Association between atherogenic risk-modulating proteins and endothelium-dependent flow-mediated dilation in coronary artery disease patients.

PURPOSE: Endothelial dysfunction is an early and integral event in the development of atherosclerosis and coronary artery disease (CAD). Reduced NO bioavailability, oxidative stress, vasoconstriction, inflammation and senescence are all implicated in endothelial dysfunction. However, there are limited data examining associations between these pathways and direct in vivo bioassay measures of endothelial function in CAD patients. This study aimed to examine the relationships between in vivo measures of vascular function and the expression of atherogenic risk-modulating proteins in endothelial cells (ECs) isolated from the radial artery of CAD patients. METHODS: Fifty-six patients with established CAD underwent trans-radial catheterization. Prior to catheterization, radial artery vascular function was assessed using a) flow-mediated dilation (FMD), and b) exercise-induced dilation in response to handgrip (HE%). Freshly isolated ECs were obtained from the radial artery during catheterization and protein content of eNOS, NAD(P)H oxidase subunit NOX2, NFκB, ET-1 and the senescence markers p53, p21 and p16 were evaluated alongside nitrotyrosine abundance and eNOS Ser1177 phosphorylation. RESULTS: FMD was positively associated with eNOS Ser1177 phosphorylation (r = 0.290, P = 0.037), and protein content of p21 (r = 0.307, P = 0.027) and p16 (r = 0.426, P = 0.002). No associations were found between FMD and markers of oxidative stress, vasoconstriction or inflammation. In contrast to FMD, HE% was not associated with any of the EC proteins. CONCLUSION: These data revealed a difference in the regulation of endothelium-dependent vasodilation measured in vivo between patients with CAD compared to previously reported data in subjects without a clinical diagnosis, suggesting that eNOS Ser1177 phosphorylation may be the key to maintain vasodilation in CAD patients.

Young, healthy males and females present cardiometabolic protection against the detrimental effects of a 7-day high-fat high-calorie diet.

PURPOSE: High-fat, high-calorie (HFHC) diets have been used as a model to investigate lipid-induced insulin resistance. Short-term HFHC diets reduce insulin sensitivity in young healthy males, but to date, no study has directly compared males and females to elucidate sex-specific differences in the effects of a HFHC diet on functional metabolic and cardiovascular outcomes. METHODS: Eleven males (24 ± 4 years; BMI 23 ± 2 kg.m-2; V̇O2 peak 62.3 ± 8.7 ml.min-1.kg-1FFM) were matched to 10 females (25 ± 4 years; BMI 23 ± 2 kg.m-2; V̇O2 peak 58.2 ± 8.2 ml.min-1.kg-1FFM). Insulin sensitivity, measured via oral glucose tolerance test, metabolic flexibility, arterial stiffness, body composition and blood lipids and liver enzymes were measured before and after 7 days of a high-fat (65% energy) high-calorie (+ 50% kcal) diet. RESULTS: The HFHC diet did not change measures of insulin sensitivity, metabolic flexibility or arterial stiffness in either sex. There was a trend towards increased total body fat mass (kg) after the HFHC diet (+ 1.8% and + 2.3% for males and females, respectively; P = 0.056). In contrast to females, males had a significant increase in trunk to leg fat mass ratio (+ 5.1%; P = 0.005). CONCLUSION: Lean, healthy young males and females appear to be protected from the negative cardio-metabolic effects of a 7-day HFHC diet. Future research should use a prolonged positive energy balance achieved via increased energy intake and reduced energy expenditure to exacerbate negative metabolic and cardiovascular functional outcomes to determine whether sex-specific differences exist under more metabolically challenging conditions.

Probiotic supplementation increases carbohydrate metabolism in trained male cyclists: a randomized, double-blind, placebo-controlled crossover trial.

We hypothesized that probiotic supplementation (PRO) increases the absorption and oxidation of orally ingested maltodextrin during 2 h endurance cycling, thereby sparing muscle glycogen for a subsequent time trial (simulating a road race). Measurements were made of lipid and carbohydrate oxidation, plasma metabolites and insulin, gastrointestinal (GI) permeability, and subjective symptoms of discomfort. Seven male cyclists were randomized to PRO (bacterial composition given in methods) or placebo for 4 wk, separated by a 14-day washout period. After each period, cyclists consumed a 10% maltodextrin solution (initial 8 mL/kg bolus and 2 mL/kg every 15 min) while exercising for 2 h at 55% maximal aerobic power output, followed by a 100-kJ time trial. PRO resulted in small increases in peak oxidation rates of the ingested maltodextrin (0.84 ± 0.10 vs. 0.77 ± 0.09 g/min; P = 0.016) and mean total carbohydrate oxidation (2.20 ± 0.25 vs. 1.87 ± 0.39 g/min; P = 0.038), whereas fat oxidation was reduced (0.40 ± 0.11 vs. 0.55 ± 0.10 g/min; P = 0.021). During PRO, small but significant increases were seen in glucose absorption, plasma glucose, and insulin concentration and decreases in nonesterified fatty acid and glycerol. Differences between markers of GI damage and permeability and time-trial performance were not significant (P > 0.05). In contrast to the hypothesis, PRO led to minimal increases in absorption and oxidation of the ingested maltodextrin and small reductions in fat oxidation, whereas having no effect on subsequent time-trial performance.

Home-based high-intensity interval training reduces barriers to exercise in people with type 1 diabetes.

NEW FINDINGS: What is the topic of this review? This symposium review provides an overview of the recent work investigating whether a virtually monitored home-based high-intensity interval training (Home-HIT) intervention reduces the fear of hypoglycaemia and other common barriers to exercise in people with type 1 diabetes. What advances does it highlight? Home-HIT seems to offer a strategy to reduce fear of hypoglycaemia, while simultaneously removing other known barriers that prevent people with type 1 diabetes from taking up exercise, because it is time efficient, requires no travel time or costs associated with gym memberships, and allows people to exercise in their chosen environment. ABSTRACT: People with type 1 diabetes (T1D) are recommended to engage in regular exercise for a variety of health and fitness reasons. However, many lead a sedentary lifestyle and fail to meet the physical activity guidelines, in part because of the challenge of managing blood glucose concentration and fear of hypoglycaemia. A number of strategies designed to help people with T1D to manage their blood glucose during and after exercise have been investigated. Although many of these strategies show promise in facilitating blood glucose management during and after exercise, they do not target the many other common barriers to exercise that people with T1D face, such as difficulty with cost and travel time to gyms, limited access to exercise bikes and treadmills, and a possible dislike of exercising in front of others in public places. In this symposium review, we provide an overview of ongoing research into a virtually monitored home-based high-intensity interval training (Home-HIT) programme that is designed to reduce these other common barriers to exercise. The conclusion of this review is that Home-HIT seems to offer a strategy to reduce fear of hypoglycaemia, while simultaneously removing other known barriers preventing people with T1D from taking up exercise, such as being time efficient, requiring no travel time or costs associated with gym memberships, and giving them the opportunity to exercise in their chosen environment, reducing the embarrassment experienced by some when exercising in public.

Home-hit improves muscle capillarisation and eNOS/NAD(P)Hoxidase protein ratio in obese individuals with elevated cardiovascular disease risk.

KEY POINTS: Obesity and sedentary behaviour are associated with capillary rarefaction and impaired muscle microvascular vasoreactivity, due to reduced nitric oxide bioavailability. Low-volume high-intensity interval training (HIT) is a time-efficient alternative to traditional moderate-intensity continuous training (MICT), but its effect on the muscle microvasculature has not been studied. The applicability of current laboratory- and gym-based HIT protocols for obese individuals with low fitness and mobility has been disputed by public health experts, who cite the strenuous nature and complex protocols as major barriers. Therefore, we developed a virtually supervised HIT protocol targeting this group that can be performed at home without equipment (Home-HIT). This study is the first to show that 12 weeks of virtually supervised Home-HIT in obese individuals with elevated cardiovascular disease risk leads to similar increases in capillarisation and eNOS/NAD(P)Hoxidase protein ratio within the muscle microvascular endothelium as virtually supervised home-based MICT and laboratory-based HIT, while reducing many of the major barriers to exercise. ABSTRACT: This study investigated the effect of a novel virtually supervised home-based high-intensity interval training (HIT) (Home-HIT) intervention in obese individuals with elevated cardiovascular disease (CVD) risk on capillarisation and muscle microvascular eNOS/NAD(P)Hoxidase ratio. Thirty-two adults with elevated CVD risk (age 36 ± 10 years; body mass index 34.3 ± 5 kg m-2 ; V̇O2peak 24.6 ± 5.7 ml kg min-1 ), completed one of three 12-week training programmes: Home-HIT (n = 9), laboratory-based supervised HIT (Lab-HIT; n = 10) or virtually supervised home-based moderate-intensity continuous training (Home-MICT; n = 13). Muscle biopsies were taken before and after training to assess changes in vascular enzymes, capillarisation, mitochondrial density, intramuscular triglyceride content and GLUT4 protein expression using quantitative immunofluorescence microscopy. Training increased V̇O2peak (P < 0.001), whole-body insulin sensitivity (P = 0.033) and flow-mediated dilatation (P < 0.001), while aortic pulse wave velocity decreased (P < 0.001) in all three groups. Immunofluorescence microscopy revealed comparable increases in total eNOS content in terminal arterioles and capillaries (P < 0.001) in the three conditions. There was no change in eNOS ser1177 phosphorylation (arterioles P = 0.802; capillaries P = 0.311), but eNOS ser1177 /eNOS content ratio decreased significantly following training in arterioles and capillaries (P < 0.001). Training decreased NOX2 content (arterioles P < 0.001; capillaries P < 0.001), but there was no change in p47phox content (arterioles P = 0.101; capillaries P = 0.345). All measures of capillarisation increased (P < 0.05). There were no between-group differences. Despite having no direct supervision during exercise, virtually supervised Home-HIT resulted in comparable structural and endothelial enzymatic changes in the skeletal muscle microvessels to the traditional training methods. We provide strong evidence that Home-HIT is an effective novel strategy to remove barriers to exercise and improve health in an obese population at risk of CVD.

Passive heat therapy in sedentary humans increases skeletal muscle capillarization and eNOS content but not mitochondrial density or GLUT4 content.

Passive heat therapy (PHT) has been proposed as an alternative intervention to moderate-intensity continuous training (MICT) in individuals who are unable or unwilling to exercise. This study aimed to make the first comparison of the effect of PHT and MICT on 1) skeletal muscle capillarization and endothelial-specific endothelial nitric oxide synthase (eNOS) content and 2) mitochondrial density, glucose transporter 4 (GLUT4), and intramuscular triglyceride (IMTG) content. Twenty young sedentary males (21 ± 1 yr, body mass index 25 ± 1 kg/m2) were allocated to either 6 wk of PHT (n = 10; 40-50 min at 40°C in a heat chamber, 3×/wk) or MICT (n = 10; time-matched cycling at ~65% V̇o2peak). Muscle biopsies were taken from the vastus lateralis muscle before and after training. Immunofluorescence microscopy was used to assess changes in skeletal muscle mitochondrial density (mitochondrial marker cytochrome c oxidase subunit 4), GLUT4, and IMTG content, capillarization, and endothelial-specific eNOS content. V̇o2peak and whole body insulin sensitivity were also assessed. PHT and MICT both increased capillary density (PHT 21%; MICT 12%), capillary-fiber perimeter exchange index (PHT 15%; MICT 12%) (P < 0.05), and endothelial-specific eNOS content (PHT 8%; MICT 12%) (P < 0.05). However, unlike MICT (mitochondrial density 40%; GLUT4 14%; IMTG content 70%) (P < 0.05), PHT did not increase mitochondrial density (11%, P = 0.443), GLUT4 (7%, P = 0.217), or IMTG content (1%, P = 0.957). Both interventions improved aerobic capacity (PHT 5%; MICT 7%) and whole body insulin sensitivity (PHT 15%; MICT 36%) (P < 0.05). Six-week PHT in young sedentary males increases skeletal muscle capillarization and eNOS content to a similar extent as MICT; however, unlike MICT, PHT does not affect skeletal muscle mitochondrial density, GLUT4, or IMTG content. NEW & NOTEWORTHY The effect of 6-wk passive heat therapy (PHT) compared with moderate-intensity continuous training (MICT) was investigated in young sedentary males. PHT induced similar increases in skeletal muscle capillarization and endothelial-specific endothelial nitric oxide synthase content to MICT. Unlike MICT, PHT did not improve skeletal muscle mitochondrial density, glucose transporter 4, or intramuscular triglyceride content. These microvascular adaptations were paralleled by improvements in V̇o2peak and insulin sensitivity, suggesting that microvascular adaptations may contribute to functional improvements following PHT.

Hormone-sensitive lipase preferentially redistributes to lipid droplets associated with perilipin-5 in human skeletal muscle during moderate-intensity exercise.

KEY POINTS: Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) are the key enzymes involved in intramuscular triglyceride (IMTG) lipolysis. In isolated rat skeletal muscle, HSL translocates to IMTG-containing lipid droplets (LDs) following electrical stimulation, but whether HSL translocation occurs in human skeletal muscle during moderate-intensity exercise is currently unknown. Perilipin-2 (PLIN2) and perilipin-5 (PLIN5) proteins have been implicated in regulating IMTG lipolysis by interacting with HSL and ATGL in cell culture and rat skeletal muscle studies. This study investigated the hypothesis that HSL (but not ATGL) redistributes to LDs during moderate-intensity exercise in human skeletal muscle, and whether the localisation of these lipases with LDs was affected by the presence of PLIN proteins on the LDs. HSL preferentially redistributed to PLIN5-associated LDs whereas ATGL distribution was not altered with exercise; this is the first study to illustrate the pivotal step of HSL redistribution to PLIN5-associated LDs following moderate-intensity exercise in human skeletal muscle. ABSTRACT: Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) control skeletal muscle lipolysis. ATGL is present on the surface of lipid droplets (LDs) containing intramuscular triglyceride (IMTG) in both the basal state and during exercise. HSL translocates to LD in ex vivo electrically stimulated rat skeletal muscle. Perilipin-2- and perilipin-5-associated lipid droplets (PLIN2+ and PLIN5+ LDs) are preferentially depleted during exercise in humans, indicating that these PLINs may control muscle lipolysis. We aimed to test the hypothesis that in human skeletal muscle in vivo HSL (but not ATGL) is redistributed to PLIN2+ and PLIN5+ LDs during moderate-intensity exercise. Muscle biopsies from 8 lean trained males (age 21 ± 1 years, BMI 22.6 ± 1.2 kg m-2 and V̇O2 peak 48.2 ± 5.0 ml min-1  kg-1 ) were obtained before and immediately following 60 min of cycling exercise at ∼59% V̇O2 peak . Cryosections were stained using antibodies targeting ATGL, HSL, PLIN2 and PLIN5. LDs were stained using BODIPY 493/503. Images were obtained using confocal immunofluorescence microscopy and object-based colocalisation analyses were performed. Following exercise, HSL colocalisation to LDs increased (P < 0.05), and was significantly greater to PLIN5+ LDs (+53%) than to PLIN5- LDs (+34%) (P < 0.05), while the increases in HSL colocalisation to PLIN2+ LDs (+16%) and PLIN2- LDs (+28%) were not significantly different. Following exercise, the fraction of LDs colocalised with ATGL (0.53 ± 0.04) did not significantly change (P < 0.05) and was not affected by PLIN association to the LDs. This study presents the first evidence of exercise-induced HSL redistribution to LDs in human skeletal muscle and identifies PLIN5 as a facilitator of this mechanism.

In It Together: A Qualitative Evaluation of Participant Experiences of a 10-Week, Group-Based, Workplace HIIT Program for Insufficiently Active Adults.

Using guidance from the reach, efficacy, adoption, implementation, and maintenance evaluation framework, we aimed to qualitatively evaluate the participant experiences of a workplace high-intensity interval training (HIIT) intervention. Twelve previously insufficiently active individuals (four males and eight females) were interviewed once as part of three focus groups. Perceptions of program satisfaction, barriers to and facilitators of adherence, and persistence to exercise were explored. HIIT initiates interest because of its novelty, provides a sense of accomplishment, and overcomes the barriers of perceived lack of time. The feeling of relatedness between the participants can attenuate negative unpleasant responses during the HIIT sessions. HIIT, in this workplace setting, is an acceptable intervention for physically inactive adults. However, participants were reluctant to maintain the same mode of exercise, believing that HIIT sessions were for the very fit.

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

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.

Increased muscle blood supply and transendothelial nutrient and insulin transport induced by food intake and exercise: effect of obesity and ageing.

This review concludes that a sedentary lifestyle, obesity and ageing impair the vasodilator response of the muscle microvasculature to insulin, exercise and VEGF-A and reduce microvascular density. Both impairments contribute to the development of insulin resistance, obesity and chronic age-related diseases. A physically active lifestyle keeps both the vasodilator response and microvascular density high. Intravital microscopy has shown that microvascular units (MVUs) are the smallest functional elements to adjust blood flow in response to physiological signals and metabolic demands on muscle fibres. The luminal diameter of a common terminal arteriole (TA) controls blood flow through up to 20 capillaries belonging to a single MVU. Increases in plasma insulin and exercise/muscle contraction lead to recruitment of additional MVUs. Insulin also increases arteriolar vasomotion. Both mechanisms increase the endothelial surface area and therefore transendothelial transport of glucose, fatty acids (FAs) and insulin by specific transporters, present in high concentrations in the capillary endothelium. Future studies should quantify transporter concentration differences between healthy and at risk populations as they may limit nutrient supply and oxidation in muscle and impair glucose and lipid homeostasis. An important recent discovery is that VEGF-B produced by skeletal muscle controls the expression of FA transporter proteins in the capillary endothelium and thus links endothelial FA uptake to the oxidative capacity of skeletal muscle, potentially preventing lipotoxic FA accumulation, the dominant cause of insulin resistance in muscle fibres.

Sprint interval and moderate-intensity continuous training have equal benefits on aerobic capacity, insulin sensitivity, muscle capillarisation and endothelial eNOS/NAD(P)Hoxidase protein ratio in obese men.

KEY POINTS: Skeletal muscle capillary density and vasoreactivity are reduced in obesity, due to reduced nitric oxide bioavailability. Sprint interval training (SIT) has been proposed as a time efficient alternative to moderate-intensity continuous training (MICT), but its effect on the skeletal muscle microvasculature has not been studied in obese individuals. We observed that SIT and MICT led to equal increases in capillarisation and endothelial eNOS content, while reducing endothelial NOX2 content in microvessels of young obese men. We conclude that SIT is equally effective at improving skeletal muscle capillarisation and endothelial enzyme balance, while being a time efficient alternative to traditional MICT. ABSTRACT: Sprint interval training (SIT) has been proposed as a time efficient alternative to moderate-intensity continuous training (MICT), leading to similar improvements in skeletal muscle capillary density and microvascular function in young healthy humans. In this study we made the first comparisons of the muscle microvascular response to SIT and MICT in an obese population. Sixteen young obese men (age 25 ± 1 years, BMI 34.8 ± 0.9 kg m(-2) ) were randomly assigned to 4 weeks of MICT (40-60 min cycling at ∼65% V̇O2 peak , 5 times per week) or constant load SIT (4-7 constant workload intervals of 200% Wmax 3 times per week). Muscle biopsies were taken before and after training from the m. vastus lateralis to measure muscle microvascular endothelial eNOS content, eNOS serine(1177) phosphorylation, NOX2 content and capillarisation using quantitative immunofluorescence microscopy. Maximal aerobic capacity (V̇O2 peak ), whole body insulin sensitivity and arterial stiffness were also assessed. SIT and MICT increased skeletal muscle microvascular eNOS content and eNOS ser(1177) phosphorylation in terminal arterioles and capillaries (P < 0.05), but the latter effect was eliminated when normalised to eNOS content (P = 0.217). SIT and MICT also reduced microvascular endothelial NOX2 content (P < 0.05) and both increased capillary density and capillary-fibre perimeter exchange index (P < 0.05). In parallel, SIT and MICT increased V̇O2 peak (P < 0.05) and whole body insulin sensitivity (P < 0.05), and reduced central artery stiffness (P < 0.05). As no significant differences were observed between SIT and MICT it is concluded that SIT is a time efficient alternative to MICT to improve aerobic capacity, insulin sensitivity and muscle capillarisation and endothelial eNOS/NAD(P)Hoxidase protein ratio in young obese men.

Habitual physical activity is associated with the maintenance of neutrophil migratory dynamics in healthy older adults.

BACKGROUND: Dysfunctional neutrophils with advanced age are a hallmark of immunosenescence. Reduced migration and bactericidal activity increase the risk of infection. It remains unclear why neutrophil dysfunction occurs with age. Physical activity and structured exercise have been suggested to improve immune function in the elderly. The aim of this study was to assess a comprehensive range of neutrophil functions and determine their association with habitual physical activity. METHOD: Physical activity levels were determined in 211 elderly (67±5years) individuals by 7-days of accelerometry wear. Twenty of the most physically active men and women were matched for age and gender to twenty of the least physically active individuals. Groups were compared for neutrophil migration, phagocytosis, oxidative burst, cell surface receptor expression, metabolic health parameters and systemic inflammation. Groups were also compared against ten young participants (23±4years). RESULTS: The most active group completed over twice as many steps/day as the least active group (p<0.001), had lower BMI's (p=0.007) and body fat percentages (p=0.029). Neutrophils migrated towards IL-8 better in the most active group compared to the least active (p<0.05) and was comparable to that of the young (p>0.05). These differences remained after adjusting for BMI, body fat and plasma metabolic markers which were different between groups. Correlations revealed that steps/day, higher adiponectin and lower insulin were positively associated with migratory ability (p<0.05). There was no difference in expression of the chemokine receptors CXCR1 or CXCR2 (p>0.05 for both). CD11b was higher in the most active group compared to the least active (p=0.048). No differences between activity groups or young controls were observed for neutrophil phagocytosis or oxidative burst in response to Escherichia coli (p>0.05). The young group had lower concentrations of IL-6, IL-8, MCP-1, CRP, IL-10 and IL-13 (p<0.05 for all) with no differences between the two older groups. CONCLUSION: These data suggest that impaired neutrophil migration, but not bactericidal function, in older adults may be, in part, the result of reduced physical activity. A 2-fold difference in physical activity is associated with better preserved neutrophil migratory dynamics in healthy older people. As a consequence increasing habitual physical activity may be beneficial for neutrophil mediated immunity.

Effect of resistance training on microvascular density and eNOS content in skeletal muscle of sedentary men.

OBJECTIVE: The effects of RT on muscle mass, strength, and insulin sensitivity are well established, but the underlying mechanisms are only partially understood. The main aim of this study was to investigate whether RT induces changes in endothelial enzymes of the muscle microvasculature, which would increase NO bioavailability and could contribute to improved insulin sensitivity. METHODS: Eight previously sedentary males (age 20 ± 0.4 years, BMI 24.5 ± 0.9 kg/m(2) ) completed six weeks of RT 3x/week. Muscle biopsies were taken from the m. vastus lateralis and microvascular density; and endothelial-specific eNOS content, eNOS Ser(1177) phosphorylation, and NOX2 content were assessed pre- and post-RT using quantitative immunofluorescence microscopy. Whole-body insulin sensitivity (measured as Matsuda Index), microvascular Kf (functional measure of the total available endothelial surface area), and arterial stiffness (AIx, central, and pPWV) were also measured. RESULTS: Measures of microvascular density, microvascular Kf , microvascular eNOS content, basal eNOS phosphorylation, and endothelial NOX2 content did not change from pre-RT to post-RT. RT increased insulin sensitivity (p < 0.05) and reduced resting blood pressure and AIx (p < 0.05), but did not change central or pPWV. CONCLUSIONS: RT did not change any measure of muscle microvascular structure or function.

Paxillin and focal adhesion kinase colocalise in human skeletal muscle and its associated microvasculature.

Focal adhesion kinase (FAK) and paxillin are functionally linked hormonal- and mechano-sensitive proteins. We aimed to describe paxillin's subcellular distribution using widefield and confocal immunofluorescence microscopy and test the hypothesis that FAK and paxillin colocalise in human skeletal muscle and its associated microvasculature. Percutaneous muscle biopsies were collected from the m. vastus lateralis of seven healthy males, and 5-µm cryosections were stained with anti-paxillin co-incubated with anti-dystrophin to identify the sarcolemma, anti-myosin heavy chain type I for fibre-type differentiation, anti-dihydropyridine receptor to identify T-tubules, lectin UEA-I to identify the endothelium of microvessels and anti-α-smooth muscle actin to identify vascular smooth muscle cells (VSMC). Colocalisation of anti-paxillin with anti-dystrophin or anti-FAK was quantified using Pearson's correlation coefficient on confocal microscopy images. Paxillin was primarily present in (sub)sarcolemmal regions of skeletal muscle fibres where it colocalised with dystrophin (r = 0.414 ± 0.026). The (sub)sarcolemmal paxillin immunofluorescence intensity was ~2.4-fold higher than in sarcoplasmic regions (P < 0.001) with sarcoplasmic paxillin immunofluorescence intensity ~10 % higher in type I than in type II fibres (P < 0.01). In some longitudinally orientated fibres, paxillin formed striations that corresponded to the I-band region. Paxillin immunostaining was highest in endothelial and VSMC and distributed heterogeneously in both cell types. FAK and paxillin colocalised at (sub)sarcolemmal regions and within the microvasculature (r = 0.367 ± 0.036). The first images of paxillin in human skeletal muscle suggest paxillin is present in (sub)sarcolemmal and I-band regions of muscle fibres and within the microvascular endothelium and VSMC. Colocalisation of FAK and paxillin supports their suggested role in hormonal and mechano-sensitive signalling.

Sprint interval and endurance training are equally effective in increasing muscle microvascular density and eNOS content in sedentary males.

Sprint interval training (SIT) has been proposed as a time efficient alternative to endurance training (ET) for increasing skeletal muscle oxidative capacity and improving certain cardiovascular functions. In this study we sought to make the first comparisons of the structural and endothelial enzymatic changes in skeletal muscle microvessels in response to ET and SIT. Sixteen young sedentary males (age 21 ± SEM 0.7 years, BMI 23.8 ± SEM 0.7 kg m(-2)) were randomly assigned to 6 weeks of ET (40-60 min cycling at ∼65% , 5 times per week) or SIT (4-6 Wingate tests, 3 times per week). Muscle biopsies were taken from the m. vastus lateralis before and following 60 min cycling at 65% to measure muscle microvascular endothelial eNOS content, eNOS serine(1177) phosphorylation, NOX2 content and capillarisation using quantitative immunofluorescence microscopy. Whole body insulin sensitivity, arterial stiffness and blood pressure were also assessed. ET and SIT increased skeletal muscle microvascular eNOS content (ET 14%; P < 0.05, SIT 36%; P < 0.05), with a significantly greater increase observed following SIT (P < 0.05). Sixty minutes of moderate intensity exercise increased eNOS ser(1177) phosphorylation in all instances (P < 0.05), but basal and post-exercise eNOS ser(1177) phosphorylation was lower following both training modes. All microscopy measures of skeletal muscle capillarisation (P < 0.05) were increased with SIT or ET, while neither endothelial nor sarcolemmal NOX2 was changed. Both training modes reduced aortic stiffness and increased whole body insulin sensitivity (P < 0.05). In conclusion, in sedentary males SIT and ET are effective in improving muscle microvascular density and eNOS protein content.

Immunofluorescence microscopy to assess enzymes controlling nitric oxide availability and microvascular blood flow in muscle.

OBJECTIVE: The net production of NO by the muscle microvascular endothelium is a key regulator of muscle microvascular blood flow. Here, we describe the development of a method to quantify the protein content and phosphorylation of endothelial NO synthase (eNOS content and eNOS ser(1177) phosphorylation) and NAD(P)H oxidase expression. METHODS: Human muscle cryosections were stained using antibodies targeting eNOS, p-eNOS ser(1177) and NOX2 in combination with markers of the endothelium and the sarcolemma. Quantitation was achieved by analyzing fluorescence intensity within the area stained positive for the microvascular endothelium. Analysis was performed in duplicate and repeated five times to investigate CV. In addition, eight healthy males (age 21 ± 1 year, BMI 24.4 ± 1.0 kg/m(2)) completed one hour of cycling exercise at ~65%VO(2max) . Muscle biopsies were taken from the m. vastus lateralis before and immediately after exercise and analyzed using the new methods. RESULTS: The CV of all methods was between 6.5 and 9.5%. Acute exercise increased eNOS serine(1177) phosphorylation (fold change 1.29 ± 0.05, p < 0.05). CONCLUSIONS: These novel methodologies will allow direct investigations of the molecular mechanisms underpinning the microvascular responses to insulin and exercise, the impairments that occur in sedentary, obese and elderly individuals and the effect of lifestyle interventions.

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.

What Happened in 'The HERizon Project'?-Process Evaluation of a Multi-Arm Remote Physical Activity Intervention for Adolescent Girls.

This mixed-methods process evaluation examines the reach, recruitment, fidelity, adherence, acceptability, mechanisms of impact, and context of remote 12-week physical activity (PA) interventions for adolescent girls named The HERizon Project. The study was comprised of four arms-a PA programme group, a behaviour change support group, a combined group, and a comparison group. Data sources included intervention deliverer and participant logbooks (100 and 71% respective response rates, respectively), exit surveys (72% response rate), and semi-structured focus groups/interviews conducted with a random subsample of participants from each of the intervention arms (n = 34). All intervention deliverers received standardised training and successfully completed pre-intervention competency tasks. Based on self-report logs, 99% of mentors adhered to the call guide, and 100% of calls and live workouts were offered. Participant adherence and intervention receipt were also high for all intervention arms. Participants were generally satisfied with the intervention components; however, improvements were recommended for the online social media community within the PA programme and combined intervention arms. Autonomy, sense of accomplishment, accountability, and routine were identified as factors facilitating participant willingness to adhere to the intervention across all intervention arms. Future remote interventions should consider structured group facilitation to encourage a genuine sense of community among participants.