Stefan P. Mortensen

Resveratrol blunts the positive effects of exercise training on cardiovascular health in aged men

•  In rodents, resveratrol has been shown to enhance training‐induced changes in cardiovascular function, exercise performance and the retardation of atherosclerosis. We examined the effect of 8 weeks of exercise training with and without concomitant resveratrol supplementation in aged men. •  Exercise training potently improved blood pressure, blood cholesterol, maximal oxygen uptake and the plasma lipid profile. •  Resveratrol supplementation reduced the positive effect of exercise training on blood pressure, blood cholesterol and maximal oxygen uptake and did not affect the retardation of atherosclerosis. •  Whereas exercise training improved formation of the vasodilator prostacyclin, concomitant resveratrol supplementation caused a shift in vasoactive systems favouring vasoconstriction. •  The present study is the first to demonstrate negative effects of resveratrol on training‐induced improvements in cardiovascular health parameters in humans and adds to the growing body of evidence questioning the positive effects of resveratrol supplementation in humans.

Erythrocytes and the regulation of human skeletal muscle blood flow and oxygen delivery: role of erythrocyte count and oxygenation state of haemoglobin

Blood flow to dynamically contracting myocytes is regulated to match O2 delivery to metabolic demand. The red blood cell (RBC) itself functions as an O2 sensor, contributing to the control of O2 delivery by releasing the vasodilators ATP and S‐nitrosohaemoglobin with the offloading of O2 from the haemoglobin molecule. Whether RBC number is sensed remains unknown. To investigate the role of RBC number, in isolation and in combination with alterations in blood oxygenation, on muscle and systemic perfusion, we measured local and central haemodynamics during one‐legged knee‐extensor exercise (∼50% peak power) in 10 healthy males under conditions of normocythaemia (control), anaemia, anaemia + plasma volume expansion (PVX), anaemia + PVX + hypoxia, polycythaemia, polycythaemia + hyperoxia and polycythaemia + hypoxia, which changed either RBC count alone or both RBC count and oxyhaemoglobin. Leg blood flow (LBF), cardiac output (Q) and vascular conductance did not change with either anaemia or polycythaemia alone. However, LBF increased with anaemia + PVX (28 ± 4%) and anaemia + PVX + hypoxia (46 ± 6%) and decreased with polycythaemia + hyperoxia (18 ± 5%). LBF and Q with anaemia + PVX + hypoxia (8.0 ± 0.5 and 15.8 ± 0.7 l min−1, respectively) equalled those during maximal knee‐extensor exercise. Collectively, LBF and vascular conductance were intimately related to leg arterial–venous (a–v) O2 difference (r2= 0.89–0.93; P < 0.001), suggesting a pivotal role of blood O2 gradients in muscle microcirculatory control. The systemic circulation accommodated to the changes in muscle perfusion. Our results indicate that, when coping with severe haematological challenges, local regulation of skeletal muscle blood flow and O2 delivery primarily senses alterations in the oxygenation state of haemoglobin and, to a lesser extent, alterations in the number of RBCs and haemoglobin molecules.

Restrictions in systemic and locomotor skeletal muscle perfusion, oxygen supply and VO2 during high-intensity whole-body exercise in humans.

Perfusion to exercising skeletal muscle is regulated to match O2 delivery to the O2 demand, but this regulation might be compromised during or approaching maximal whole‐body exercise as muscle blood flow for a given work rate is blunted. Whether muscle perfusion is restricted when there is an extreme metabolic stimulus to vasodilate during supramaximal exercise remains unknown. To examine the regulatory limits of systemic and muscle perfusion in exercising humans, we measured systemic and leg haemodynamics, O2 transport, and , and estimated non‐locomotor tissue perfusion during constant load supramaximal cycling (498 ± 16 W; 110% of peak power; mean ±s.e.m.) in addition to both incremental cycling and knee‐extensor exercise to exhaustion in 13 trained males. During supramaximal cycling, cardiac output (), leg blood flow (LBF), and systemic and leg O2 delivery and reached peak values after 60–90 s and thereafter levelled off at values similar to or ∼6% (P < 0.05) below maximal cycling, while upper body blood flow remained unchanged (∼5.5 l min−1). In contrast, and LBF increased linearly until exhaustion during one‐legged knee‐extensor exercise accompanying increases in non‐locomotor tissue blood flow to ∼12 l min−1. At exhaustion during cycling compared to knee‐extensor exercise, , LBF, leg vascular conductance, leg O2 delivery and leg for a given power were reduced by 32–47% (P < 0.05). In conclusion, locomotor skeletal muscle perfusion is restricted during maximal and supramaximal whole–body exercise in association with a plateau in and limb vascular conductance. These observations suggest that limits of cardiac function and muscle vasoconstriction underlie the inability of the circulatory system to meet the increasing metabolic demand of skeletal muscles and other tissues during whole‐body exercise.

Vasodilator interactions in skeletal muscle blood flow regulation

Abstract  During exercise, oxygen delivery to skeletal muscle is elevated to meet the increased oxygen demand. The increase in blood flow to skeletal muscle is achieved by vasodilators formed locally in the muscle tissue, either on the intraluminal or on the extraluminal side of the blood vessels. A number of vasodilators have been shown to bring about this increase in blood flow and, importantly, interactions between these compounds seem to be essential for the precise regulation of blood flow. Two compounds stand out as central in these vasodilator interactions: nitric oxide (NO) and prostacyclin. These two vasodilators are both stimulated by several compounds, e.g. adenosine, ATP, acetylcholine and bradykinin, and are affected by mechanically induced signals, such as shear stress. NO and prostacyclin have also been shown to interact in a redundant manner where one system can take over when formation of the other is compromised. Although numerous studies have examined the role of single and multiple pharmacological inhibition of different vasodilator systems, and important vasodilators and interactions have been identified, a large part of the exercise hyperaemic response remains unexplained. It is plausible that this remaining hyperaemia may be explained by cAMP‐ and cGMP‐independent smooth muscle relaxation, such as effects of endothelial derived hyperpolarization factors (EDHFs) or through metabolic modulation of sympathetic effects. The nature and role of EDHF as well as potential novel mechanisms in muscle blood flow regulation remain to be further explored to fully elucidate the regulation of exercise hyperaemia.

ATP-induced vasodilation and purinergic receptors in the human leg: roles of nitric oxide, prostaglandins, and adenosine

Plasma ATP is thought to contribute to the local regulation of skeletal muscle blood flow. Intravascular ATP infusion can induce profound limb muscle vasodilatation, but the purinergic receptors and downstream signals involved in this response remain unclear. This study investigated: 1) the role of nitric oxide (NO), prostaglandins, and adenosine as mediators of ATP-induced limb vasodilation and 2) the expression and distribution of purinergic P(2) receptors in human skeletal muscle. Systemic and leg hemodynamics were measured before and during 5-7 min of femoral intra-arterial infusion of ATP [0.45-2.45 micromol/min] in 19 healthy male subjects with and without coinfusion of N(G)-monomethyl-l-arginine (l-NMMA; NO formation inhibitor; 12.3 +/- 0.3 (SE) mg/min), indomethacin (INDO; prostaglandin formation blocker; 613 +/- 12 microg/min), and/or theophylline (adenosine receptor blocker; 400 +/- 26 mg). During control conditions, ATP infusion increased leg blood flow (LBF) from baseline conditions by 1.82 +/- 0.14 l/min. When ATP was coinfused with either l-NMMA, INDO, or l-NMMA + INDO combined, the increase in LBF was reduced by 14 +/- 6, 15 +/- 9, and 39 +/- 8%, respectively (all P < 0.05), and was associated with a parallel lowering in leg vascular conductance and cardiac output and a compensatory increase in leg O(2) extraction. Infusion of theophylline did not alter the ATP-induced leg hyperemia or systemic variables. Real-time PCR analysis of the mRNA content from the vastus lateralis muscle of eight subjects showed the highest expression of P(2Y2) receptors of the 10 investigated P(2) receptor subtypes. Immunohistochemistry showed that P(2Y2) receptors were located in the endothelium of microvessels and smooth muscle cells, whereas P(2X1) receptors were located in the endothelium and the sacrolemma. Collectively, these results indicate that NO and prostaglandins, but not adenosine, play a role in ATP-induced vasodilation in human skeletal muscle. The expression and localization of the nucleotide selective P(2Y2) and P(2X1) receptors suggest that these receptors may mediate ATP-induced vasodilation in skeletal muscle.

Haemodynamic responses to exercise, ATP infusion and thigh compression in humans: insight into the role of muscle mechanisms on cardiovascular function

The muscle pump and muscle vasodilatory mechanims are thought to play important roles in increasing and maintaining muscle perfusion and cardiac output during exercise, but their actual contributions remain uncertain. To evaluate the role of the skeletal muscle pump and vasodilatation on cardiovascular function during exercise, we determined leg and systemic haemodynamic responses in healthy men during (1) incremental one‐legged knee‐extensor exercise, (2) step‐wise femoral artery ATP infusion at rest, (3) passive exercise (n= 10), (4) femoral vein or artery ATP infusion (n= 6), and (5) cyclic thigh compressions at rest and during passive and voluntary exercise (n= 7). Incremental exercise resulted in progressive increases in leg blood flow (ΔLBF 7.4 ± 0.7 l min−1), cardiac output ( 8.7 ± 0.7 l min−1), mean arterial pressure (ΔMAP 51 ± 5 mmHg), and leg and systemic oxygen delivery and . Arterial ATP infusion resulted in similar increases in , LBF, and systemic and leg oxygen delivery, but central venous pressure and muscle metabolism remained unchanged and MAP was reduced. In contrast, femoral vein ATP infusion did not alter LBF, or MAP. Passive exercise also increased blood flow (ΔLBF 0.7 ± 0.1 l min−1), yet the increase in muscle and systemic perfusion, unrelated to elevations in aerobic metabolism, accounted only for ∼5% of peak exercise hyperaemia. Likewise, thigh compressions alone or in combination with passive exercise increased blood flow (ΔLBF 0.5–0.7 l min−1) without altering , MAP or . These findings suggest that the skeletal muscle pump is not obligatory for sustaining venous return, central venous pressure, stroke volume and or maintaining muscle blood flow during one‐legged exercise in humans. Further, its contribution to muscle and systemic peak exercise hyperaemia appears to be minimal in comparison to the effects of muscle vasodilatation.

Lifelong physical activity preserves functional sympatholysis and purinergic signalling in the ageing human leg

•  Ageing is associated with a reduced exercise hyperaemia and impaired ability to override sympathetic vasoconstrictor activity (functional sympatholysis). •  We find that sedentary elderly have a lower vasodilator response to ACh and ATP in the leg compared to young, but also that this age‐related reduction is partially (ACh) or completely (ATP) offset in lifelong physically active elderly subjects. •  An increase in sympathetic vasoconstrictor activity induced by tyramine reduces exercise hyperaemia in sedentary elderly, but not active elderly and young subjects. •  Interstitial ATP levels during exercise and P2Y2 receptor content are more related to the physical activity level than age. •  Physical activity can prevent the age‐related impairment in functional sympatholysis and maintain a sufficient O2 delivery during moderate intensity exercise despite a loss of endothelial function.

Muscle interstitial ATP and norepinephrine concentrations in the human leg during exercise and ATP infusion

ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanisms remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on leg muscle interstitial ATP and norepinephrine (NE) concentrations to gain insight into the interstitial and intravascular mechanisms by which ATP causes muscle vasodilation and sympatholysis. Leg hemodynamics and muscle interstitial nucleotide and NE concentrations were measured during 1) femoral arterial ATP infusion (0.42 +/- 0.04 and 2.26 +/- 0.52 micromol/min; mean +/- SE) and 2) one-leg knee-extensor exercise (18 +/- 0 and 37 +/- 2 W) in 10 healthy men. Arterial ATP infusion and exercise increased leg blood flow (LBF) in the experimental leg from approximately 0.3 l/min at baseline to 4.2 +/- 0.3 and 4.6 +/- 0.5 l/min, respectively, whereas it was reduced or unchanged in the control leg. During arterial ATP infusion, muscle interstitial ATP, ADP, AMP, and adenosine concentrations remained unchanged in both legs, but muscle interstitial NE increased from approximately 5.9 nmol/l at baseline to 8.3 +/- 1.2 and 8.7 +/- 0.7 nmol/l in the experimental and control leg, respectively (P < 0.05), in parallel to a reduction in arterial pressure (P < 0.05). During exercise, however, interstitial ATP, ADP, AMP, and adenosine concentrations increased in the contracting muscle (P < 0.05), but not in inactive muscle, whereas interstitial NE concentrations increased similarly in both active and inactive muscles. These results suggest that the vasodilatory and sympatholytic effects of intraluminal ATP are mainly mediated via endothelial purinergic receptors. Intraluminal ATP and muscle contractions appear to modulate sympathetic nerve activity by inhibiting the effect of NE rather than blunting its local concentration.

Intravascular ADP and soluble nucleotidases contribute to acute prothrombotic state during vigorous exercise in humans

Extracellular ATP and ADP trigger vasodilatatory and prothrombotic signalling events in the vasculature. Here, we tested the hypothesis that nucleotide turnover is activated in the bloodstream of exercising humans thus contributing to the enhanced platelet reactivity and haemostasis. Right atrial, arterial and venous blood samples were collected from endurance‐trained athletes at rest, during submaximal and maximal cycle ergometer exercise, and after early recovery. ATP‐specific bioluminescent assay, together with high‐performance liquid chromatographic analysis, revealed that plasma ATP and ADP concentrations increased up to 2.5‐fold during maximal exercise. Subsequent flow cytometric analysis showed that plasma from exercising subjects significantly up‐regulated the surface expression of P‐selectin in human platelets and these prothrombotic effects were diminished after scavenging plasma nucleotides with exogenous apyrase. Next, using thin layer chromatographic assays with [γ‐32P]ATP and 3H/14C‐labelled nucleotides, we showed that two soluble nucleotide‐inactivating enzymes, nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate diphosphohydrolase, constitutively circulate in human bloodstream. Strikingly, serum nucleotide pyrophosphatase and hydrolase activities rose during maximal exercise by 20–25 and 80–100%, respectively, and then declined after 30 min recovery. Likewise, soluble nucleotidases were transiently up‐regulated in the venous blood of sedentary subjects during exhaustive exercise. Human serum also contains 5′‐nucleotidase, adenylate kinase and nucleoside diphosphate (NDP) kinase; however, these activities remain unchanged during exercise. In conclusion, intravascular ADP significantly augments platelet activity during strenuous exercise and these prothrombotic responses are counteracted by concurrent release of soluble nucleotide‐inactivating enzymes. These findings provide a novel insight into the mechanisms underlying the enhanced risk of occlusive thrombus formation under exercising conditions.

Interstitial and plasma adenosine stimulate nitric oxide and prostacyclin formation in human skeletal muscle.

One major unresolved issue in muscle blood flow regulation is that of the role of circulating versus interstitial vasodilatory compounds. The present study determined adenosine-induced formation of NO and prostacyclin in the human muscle interstitium versus in femoral venous plasma to elucidate the interaction and importance of these vasodilators in the 2 compartments. To this end, we performed experiments on humans using microdialysis technique in skeletal muscle tissue, as well as the femoral vein, combined with experiments on cultures of microvascular endothelial versus skeletal muscle cells. In young healthy humans, microdialysate was collected at rest, during arterial infusion of adenosine, and during interstitial infusion of adenosine through microdialysis probes inserted into musculus vastus lateralis. Muscle interstitial NO and prostacyclin increased with arterial and interstitial infusion of adenosine. The addition of adenosine to skeletal muscle cells increased NO formation (fluorochrome 4-amino-5-methylamino-2′,7-difluorescein fluorescence), whereas prostacyclin levels remained unchanged. The addition of adenosine to microvascular endothelial cells induced an increase in NO and prostacyclin levels. These findings provide novel insight into the role of adenosine in skeletal muscle blood flow regulation and vascular function by revealing that both interstitial and plasma adenosine have a stimulatory effect on NO and prostacyclin formation. In addition, both skeletal muscle and microvascular endothelial cells are potential mediators of adenosine-induced formation of NO in vivo, whereas only endothelial cells appear to play a role in adenosine-induced formation of prostacyclin.