Michael D. Delp

Effects of ageing and exercise training on endothelium‐dependent vasodilatation and structure of rat skeletal muscle arterioles

Ageing reduces endothelium‐dependent vasodilatation in humans and animals, and in humans, exercise training reverses the ageing‐associated reduction in endothelium‐dependent vasodilatation. The purpose of this study was to determine the mechanism(s) by which 10–12 weeks of treadmill exercise enhances endothelium‐dependent vasodilatation in muscles of differing fibre composition from young and old rats. Three‐ and 22‐month‐old male Fischer 344 rats were assigned to young sedentary, young exercise‐trained, old sedentary, or old exercise‐trained groups. Arterioles were isolated from the soleus and gastrocnemius muscles; luminal diameter changes were determined in response to the endothelium‐dependent vasodilator acetylcholine (ACh, 10−9–10−4 mol l−1) alone and in the presence of the nitric oxide synthase (NOS) inhibitor l‐NAME (10−5 mol l−1) or the combination of l‐NAME and the cyclooxygenase inhibitor indomethacin (10−5 mol l−1). Training ameliorated the ageing‐induced reduction in endothelium‐dependent vasodilatation in soleus muscle arterioles. Treatment with l‐NAME alone and in combination with indomethacin abolished differences in ACh vasodilatation occurring with ageing and training. Expression of endothelial NOS (eNOS) mRNA in soleus arterioles was unaltered by ageing, whereas eNOS protein was increased with age; training elevated both eNOS mRNA and protein. In gastrocnemius muscle arterioles, ageing did not alter maximal vasodilatation, but ageing and training increased maximal arteriolar diameter. These results demonstrate that ageing‐induced reductions and training‐induced enhancement of endothelial vasodilatation both occur through the nitric oxide signalling mechanism in highly oxidative skeletal muscle, but ageing and training do not appear to act on the same portion of the signalling cascade.

Effects of ageing and exercise training on eNOS uncoupling in skeletal muscle resistance arterioles

Reduced availability of tetrahydrobiopterin (BH4) contributes to the age‐related decline of nitric oxide (NO)‐mediated vasodilatation of soleus muscle arterioles. Depending on availability of substrate and/or necessary co‐factors, endothelial nitric oxide synthase (eNOS) can generate NO and/or superoxide (O2−). We evaluated the effects of age and chronic exercise on flow‐induced vasodilatation and levels of NO and O2− in soleus muscle arterioles. Young (3 months) and old (22 months) male rats were exercise trained or remained sedentary (SED) for 10 weeks. Flow‐stimulated NO and O2−, as well as BH4 and l‐arginine content, were determined in soleus muscle arterioles. Flow‐induced vasodilatation was assessed under control conditions and during the blockade of O2− and/or hydrogen peroxide. Exercise training enhanced flow‐induced vasodilatation in arterioles from young and old rats. Old age reduced, and exercise training restored, BH4 content and flow‐stimulated NO availability. Flow‐stimulated, eNOS‐derived O2− levels were higher in arterioles from old SED compared to those from young SED rats. Exercise training increased flow‐stimulated eNOS‐derived O2− levels in arterioles from young but not old rats. O2− scavenging with Tempol reduced flow‐induced vasodilatation from all groups except young SED rats. Addition of catalase to Tempol‐treated arterioles eliminated flow‐induced vasodilatation in arterioles from all groups. Catalase reduced flow‐induced vasodilatation from all groups. In Tempol‐treated arterioles, flow‐induced vasodilatation was restored by deferoxamine, an iron chelator. These data indicate that uncoupling of eNOS contributes to the age‐related decline in flow‐induced vasodilatation; however, reactive oxygen species are required for flow‐induced vasodilatation in soleus muscle arterioles from young and old rats.

Regional variations of contractile activity in isolated rat lymphatics.

Objective: To evaluate lymphatic contractile activity in different regions of the lymphatic system in a single animal model (the rat thoracic duct, mesenteric, cervical, and femoral lymphatics) in response to changes in lymph pressure and flow.

Ageing diminishes endothelium-dependent vasodilatation and tetrahydrobiopterin content in rat skeletal muscle arterioles.

Ageing reduces endothelium‐dependent vasodilatation through an endothelial nitric oxide synthase (NOS) signalling pathway. The purpose of this study was to determine whether arginase activity diminishes endothelium‐dependent vasodilatation in skeletal muscle arterioles from old rats, and whether NOS substrate (l‐arginine) and cofactor (tetrahydrobiopterin; BH4) concentrations are reduced. First‐order arterioles were isolated from the soleus muscle of young (6 months old) and old (24 months old) male Fischer 344 rats. In vitro changes in luminal diameter in response to stepwise increases in flow were determined in the presence of the NOS inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME, 10−5 mol l−1), the arginase inhibitor Nω‐hydroxy‐nor‐l‐arginine (NOHA, 5 × 10−4 mol l−1), exogenous l‐arginine (3 × 10−3 mol l−1) or the precursor for BH4 synthesis sepiapterin (1 μmol l−1). Arteriolar l‐arginine and BH4 content were determined via HPLC. Ageing decreased flow‐mediated vasodilatation by 52%, and this difference was abolished with NOS inhibition. Neither inhibition of arginase activity nor addition of exogenous l‐arginine had any effect on flow‐mediated vasodilatation; arteriolar l‐arginine content was also not different between age groups. BH4 content was lower in arterioles from old rats (94 ± 8 fmol (mg tissue)−1) relative to controls (234 ± 21 fmol (mg tissue)−1), and sepiapterin elevated flow‐mediated vasodilatation in arterioles from old rats. These results demonstrate that the impairment of endothelium‐dependent vasodilatation induced by old age is due to an altered nitric oxide signalling mechanism in skeletal muscle arterioles, but is not the result of increased arginase activity and limited l‐arginine substrate. Rather, the age‐related deficit in flow‐mediated vasodilatation appears to be the result, in part, of limited BH4 bioavailability.

Exercise increases blood flow to locomotor, vestibular, cardiorespiratory and visual regions of the brain in miniature swine

1 The purpose of these experiments was to use radiolabelled microspheres to measure blood flow distribution within the brain, and in particular to areas associated with motor function, maintenance of equilibrium, cardiorespiratory control, vision, hearing and smell, at rest and during exercise in miniature swine. Exercise consisted of steady‐state treadmill running at intensities eliciting 70 and 100 % maximal oxygen consumption (V̇O2,max). 2 Mean arterial pressure was elevated by 17 and 26 % above that at rest during exercise at 70 and 100 %V̇O2,max, respectively. 3 Mean brain blood flow increased 24 and 25 % at 70 and 100 %V̇O2,max, respectively. Blood flow was not locally elevated to cortical regions associated with motor and somatosensory functions during exercise, but was increased to several subcortical areas that are involved in the control of locomotion. 4 Exercise elevated perfusion and diminished vascular resistance in several regions of the brain related to the maintenance of equilibrium (vestibular nuclear area, cerebellar ventral vermis and floccular lobe), cardiorespiratory control (medulla and pons), and vision (dorsal occipital cortex, superior colliculi and lateral geniculate body). Conversely, blood flow to regions related to hearing (cochlear nuclei, inferior colliculi and temporal cortex) and smell (olfactory bulbs and rhinencephalon) were unaltered by exercise and associated with increases in vascular resistance. 5 The data indicate that blood flow increases as a function of exercise intensity to several areas of the brain associated with integrating sensory input and motor output (anterior and dorsal cerebellar vermis) and the maintenance of equilibrium (vestibular nuclei). Additionally, there was an intensity‐dependent decrease of vascular resistance in the dorsal cerebellar vermis.

Site- and compartment-specific changes in bone with hindlimb unloading in mature adult rats

The purpose of this study was to examine site- and compartment-specific changes in bone induced by hindlimb unloading (HU) in the mature adult male rat (6 months old). Tibiae, femora, and humeri were removed after 14, 21, and 28 days of HU for determination of bone mineral density (BMD) and geometry by peripheral quantitative computed tomography (pQCT), mechanical properties, and bone formation rate (BFR), and compared with baseline (0 day) and aging (28 day) controls. HU resulted in 20%-21% declines in cancellous BMD at the proximal tibia and femoral neck after 28 day HU vs. 0 day controls (CON). Cortical shell BMD at these sites was greater (by 4%-6%) in both 28 day HU and 28 day CON vs. 0 day CON animals, and nearly identical to that gain seen in the weight-bearing humerus. Mechanical properties at the proximal tibia exhibited a nonsignificant decline after HU vs. those of 0 day CON rats. At the femoral neck, a 10% decrement was noted in ultimate load in 28 day HU rats vs. 28 day CON animals. Middiaphyseal tibial bone increased slightly in density and area during HU; no differences in structural and material properties between 28 day HU and 28 day CON rats were noted. BFR at the tibial midshaft was significantly lower (by 90%) after 21 day HU vs. 0 day CON; this decline was maintained throughout 28 day HU. These results suggest there are compartment-specific differences in the mature adult skeletal response to hindlimb unloading, and that the major impact over 28 days of unloading is on cancellous bone sites. Given the sharp decline in BFR for midshaft cortical bone, it appears likely that deficits in BMD, area, or mechanical properties would develop with longer duration unloading.

Effects of aging on microvascular oxygen pressures in rat skeletal muscle

Aging alters skeletal muscle vascular geometry and control such that the dynamics of muscular blood flow (Q) and O2 delivery (Q(O2)) may be impaired across the rest-exercise transition. If, at the onset of muscle contractions, Q dynamics are slowed disproportionately to those of muscle O2 uptake (V(O2), microvascular PO2 (PO2m) would be reduced and blood-tissue O2 transfer compromised. This investigation determined the effects of aging on PO2m (a direct reflection of the Q(O2)-to-V(O2) ratio), at rest and across the rest-contractions transition in the spinotrapezius of young (approximately 6 months, n = 9) and old (>24 months, n = 10) male Fisher 344/Brown Norway hybrid rats. Phosphorescence quenching techniques were used to quantify PO2m, and test the hypothesis that, across the rest-contractions (twitch, 1 Hz; 4-6 V, 240 s) transition, aging would transiently reduce the Q(O2)-to-V(O2) ratio causing a biphasic profile in which PO2m fell below steady-state contracting values. Old rats had a lower pre-contraction baseline PO2m than young (27.1+/-1.9 versus 33.8+/-1.6 mmHg, P<0.05, respectively). In addition, in old rats PO2m demonstrated a pronounced difference between the absolute nadir and end-contracting values (2.5+/-0.9 mmHg), which was absent in young rats. In conclusion, unlike their young counterparts, old rats exhibited a transiently reduced PO2m across the rest-contractions transition that may impair blood-tissue O2 exchange and elevate the O2 deficit, thereby contributing to premature fatigue.

Control of skeletal muscle perfusion at the onset of dynamic exercise

At the onset of exercise there is a rapid increase in skeletal muscle vascular conductance and blood flow. Several mechanisms involved in the regulation of muscle perfusion have been proposed to initiate this hyperemic response, including neural, metabolic, endothelial, myogenic, and muscle pump mechanisms. Investigators utilizing pharmacological blockade of cholinergic muscarinic receptors and sympathectomy have concluded that neither sympathetic cholinergic nor adrenergic neural mechanisms are involved in the initial hyperemia. Studies have also shown that the time course for vasoactive metabolite release, diffusion, accumulation, and action is too long to account for the rapid increase in vascular conductance at the initiation of exercise. Furthermore, there is little or no evidence to support an endothelium or myogenic mechanism as the initiating factor in the muscle hyperemia. Thus, the rise in muscle blood flow does not appear to be explained by known neural, metabolic, endothelial, or myogenic influences. However, the initial hyperemia is consistent with the mechanical effects of the muscle pump to increase the arteriovenous pressure gradient across muscle. Because skeletal muscle blood flow is regulated by multiple and redundant mechanisms, it is likely that neural, metabolic, and possibly endothelial factors become important modulators of mechanically induced exercise hyperemia following the first 5-10 s of exercise.

Ageing and exercise training alter adrenergic vasomotor responses of rat skeletal muscle arterioles

Ageing is associated with increased leg vascular resistance and reductions in leg blood flow during rest and exercise, potentially predisposing older adults to a host of functional and cardiovascular complications. The purpose of these studies was to examine the effects and possible mechanisms of ageing and exercise training on arteriolar adrenergic vasoreactivity. Young and old male Fischer 344 rats were divided into young sedentary (YS), old sedentary (OS), young exercise‐trained (YT) or old exercise‐trained (OT) groups, where training consisted of chronic treadmill exercise. Isolated soleus (SOL) and gastrocnemius (GAS) muscle arterioles were studied in vitro. Responses to noradrenaline in endothelium‐intact and endothelium‐denuded arterioles, as well as during nitric oxide synthase (NOS) inhibition were determined. Vasodilator responses to isoproterenol and forskolin were also determined. Results: Noradrenaline‐mediated vasoconstriction was increased in SOL arterioles with ageing, and exercise training in old rats attenuated α‐adrenergic vasoconstriction in arterioles from both muscle types. Removal of the endothelium and NOS inhibition eliminated these ageing and training effects. Isoproterenol‐mediated vasodilatation was impaired with ageing in SOL and GAS arterioles, and exercise training had little effect on this response. Forskolin‐induced vasodilatation was not affected by age. The data demonstrate that ageing augments α‐adrenergic vasoconstriction while exercise training attenuates this response, and both of these alterations are mediated through an endothelial α‐receptor‐NOS‐signalling pathway. In contrast, ageing diminishes β‐receptor‐mediated vasodilatation, but this impairment is specific to the smooth muscle. These studies indicate that α‐ and β‐adrenergic mechanisms may serve to increase systemic vascular resistance with ageing, and that the effects of exercise training on adrenergic vasomotor properties could contribute to the beneficial effects of exercise on cardiovascular disease.

Differential effects of training on the control of skeletal muscle perfusion

Endurance and high-intensity sprint training have been shown to alter skeletal muscle blood flow and factors that govern muscle perfusion under various conditions. Neither endurance nor sprint training alter skeletal muscle perfusion at rest but can result in an increase in muscle blood flow during the anticipation of exercise. The magnitude of the anticipatory increases in muscle blood flow is dependent on the intensity and duration of the prior training bouts and results from elevations in mean arterial pressure and decreases in vascular resistance in skeletal muscle. The decrements in skeletal muscle vascular resistance appear to be mediated through increases in muscle sympathetic cholinergic nerve activity or decreases in muscle sympathetic adrenergic nerve activity. During submaximal exercise, total muscle blood flow is either unchanged or slightly lower. However, a redistribution of muscle blood flow may occur following aerobic training, resulting in an enhanced perfusion of high-oxidative skeletal muscles and less flow going to low-oxidative muscles. The increased perfusion of the high-oxidative muscles may result from various factors including: a) increased recruitment of high-oxidative motor units, b) increased local release of metabolic vasodilator substances, c) qualitative changes in the metabolic substances released, d) decreased muscle sympathetic nerve activity, e) diminished sensitivity of the arterial vasculature to norepinephrine or other vasoconstrictor agents, f) enhanced endothelium-mediated dilation in the resistance vasculature, and g) an increased effectiveness of the skeletal muscle pump. Conversely, the decreases in blood flow to low-oxidative muscles may result from an enhanced autoregulatory responsiveness of the resistance vasculature. Endurance and sprint training increase muscle perfusion during exercise at VO2max: this primarily appears to be the result of an enhanced pumping capacity of the heart to increase in maximal cardiac output. Many of the training-induced alterations in muscle blood flow and vascular structure are localized in the muscles that are most active during the training bouts. Therefore, differences in muscle recruitment patterns that occur with low-intensity endurance exercise and high-intensity sprint exercise may account for differences observed between these two training regimens.