Effect of aging on carotid baroreflex control of blood pressure and leg vascular conductance in women

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1 Am J Physiol Heart Circ Physiol 36: H1417 H142, 214. First published March 28, 214; doi:1.112/ajpheart CALL FOR PAPERS to the Basics Sex and Gender Differences in Cardiovascular Physiology Back Effect of aging on carotid baroreflex control of blood pressure and leg vascular conductance in women Daniel P. Credeur, 1 Seth W. Holwerda, 1 Leryn J. Boyle, 2 Lauro C. Vianna, 4 Areum K. Jensen, 1 and Paul J. Fadel 1,3 Departments of 1 Medical Pharmacology and Physiology, and 2 Nutrition and Exercise Physiology, 3 Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; 4 Department of Physiology and Pharmacology, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil Submitted 16 January 214; accepted in final form 24 March 214 Credeur DP, Holwerda SW, Boyle LJ, Vianna LC, Jensen AK, Fadel PJ. Effect of aging on carotid baroreflex control of blood pressure and leg vascular conductance in women. Am J Physiol Heart Circ Physiol 36: H1417 H142, 214. First published March 28, 214; doi:1.112/ajpheart Recent work suggests that -adrenergic vasodilation offsets -adrenergic vasoconstriction in young women, but this effect is lost after menopause. Given these age-related vascular changes, we tested the hypothesis that older women would exhibit a greater change in vascular conductance following baroreflex perturbation compared with young women. In 1 young (21 1 yr) and 1 older (62 2 yr) women, mean arterial pressure (MAP; Finometer), heart rate (HR), cardiac output (CO; Modelflow), total vascular conductance (TVC), and leg vascular conductance (LVC, duplex-doppler ultrasound) were continuously measured in response to -s pulses of neck suction (NS; 6 Torr) and neck pressure (NP; 4 Torr) to simulate carotid hypertension and hypotension, respectively. Following NS, decreases in MAP were similar between groups; however, MAP peak response latency was slower in older women (P.). Moreover, at the time of peak MAP, increases in LVC (young, %LVC vs. older, %LVC; P.) and TVC were greater in older women, whereas young women exhibited larger decreases in HR and CO (young, 1 3% CO vs. older,.8 2% CO; P.). Following NP, increases in MAP were blunted (young, 14 1 mmhg vs. older, 8 1 mmhg; P.) in older women, whereas MAP response latencies were similar. Interestingly, decreases in LVC and TVC were similar between groups, but HR and CO (young, 7. 2% CO vs. older, 4. 2% CO; P.) responses were attenuated in older women. These findings suggest that older women have greater reliance on vascular conductance to modulate MAP via carotid baroreflex, whereas young women rely more on cardiac responsiveness. Furthermore, older women demonstrate a blunted ability to increase MAP to hypotensive stimuli. vascular responsiveness; baroreceptors; blood flow; total vascular conductance; cardiac output Address for reprint requests and other correspondence: P. Fadel, Dalton Cardiovascular Research Center and Dept. of Medical Pharmacology and Physiology, Univ. of Missouri, Columbia, MO 6212 ( THE ARTERIAL BAROREFLEX IS critical for the beat-to-beat regulation of arterial blood pressure (BP) (13, 37). Although several reports indicate that aging is associated with impaired cardiac baroreflex function (16, 17, 26, 3), studies investigating baroreflex control of sympathetic nerve activity demonstrate equivocal results showing preserved (6, 26), impaired (27, 4), or even augmented sympathetic responses in older subjects (4). In regards to arterial baroreflex control of BP, our laboratory has reported augmented responses to a hypertensive stimulus but blunted pressor responses to acute hypotension in older compared with younger subjects (14, 1). Importantly, this previous work on baroreflex function and aging in humans has been performed primarily in men, and surprisingly limited data exist for women, particularly older postmenopausal women. Given that the elderly population is increasing exponentially and the number of postmenopausal women is estimated to more than double by the year 2 (29), an understanding of age-related changes in baroreflex function in women is warranted. Previous studies have reported that alterations in vasomotor activity are the primary means by which the carotid baroreflex regulates BP (3, 8, 32, 33). In dogs, the pressor response to bilateral carotid occlusion was mediated solely by changes in total vascular conductance (TVC) (3). Similarly, human studies in young men have demonstrated that BP responses to carotid baroreflex perturbation were predominantly attributable to vascular changes (i.e., TVC) with a minimal contribution from cardiac output (CO) (32, 33). However, in contrast, we recently found that young women rely more on heart rate (HR) and subsequent changes in CO to modulate BP via the baroreflex, particularly in response to carotid hypertensive stimuli (2). Whether these sex differences that manifest in young women are present in older women remains unknown. To date, no studies have reported the mechanism (i.e., cardiac vs. vascular) of carotid baroreflex-mediated changes in BP in older postmenopausal women. This becomes important considering recent work suggesting that sympathetic control of the vasculature is altered with age in women (19). Indeed, the ability of -adrenergic vasodilation to offset -adrenergic vasoconstriction that is seen in young women has been shown to be lost in postmenopausal women (19). Also, unlike young women, postmenopausal women exhibit a positive relationship between muscle sympathetic nerve activity (MSNA) and total peripheral resistance (19). These data support the notion that sympathetic control of the vasculature differs with age in women. However, the impact of these age-related vascular changes on baroreflex control of vascular conductance and BP remains unknown /14 Copyright 214 the American Physiological Society H1417

2 H1418 Therefore, we sought to examine the influence of age on arterial baroreflex control of vascular conductance and BP in women. Using the variable pressure neck chamber technique, we first examined whether leg vascular conductance (LVC) and BP responses to simulated carotid hypertension and hypotension were different between young and older postmenopausal women. We then determined whether the contributions of CO and TVC to BP responses evoked by carotid baroreflex perturbation were different in young compared with older women. We hypothesized that older women would exhibit a greater change in LVC and TVC following baroreflex perturbation compared with young women, indicative of a greater reliance on vascular changes to regulate arterial BP. MATERIALS AND METHODS Subjects Ten young and ten older healthy women participated in this study. All subjects were recruited from the University of Missouri and surrounding Columbia, MO community. All experimental procedures and protocols conformed to the Declaration of Helsinki and were approved by the Health Sciences Institutional Review Board of the University of Missouri. Each subject provided written informed consent before participating in any facet of the study. Young women were studied during the early follicular phase of their menstrual cycle (days 2 ). All older women were postmenopausal and not taking hormone replacement therapy. Subjects were nonsmokers and recreationally active, but importantly none were endurance exercise trained. Each subject completed a medical health history questionnaire and underwent a screening to assure that they were healthy and free of any chronic disease. No subject had a history or symptoms of cardiovascular, pulmonary, metabolic, or neurological disease, and none were using prescribed or over-the-counter medications. However, given the greater risk for cardiovascular disease with aging, a 12-h fasting blood chemistry screening was also performed in the older women, similar to our previous aging studies (14 16). Experimental Procedures Neck suction and neck pressure. Five-second pulses of 6 Torr neck suction (NS) and 4 Torr neck pressure (NP) were applied to selectively load (simulated carotid hypertension) and unload (simulated carotid hypotension) the carotid baroreceptors, respectively, using the variable pressure neck chamber as described previously (9, 14, 2, 32). The -s periods of NS and NP provide reflex activation of carotid baroreceptors independent of extracarotid baroreceptors (i.e., reflex adjustments elicited by aortic and cardiopulmonary baroreceptors) (34, 37). Also, of importance for the present study, unlike pharmacological infusions (i.e., modified Oxford), the application of NS-NP allows for the examination of peripheral vascular responses during baroreflex perturbation (8, 9, 37), the primary outcome variable studied. Briefly, a malleable lead neck collar was fitted around the anterior two-thirds of the neck with each NS and NP stimulus being delivered ms after the second consecutive R-R interval that did not vary by ms using customized computer-controlled software (2, 34). A variable pressure source was used to generate the changes in neck collar pressure and delivered to the collar through large-bore two-way solenoid valves (Asco, Florham Park, NJ). To accurately quantify the stimulus applied, a pressure transducer (Validyne, Northridge, CA) was connected to a port on the collar. To minimize respiratory-related modulation of HR, the -s pulses of NS and NP were delivered to the carotid sinus during a brief 12 1-s breath hold at end expiration (7, 1, 2). With the use of the variable pressure neck chamber to selectively describe carotid baroreflex control, the assumption is made that the aortic baroreflex operates in parallel with the carotid baroreflex and, therefore, will respond similarly (9, 1, 36, 37). Experimental Measurements Cardiovascular measures. HR was continuously monitored using a standard lead II surface ECG (Q71; Quinton, Bothell, WA). Beatto-beat BP was measured using photoplethysmography obtained from the left middle finger (Finometer; Finapres Medical Systems, Amsterdam, The Netherlands). Before Finometer recordings were obtained, the return-to-flow calibration on the Finometer device was performed. Additionally, diastolic BP of the Finometer was matched with diastolic BP measurements obtained from the brachial artery of the right arm by automated sphygmanometry (Welch Allyn, Skaneateles Falls, NY) to assure accurate mean arterial pressure (MAP) measures in response to carotid baroreflex perturbation. Respiratory movements were monitored using a strain-gauge pneumobelt placed around the abdomen (Pneumotrace; UFI, Morro Bay, CA). Leg blood flow. Femoral artery diameter and blood velocity were measured via duplex-doppler ultrasound in the right leg, as previously described in detail by our laboratory (12, 41). Briefly, the femoral artery was imaged longitudinally, distal to the inguinal crease 2 3 cm proximal from the bifurcation of the superficial and deep femoral artery branches with a high-resolution ultrasound system (Logiq 7; GE, Milwaukee, WI). Diameter and velocity were simultaneously and continuously measured using a 1-MHz linear array transducer probe in pulse-wave mode operating at a frequency of MHz and insonation angle of 6. Measurements were performed with the velocity cursor placed midvessel and sample volume encompassing the entire vessel lumen but not extending beyond it. To ensure acquisition of stationary images, the transducer was stabilized during all experiments using a custom-designed clamp. Familiarization Sessions All subjects were familiarized with the study procedures and measurements before the actual experimental visit. Familiarization sessions included screening subjects to identify the location of the carotid sinus bifurcation using B-Mode ultrasound to ensure that the neck collar fully enclosed the carotid sinuses. Although transmission of NS and NP to the carotid sinus has been shown to be near complete, there is variability in the location of the carotid sinuses that requires consideration (3). In addition, older women underwent duplex- Doppler ultrasound imaging within the University Radiology Department to screen for any significant carotid artery plaques before we performed NS and NP. In another session, appropriate neck chamber placement was determined by fitting the subjects based on carotid sinus location and observed neck size. Practice trials of NS and NP were then performed to determine directionally appropriate and consistent HR and BP responses. In all cases, two sessions were performed to assure subject familiarity and clarity of responses. In addition, the femoral artery was imaged to ensure the attainment of an adequate signal and to allow the subject to become comfortable with this measurement. Experimental Protocol Before the experimental session, subjects were instructed to fast for 3 h and refrain from caffeine intake for 12 h and strenuous physical activity and alcohol for at least 24 h. Subjects were positioned supine in a temperature-controlled room ( 23 C) and instrumented for continuous measures of BP, HR, and leg blood flow (LBF). After instrumentation, the neck collar was placed around the anterior twothirds of the subject s neck, and min of resting baseline data were recorded. Carotid baroreflex-mediated changes in BP, HR, and LBF were then determined by applying random-ordered single -s pulses of NS and NP as described in detail in Neck suction and neck pressure. Ten trials for each level of NS and NP were performed with AJP-Heart Circ Physiol doi:1.112/ajpheart

3 H1419 a minimum of 4 s of recovery allotted between trials to allow all physiological variables to return to prestimulus values. The rationale for performing ten trials of NS and NP was to better characterize individual carotid baroreflex responses for all cardiovascular variables and to account for the variability in beat-to-beat blood flow and vascular conductance measures observed in preliminary studies. Data Analysis The ECG, arterial BP waveform and respiratory signals were acquired in PowerLab (AD Instruments, Colorado Springs, CO) to allow continuous visual monitoring of data during the experiment. In addition, all experimental measurements were acquired into a custom LabVIEW program interfaced with video output of the Doppler ultrasound machine as described previously (11, 12, 39). The ECG, BP, and neck chamber pressure signals were sampled at 1 khz and embedded as data streams into an AVI file containing video images of the femoral artery with corresponding blood-velocity waveform output from the ultrasound at a sampling rate of 3 Hz. A customdesigned edge detection and wall-tracking software (LabVIEW; National Instruments, Austin, TX) was used to determine beat-by-beat arterial diameters and weighted mean velocity offline from the captured video output (11, 12, 39). These data were processed using a second custom LabVIEW program, which generated synchronized beat-by-beat data of all recorded variables gated by the R-wave of the ECG. Femoral diameter and blood velocity measurements were used to calculate LBF as (d/2) 2 V mean 6, where V mean is mean blood velocity (cm/s) and d is arterial diameter (cm). LVC was calculated as LBF/MAP (ml min 1 mmhg 1 ). Stroke volume (SV) and CO were estimated from the arterial BP waveform using the Modelflow method through Beatscope (TNO- TPD; Biomedical Instrumentation, Amsterdam, Netherlands), which incorporates age, sex, weight, and height (21, 22, 2). The estimated SV and CO were aligned with the LabVIEW program output via changes in cardiac interval time. Previous work in our laboratory has demonstrated that beat-to-beat changes in CO to NS and NP derived from Modelflow provide comparable results to direct Doppler echocardiography-derived measurements (2). Total vascular conductance was calculated as TVC CO/MAP. Characterization of carotid baroreflex response variables. Carotid baroreflex-mediated changes in all variables were calculated from the cardiac cycle immediately preceding the start of NS and NP (i.e., prestimulus) and plotted on a beat-by-beat scale. Importantly, with the application of NS-NP, the peak HR response typically occurs within 2 4 s, which is during the stimulus, whereas the peak MAP response does not occur until 6 8 s, which is after the -s stimulus and at a time where HR is returning back toward prestimulus values. These latencies are well documented (9, 1, 36, 37); however, recently we reported shorter response times for carotid baroreflex-mediated MAP responses in young women (2). Nevertheless, given the temporal differences in response variables, we present beat-to-beat data from both during and after the stimulus to show the temporal profile and peak response latencies of all variables. Of note, all figures present responses during the -s stimulus time and the s that follow. This timeframe encompasses peak responses for all measured variables and also includes the off response for some variables (e.g., HR), which is the time point in which the response variable starts to return from its peak toward prestimulus values. These data are important to show the transition that occurs from an initial MAP response driven by HR and CO to a more vascular-mediated MAP response (9, 1, 36, 37). Changes in all cardiovascular variables in response to the 1 individual trials of NS and NP were averaged for each subject to provide individual mean responses, which were subsequently averaged for a group mean response. Data are presented as absolute and percent changes from prestimulus values. In addition, to examine potential latency differences in carotid baroreflex-mediated responses, the times to peak HR and MAP were calculated, as previously performed (1, 2). Characterization of carotid baroreflex response variables at the time of peak MAP. To more completely understand the primary contributors to the carotid baroreflex-mediated MAP responses, changes in LVC, TVC, and CO were calculated at the time point of the nadir and peak MAP response to NS and NP, respectively. This was done because our focus for reporting group differences is on the peak MAP response, which reflects the overall effectiveness of the carotid baroreflex to respond to any given perturbation (9, 1, 36, 37). For this analysis, nadir and peak changes of all response variables were determined at the cardiac cycle in which the peak change in MAP occurred. Such an analysis was also important because of the observed differences in MAP response latencies to NS between the younger and older women (see RESULTS). Percent contribution of CO and TVC to peak carotid baroreflexmediated MAP response. The percent contribution of CO and TVC to the peak MAP response to NS and NP was also quantified according to previous methods (1, 3, 2, 33). In brief, the individual contribution of CO and TVC to the peak MAP response elicited by either NS or NP was determined by calculating the predicted level of MAP if only the individual changes in CO or TVC occurred and other parameters remained at control values. In other words, when the contribution of TVC was calculated, the CO was held constant at its prestimulus value, and then the MAP response was recalculated. This predicted change in MAP due to TVC alone was then divided by the actual change in MAP to derive the percent contribution of TVC. The same calculations were performed for CO. The following formulas were used: 1) TVC control CO control/map control, 2) MAP CO CO/TVC control, 3) MAP TVC CO control/tvc, 4) Predicted change in MAP CO MAP CO MAP control, ) Predicted change in MAP TVC MAP TVC MAP control, 6) Actual change in MAP MAP MAP control, 7) Percent contribution of CO (predicted change in MAP CO)/(actual change in MAP) 1, and 8) Percent contribution of TVC (predicted change in MAP TVC)/(actual change in MAP) 1, where MAP CO is the MAP response to carotid baroreflex stimulation due to CO alone, MAP TVC is the MAP response to carotid baroreflex stimulation due to TVC alone, MAP control is the MAP value before NS or NP, CO control is the CO value before NS or NP, and TVC control is the TVC value before NS or NP. Statistical Analysis All data are presented as means SE. Statistical analyses were conducted using SigmaStat (Jandel Scientific Software, Chicago, IL). Unpaired t-tests were used to compare group differences in cardiovascular responses to NS and NP. A two-way repeated-measures ANOVA (group time) was performed to examine group differences in the temporal responses of cardiovascular variables to NS and NP. Bonferroni corrections were used for post hoc analyses when appropriate. Statistical significance was set at P.. RESULTS Baseline Subject Characteristics General baseline characteristics for young and older women are summarized in Table 1. The mean age difference between groups was 41 yr. Both young and older women had a similar body mass index, HR, and BP at rest. Older women had a larger femoral artery diameter (.82.2 cm in young vs cm in older women, P.), but resting LBF and LVC were comparable between groups (both P.). For older women, the average time they were considered postmenopausal was 9 2 yr, and fasting blood chemistry (Table 1) indicated that triglycerides, cholesterol, lipoproteins, and glucose concentrations were within the normal range for healthy adults (18). AJP-Heart Circ Physiol doi:1.112/ajpheart

4 H142 Table 1. Baseline characteristics Young (n 1) Older (n 1) Age, yr BMI, kg/m Triglycerides, mg/dl Cholesterol, mg/dl HDL, mg/dl LDL, mg/dl BUN, mg/dl Plasma Na, meq/l Plasma K, meq/ Glucose, mg/dl Heart rate, beats/min Systolic BP, mmhg Diastolic BP, mmhg MAP, mmhg Femoral diameter, cm LBF, ml/min LVC, ml min 1 mmhg Values are means SE. BMI, body mass index; HDL, high-density lipoprotein; LDL, low-density lipoprotein; BUN, blood urea nitrogen; BP, blood pressure; MAP, mean arterial pressure; LBF, leg blood flow; LVC, leg vascular conductance. Significantly different from young women (P.). Baroreflex Responses to Simulated Carotid Hypertension Table 2 summarizes prestimulus and peak responses for all cardiovascular variables at the time of the peak MAP response to NS in young and older women. The beat-to-beat and peak MAP, CO, and LVC responses to NS for both groups are presented in Fig. 1. In response to NS, the nadir decrease in MAP was similar between groups (P.); however, the carotid baroreflex response latencies to the nadir HR (1.7.2 s in young vs s in older women, P.) and MAP (4.2. s in young vs s in older women, P.) were significantly shorter in young compared with older women. This temporal difference, which can be appreciated in the beat-to-beat data presentation in Figs. 1 and 3, represents a fundamental difference in baroreflex control in women as they age. Indeed, at the time of the nadir MAP response, the changes in LVC ( % in young vs % in older women, P.) and TVC ( % in young vs % in older women, P.) were significantly greater in the older women. In contrast, older women demonstrated a blunted reduction in CO compared with young women ( 1 3% in young vs..8 2% in older women, P.). This appeared to be mediated by a reduction in the nadir HR response in older women ( 14 1 beats/min in young vs. 9 2 beats/min in older women; P.), as SV responses were not different between groups (P.). Thus, in young women, the rapid and robust HR and CO responses to NS are a critical component to the MAP response to carotid hypertension, contributing to a faster MAP response, whereas the reduction of this cardiac responsiveness with age appears to contribute to a slower and primarily vascular-mediated MAP response in older women (Figs. 1 and 3). Figures 1 and 3 also show that the timing of peak HR and CO were generally aligned with nadir MAP responses in the young women. In other words, 84% of the nadir MAP response to NS was manifest when HR and CO reached peak response values. This temporal pattern clearly demonstrates HR and CO to be a major driver of MAP changes to a hypertensive stimulus in young women. In contrast, although HR and CO may contribute to the initial decrease in MAP to NS in older women (Figs. 1 and 3), a heightened vascular response appears to primarily drive the carotid baroreflexmediated MAP decrease. Indeed, peak LVC and TVC increases were closely aligned with peak MAP responses in older women (Figs. 1 and 3), a time in which CO had returned toward prestimulus values. Thus, to achieve a similar MAP, older women demonstrated a significantly greater contribution of TVC (young women 12 22% vs. older women 98 14%, P.) to the nadir MAP response, whereas young women exhibited a greater contribution of CO (young women 79 16% vs. older women 1 23%, P.) (Fig. A). Baroreflex Responses to Simulated Carotid Hypotension Table 2 summarizes prestimulus and peak responses for all cardiovascular variables at the time of the peak MAP response to NP in young and older women. The beat-to-beat and peak MAP, CO, and LVC responses to NP for both groups are presented in Fig. 2. In response to NP, the peak increase in MAP was blunted in older women (P.), whereas carotid baroreflex response latencies to the time of peak HR (3..3 s in young vs s in older women, P.) and MAP (..2 s in young vs..7.6 s in older women, P.) were comparable between groups. Interestingly, despite the similar latencies, young women showed a greater temporal reliance on CO to drive the initial MAP response to NP, as the older women had negligible changes in CO (Figs. 2 and 4). Indeed, at the time of the peak MAP response, young women exhibited a greater increase in CO compared with older women ( 7. 2% in young vs. 4. 2% in older women, P.). This appeared to be mediated by a greater peak HR response in young women ( 11 1 beats/min in young vs. 3 1 beats/min in older women, P.), as SV responses were not different between groups (P.). The greater reliance on CO in young women can clearly be seen in the temporal responses in Fig. 2, where the peak CO response coincides closely with the peak MAP response. Indeed, the timing of peak CO (and HR; Fig. 4) was generally aligned with Table 2. Summary data for prestimulus and peak values to neck suction and neck pressure Prestimulus Peak Prestimulus Peak Neck suction ( 6 Torr) MAP, mmhg Heart rate, beats/min SV, ml/beat CO, l/min TVC, ml min 1 mmhg LVC, ml min 1 mmhg Neck pressure ( 4 Torr) MAP, mmhg Heart rate, beats/min SV, ml/beat CO, l/min TVC, ml min 1 mmhg LVC, ml min 1 mmhg Values are means SE. All cardiovascular response values except for heart rate are taken at the time of the peak MAP response. SV, stroke volume; CO, cardiac output; TVC, total vascular conductance. Significantly different from prestimulus values; Significantly different from young women (P.). AJP-Heart Circ Physiol doi:1.112/ajpheart

5 H1421 A -6 Torr Group: P=.169 Time: P<.1 Interaction: P<.1 - ΔMAP (mmhg) - ΔMAP% B Torr Group: P=.444 Time: P<.1 Interaction: P<.1 1 ΔCO (L min -1 ). -. ΔCO% - -1 Fig. 1. Summary data showing carotid baroreflexmediated changes in mean arterial pressure (MAP) (A), cardiac output (CO) (B) and leg vascular conductance (LVC) (C) to neck suction (NS) ( 6 Torr) in young and older women. Left: temporality of beat-to-beat changes. Right: peak percent changes at the time of the peak MAP response. Values are means SE. Significantly different from younger women (P.) C Torr Group: P<.1 Time: P<.1 Interaction: P=.39 3 ΔLVC (ml min -1 / mmhg) ΔLVC% peak MAP responses in the young women such that 72% of the peak MAP response to NP was manifest when HR and CO reached peak response values. These temporal responses also show a gradual shift from a primarily CO-driven MAP response in young women to a more vascular response (Figs. 2 and 4), as HR and CO return toward prestimulus values, whereas older women appear to primarily rely on vascular conductance to change MAP. In this regard, although, at the time of the peak MAP response to NP, decreases in LVC ( % in young vs % in older women, P.1) and TVC (.9 1.8% in young vs % in older women; P.17) were similar between groups, there was a trend for older women to rely more on vascular responses to NP (Figs. 2 and 4). This was most apparent in the percent contribution of TVC at the time of peak MAP response (young women 4 1% vs. older women %, P.3) (Fig. B). Nevertheless, as was the case for NS, the percent contribution of CO at the time of peak MAP response was significantly greater in young women (young women 4 1% vs. older women 46 23%, P.) (Fig. B). DISCUSSION Herein, we have for the first time examined the influence of age on pressor and depressor responses following carotid baroreflex perturbation in women. The major novel finding is that older women rely more on vascular responses to modulate BP, with baroreflex-mediated cardiac responses being blunted compared with young women. Indeed, young women appear to AJP-Heart Circ Physiol doi:1.112/ajpheart

6 H1422 A 1 +4 Torr NP Group: P<.1 Time: P<.1 Interaction: P=.1 2 ΔMAP (mmhg) 1 ΔMAP% Fig. 2. Summary data showing carotid baroreflexmediated changes in MAP (A), CO (B), and LVC (C) to neck pressure (NP) ( 4 Torr) in young and older women. Left: temporality of beat-to-beat changes. Right: peak percent changes at the time of the peak MAP response. Values are means SE. Significantly different from younger women (P.). B ΔCO (L min -1 ) C ΔLVC (ml min -1 / mmhg) Torr NP Torr NP Group: P<.1 Time: P<.1 Interaction: P=.27 Group: P=.263 Time: P<.1 Interaction: P=.98 ΔLVC% ΔCO% be more dependent on cardiac responsiveness to respond to carotid baroreflex perturbations. Thus with aging in women there appears to be a shift to a greater reliance on the vasculature to defend against changes in BP. However, the vascular responses to hypotension appear insufficient to compensate for a reduction in cardiac responsiveness, as the older women demonstrated a blunted BP response to simulated carotid hypotension. Overall, our findings indicate that young and older women depend on different mechanisms to modulate BP via the arterial baroreflex with older women having a preserved ability to defend BP during hypertensive stimuli but not hypotensive stimuli. Recent work has reported a positive relationship between resting MSNA and total peripheral resistance in older women but not younger women. However, this relationship was present in young women following removal of -adrenergic-mediated dilation with systemic propranolol, suggesting that -adrenergic receptors normally offset -adrenergic vasoconstriction in young women (19). In the present study, we reasoned that this would affect how young and older women modulate BP via the arterial baroreflex. Indeed, the application of NS to simulate carotid hypertension caused minimal increases in LVC and TVC in young women, whereas older women had significant elevations in vascular conductance. Because removal of sympathetic tone is a primary means by which the baroreflex defends against hypertensive challenges, these findings are in general agreement with a recent study by Schmitt et al. (38), demonstrating that young women have a lower -adrenergic support of BP compared with young men, which may be due to the aforementioned greater -adrenergicmediated dilation. This may also be due, in part, to the well-documented low resting MSNA levels in young women (2, 23, 28, 31). Likewise, given that older postmenopausal women have high resting MSNA (2, 28, 31), it is reasonable AJP-Heart Circ Physiol doi:1.112/ajpheart

7 A 1 1 ΔMAP and HR B ΔMAP and HR MAP (mmhg) HR (bpm) TVC (ml/min mmhg -1 ) -6 Torr NS Torr NS ΔTVC% ΔTVC% fact, older women had marginal to no changes in CO to NP. However, in contrast to NS, there did not appear to be an adequate compensation via the vasculature, as the older women exhibited a blunted carotid baroreflex-mediated increase in BP. Nonetheless, there was a trend for older women to have greater changes in total vascular conductance in response to NP (Fig. B). This is in line with the recent work of Hart et al. (19) demonstrating that -adrenergic vasodilation can offset sympathetic vasoconstriction in young women, and this effect was lost after menopause. Indeed, on the basis of these data, it would be predicted that vascular responses to sympathetic activation with NP would be greater in the older women. The lack of a statistically significant group difference in vascular conductance, along with the blunted BP response to NP, raises the question of whether the increase in MSNA in response to carotid hypotension is different between younger and older women. Studinger et al. (4) recently demonstrated that older subjects have a lower integrated-baroreflex gain to pharmacological reductions in arterial BP (sodium nitroprusside) compared with young subjects; however, direct comparisons be- A 1 1 MAP (mmhg) HR (bpm) TVC (ml/min mmhg -1 ) +4 Torr NP 3 2 H Fig. 3. Summary data showing temporality of beat-to-beat carotid baroreflexmediated changes in MAP (red line), heart rate (HR) (blue line), and total vascular conductance (TVC) (green line) to NS in young (A) and older women (B). The vertical gray lines represent mean latency for the peak MAP response in each group. Values are means SE. to suggest that the larger increase in LVC was attributable to the ability to remove this greater sympathetically mediated tone. Regardless, the BP responses to carotid hypertension were similar between groups owing to a greater cardiac response in the younger compared with older women. These findings are in line with a previous study from our laboratory (2) demonstrating that young women rely primarily on cardiac responses to modulate BP during simulated carotid hypertension compared with young men. The reason for the greater cardiac responsiveness in young women is unclear, but evidence from animal studies suggests that young female rats demonstrate a greater release of acetylcholine following vagal nerve stimulation compared with male rats, which could explain the greater bradycardia to NS (). In addition, the wellknown decrease in cardiac parasympathetic tone with age (6, 1, 4) likely contributes to the blunted HR responses in older women. Thus it appears that the age-related decline in cardiac baroreflex responsiveness in women can be compensated for by a greater vascular response to preserve BP responses to hypertensive stimuli. In response to carotid hypotension with NP, there was also a greater reliance on CO in young women to modulate BP. In ΔMAP and HR B 1 ΔMAP and HR Torr NP Fig. 4. Summary data showing temporality of beat-to-beat carotid baroreflexmediated changes in MAP (red line), HR (blue line), and TVC (green line) to NP ( 4 Torr) in young (A) and older women (B). The vertical gray lines represent mean latency for the peak MAP response in each group. Values are means SE ΔTVC% ΔTVC% AJP-Heart Circ Physiol doi:1.112/ajpheart

8 H1424 A -6 Torr NS % Contribution to Peak MAP CO TVC B +4 Torr NP tween young and older women were not made. Thus future studies comparing baroreflex-mediated MSNA responses in young and older women are warranted. It is also plausible that older women are operating at a point closer to the threshold of their full sigmoidal carotid baroreflex MAP stimulus response curve and away from the centering point, which would minimize responses to hypotensive challenges. We previously showed this to be the case with aging (14); however, this relocation of the operating point also augmented responses to hypertensive challenges in older subjects, and we did not observe this in the older women in the present study compared with young woman. Thus we will remain cautious in this interpretation as it relates to the present work because sex differences were not considered in our prior work. Nevertheless, the current findings demonstrate a blunted ability of older women to defend against hypotensive stimuli that was attributable, in part, to diminished cardiac responses compared with younger women. % Contribution to Peak MAP CO P=.3 TVC Fig.. Summary data illustrating the relative contributions of CO and TVC to peak MAP response elicited by 6 Torr NS (A) and 4 Torr NP (B). Values are means SE. Significantly different from younger women (P.). Perspectives The current findings have important implications for baroreflex control of the vasculature and subsequent regulation of BP and changes with advancing age in women. A properly functioning arterial baroreflex is critical for the beat-to-beat regulation of arterial BP (13, 37). Our previous work demonstrated that young women rely predominantly on cardiac changes to modulate arterial BP during a hypertensive stimulus compared with young men (2). We now demonstrate that with advancing age women appear to transition toward a greater reliance on changes in vascular conductance to modulate BP, which may be attributed to a loss of -adrenergic-mediated vasodilation (19) and/or decreased -adrenergic sensitivity (19), along with the well-known reduction in cardiac vagal tone with age (14). Considering the maintained MAP responses to simulated carotid hypertension, it is plausible that the age-related vascular changes in women may signify a compensatory mechanism to preserve arterial BP regulation to a hypertensive stimulus. On the contrary, advancing age is associated with a blunted ability of the arterial baroreflex to defend BP following a hypotensive challenge in older women. Thus it appears that there is a lack of adequate compensation for the attenuated cardiac baroreflex responsiveness to hypotension via a sympathetically mediated peripheral vasoconstriction in older women. This has implications for the greater prevalence of orthostatic intolerance seen in this population and warrants further investigation (19). Another important area for future study is the influence female sex hormones may have on baroreflex control of the vasculature. Although we recently reported minimal differences in baroreflex responsiveness (24) across the menstrual cycle in young women, a recent study showed potential counteractive responses between sex hormones such that high estrogen augmented decreases in vascular conductance to NP, whereas high progesterone blunted the reduction in vascular conductance (2). The latter study used a gonadotropin-releasing hormone antagonist in young women to suppress endogenous hormone production and then supplemented with each hormone alone and in combination, which may also have important implications for hormone replacement therapy in postmenopausal women. Nevertheless, future studies examining the potential influence of sex hormones on baroreflex control of vascular conductance and its effects on BP regulation are needed in both younger and older women. Conclusions In summary, these data indicate that aging in women is associated with an alteration in carotid baroreflex control of BP, such that young women rely more on cardiac responses, whereas older women demonstrate a greater reliance on the vasculature to modulate BP. This transition to a dependence on baroreflex-mediated vascular responses to defend against changes in BP is likely due to diminished cardiac responses and age-related vascular changes in women (1, 16, 19). However, although increases in vascular conductance were able to preserve baroreflex responses to simulated carotid hypertension, the vascular responses to hypotension appear insufficient to compensate for the reductions in cardiac responsiveness, as older women exhibit a blunted BP response to simulated carotid hypotension. ACKNOWLEDGMENTS The authors appreciate the time and effort put in by all volunteer subjects. We also thank Dr. Seth Fairfax for assistance with the data analysis program. GRANTS This work was supported by the National Heart, Lung, and Blood Institute (NHLBI) Grant RO1-HL (P. Fadel), National Institutes of Health (NIH) Grant T32-T32AR (D. Credeur and S. Holwerda), and American Heart Association (AHA) Grant 12Pre (L. Boyle). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: D.P.C., S.W.H., L.C.V., A.K.J., and P.J.F. conception and design of research; D.P.C., S.W.H., L.J.B., L.C.V., and A.K.J. performed experiments; D.P.C., S.W.H., and L.C.V. analyzed data; D.P.C., S.W.H., L.C.V., and P.J.F. interpreted results of experiments; D.P.C. prepared figures; D.P.C. drafted manuscript; D.P.C., S.W.H., L.J.B., L.C.V., A.K.J., and P.J.F. edited and revised manuscript; D.P.C., S.W.H., L.J.B., L.C.V., A.K.J., and P.J.F. approved final version of manuscript. AJP-Heart Circ Physiol doi:1.112/ajpheart

9 H142 REFERENCES 1. Augustyniak RA, Ansorge EJ, O Leary DS. Muscle metaboreflex control of cardiac output and peripheral vasoconstriction exhibit different latencies. Am J Physiol Heart Circ Physiol 278: H3 H37, Brunt VE, Miner JA, Kaplan PF, Halliwill JR, Strycker LA, Minson CT. Short-term administration of progesterone and estradiol independently alter carotid-vasomotor, but not carotid-cardiac, baroreflex function in young women. Am J Physiol Heart Circ Physiol 3: H141 H149, Collins HL, Augustyniak RA, Ansorge EJ, O Leary DS. Carotid baroreflex pressor responses at rest and during exercise: cardiac output vs. regional vasoconstriction. Am J Physiol Heart Circ Physiol 28: H642 H648, Davy KP, Seals DR, Tanaka H. Augmented cardiopulmonary and integrative sympathetic baroreflexes but attenuated peripheral vasoconstriction with age. Hypertension 32: , Du XJ, Dart AM, Riemersma RA. Sex differences in the parasympathetic nerve control of rat heart. Clin Exp Pharmacol Physiol 21: , Ebert TJ, Morgan BJ, Barney JA, Denahan T, Smith JJ. Effects of aging on baroreflex regulation of sympathetic activity in humans. Am J Physiol Heart Circ Physiol 263: H798 H83, Eckberg DL, Kifle YT, Roberts VL. Phase relationship between normal human respiration and baroreflex responsiveness. J Physiol 34: 489 2, Fadel PJ. Arterial baroreflex control of the peripheral vasculature in humans: rest and exercise. Med Sci Sports Exerc 4: 2 262, Fadel PJ, Ogoh S, Keller DM, Raven PB. Recent insights into carotid baroreflex function in humans using the variable pressure neck chamber. Exp Physiol 88: , Fadel PJ, Raven PB. Human investigations into the arterial and cardiopulmonary baroreflexes during exercise. Exp Physiol 97: 39, Fairfax ST, Holwerda SW, Credeur DP, Zuidema MY, Medley JH, Dyke PC 2nd, Wray DW, Davis MJ, Fadel PJ. The role of alphaadrenergic receptors in mediating beat-by-beat sympathetic vascular transduction in the forearm of resting man. J Physiol 91: , Fairfax ST, Padilla J, Vianna LC, Davis MJ, Fadel PJ. Spontaneous bursts of muscle sympathetic nerve activity decrease leg vascular conductance in resting humans. Am J Physiol Heart Circ Physiol 34: H79 H766, Fisher JP, Kim A, Hartwich D, Fadel PJ. New insights into the effects of age and sex on arterial baroreflex function at rest and during dynamic exercise in humans. Auton Neurosci 172: 13 22, Fisher JP, Kim A, Young CN, Fadel PJ. Carotid baroreflex control of arterial blood pressure at rest and during dynamic exercise in aging humans. Am J Physiol Regul Integr Comp Physiol 299: R1241 R1247, Fisher JP, Kim A, Young CN, Ogoh S, Raven PB, Secher NH, Fadel PJ. Influence of ageing on carotid baroreflex peak response latency in humans. J Physiol 87: , Fisher JP, Ogoh S, Ahmed A, Aro MR, Gute D, Fadel PJ. Influence of age on cardiac baroreflex function during dynamic exercise in humans. Am J Physiol Heart Circ Physiol 293: H777 H783, Gribbin B, Pickering TG, Sleight P, Peto R. Effect of age and high blood pressure on baroreflex sensitivity in man. Circ Res 29: , Grundy SM. Primary prevention of coronary heart disease: integrating risk assessment with intervention. Circulation 1: , Hart EC, Charkoudian N, Wallin BG, Curry TB, Eisenach J, Joyner MJ. Sex and ageing differences in resting arterial pressure regulation: the role of the beta-adrenergic receptors. J Physiol 89: , Hart EC, Wallin BG, Curry TB, Joyner MJ, Karlsson T, Charkoudian N. Hysteresis in the sympathetic baroreflex: role of baseline nerve activity. J Physiol 89: , Jansen JR, Schreuder JJ, Mulier JP, Smith NT, Settels JJ, Wesseling KH. A comparison of cardiac output derived from the arterial pressure wave against thermodilution in cardiac surgery patients. Br J Anaesth 87: , Jansen JR, Wesseling KH, Settels JJ, Schreuder JJ. Continuous cardiac output monitoring by pulse contour during cardiac surgery. Eur Heart J 11, Suppl I: 26 32, Jones PP, Snitker S, Skinner JS, Ravussin E. Gender differences in muscle sympathetic nerve activity: effect of body fat distribution. Am J Physiol Endocrinol Metab 27: E363 E366, Kim A, Deo SH, Fisher JP, Fadel PJ. Effect of sex and ovarian hormones on carotid baroreflex resetting and function during dynamic exercise in humans. J Appl Physiol 112: , Kim A, Deo SH, Vianna LC, Balanos GM, Hartwich D, Fisher JP, Fadel PJ. Sex differences in carotid baroreflex control of arterial blood pressure in humans: relative contribution of cardiac output and total vascular conductance. Am J Physiol Heart Circ Physiol 31: H244 H246, Matsukawa T, Sugiyama Y, Mano T. Age-related changes in baroreflex control of heart rate and sympathetic nerve activity in healthy humans. J Auton Nerv Syst 6: , Matsukawa T, Sugiyama Y, Watanabe T, Kobayashi F, Mano T. Baroreflex control of muscle sympathetic nerve activity is attenuated in the elderly. J Auton Nerv Syst 73: , Matsukawa T, Sugiyama Y, Watanabe T, Kobayashi F, Mano T. Gender difference in age-related changes in muscle sympathetic nerve activity in healthy subjects. Am J Physiol Regul Integr Comp Physiol 27: R16 R164, Meyer MR, Haas E, Barton M. Need for research on estrogen receptor function: importance for postmenopausal hormone therapy and atherosclerosis. Gend Med, Suppl A: S19 S33, Monahan KD. Effect of aging on baroreflex function in humans. Am J Physiol Regul Integr Comp Physiol 293: R3 R12, Ng AV, Callister R, Johnson DG, Seals DR. Age and gender influence muscle sympathetic nerve activity at rest in healthy humans. Hypertension 21: 498 3, Ogoh S, Fadel PJ, Monteiro F, Wasmund WL, Raven PB. Haemodynamic changes during neck pressure and suction in seated and supine positions. J Physiol 4: , Ogoh S, Fadel PJ, Nissen P, Jans O, Selmer C, Secher NH, Raven PB. Baroreflex-mediated changes in cardiac output and vascular conductance in response to alterations in carotid sinus pressure during exercise in humans. J Physiol : , Potts JT, Shi XR, Raven PB. Carotid baroreflex responsiveness during dynamic exercise in humans. Am J Physiol Heart Circ Physiol 26: H1928 H1938, Querry RG, Smith SA, Stromstad M, Ide K, Secher NH, Raven PB. Anatomical and functional characteristics of carotid sinus stimulation in humans. Am J Physiol Heart Circ Physiol 28: H239 H2398, Raven PB, Fadel PJ, Ogoh S. Arterial baroreflex resetting during exercise: a current perspective. Exp Physiol 91: 37 49, Raven PB, Potts JT, Shi X. Baroreflex regulation of blood pressure during dynamic exercise. Exerc Sport Sci Rev 2: , Schmitt JA, Joyner MJ, Charkoudian N, Wallin BG, Hart EC. Sex differences in alpha-adrenergic support of blood pressure. Clin Auton Res 2: , Simmons GH, Padilla J, Young CN, Wong BJ, Lang JA, Davis MJ, Laughlin MH, Fadel PJ. Increased brachial artery retrograde shear rate at exercise onset is abolished during prolonged cycling: role of thermoregulatory vasodilation. J Appl Physiol 11: , Studinger P, Goldstein R, Taylor JA. Age- and fitness-related alterations in vascular sympathetic control. J Physiol 87: , Young CN, Deo SH, Padilla J, Laughlin MH, Fadel PJ. Pro-atherogenic shear rate patterns in the femoral artery of healthy older adults. Atherosclerosis 211: , 21. AJP-Heart Circ Physiol doi:1.112/ajpheart