NOTARIUS, CATHERINE F.; MURAI, HISAYOSHI; MORRIS, BEVERLEY L.; FLORAS, JOHN S.
In healthy young adults, the reflex response to orthostasis simulated by graded lower body negative pressure (LBNP) includes dose-related increases in muscle sympathetic nerve activity (MSNA) and plasma norepinephrine (PNE), resulting in progressive forearm vasoconstriction (29,33,38). Infusions of norepinephrine as well as tyramine (stimulating an endogenous release of norepinephrine) in such subjects demonstrate that the reflex forearm vasoconstrictor response is inversely related to resting sympathetic tone, as estimated by MSNA (3). In aerobically trained men, known to have increased resting MSNA and PNE (1,25,32), the reflex increase in forearm vascular resistance (FVR) with graded LBNP is blunted compared with sedentary men (20). However, because MSNA was not measured, the effect of such training on the FVR–MSNA relationship was not assessed in those experiments.
Aging studies focusing on changes in neurovascular transduction report attenuated reflex sympathetic peripheral vasoconstriction (4) in older men, reduced postjunctional α-1 adrenoceptor responsiveness to norepinephrine in the forearm (6,13), and diminished responsiveness of both α-1 and 2 receptors in the leg circulation (31). However, these studies did not assess or control the potential influence of aerobic fitness capacity on these measures of neurovascular coupling.
Regular endurance exercise prevents or improves age-related declines in endothelium-dependent vasodilation (5), arterial compliance (36), and cardiovagal baroreflex sensitivity (21). Although MSNA and PNE are increased in fit subjects, blood pressure is not (1,25,32). This suggests a protective effect of regular endurance exercise, which may involve diminished neurovascular coupling, perhaps due to increased nitric oxide bioavailability at rest (32), analogous to the “functional sympatholysis” observed during and after acute exercise (12).
The purpose of this study was to assess the influence of aerobic exercise capacity on such neurovascular coupling in middle age. We studied fit and sedentary middle-age men to test the hypothesis that during LBNP, the FVR–MSNA relationship would be steeper in sedentary versus fit subjects.
The experimental protocol was approved by the University Health Network Research Ethics Board. Informed consent was obtained in writing from each subject before his participation, according to the University Health Network Principles of Clinical Research Practice.
Twenty heathy male volunteers, none taking medication, were recruited. For a desired power of 0.8 and an r value of 0.8 in the regression analysis, with an α level of 0.05, the number of subjects required in each group was calculated as 10 (SigmaStat™ for Windows, Ver. 1.0; Jandel Scientific Corp., San Rafael, CA). A graded exercise test was administered using a cycle ergometer with ramped increments of 17 W·min−1 until pedal speed could no longer be maintained and the RER (V˙CO2/V˙O2) exceeded 1.1. Expired gases and volumes were measured breath by breath to establish oxygen consumption at peak exercise (V˙O2peak; open circuit spirometry, Vmax Series 229; SensorMedics, San Diego, CA). V˙O2peak was expressed both as liters per minute and milliliters per kilogram per minute and, to account for age, male sex, body weight, and height, as percent of predicted V˙O2peak (16). Subjects were categorized as fit (FIT, n = 10) or sedentary (SED, n = 10) on the basis of whether the peak oxygen uptake achieved was greater or less than 100% of predicted. All subjects in the FIT group reported performing regular running or cycling exercise 3–7 d·wk−1, whereas 8 of 10 SED subjects did not report undergoing any regular exercise, and the remaining two subjects reported walking for 20 min, 2 d·wk−1.
On a subsequent day, subjects were placed supine into a metal LBNP chamber equipped with a resealable trap door to permit measurement of MSNA from the right fibular nerve. During the last 2 min of each LBNP level, venous blood was sampled from an indwelling antecubital venous catheter and submitted to PNE determination by high-pressure liquid chromatography (9). Blood pressure was monitored every minute from the right arm by an automated device (Dinamap Pro 100; Critikon LLC, Tampa, FL). HR was derived from lead II of the ECG. Mean arterial pressure was calculated as 1/3 pulse pressure + diastolic blood pressure.
Multiunit recordings of postganglionic MSNA were obtained with a unipolar tungsten electrode inserted selectively into a muscle–nerve fascicle of the right peroneal (fibular) nerve as described previously (26).
Venous occlusion plethysmography
The nondominant arm was elevated and supported so that the proximal forearm rested about 10 cm above the anterior chest wall. Forearm blood flow (FBF) was measured by plethysmography using a mercury-in-silastic strain gauge (D. E. Hokanson, Inc., Bellevue, WA) (18) placed approximately 5 cm below the antecubital crease. During measurement of blood flow, circulation to the hand was arrested by inflation of a wrist cuff to 180–200 mm Hg, and a venous occlusion cuff on the upper arm was inflated for 10 s to 40 mm Hg.
Signals were output to a recorder (Gould Viper-TA; Gould Instrument Systems, Madison, WI), sampled at a frequency of 200 Hz (with the exception of the ECG, 1000 Hz), and, after analog-to-digital conversion, stored in a personal computer for subsequent analysis. Sympathetic nerve bursts were detected by a customized computer analysis program based on the LabVIEW® software (National Instruments, Austin, TX) with expert observer editing. MSNA was expressed as burst frequency (bursts per minute) and burst incidence (bursts per 100 heart beats). FBF (in milliliters per 100 g·min−1) was calculated from the mean of four to eight measurements made at 15-s intervals during 2 min. FVR was calculated as mean arterial pressure / FBF, with forearm vascular conductance (FVC) being the reciprocal of FVR.
After this setup was complete and after a stabilization period, a 7-min baseline recording including 2 min of FBF was acquired. Thereafter, 4 min each of intermittent LBNP at −5, −10, −20, and −40 mm Hg was applied in a random order, with each stimulus separated by 10 min. Values obtained during the last 2 min of each LBNP level were each averaged to determine mean individual responses to each stimulus.
Data are presented as mean ± SE. Unpaired t-tests were performed to test for differences between group means for dependent variables measured at rest. A repeated-measures two-way ANOVA was used to examine the main effects of group (FIT and SED) and LBNP (−5, −10, −20, −40) and any potential interaction (group × LBNP) (SigmaStat™ for Windows, Ver. 1.0, Jandel Scientific Corp.) on dependent variables measured from minute 2 to 4 during LBNP levels. When a significant main effect was identified, point differences between LBNP levels or groups were detailed by the post hoc Student–Newman–Keuls test.
Neurovascular transduction was assessed by the MSNA (stimulus) and FVR (response) relationship during LBNP and derived separately for each group using linear regression to test for a statistically significant within-group association. We included the first 2 min of MSNA and FVR during each level of LBNP in addition to that during the next (and last) 2 min of LBNP in these regression equations. We also performed a multiple linear regression, which included cardiovascular fitness level, subjects, and MSNA as independent variables in the model, with the natural log of FVR (lnFVR) as the dependent variable. FVR, a derived ratio, was transformed to its natural log in this latter analysis to satisfy the assumptions of normal distribution of data and equal variance in this model.
Descriptive and baseline data
Age, height, baseline MSNA burst frequency, FBF, and calculated FVR and conductance were similar in the two groups, but by design, both absolute and normalized peak oxygen uptake were significantly less in the SED men (mean peak V˙O2 of 85% vs 129% in FIT subjects, P < 0.001) (Table 1). Compared with the FIT group, SED men had significantly higher resting HR (P = 0.004), systolic blood pressure (P = 0.04), and body weight (P < 0.001) but lower PNE concentrations (P < 0.05).
Response to LBNP
In all subjects, blood pressure was stable throughout the lower levels of LBNP. At −40 mm Hg, there was a slight but significant drop in systolic blood pressure compared with baseline (P = 0.03) in both groups (Table 2). Systolic blood pressure tended to be lower in the FIT group throughout the LBNP protocol (group effect, P = 0.06; LBNP effect, P < 0.001).
In the SED group, HR was higher not only at rest but also throughout LBNP (P < 0.002) compared with the FIT group. However, at LBNP −20 and −40, HR was significantly and similarly increased from baseline in both groups (P < 0.05) (Table 2).
MSNA burst frequency rose during LBNP. These values were virtually identical in the two groups. PNE, which increased during LBNP in both groups (LBNP effect, P < 0.001), was higher in the FIT subjects at all levels (group effect, P = 0.043).
In both groups, LBNP lowered FBF, increased FVR, and lowered FVC significantly (main effect LBNP, P = 0.002, 0.003, and 0.008, respectively). There were no significant differences in these values at any given level of LBNP between groups, except for a tendency toward a lower FBF during LBNP in the FIT group (main effect group, P = 0.06) (Table 2).
When the relationship between FVR and MSNA burst frequency was plotted across all LBNP levels, a significant positive relationship emerged in the SED group (r = 0.44, P < 0.001). The regression equation for this group was the following:
Equation (Uncited)Image Tools
By contrast, there was no relationship between these variables in the FIT group (r = 0.19, P = 0.10) (Fig. 1). Thus, the principal difference between groups was the dissociation in the FIT group between the effect of LBNP on sympathetic vasoconstrictor tone and FVR. Similarly, after FVR was transformed to its natural log to satisfy tests for normality and equal variance of the aggregate data, multiple regression analysis revealed two independent predictors of lnFVR: MSNA (P < 0.001) and fitness level (P = 0.04). The equation for this model is the following:
Equation (Uncited)Image Tools
The purpose of this cross-sectional study was to determine whether the level of prevailing cardiovascular fitness influences the relationship previously reported between MSNA and FVR responses to graded LBNP in healthy SED men (4,29). Importantly, because SED and FIT men had similar MSNA burst frequency at rest and during all levels of LBNP, we were able to study the relationship between FVR and MSNA without the need to adjust for baseline differences in this independent variable. This reflected the higher body mass index of the SED group (1,10).
In SED men, we observed, as anticipated, a significant relationship between MSNA and FVR across all levels of LBNP (Fig. 1). By contrast, in FIT men, this relationship was absent, despite almost identical mean sympathetic responses to LBNP in the FIT and SED groups.
Taken together, these findings are consistent with our hypothesis that in middle age, fitness uncouples the vasoconstrictor response to reflex increases in sympathetic nerve firing rate present in SED men. Sympathetic nerve traffic directed to skeletal muscle increases with age (4,24), with the potential consequence of augmented vascular resistance. It is higher in older FIT than in SED subjects, yet resting blood pressure is similar (1,25,27,32). Altered neurovascular transduction as observed in the present experiment conceivably could protect FIT middle-age men against age-related increases in sympathetic vasoconstrictor discharge.
In response to increasing levels of LBNP, a significant positive relationship has been described between MSNA and FVR in both young and older subjects (4,28,29).
Others have shown higher blood pressure both at baseline and during LBNP in older subjects but similar HR, FVR, PNE, and central venous pressure (CVP) responses to graded LBNP (stepwise from 0 to −50 mm Hg) in young and older healthy subjects (37). In men, aging is associated with declines in peak oxygen consumption and physical activity (30), vasodilatory capacity at rest, and functional sympatholysis during exercise (7). Aging is also accompanied by increases in arterial stiffness and endothelial dysfunction (5,6,21,36). The latter may reflect increased vascular oxidative stress with reduced nitric oxide bioavailability (7,34). Regular exercise may reverse some of these aging effects (5). However, few studies have considered their findings in the context of aerobic fitness.
Cardiorespiratory fitness is an independent risk factor for both coronary heart disease (40) and hypertension (2). Previous investigations have established that in both young and middle-age FIT men compared with SED men, there is a steeper relationship between changes in FVR relative to changes in central venous pressure in response to nonhypotensive LBNP. Although MSNA was not recorded in these experiments, this finding suggests that fitness augments the tonic inhibitory influence of cardiopulmonary baroreceptors on sympathetic outflow (15,35). Longitudinal studies, on the other hand, report a reduced FVR/CVP slope during nonhypotensive LBNP, which has been attributed to exercise training–induced increases in blood volume (19) and reduced cardiopulmonary baroreflex responsiveness.
In the present study, the dissociation between FVR and MSNA in FIT middle-age men, in contrast to their SED counterparts, may result from an alteration in neurovascular transduction, which could favorably influence disease risk despite the increased sympathetic outflow in middle-age men (2,11,30,40). Three months of aerobic exercise training enhanced nitric oxide bioavailability in middle-age and older adults but did not alter resting blood flow because of a concomitant increase in PNE, assumed to be a surrogate for sympathetic activity (32). If the level of fitness influences the relationship between MSNA and skeletal muscle vascular resistance by attenuating age-related declines in endothelium-dependent vasodilation (5) and arterial compliance (36), greater sympathetic vasoconstrictor tone in FIT individuals may be required to offset training-induced vascular changes so as to maintain blood flow and vascular resistance. Importantly, direct effects of physical training on the vasculature, such as enhancement of nitric oxide bioavailability and improved vascular reactivity, may explain why reductions in conventional coronary risk factors do not explain all of the benefits of regular exercise (11,22).
We focused on burst frequency as our primary independent variable because this defines local norepinephrine release and the magnitude of the local vasoconstrictor response (8,39). However, it is of interest that the FIT group had higher MSNA burst incidence, as observed by other groups (1,25), suggesting altered central regulation of resting MSNA.
The FIT group had both a lower body mass and a lower baseline blood pressure compared with the SED group, reflecting the beneficial effects of regular physical activity on the maintenance of optimal body mass and blood pressure, yet FIT and SED subjects had similar baseline MSNA and FVR.
For technical reasons, it was not possible to study MSNA and blood flow during LBNP in the same limb. We therefore measured MSNA in the leg and assessed blood flow and calculated vascular resistance in the forearm. Previous works support a similar sympathetic outflow in the arm and leg, both at rest and in response to LBNP (28) as well as comparable changes in blood flow response to LBNP (14).
Consistent with our hypothesis, when reflex sympathetic vasoconstrictor discharge was elicited by graded LBNP, there was a significant positive FVR–MSNA relationship in SED middle-age men but not in age-matched FIT men.
These novel findings underscore the importance of considering fitness status or aerobic exercise capacity when using vascular resistance as an indicator of sympathetic outflow or when studying the effects of age on blood flow and vascular function since physical conditioning in middle-age men modifies the usual age-related neurohemodynamic relationship. Because middle-age subjects are often used as healthy age-matched control subjects in studies of a specific disease, our findings would argue for the importance of selecting SED rather than regularly exercising subjects when considering appropriate controls for study patients experiencing cardiovascular, respiratory, or other chronic diseases accompanied by impaired exercise tolerance. Our findings may also help explain the concordance between MSNA and blood pressure after the age of 40 in both men and women (17,23) and why a SED lifestyle might increase the risk of developing hypertension (2). One could speculate that a neurovascular disconnect may actually protect FIT middle-age men against the possible adverse effect of both age- and fitness-induced increases in basal sympathetic nervous outflow on vascular resistance and resting blood pressure, potentially reducing their cardiovascular risk.
This study was supported by grants-in-aid from the Heart and Stroke Foundation of Ontario (T4938, NA6298).
Dr. Floras is a career investigator of the Heart and Stroke Foundation of Ontario and holds the Canada Research Chair in Integrative Cardiovascular Biology.
The authors have no conflict of interest to declare.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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