity and glucose tolerance. In sedentary individuals this balance is less favourable and we hypothesise that it is because of this adaptation that the sedentary state comes with a high risk for the development of obesity, metabolic syndrome and type 2 diabetes, conditions in which a low NO bioavailability leads to severe impairments in insulin sensitivity, glucose tolerance, anabolic resistance, and exercise tolerance. Exercise training interventions of 4–6 weeks in sedentary individuals with and without obesity have led to an improved balance between eNOS and NAD(P)Hoxidase protein content.

Figure 2. Image analysis for quantitative immunofluorescence

The skeletal muscle microvascular endothelium is identified using the endothelial marker UEA‐I Lectin FITC conjugated (A). Using this endothelial image an endothelial mask is created using image analysis software (Image Pro Plus 5.1; B). The endothelial enzyme being investigated is then identified using an appropriate validated antibody (in this case eNOS; C). The endothelial outline is then transferred to the corresponding endothelial enzyme image. The fluorescence intensity of the endothelial image is then quantified within the endothelial outline. E and F, terminal arterioles are differentiated from capillaries; an arteriole is marked with an arrow. E, anti‐alpha smooth muscle actin is used as an identifier of terminal arterioles; F, the endothelium of capillaries and terminal arterioles is marked with UEA‐I Lectin FITC conjugated.

Figure 3. Effects of endurance training on eNOS and NOX2 content and eNOS ser¹¹⁷⁷ phosphorylation in lean sedentary individuals

A, C and E, representative widefield microscopy images of skeletal muscle before (a, b) and after (c, d) endurance training. Panels a and c, the skeletal muscle microvascular endothelium was revealed using Ulex Europaeus‐FITC conjugated lectin (green). Ab and d, skeletal muscle eNOS expression (red). Ca and b, skeletal muscle eNOS ser¹¹⁷⁷ phosphorylation (red). Ea and b, skeletal muscle NOX2 expression (red). Bar represents 20 μm. B, D and F, mean fluorescence intensity is summarised. The mean level of eNOS, eNOS ser¹¹⁷⁷ or NOX2 before training was assigned a value of 1, and the relative intensity post training was calculated. Data are means ± SEM for 8 (eNOS) or 7 (eNOS ser¹¹⁷⁷ and NOX2) participants *P < 0.05, significant main effect of training. Adapted with permission from Cocks et al. (2013 a).