Machine learning determination of applied behavioral analysis treatment plan type.

BACKGROUND: Applied behavioral analysis (ABA) is regarded as the gold standard treatment for autism spectrum disorder (ASD) and has the potential to improve outcomes for patients with ASD. It can be delivered at different intensities, which are classified as comprehensive or focused treatment approaches. Comprehensive ABA targets multiple developmental domains and involves 20-40 h/week of treatment. Focused ABA targets individual behaviors and typically involves 10-20 h/week of treatment. Determining the appropriate treatment intensity involves patient assessment by trained therapists, however, the final determination is highly subjective and lacks a standardized approach. In our study, we examined the ability of a machine learning (ML) prediction model to classify which treatment intensity would be most suited individually for patients with ASD who are undergoing ABA treatment. METHODS: Retrospective data from 359 patients diagnosed with ASD were analyzed and included in the training and testing of an ML model for predicting comprehensive or focused treatment for individuals undergoing ABA treatment. Data inputs included demographics, schooling, behavior, skills, and patient goals. A gradient-boosted tree ensemble method, XGBoost, was used to develop the prediction model, which was then compared against a standard of care comparator encompassing features specified by the Behavior Analyst Certification Board treatment guidelines. Prediction model performance was assessed via area under the receiver-operating characteristic curve (AUROC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). RESULTS: The prediction model achieved excellent performance for classifying patients in the comprehensive versus focused treatment groups (AUROC: 0.895; 95% CI 0.811-0.962) and outperformed the standard of care comparator (AUROC 0.767; 95% CI 0.629-0.891). The prediction model also achieved sensitivity of 0.789, specificity of 0.808, PPV of 0.6, and NPV of 0.913. Out of 71 patients whose data were employed to test the prediction model, only 14 misclassifications occurred. A majority of misclassifications (n = 10) indicated comprehensive ABA treatment for patients that had focused ABA treatment as the ground truth, therefore still providing a therapeutic benefit. The three most important features contributing to the model's predictions were bathing ability, age, and hours per week of past ABA treatment. CONCLUSION: This research demonstrates that the ML prediction model performs well to classify appropriate ABA treatment plan intensity using readily available patient data. This may aid with standardizing the process for determining appropriate ABA treatments, which can facilitate initiation of the most appropriate treatment intensity for patients with ASD and improve resource allocation.

Daily blueberry consumption for 12 weeks improves endothelial function in postmenopausal women with above-normal blood pressure through reductions in oxidative stress: a randomized controlled trial.

Estrogen-deficient postmenopausal women have oxidative stress-mediated suppression of endothelial function that is exacerbated by high blood pressure. Previous research suggests blueberries may improve endothelial function through reductions in oxidative stress, while also exerting other cardiovascular benefits. The objective of this study was to examine the efficacy of blueberries to improve endothelial function and blood pressure in postmenopausal women with above-normal blood pressure, and to identify potential mechanisms for improvements in endothelial function. A randomized, double-blind, placebo-controlled, parallel-arm clinical trial was performed, where postmenopausal women aged 45-65 years with elevated blood pressure or stage 1-hypertension (total n = 43, endothelial function n = 32) consumed 22 g day-1 of freeze-dried highbush blueberry powder or placebo powder for 12 weeks. Endothelial function was assessed at baseline and 12 weeks through ultrasound measurement of brachial artery flow-mediated dilation (FMD) normalized to shear rate area under the curve (FMD/SRAUC) before and after intravenous infusion of a supraphysiologic dose of ascorbic acid to evaluate whether FMD improvements were mediated by reduced oxidative stress. Hemodynamics, arterial stiffness, cardiometabolic blood biomarkers, and plasma (poly)phenol metabolites were assessed at baseline and 4, 8, and 12 weeks, and venous endothelial cell protein expression was assessed at baseline and 12 weeks. Absolute FMD/SRAUC was 96% higher following blueberry consumption compared to baseline (p < 0.05) but unchanged in the placebo group (p > 0.05), and changes from baseline to 12 weeks were greater in the blueberry group than placebo (+1.09 × 10-4 ± 4.12 × 10-5vs. +3.82 × 10-6 ± 1.59 × 10-5, p < 0.03, respectively). The FMD/SRAUC response to ascorbic acid infusion was lower (p < 0.05) at 12 weeks compared to baseline in the blueberry group with no change in the placebo group (p > 0.05). The sum of plasma (poly)phenol metabolites increased at 4, 8, and 12 weeks in the blueberry group compared to baseline, and were higher than the placebo group (all p < 0.05). Increases in several plasma flavonoid and microbial metabolites were also noted. No major differences were found for blood pressure, arterial stiffness, blood biomarkers, or endothelial cell protein expression following blueberry consumption. These findings suggest daily consumption of freeze-dried blueberry powder for 12 weeks improves endothelial function through reduced oxidative stress in postmenopausal women with above-normal blood pressure. The clinical trial registry number is NCT03370991 (https://clinicaltrials.gov).

Machine Learning Approach for Improved Longitudinal Prediction of Progression from Mild Cognitive Impairment to Alzheimer's Disease.

Mild cognitive impairment (MCI) is cognitive decline that can indicate future risk of Alzheimer's disease (AD). We developed and validated a machine learning algorithm (MLA), based on a gradient-boosted tree ensemble method, to analyze phenotypic data for individuals 55-88 years old (n = 493) diagnosed with MCI. Data were analyzed within multiple prediction windows and averaged to predict progression to AD within 24-48 months. The MLA outperformed the mini-mental state examination (MMSE) and three comparison models at all prediction windows on most metrics. Exceptions include sensitivity at 18 months (MLA and MMSE each achieved 0.600); and sensitivity at 30 and 42 months (MMSE marginally better). For all prediction windows, the MLA achieved AUROC ≥ 0.857 and NPV ≥ 0.800. With averaged data for the 24-48-month lookahead timeframe, the MLA outperformed MMSE on all metrics. This study demonstrates that machine learning may provide a more accurate risk assessment than the standard of care. This may facilitate care coordination, decrease healthcare expenditures, and maintain quality of life for patients at risk of progressing from MCI to AD.

Rho-kinase inhibition improves haemodynamic responses and circulating ATP during hypoxia and moderate intensity handgrip exercise in healthy older adults.

Skeletal muscle haemodynamics and circulating adenosine triphosphate (ATP) responses during hypoxia and exercise are blunted in older (OA) vs. young (YA) adults, which may be associated with impaired red blood cell (RBC) ATP release. Rho-kinase inhibition improves deoxygenation-induced ATP release from OA isolated RBCs. We tested the hypothesis that Rho-kinase inhibition (via fasudil) in vivo would improve local haemodynamic and ATP responses during hypoxia and exercise in OA. Healthy YA (25 ± 3 years; n = 12) and OA (65 ± 5 years; n = 13) participated in a randomized, double-blind, placebo-controlled, crossover study on two days (≥5 days between visits). A forearm deep venous catheter was used to administer saline/fasudil and sample venous plasma ATP ([ATP]V ). Forearm vascular conductance (FVC) and [ATP]V were measured at rest, during isocapnic hypoxia (80% SpO2${S_{{\rm{p}}{{\rm{O}}_{\rm{2}}}}}$ ), and during graded rhythmic handgrip exercise that was similar between groups (5, 15 and 25% maximum voluntary contraction (MVC)). Isolated RBC ATP release was measured during normoxia/hypoxia. With saline, ΔFVC was lower (P < 0.05) in OA vs. YA during hypoxia (∼60%) and during 15 and 25% MVC (∼25-30%), and these impairments were abolished with fasudil. Similarly, [ATP]V and ATP effluent responses from normoxia to hypoxia and rest to 25% MVC were lower in OA vs. YA and improved with fasudil (P < 0.05). Isolated RBC ATP release during hypoxia was impaired in OA vs. YA (∼75%; P < 0.05), which tended to improve with fasudil in OA (P = 0.082). These data suggest Rho-kinase inhibition improves haemodynamic responses to hypoxia and moderate intensity exercise in OA, which may be due in part to improved circulating ATP. KEY POINTS: Skeletal muscle blood flow responses to hypoxia and exercise are impaired with age. Blunted increases in circulating ATP, a vasodilator, in older adults may contribute to age-related impairments in haemodynamics. Red blood cells (RBCs) are a primary source of circulating ATP, and treating isolated RBCs with a Rho-kinase inhibitor improves age-related impairments in deoxygenation-induced RBC ATP release. In this study, treating healthy older adults systemically with the Rho-kinase inhibitor fasudil improved blood flow and circulating ATP responses during hypoxia and moderate intensity handgrip exercise compared to young adults, and also tended to improve isolated RBC ATP release. Improved blood flow regulation with fasudil was also associated with increased skeletal muscle oxygen delivery during hypoxia and exercise in older adults. This is the first study to demonstrate that Rho-kinase inhibition can significantly improve age-related impairments in haemodynamic and circulating ATP responses to physiological stimuli, which may have therapeutic implications.

Carbohydrate ingestion attenuates cognitive dysfunction following long-duration exercise in the heat in humans.

INTRODUCTION: To determine if electrolyte or carbohydrate supplementation vs. water would limit the magnitude of dehydration and decline in cognitive function in humans following long-duration hyperthermic-exercise. METHODS: 24 subjects performed 3 visits of 2 h walking (3mph/7% grade) in an environmental chamber (33 °C/10% relative humidity). In random order, subjects consumed water (W), electrolytes (Gatorade Zero; E), or electrolytes+carbohydrates (Gatorade; E+C). Throughout exercise (EX), subjects carried a 23 kg pack and drank ad-libitum. Pre-and post-EX, body mass (BM) and plasma osmolality (pOsm) were measured. Physiological Strain Index (PSI) and core temperature (TC) were recorded every 15 min. Plasma glucose (GLU) was measured every 30 min. Cognitive processing (SCWT) was measured post-EX and compared to baseline (BL). A subset of 8 subjects performed a normothermic (N) protocol (21 °C/ambient humidity) to ascertain how the exercise stimulus influenced hydration status and cognition without heat. RESULTS: There were no significant differences between fluid conditions (W, E, E+C) for BM loss (Δ2.5 ± 0.2, 2.5 ± 0.2, 2.3 ± 0.2 kg), fluid consumption (1.9 ± 0.2, 1.9 ± 0.2, 1.8 ± 0.2L), pOsm (Δ1.5 ± 2.7, 2.2 ± 2.4, 2.0 ± 1.5 mmol/L), peak-PSI (7.5 ± 0.4, 7.0 ± 0.6, 7.9 ± 0.5), and peak-TC (38.7 ± 0.1, 38.6 ± 0.2, 38.8 ± 0.2 °C). GLU decreased significantly in W and E, whereas it increased above BL in E+C at 60, 90, and 120 min (P < 0.05). Compared to BL values (43.6 ± 26 ms), SCWT performance significantly decreased in all conditions (463 ± 93, 422 ± 83, 140 ± 52 ms, P < 0.05). Importantly, compared to W and E, the impairment in SCWT was significantly attenuated in E+C (P < 0.05). As expected, when compared to the heat-stress protocol (W, E, E+C), N resulted in lower BM loss, fluid consumption, and peak-PSI (1.1 ± 0.1 kg, 1.2 ± 0.7L, 4.8, respectively), and improved SCWT performance. CONCLUSIONS: These data are the first to suggest that, independent of supplementation variety, cognitive processing significantly decreases immediately following long-duration exercise in the heat in healthy humans. Compared to water and fluids supplemented with only electrolytes, fluids supplemented with carbohydrates significantly blunts this decrease in cognitive function.

ATP and acetylcholine interact to modulate vascular tone and α1-adrenergic vasoconstriction in humans.

The vascular endothelium senses and integrates numerous inputs to regulate vascular tone. Recent evidence reveals complex signal processing within the endothelium, yet little is known about how endothelium-dependent stimuli interact to regulate blood flow. We tested the hypothesis that combined stimulation of the endothelium with adenosine triphosphate (ATP) and acetylcholine (ACh) elicits greater vasodilation and attenuates α1-adrenergic vasoconstriction compared with combination of ATP or ACh with the endothelium-independent dilator sodium nitroprusside (SNP). We assessed forearm vascular conductance (FVC) in young adults (6 women, 7 men) during local intra-arterial infusion of ATP, ACh, or SNP alone and in the following combinations: ATP + ACh, SNP + ACh, and ATP + SNP, wherein the second dilator was coinfused after attaining steady state with the first dilator. By design, each dilator evoked a similar response when infused separately (ΔFVC, ATP: 48 ± 4; ACh: 57 ± 6; SNP: 53 ± 6 mL·min-1·100 mmHg-1; P ≥ 0.62). Combined infusion of the endothelium-dependent dilators evoked greater vasodilation than combination of either dilator with SNP (ΔFVC from first dilator, ATP + ACh: 45 ± 9 vs. SNP + ACh: 18 ± 7 and ATP + SNP: 26 ± 4 mL·min-1·100 mmHg-1, P < 0.05). Phenylephrine was subsequently infused to evaluate α1-adrenergic vasoconstriction. Phenylephrine elicited less vasoconstriction during infusion of ATP or ACh versus SNP (ΔFVC, -25 ± 3 and -29 ± 4 vs. -48 ± 3%; P < 0.05). The vasoconstrictor response to phenylephrine was further diminished during combined infusion of ATP + ACh (-13 ± 3%; P < 0.05 vs. ATP or ACh alone) and was less than that observed when either dilator was combined with SNP (SNP + ACh: -26 ± 3%; ATP + SNP: -31 ± 4%; both P < 0.05 vs. ATP + ACh). We conclude that endothelium-dependent agonists interact to elicit vasodilation and limit α1-adrenergic vasoconstriction in humans.NEW & NOTEWORTHY The results of this study highlight the vascular endothelium as a critical site for integration of vasomotor signals in humans. To our knowledge, this is the first study to demonstrate that combined stimulation of the endothelium with ATP and ACh results in enhanced vasodilation compared with combination of either ATP or ACh with an endothelium-independent dilator. Furthermore, we show that ATP and ACh interact to modulate α1-adrenergic vasoconstriction in human skeletal muscle in vivo.

Comprehensive assessment of cardiovascular structure and function and disease risk in middle-aged ultra-endurance athletes.

BACKGROUND AND AIMS: Recent studies suggest that long-term endurance training may be damaging to the heart, thus increasing cardiovascular disease (CVD) risk. However, studies utilizing cardiac imaging are conflicting and lack measures of central and peripheral vascular structure and function, which are also independently predictive of CVD events. METHODS: We performed a comprehensive assessment of cardiovascular structure and function in long-term (≥ 10 years) ultra-endurance athletes (ATH, 14 M/11 F, 50 ± 1 y) and physically active controls (CON, 9 M/9 F, 49 ± 2 y). RESULTS: As expected, left ventricular mass and end-diastolic volume (echocardiography) were greater in ATH vs CON, whereas there was no difference in cardiac function at rest. Coronary artery calcium scores (computed tomography) were not statistically different between groups. There was no evidence of myocardial fibrosis (contrast magnetic resonance imaging) in any subject. Aortic stiffness (carotid-femoral pulse wave velocity) was lower in ATH vs CON (6.2 ± 0.2 vs 6.9 ± 0.2 m/s, p < 0.05), whereas carotid intima-media thickness (ultrasound) was not different between groups. Peripheral vascular endothelial function (flow-mediated vasodilation of the brachial artery) and microvascular function (peak blood velocity) in response to 5 min of forearm ischemia were not different between groups. Furthermore, there was no difference in 10-year coronary heart disease risk (ATH; 2.3 ± 0.5 vs CON; 1.6 ± 0.2%, p > 0.05). CONCLUSIONS: Our data indicate that middle-aged ultra-endurance ATH do not have marked signs of widespread cardiovascular dysfunction or elevated CHD risk compared to CON meeting physical activity guidelines.

Self-selected fluid volume and flavor strength does not alter fluid intake, body mass loss, or physiological strain during moderate-intensity exercise in the heat.

INTRODUCTION: The purpose of this study was to determine the effects of ad libitum flavor and fluid intake on changes in body mass (BM) and physiological strain during moderate intensity exercise in the heat. METHODS: Ten subjects (24±3yrs, 7M/3F) performed 60 min of treadmill walking at 1.3 m/s and 7% grade in an environmental chamber set to 33 °C and 10% relative humidity while carrying a 22.7 kg pack on two different occasions. Subjects consumed either plain water or water plus flavor (Infuze), ad libitum, at each visit. Pre and post exercise, fluid consumption (change in fluid reservoir weight) and BM (nude) were measured. During exercise, heart rate (HR), systolic blood pressure (SBP), rate of perceived exertion (RPE), oxygen consumption (VO2), respiratory exchange ratio (RER), core temperature (TC), and physiological strain index (PSI) were recorded every 15 min during exercise. RESULTS: No significant differences were observed for fluid consumption between fluid conditions (512 ± 97.2 mL water vs. 414.3 ± 62.5 mL Infuze). Despite a significant decrease from baseline, there were no significant differences in overall change of BM (Δ -1.18 vs. -0.64 Kg) or percent body weight loss for water and Infuze conditions, respectively (1.58 ± 0.6 and 0.79 ± 0.2%). Furthermore, there were no significant differences in HR (144 ± 6 vs. 143 ± 8 bpm), SBP (157 ± 5 vs. 155 ± 5 mmHg), RPE, VO2 (27.4 ± 0.9 vs. 28.1 ± 1.2 ml/Kg/min), RER, TC (38.1 ± 0.1 vs. 37.0 ± 0.1 °C), and peak PSI (5.4 ± 0.4 vs. 5.7 ± 0.8) between conditions. CONCLUSIONS: Offering individuals the choice to actively manipulate flavor strength did not significantly influence ad libitum fluid consumption, fluid loss, or physiological strain during 60 min of moderate intensity exercise in the heat.

Augmentation of endothelium-dependent vasodilatory signalling improves functional sympatholysis in contracting muscle of older adults.

KEY POINTS: The ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction (functional sympatholysis) is critical for maintaining blood flow during exercise-mediated sympathoexcitation. Functional sympatholysis and endothelial function are impaired with ageing, resulting in compromised blood flow and oxygen delivery to contracting skeletal muscle during exercise. In the present study, intra-arterial infusion of ACh or ATP to augment endothelium-dependent signalling during exercise attenuated α1 -adrenergic vasoconstriction in the contracting muscle of older adults. The vascular signalling mechanisms capable of functional sympatholysis are preserved in healthy ageing, and thus the age-related impairment in functional sympatholysis probably results from the loss of a functional signal (e.g. plasma [ATP]) as opposed to an intrinsic endothelial dysfunction. ABSTRACT: The ability of contracting skeletal muscle to attenuate sympathetic α-adrenergic vasoconstriction ('functional sympatholysis') is impaired with age. In young adults, increasing endothelium-dependent vasodilatory signalling during mild exercise augments sympatholysis. In the present study, we tested the hypothesis that increasing endothelium-dependent signalling during exercise in older adults can improve sympatholysis. In 16 older individuals (Protocol 1, n = 8; Protocol 2, n = 8), we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to local intra-arterial infusion of phenylephrine (PE; α1 -agonist) during (i) infusion of an endothelium-dependent vasodilator alone (Protocol 1: ACh or Protocol 2: low dose ATP); (ii) mild handgrip exercise (5% maximum voluntary contraction; MVC); (iii) moderate handgrip exercise (15% MVC); and (iv) mild or moderate handgrip exercise + infusion of ACh or ATP to augment endothelium-dependent signalling. PE caused robust vasoconstriction in resting skeletal muscle during control vasodilator infusions (ΔFVC: ACh: -31 ± 3 and ATP: -30 ± 4%). PE-mediated vasoconstriction was not attenuated by mild or moderate intensity exercise (ΔFVC: 5% MVC: -30 ± 9; 15% MVC: -33 ± 8%; P > 0.05 vs. control ACh and ATP), indicative of impaired sympatholysis, and ACh or ATP infusion during mild exercise did not impact this response. However, augmentation of endothelium-dependent signalling via infusion of ACh or ATP during moderate intensity exercise attenuated PE-mediated vasoconstriction (ΔFVC: -13 ± 1 and -19 ± 5%, respectively; P < 0.05 vs. all conditions). Our findings demonstrate that, given a sufficient stimulus, endothelium-dependent sympatholysis remains intact in older adults. Strategies aimed at activating such pathways represent a viable approach for improving sympatholysis and thus tissue blood flow and oxygen delivery in older adults.

KIR channel activation links local vasodilatation with muscle fibre recruitment during exercise in humans.

KEY POINTS: During exercise, blood flow to working skeletal muscle increases in parallel with contractile activity such that oxygen delivery is sufficient to meet metabolic demand. K+ released from active skeletal muscle fibres could facilitate vasodilatation in proportion to the degree of muscle fibre recruitment. Once released, K+ stimulates inwardly rectifying K+ (KIR ) channels on the vasculature to elicit an increase in blood flow. In the present study, we demonstrate that KIR channels mediate the rapid vasodilatory response to an increase in exercise intensity. We also show that KIR channels augment vasodilatation during exercise which demands greater muscle fibre recruitment independent of the total amount of work performed. These results suggest that K+ plays a key role in coupling the magnitude of vasodilatation to the degree of contractile activity. Ultimately, the findings from this study help us understand the signalling mechanisms that regulate muscle blood flow in humans. ABSTRACT: Blood flow to active skeletal muscle is augmented with greater muscle fibre recruitment. We tested whether activation of inwardly rectifying potassium (KIR ) channels underlies vasodilatation with elevated muscle fibre recruitment when work rate is increased (Protocol 1) or held constant (Protocol 2). We assessed forearm vascular conductance (FVC) during rhythmic handgrip exercise under control conditions and during local inhibition of KIR channels (intra-arterial BaCl2 ). In Protocol 1, healthy volunteers performed mild handgrip exercise for 3 min, then transitioned to moderate intensity for 30 s. BaCl2 eliminated vasodilatation during the first contraction at the moderate workload (ΔFVC, BaCl2 : -1 ± 17 vs. control: 30 ± 28 ml min-1  100 mmHg-1 ; n = 9; P = 0.004) and attenuated the 30 s area under the curve by 56 ± 14% (n = 9; P < 0.0001). In Protocol 2, participants performed two exercise bouts in which muscle fibre recruitment was manipulated while total contractile work was held constant via reciprocal changes in contraction frequency: (1) low fibre recruitment, with contractions at 12.5% maximal voluntary contraction once every 4 s and (2) high fibre recruitment, with contractions at 25% maximal voluntary contraction once every 8 s. Under control conditions, steady-state FVC was augmented in high vs. low fibre recruitment (211 ± 90 vs. 166 ± 73 ml min-1 ⋅100 mmHg-1 ; n = 10; P = 0.0006), whereas BaCl2 abolished the difference between high and low fibre recruitment (134 ± 59 vs. 134 ± 63 ml min-1  100 mmHg-1 ; n = 10; P = 0.85). These findings demonstrate that KIR channel activation is a key mechanism linking local vasodilatation with muscle fibre recruitment during exercise.

Rapid-onset vasodilator responses to exercise in humans: Effect of increased baseline blood flow.

NEW FINDINGS: What is the central question of this study? What is the effect of an elevated baseline blood flow, induced by high-dose intra-arterial infusion of either adenosine or ATP, on the rapid-onset vasodilatory response to a single forearm muscle contraction? What is the main finding and its importance? The peak response to a single contraction is unaffected by augmented baseline blood flow, and thus, is likely to be attributable to a feedforward vasodilatory mechanism. ABSTRACT: The hyperaemic responses to single muscle contractions are proportional to exercise intensity, which, in turn, is proportional to tissue metabolic demand. Hence, we tested the hypothesis that the rapid-onset vasodilatory response after a single muscle contraction would be unaffected when baseline blood flow was increased via high-dose intra-arterial infusion of either adenosine (ADO) or ATP. Twenty-four healthy young participants (28 ± 1 years) performed a single forearm contraction (20% maximal voluntary contraction) 75 min after commencement of a continuous infusion of ADO (n = 6), ATP (n = 8) or saline (control; n = 10). Brachial artery diameter and blood velocity were measured using Doppler ultrasound. Resting forearm vascular conductance (FVC; in millilitres per minute per 100 mmHg per decilitre of forearm volume) was significantly higher during ADO (33 ± 17) and ATP infusion (33 ± 17) compared with the control infusion (8 ± 3; P < 0.05). The peak FVCs post-contraction during ADO and ATP infusions were significantly greater than during the control infusion (P < 0.05), but not different from one another. The peak change in FVC from baseline was similar in all three conditions (control, 14 ± 1; ADO, 24 ± 2; and ATP, 23 ± 6; P = 0.15). Total FVC (area under the curve) did not differ significantly between ADO and ATP (333 ± 69 and 440 ± 125); however, total FVC during ATP infusion was significantly greater compared with the control value (150 ± 19; P < 0.05). We conclude that the peak response to a single contraction is unaffected by augmented baseline blood flow and is therefore likely to be attributable to a feedforward vasodilatory mechanism.

Reduced deformability contributes to impaired deoxygenation-induced ATP release from red blood cells of older adult humans.

KEY POINTS: Red blood cells (RBCs) release ATP in response to deoxygenation, which can increase blood flow to help match oxygen supply with tissue metabolic demand. This release of ATP is impaired in RBCs from older adults, but the underlying mechanisms are unknown. In this study, improving RBC deformability in older adults restored deoxygenation-induced ATP release, whereas decreasing RBC deformability in young adults reduced ATP release to the level of that of older adults. In contrast, treating RBCs with a phosphodiesterase 3 inhibitor did not affect ATP release in either age group, possibly due to intact intracellular signalling downstream of deoxygenation as indicated by preserved cAMP and ATP release responses to pharmacological Gi protein activation in RBCs from older adults. These findings are the first to demonstrate that the age-related decrease in RBC deformability is a primary mechanism of impaired deoxygenation-induced ATP release, which may have implications for treating impaired vascular control with advancing age. ABSTRACT: In response to haemoglobin deoxygenation, red blood cells (RBCs) release ATP, which binds to endothelial purinergic receptors and stimulates vasodilatation. This ATP release is impaired in RBCs from older vs. young adults, but the underlying mechanisms are unknown. Using isolated RBCs from young (24 ± 1 years) and older (65 ± 2 years) adults, we tested the hypothesis that age-related changes in RBC deformability (Study 1) and cAMP signalling (Study 2) contribute to the impairment. RBC ATP release during normoxia ( PO2 ∼112 mmHg) and hypoxia ( PO2 ∼20 mmHg) was quantified with the luciferin-luciferase technique following RBC incubation with Y-27632 (Rho-kinase inhibitor to increase deformability), diamide (cell-stiffening agent), cilostazol (phosphodiesterase 3 inhibitor), or vehicle control. The mean change in RBC ATP release from normoxia to hypoxia in control conditions was significantly impaired in older vs. young (∼50% vs. ∼120%; P < 0.05). RBC deformability was also lower in older vs. young as indicated by a higher RBC transit time (RCTT) measured by blood filtrometry (RCTT: 8.541 ± 0.050 vs. 8.234 ± 0.098 a.u., respectively; P < 0.05). Y-27632 improved RBC deformability (RCTT: 8.228 ± 0.083) and ATP release (111.7 ± 17.2%) in older and diamide decreased RBC deformability (RCTT: 8.955 ± 0.114) and ATP release (67.4 ± 11.8%) in young (P < 0.05), abolishing the age group differences (P > 0.05). Cilostazol did not change ATP release in either age group (P > 0.05), and RBC cAMP and ATP release to pharmacological Gi protein activation was similar in both groups (P > 0.05). We conclude that decreased RBC deformability is a primary contributor to age-related impairments in RBC ATP release, which may have implications for impaired vascular control with advancing age.

Escape, lysis, and feedback: endothelial modulation of sympathetic vasoconstriction.

The sympathetic nervous system exerts a vasoconstrictor influence over peripheral vascular beds that is counter-regulated by local vascular signaling mechanisms (i.e. sympathetic escape, sympatholysis, and myoendothelial feedback). The endothelium has emerged as a primary site for the regulation of sympathetic vasoconstriction through highly specialized cellular connections called myoendothelial projections (MEPs) that facilitate electrical coupling of endothelial and vascular smooth muscle cells. Endothelial derived hyperpolarization (EDH) via activation of IKCa channels is an important component of MEP-mediated feedback regulation of sympathetic vasoconstriction in animal models. Recent pharmacological data highlight the unique ability of EDH signaling to attenuate sympathetic vasoconstriction in humans.

Sustained exercise hyperemia during prolonged adenosine infusion in humans.

The contribution of Adenosine (ADO) to exercise hyperemia remains controversial and it is unknown whether ADO can evoke the prolonged vasodilation seen during exercise bouts. Therefore, we tested hypotheses in the human forearm during 3 h of intra-arterial high dose ADO infusion: (1) skeletal muscle blood flow would wane over time; (2) exercise hyperemic responses during ADO administration would be unaffected compared to baseline. Using sodium nitroprusside (SNP), we tested parallel hypotheses regarding nitric oxide (NO) in a separate group of participants. Seventeen young healthy participants (ADO: n = 9; SNP: n = 8) performed multiple rhythmic handgrip exercise bouts (20% of maximum), two during saline and five during 3 h of continuous drug infusion. Five minutes of ADO infusion resulted in a ~5-fold increase in forearm vascular conductance (FVC; 4.8 ± 0.6 vs. 24.2 ± 3.2 mL/min/100 mmHg, P < 0.05). SNP caused a ~4-fold increase (4.4 ± 0.6 vs. 16.6 ± 2 mL/min/100 mmHg, P < 0.05). FVC did not wane over time with ADO (24.2 ± 3.2 and 22 ± 1.2 mL/min/100 mmHg [P > 0.05]) or SNP (16.6 ± 2 and 14.1 ± 2.4 mL/min/ 100 mmHg [P > 0.05]) at 5 versus 150 min. Superimposed exercise during ADO or SNP infusions evoked marked and consistent additional dilation over the course of the infusions. Our findings demonstrate that in humans there is no reduction in endothelial or vascular smooth muscle responsiveness to the exogenous vasodilatory metabolites ADO and NO. Additionally, even in the presence of an exogenous vasodilator, superimposed exercise can cause significant hyperemia.

Amplification of endothelium-dependent vasodilatation in contracting human skeletal muscle: role of KIR channels.

KEY POINTS: In humans, the vasodilatory response to skeletal muscle contraction is mediated in part by activation of inwardly rectifying potassium (KIR ) channels. Evidence from animal models suggest that KIR channels serve as electrical amplifiers of endothelium-dependent hyperpolarization (EDH). We found that skeletal muscle contraction amplifies vasodilatation to the endothelium-dependent agonist ACh, whereas there was no change in the vasodilatory response to sodium nitroprusside, an endothelium-independent nitric oxide donor. Blockade of KIR channels reduced the exercise-induced amplification of ACh-mediated vasodilatation. Conversely, pharmacological activation of KIR channels in quiescent muscle via intra-arterial infusion of KCl independently amplified the vasodilatory response to ACh. This study is the first in humans to demonstrate that specific endothelium-dependent vasodilatory signalling is amplified in the vasculature of contracting skeletal muscle and that KIR channels may serve as amplifiers of EDH-like vasodilatory signalling in humans. ABSTRACT: The local vasodilatory response to muscle contraction is due in part to the activation of inwardly rectifying potassium (KIR ) channels. Evidence from animal models suggest that KIR channels function as 'amplifiers' of endothelium-dependent vasodilators. We tested the hypothesis that contracting muscle selectively amplifies endothelium-dependent vasodilatation via activation of KIR channels. We measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to local intra-arterial infusion of ACh (endothelium-dependent dilator) during resting conditions, handgrip exercise (5% maximum voluntary contraction) or sodium nitroprusside (SNP; endothelium-independent dilator) which served as a high-flow control condition (n = 7, young healthy men and women). Trials were performed before and after blockade of KIR channels via infusion of barium chloride. Exercise augmented peak ACh-mediated vasodilatation (ΔFVC saline: 117 ± 14; exercise: 236 ± 21 ml min-1 (100 mmHg)-1 ; P < 0.05), whereas SNP did not impact ACh-mediated vasodilatation. Blockade of KIR channels attenuated the exercise-induced augmentation of ACh. In eight additional subjects, SNP was administered as the experimental dilator. In contrast to ACh, exercise did not alter SNP-mediated vasodilatation (ΔFVC saline: 158 ± 35; exercise: 121 ± 22 ml min-1 (100 mmHg)-1 ; n.s.). Finally, in a subset of six subjects, direct pharmacological activation of KIR channels in quiescent muscle via infusion of KCl amplified peak ACh-mediated vasodilatation (ΔFVC saline: 97 ± 15, KCl: 142 ± 16 ml min-1  (100 mmHg)-1 ; respectively; P < 0.05). These findings indicate that skeletal muscle contractions selectively amplify endothelium-dependent vasodilatory signalling via activation of KIR channels, and this may be an important mechanism contributing to the normal vasodilatory response to exercise in humans.

Elevated extracellular potassium prior to muscle contraction reduces onset and steady-state exercise hyperemia in humans.

The increase in interstitial potassium (K+) during muscle contractions is thought to be a vasodilatory signal that contributes to exercise hyperemia. To determine the role of extracellular K+ in exercise hyperemia, we perfused skeletal muscle with K+ before contractions, such that the effect of any endogenously-released K+ would be minimized. We tested the hypothesis that local, intra-arterial infusion of potassium chloride (KCl) at rest would impair vasodilation in response to subsequent rhythmic handgrip exercise in humans. In 11 young adults, we determined forearm blood flow (FBF) (Doppler ultrasound) and forearm vascular conductance (FVC) (FBF/mean arterial pressure) during 4 min of rhythmic handgrip exercise at 10% of maximal voluntary contraction during 1) control conditions, 2) infusion of KCl before the initiation of exercise, and 3) infusion of sodium nitroprusside (SNP) as a control vasodilator. Infusion of KCl or SNP elevated resting FVC similarly before the onset of exercise (control: 39 ± 6 vs. KCl: 81 ± 12 and SNP: 82 ± 13 ml·min-1·100 mmHg-1; both P < 0.05 vs. control). Infusion of KCl at rest diminished the hyperemic (ΔFBF) and vasodilatory (ΔFVC) response to subsequent exercise by 22 ± 5% and 30 ± 5%, respectively (both P < 0.05 vs. control), whereas SNP did not affect the change in FBF ( P = 0.74 vs. control) or FVC ( P = 0.61 vs. control) from rest to steady-state exercise. These findings implicate the K+ ion as an essential vasodilator substance contributing to exercise hyperemia in humans. NEW & NOTEWORTHY Our findings support a significant and obligatory role for potassium signaling in the local vasodilatory and hyperemic response to exercise in humans.

Inhibition of Na+ /K+ -ATPase and KIR channels abolishes hypoxic hyperaemia in resting but not contracting skeletal muscle of humans.

KEY POINTS: Increasing blood flow (hyperaemia) to exercising muscle helps match oxygen delivery and metabolic demand. During exercise in hypoxia, there is a compensatory increase in muscle hyperaemia that maintains oxygen delivery and tissue oxygen consumption. Nitric oxide (NO) and prostaglandins (PGs) contribute to around half of the augmented hyperaemia during hypoxic exercise, although the contributors to the remaining response are unknown. In the present study, inhibiting NO, PGs, Na+ /K+ -ATPase and inwardly rectifying potassium (KIR ) channels did not blunt augmented hyperaemia during hypoxic exercise beyond previous observations with NO/PG block alone. Furthermore, although inhibition of only Na+ /K+ -ATPase and KIR channels abolished hyperaemia during hypoxia at rest, it had no effect on augmented hyperaemia during hypoxic exercise. This is the first study in humans to demonstrate that Na+ /K+ -ATPase and KIR channel activation is required for augmented muscle hyperaemia during hypoxia at rest but not during hypoxic exercise, thus providing new insight into vascular control. ABSTRACT: Exercise hyperaemia in hypoxia is augmented relative to the same exercise intensity in normoxia. During moderate-intensity handgrip exercise, endothelium-derived nitric oxide (NO) and vasodilating prostaglandins (PGs) contribute to ∼50% of the augmented forearm blood flow (FBF) response to hypoxic exercise (HypEx), although the mechanism(s) underlying the remaining response are unclear. We hypothesized that combined inhibition of NO, PGs, Na+ /K+ -ATPase and inwardly rectifying potassium (KIR ) channels would abolish the augmented hyperaemic response in HypEx. In healthy young adults, FBF responses were measured (Doppler ultrasound) and forearm vascular conductance was calculated during 5 min of rhythmic handgrip exercise at 20% maximum voluntary contraction under regional sympathoadrenal inhibition in normoxia and isocapnic HypEx (O2 saturation ∼80%). Compared to control, combined inhibition of NO, PGs, Na+ /K+ -ATPase and KIR channels (l-NMMA + ketorolac + ouabain + BaCl2; Protocol 1; n = 10) blunted the compensatory increase in FBF during HypEx by ∼50% (29 ± 6 mL min-1 vs. 62 ± 8 mL min-1 , respectively, P < 0.05). By contrast, ouabain + BaCl2 alone (Protocol 2; n = 10) did not affect this augmented hyperaemic response (50 ± 11 mL min-1 vs. 60 ± 13 mL min-1 , respectively, P > 0.05). However, the blocked condition in both protocols abolished the hyperaemic response to hypoxia at rest (P < 0.05). We conclude that activation of Na+ /K+ -ATPase and KIR channels is involved in the hyperaemic response to hypoxia at rest, although it does not contribute to the augmented exercise hyperaemia during hypoxia in humans.

Acute ingestion of dietary nitrate increases muscle blood flow via local vasodilation during handgrip exercise in young adults.

Dietary nitrate (NO3-) is converted to nitrite (NO2-) and can be further reduced to the vasodilator nitric oxide (NO) amid a low O2 environment. Accordingly, dietary NO3- increases hind limb blood flow in rats during treadmill exercise; however, the evidence of such an effect in humans is unclear. We tested the hypothesis that acute dietary NO3- (via beetroot [BR] juice) increases forearm blood flow (FBF) via local vasodilation during handgrip exercise in young adults (n = 11; 25 ± 2 years). FBF (Doppler ultrasound) and blood pressure (Finapres) were measured at rest and during graded handgrip exercise at 5%, 15%, and 25% maximal voluntary contraction (MVC) lasting 4 min each. At the highest workload (25% MVC), systemic hypoxia (80% SaO2 ) was induced and exercise continued for three additional minutes. Subjects ingested concentrated BR (12.6 mmol nitrate (n = 5) or 16.8 mmol nitrate (n = 6) and repeated the exercise bout either 2 (12.6 mmol) or 3 h (16.8 mmol) postconsumption. Compared to control, BR significantly increased FBF at 15% MVC (184 ± 15 vs. 164 ± 15 mL/min), 25% MVC (323 ± 27 vs. 286 ± 28 mL/min), and 25% + hypoxia (373 ± 39 vs. 343 ± 32 mL/min) and this was due to increases in vascular conductance (i.e., vasodilation). The effect of BR on hemodynamics was not different between the two doses of BR ingested. Forearm VO2 was also elevated during exercise at 15% and 25% MVC. We conclude that acute increases in circulating NO3- and NO2- via BR increases muscle blood flow during moderate- to high-intensity handgrip exercise via local vasodilation. These findings may have important implications for aging and diseased populations that demonstrate impaired muscle perfusion and exercise intolerance.

Sympatholytic effect of intravascular ATP is independent of nitric oxide, prostaglandins, Na+ /K+ -ATPase and KIR channels in humans.

KEY POINTS: Intravascular ATP attenuates sympathetic vasoconstriction (sympatholysis) similar to what is observed in contracting skeletal muscle of humans, and may be an important contributor to exercise hyperaemia. Similar to exercise, ATP-mediated vasodilatation occurs via activation of inwardly rectifying potassium channels (KIR ), and synthesis of nitric oxide (NO) and prostaglandins (PG). However, recent evidence suggests that these dilatatory pathways are not obligatory for sympatholysis during exercise; therefore, we tested the hypothesis that the ability of ATP to blunt α1 -adrenergic vasoconstriction in resting skeletal muscle would be independent of KIR , NO, PGs and Na+ /K+ -ATPase activity. Blockade of KIR channels alone or in combination with NO, PGs and Na+ /K+ -ATPase significantly reduced the vasodilatatory response to ATP, although intravascular ATP maintained the ability to attenuate α1 -adrenergic vasoconstriction. This study highlights similarities in the vascular response to ATP and exercise, and further supports a potential role of intravascular ATP in blood flow regulation during exercise in humans. ABSTRACT: Exercise and intravascular ATP elicit vasodilatation that is dependent on activation of inwardly rectifying potassium (KIR ) channels, with a modest reliance on nitric oxide (NO) and prostaglandin (PG) synthesis. Both exercise and intravascular ATP attenuate sympathetic α-adrenergic vasoconstriction (sympatholysis). However, KIR channels, NO, PGs and Na+ /K+ -ATPase activity are not obligatory to observe sympatholysis during exercise. To further determine similarities between exercise and intravascular ATP, we tested the hypothesis that inhibition of KIR channels, NO and PG synthesis, and Na+ /K+ -ATPase would not alter the ability of ATP to blunt α1 -adrenergic vasoconstriction. In healthy subjects, we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to intra-arterial infusion of phenylephrine (PE; α1 -agonist) during ATP or control vasodilatator infusion, before and after KIR channel inhibition alone (barium chloride; n = 7; Protocol 1); NO (l-NMMA) and PG (ketorolac) inhibition alone, or combined NO, PGs, Na+ /K+ -ATPase (ouabain) and KIR channel inhibition (n = 6; Protocol 2). ATP attenuated PE-mediated vasoconstriction relative to adenosine (ADO) and sodium nitroprusside (SNP) (PE-mediated ΔFVC: ATP: -16 ± 2; ADO: -38 ± 6; SNP: -59 ± 6%; P < 0.05 vs. ADO and SNP). Blockade of KIR channels alone or combined with NO, PGs and Na+ /K+ -ATPase, attenuated ATP-mediated vasodilatation (∼35 and ∼60% respectively; P < 0.05 vs. control). However, ATP maintained the ability to blunt PE-mediated vasoconstriction (PE-mediated ΔFVC: KIR blockade alone: -6 ± 5%; combined blockade:-4 ± 14%; P > 0.05 vs. control). These findings demonstrate that intravascular ATP modulates α1 -adrenergic vasoconstriction via pathways independent of KIR channels, NO, PGs and Na+ /K+ -ATPase in humans, consistent with a role for endothelium-derived hyperpolarization in functional sympatholysis.