On the hemodynamic consequence of the chemoreflex and muscle mechanoreflex interaction in women and men: two tales, one story

Abstract

The cardiovascular response resulting from the individual activation of the muscle mechanoreflex (MMR), or the chemoreflex (CR), is different between men and women. Whether the hemodynamic consequence resulting from the interaction of these sympathoexcitatory reflexes is also sex-dependent remains unknown. MMR and CR were activated by passive leg movement (LM) and exposure to hypoxia (O₂-CR), or hypercapnia (CO₂-CR), respectively. Twelve young men and 12 young women completed two experimental protocols: 1) resting in normoxia (P_ETO₂: ~83mmHg, P_ETCO₂: ~34mmHg), normocapnic hypoxia (P_ETO₂: ~48mmHg, P_ETCO₂: ~34mmHg), and hyperoxic hypercapnia (P_ETO₂: ~524mmHg, P_ETCO₂: ~44mmHg); 2) LM under the same gas conditions. During the MMR:O₂-CR coactivation, in men, the observed blood pressure (MAP) and cardiac output (CO) were not different (additive effect), while the observed leg blood flow (LBF) and vascular conductance (LVC) were significantly lower (hypo-additive), compared with the sum of the responses elicited by each reflex alone. In women, the observed MAP was not different (additive) while the observed CO, LBF, and LVC were significantly greater (hyper-additive), compared with the summated responses. During the MMR:CO₂-CR coactivation, in men, the observed MAP, CO, and LBF were not different (additive), while the observed LVC was significantly lower (hypo-additive), compared with the summated responses. In women, the observed MAP was significantly higher (hyper-additive), while the observed CO, LBF, and LVC were not different (additive), compared with the summated responses. The interaction of the MMR and CR has a pronounced influence on the autonomic cardiovascular control, with the hemodynamic consequences differing between men and women.

Graphical Abstract

The chemoreflex and the muscle mechanoreflex are sympathoexcitatory mechanisms which, via neural feedback to the cardiovascular centre in the medulla, mediate neurocirculatory responses during physical activity. The interaction of the peripheral chemoreflex and muscle mechanoreflex potentiates vasoconstriction in men, but potentiates vasodilatation in women (left panel). The interaction of the central chemoreflex and muscle mechanoreflex also potentiates vasoconstriction in men, whereas the reflex interaction is simply additive for the vasomotor tone in women (right panel).

INTRODUCTION

Autonomic reflexes, including the arterial baroreflex (Bristow et al., 1971), the exercise pressor reflex (McCloskey & Mitchell, 1972), and the chemoreflex (CR; Rose et al., 1983) are essential for regulating the hemodynamic response to physiological stress, such as exercise or the hypoxia of high altitude (Rowell & Blackmon, 1987). Specifically, sensory neurons, activated by mechanical and/or chemical stimuli in the periphery, project afferent signals to the brainstem and mobilize cardiovascular control centers in the medulla. These medullary nuclei have descending control of the autonomic system and reflexively respond to sensory input by modulating sympathetic and parasympathetic nervous system activity (Zucker et al., 2012; Fisher et al., 2015). Increases in sympathetic outflow augment cardiac output (CO) and vasomotor tone to raise blood pressure, which, in concert with regional vasodilatory mechanisms, ensures appropriate blood flow to muscle and organs in need (Rowell & Blackmon, 1987; Joyner & Casey, 2015). However, despite independence from each other, interactions among these autonomic reflexes can have considerable cardiovascular implications. For example, the interaction between the exercise pressor reflex and the CR causes hemodynamic consequences which cannot be explained by the combined responses evoked by each reflex alone (Wan et al., 2020).

The peripheral CR is predominantly activated by stimulation of O₂-sensitive arterial chemoreceptors, while the central CR is predominantly triggered by activation of CO₂-sensitive medullary chemoreceptors (Forster & Smith, 2010). Both CRs facilitate sympathetic outflow leading to hemodynamic changes (Somers et al., 1989; Xie et al., 2001). In addition to the CR, the exercise pressor reflex represents another sympathoexcitatory pathway. This reflex has two components, namely, the muscle metaboreflex, which is triggered by feedback from metabosensitive (mainly group IV) muscle afferents, and the muscle mechanoreflex (MMR), which is activated by feedback from mechanosensitive (mainly group III) muscle afferents (McCloskey & Mitchell, 1972; Kaufman et al., 1983). Earlier human investigations have demonstrated that both muscle metaboreflex (Mark et al., 1985) and MMR (Cui et al., 2006) can, in isolation, increase sympathetic nerve activity and elicit hemodynamic changes. Therefore, the muscle metaboreflex and/or the MMR may contribute to the hemodynamic consequences resulting from the interaction between the exercise pressor reflex and the CR (Wan et al., 2020). However, while the cardiovascular impact of the metaboreflex:CR interaction has been documented (Seals et al., 1991; Lykidis et al., 2010; Edgell & Stickland, 2014; Delliaux et al., 2015), the hemodynamic consequence of the MMR:CR interaction remains largely unknown (Bruce & White, 2012).

Healthy young men and women exhibit different hemodynamic responses to MMR (Ives et al., 2013) or CR (Casey et al., 2014) activation, a discrepancy attributable, albeit not unanimously agreed upon (Limberg et al., 2016), to an enhanced β-adrenergic responsiveness to sympathoexcitation in premenopausal women (Kneale et al., 2000; Hart et al., 2011). Indeed, at rest and during mild exercise, the CR-mediated activation of vascular β-adrenoreceptors opposes α-adrenergic vasoconstriction and accounts for the majority of the vasodilatory response to hypoxia (Weisbrod et al., 2001; Wilkins et al., 2008). However, β-adrenergic vasodilation declines with increasing intensity and becomes absent during moderate exercise in hypoxia (Wilkins et al., 2008). While this likely explains the similar cardiovascular consequences of the exercise pressor reflex:CR interaction during moderate-intensity exercise in men and women (Wan et al., 2020), the potential for a sex difference in the hemodynamic consequence of the MMR:CR interaction remains to be explored.

It was therefore the purpose of this study to evaluate the interactive effect of the MMR and CR on central and peripheral hemodynamics in men and women. The MMR was activated by stimulating mechanosensitive muscle afferents via passive leg movement (LM), and the peripheral and central CRs were activated via hypoxia (O₂-CR) and hypercapnia (CO₂-CR), respectively. We hypothesized that the reflex interaction would potentiate sympathetically-mediated hemodynamic responses, restricting vascular conductance and muscle blood flow in men, but not in women.

METHODS

Ethical approval

All experimental protocols were approved by the Institutional Review Board of the University of Utah and by the Salt Lake City Veterans Affairs Medical Center (IRB #62889). The study conformed to the standards set by Declaration of Helsinki, except for registration in a database. Following written and verbal explanation of all procedures and risks, written informed consent was obtained from each subject before enrollment in the study. Twenty-four recreationally active, healthy volunteers participated in the study (12 men and 12 women, age: 25 ± 3 vs. 27 ± 5 yr, p = 0.398; height: 180 ± 6 vs. 168 ± 5 cm, p < 0.001; mass: 81 ± 9 vs. 64 ± 5 kg, p < 0.001; body mass index: 25 ± 3 vs. 23 ± 2 kg · m⁻², p = 0.022). The participants were non-smokers, non-medicated, and asymptomatic for cardiovascular or respiratory disorders. Prior to the study visit, participants refrained from exercise for 24 h and caffeine/alcohol for 12 h and fasted for 4 h (ad libitum water intake). Female participants were premenopausal, had either normal menstruation (n = 10) or were asked to pause oral contraceptives, and studied during the early follicular phase of the menstrual cycle (i.e., within 5 days after the onset of menses; self-reported).

Experimental design

During an initial familiarization session, volunteers were accustomed to all study procedures. To determine eligibility, one of the investigators moved participants’ right lower leg, beginning with ~0° knee joint angle, through a 90° range of motion at 1 Hz. Subjects were excluded from further participation if they were not able to fully relax their leg during this maneuver. On a separate day, participants completed six experimental trials during which they rested (Rest; 3 trials), or their right leg was passively moved (i.e., LM; 3 trials) while being exposed to room air (i.e., normoxia; Norm_Rest and Norm_LM), normocapnic hypoxia (Hypo_Rest and Hypo_LM), or hyperoxic hypercapnia (Hyper_Rest and Hyper_LM) (Fig. 1A). In each trial, participants sat quietly during a 4 min lead-in period after which they either continued to rest, or LM was performed for 60 s (total time of gas exposure: 5 min). All trials were randomized and separated by a 10-min break in room air.

Figure 1. Schematic representation of the individual and the concurrent activation of the muscle mechanoreflex (MMR) and the chemoreflex (CR).

A: subjects underwent two protocols, passive leg movement (LM) or rest for 60 s, under 3 gas conditions (i.e., normoxia, normocapnic hypoxia, and hyperoxic hypercapnia). This design resulted in 6 experimental trials with Norm_Rest serving as the baseline hemodynamic response during minimal reflex activation. The straight arrows denote the comparisons utilized to estimate the hemodynamic effects of activation of the MMR (➡), activation of the CR (⇨), and coactivation of the MMR and CR (). B: examples illustrating the hemodynamic response during the experimental trials. To determine the effects of the reflex activation and coactivation, the maximal response to LM under each gas condition was pinpointed and compared with the 60-s average of Norm_Rest in each subject (i.e., ∆₁ and ∆₂); the 60-s average of Hypo_Rest or Hyper_Rest was compared with the 60-s average of Norm_Rest in each subject (i.e., ∆₃). Therefore, ∆₁, ∆₂, and ∆₃ reflect the hemodynamic changes under the influences of the MMR activation, the MMR:CR coactivation, and the CR activation, respectively. Due to the nature of hemodynamic responses during LM, a nadir response was identified for mean arterial pressure (MAP) and a peak response was identified for heart rate (HR), stroke volume (SV), cardiac output (CO), leg blood flow (LBF), and leg vascular conductance (LVC).

Procedures and measurements

Normocapnic hypoxia and hyperoxic hypercapnia.

Pulmonary gas exchange and ventilatory responses were monitored breath-by-breath using an open circuit calorimetry system (Innocor, Innovision, Glamsbjerg, Denmark). Oxyhemoglobin saturation (S_pO₂) was estimated using pulse oximetry with a forehead sensor (OxiMax N-600x, Nellcor, Minneapolis, MN, USA). To induce normocapnic hypoxia with the goal of decreasing S_pO₂ to ~85% and maintaining the partial pressure of end-tidal CO₂ (P_ETCO₂) at baseline, participants inspired a gas mixture from a Douglas bag containing 9%–10% O₂ and 2%–3% CO₂ (balance N₂). Target S_pO₂ and P_ETCO₂ were then held constant by adding pure O₂ or CO₂ to the inspirate. To induce hyperoxic hypercapnia, P_ETCO₂ was increased by 10 mmHg above the normoxic baseline level by inspiring a gas mixture of 5%–6% CO₂ (balance O₂); P_ETCO₂ was maintained by titrating pure CO₂ into the inspirate when needed.

Leg movement (LM).

In each gas condition, participants comfortably maintained an upright seated position and initially relaxed both legs in full extension. A member of the research team then moved the subjects’ right lower leg, beginning with ~0° knee joint angle, through a 90° range of motion at 1 Hz for 60 s. To minimize the startle response (Venturelli et al., 2012), the metronome used to guide the cadence was turned on during the lead-in phase, i.e., prior to the beginning of LM. The participants were made aware of the LM procedure within a ~30-s window, but not informed of the exact starting time. To ensure a similar anticipatory influence across the experimental trials, the same information and feedback were also provided during the Rest trials; all participants were blinded on whether or not LM would be performed.

Central and peripheral hemodynamics.

Mean arterial pressure (MAP) was measured beat-by-beat using finger photoplethysmography (Finapres NOVA, Finapres Medical Systems, Enschede, Netherlands). Beat-by-beat heart rate (HR) was monitored using a 3-lead electrocardiogram (CardioCard, Nasiff Associates, Central Square, NY, USA) and recorded at 1 kHz via a personal computer equipped with commercially available software (Spike2, Cambridge Electronic Design, Cambridge, UK). Stroke volume (SV) was estimated beat-by-beat from pressure waveforms assessed by the finger photoplethysmography using the Modelflow method (Finapres NOVA, Finapres Medical Systems, Enschede, Netherlands), and CO was calculated as the product of SV and HR. To determine blood flow to the moving leg (LBF), blood flow velocity and vessel diameter were measured in the right common femoral artery, distal to the inguinal ligament and proximal to the bifurcation of the deep and superficial femoral arteries, using Doppler ultrasound (LOGIQ 7, General Electric Medical Systems, Milwaukee, WI, USA). LBF was calculated as mean blood flow velocity × π × (vessel diameter ÷ 2)² × 60. All blood flow velocity measurements were performed with the probe positioned to maintain an insonation angle of 60° or less. Leg vascular conductance (LVC) was calculated as LBF ÷ MAP.

Data processing

For the pulmonary gas exchange and ventilatory responses, 60-s averages were obtained during the Rest and LM trials. The hemodynamic responses to Rest were determined as the average over the 60-s period, the hemodynamic responses to LM were analyzed second-by-second and smoothed using a 3-s rolling average (Gifford & Richardson, 2017). Since, in contrast to steady-state voluntary muscle contractions, the hemodynamic response to LM is transient, the maximal responses in normoxia, hypoxia, and hypercapnia (i.e., Norm_LM, Hypo_LM, and Hyper_LM, respectively) were identified from each participant and used for comparisons of all cardiovascular variables (Kruse et al., 2018). Based on the assumption that the CR and MMR play no role in determining the hemodynamic response to rest in normoxia (i.e., Norm_Rest; little to no reflex activity), the individual and interactive reflex effects on central and peripheral hemodynamics were defined as follows (Fig. 1B): 1) the individual effects of the MMR, the O₂-CR, and the CO₂-CR were estimated by the differences between Norm_LM and Norm_Rest (∆Norm_LM-Norm_Rest), between Hypo_Rest and Norm_Rest (∆Hypo_Rest-Norm_Rest), and between Hyper_Rest and Norm_Rest (∆Hyper_Rest-Norm_Rest), respectively; 2) the interactive effects of the MMR and O₂-CR and of the MMR and CO₂-CR during reflex coactivation were calculated as the differences between Hypo_LM and Norm_Rest (∆Hypo_LM-Norm_Rest), and between Hyper_LM and Norm_Rest (∆Hyper_LM-Norm_Rest), respectively; 3) to investigate the mode of interaction between the reflexes, the observed hemodynamic changes during the coactivation of the MMR and CR (O₂-CR or CO₂-CR) were compared with the arithmetic sum of the changes elicited by each reflex alone (∆Hypo_LM-Norm_Rest vs. ∆Norm_LM-Norm_Rest + ∆Hypo_Rest-Norm_Rest, or, ∆Hyper_LM-Norm_Rest vs. ∆Norm_LM-Norm_Rest + ∆Hyper_Rest-Norm_Rest). The definition of the interaction mode has been previously described (Wan et al., 2020): when the observed responses during the coactivation of the reflexes were larger than the sum of the responses evoked by each reflex alone, the interaction was considered ‘hyper-additive’; when the observed responses during the reflex coactivation were not different from the summated responses, the interaction was considered ‘additive’; when the observed responses were smaller than the summated responses, the interaction was considered ‘hypo-additive’.

Statistical analysis

Data were analyzed using statistical analysis software (SigmaPlot 11.0, Systat Software, Palo Alto, CA, USA). The Student’s t-test was used for the description of subject characteristics between women and men. Since this study aimed to activate the CR via hypoxia or hypercapnia and to investigate its interaction with the MMR, data obtained from the hypoxic and hypercapnic conditions were separately compared with those from the normoxic conditions. Thus, a 2 × 2 repeated-measure ANOVA, with protocols (Rest/LM) and conditions (normoxia/hypoxia or normoxia/hypercapnia) as the two factors, were calculated to test alterations in the pulmonary gas exchange and cardiorespiratory responses during the experimental trials. If the ANOVA revealed a significant main effect or interaction, the Tukey’s post hoc analysis was performed to identify the differences. To directly test our hypotheses, a priori planned comparisons were made to determine the individual/interactive effects and the interaction mode of the reflexes on the hemodynamic changes, using the Holm-Bonferroni method to correct the familywise error of multiple comparisons. All dependent variables were analyzed for women and men separately. Additionally, the female and male participants in this study were also directly compared using a 2 × 4 mixed-model ANOVA with sexes (women/men) as the between-subjects factor and trials (Norm_Rest/Norm_LM/Hypo_Rest/Hypo_LM or Norm_Rest/Norm_LM/Hyper_Rest/Hyper_LM) as the within-subject factor (results only reported in Statistical Summary Document). All the data were presented as mean ± S.D. Statistical significance was set at p < 0.05.

RESULTS

Oxyhemoglobin saturation, partial pressure of end-tidal gases, and ventilatory responses

Compared to normoxia, S_pO₂ and end-tidal partial pressure of O₂ (P_ETO₂) were significantly reduced when exposed to hypoxia in men (n = 12, Table 1) and women (n = 12, Table 2); however, while P_ETCO₂ was maintained at the normoxic level in women, there was a small (~2 mmHg), but significant, increase in P_ETCO₂ in men in hypoxia (e.g., main effect of hypoxia, p = 0.049). Furthermore, compared to normoxic conditions, minute ventilation (V̇_E), breathing frequency (f_B), and tidal volume (V_T) were significantly increased in both groups (Tables 1 & 2). When exposed to hyperoxic hypercapnia, S_pO₂, P_ETO₂, P_ETCO₂, V̇_E, f_B, and V_T were all increased significantly from normoxia in both men (n = 12, Table 3) and women (n = 12, Table 4).

Table 1.

Pulmonary gas exchange and cardiorespiratory responses to normoxia and normocapnic hypoxia at rest and during passive leg movement (LM) in men

	Normoxia		Normocapnic hypoxia		p value
	Rest	LM	Rest	LM	Main effect of LM	Main effect of hypoxia	Interaction effect
Pulmonary gas exchange
SpO2 (%)	97 ± 2	97 ± 2	85 ± 1†	84 ± 1†	0.044	<0.001	0.587
PETO2 (mmHg)	85 ± 5	81 ± 5*	47 ± 3†	47 ± 3†	0.032	<0.001	0.037
PETCO2 (mmHg)	33 ± 3	35 ± 3*	35 ± 3†	35 ± 2	0.025	0.049	0.071
Ventilatory responses
V̇E (L·min−1)	11 ± 2	13 ± 3	22 ± 10†	25 ± 12*†	0.004	0.001	0.210
fB (breaths·min−1)	15 ± 4	18 ± 5*	18 ± 6†	20 ± 4	0.019	0.012	0.377
VT (L)	0.9 ± 0.2	0.8 ± 0.1	1.2 ± 0.3†	1.3 ± 0.4†	0.601	<0.001	0.155
Hemodynamic responses
MAP (mmHg)	101 ± 15	97 ± 18	100 ± 22	99 ± 13	0.115	0.888	0.406
HR (beats·min−1)	67 ± 9	77 ± 13*	79 ± 10†	90 ± 12*†	<0.001	<0.001	0.696
SV (mL)	78 ± 12	90 ± 14*	79 ± 14	93 ± 13*	<0.001	0.627	0.353
CO (L·min−1)	5.2 ± 0.7	6.6 ± 1.4*	6.1 ± 1.1†	7.9 ± 1.4*†	<0.001	<0.001	0.397
LBF (L·min−1)	0.3 ± 0.1	1.6 ± 0.4*	0.4 ± 0.2	1.4 ± 0.4*†	<0.001	0.249	0.023
LVC (mL·min−1·mmHg)	2.8 ± 0.9	16.4 ± 5.8*	3.9 ± 2.4	14.3 ± 4.7*†	<0.001	0.422	0.041

S_pO₂: oxyhemoglobin saturation; P_ETO₂: end-tidal partial pressure of O₂; P_ETCO₂: end-tidal partial pressure of CO₂; V̇_E: minute ventilation; f_B: breathing frequency; V_T: tidal volume; MAP: mean arterial pressure; HR: heart rate; SV: stroke volume; CO: cardiac output; LBF: leg blood flow; LVC: leg vascular conductance.

^*p < 0.05 vs. rest;

^†p < 0.05 vs. normoxia. Pulmonary gas exchange and ventilatory responses are 60-s average at rest and during LM, while hemodynamic responses are 60-s average at rest and the maximal response during LM; n = 12. Data were analyzed using two-way repeated-measure ANOVA with Tukey’s post hoc test and are presented as mean ± S.D.

Table 2.

Pulmonary gas exchange and cardiorespiratory responses to normoxia and normocapnic hypoxia at rest and during passive leg movement (LM) in women

	Normoxia		Normocapnic hypoxia		p value
	Rest	LM	Rest	LM	Main effect of LM	Main effect of hypoxia	Interaction effect
Pulmonary gas exchange
SpO2 (%)	97 ± 1	97 ± 1	84 ± 1†	84 ± 1†	0.488	<0.001	0.911
PETO2 (mmHg)	85 ± 3	83 ± 5	49 ± 2†	50 ± 2†	0.570	<0.001	0.413
PETCO2 (mmHg)	32 ± 4	33 ± 1	34 ± 2	34 ± 2	0.661	0.069	0.443
Ventilatory responses
V̇E (L·min−1)	9 ± 2	11 ± 2	17 ± 7†	20 ± 5*†	0.022	<0.001	0.655
fB (breaths·min−1)	15 ± 5	17 ± 4*	17 ± 5†	21 ± 5*†	0.008	0.002	0.311
VT (L)	0.7 ± 0.2	0.6 ± 0.1	1.0 ± 0.1†	1.0 ± 0.2†	0.317	<0.001	0.981
Hemodynamic responses
MAP (mmHg)	100 ± 17	95 ± 23*	101 ± 23	99 ± 19	0.019	0.299	0.372
HR (beats·min−1)	66 ± 10	75 ± 15*	83 ± 13†	94 ± 14*†	<0.001	<0.001	0.460
SV (mL)	70 ± 14	73 ± 16	61 ± 14†	70 ± 14*	0.020	0.004	0.010
CO (L·min−1)	4.6 ± 1.1	5.0 ± 1.1*	5.0 ± 1.2†	6.2 ± 1.2*†	<0.001	<0.001	0.005
LBF (L·min−1)	0.5 ± 0.4	1.1 ± 0.5*	0.5 ± 0.4	1.4 ± 0.5*†	<0.001	0.008	0.003
LVC (mL·min−1·mmHg)	4.1 ± 2.8	11.1 ± 5.3*	4.4 ± 2.5	13.2 ± 5.0*†	<0.001	0.012	0.019

S_pO₂: oxyhemoglobin saturation; P_ETO₂: end-tidal partial pressure of O₂; P_ETCO₂: end-tidal partial pressure of CO₂; V̇_E: minute ventilation; f_B: breathing frequency; V_T: tidal volume; MAP: mean arterial pressure; HR: heart rate; SV: stroke volume; CO: cardiac output; LBF: leg blood flow; LVC: leg vascular conductance.

^*p < 0.05 vs. rest;

^†p < 0.05 vs. normoxia. Pulmonary gas exchange and ventilatory responses are 60-s average at rest and during LM, while hemodynamic responses are 60-s average at rest and the maximal response during LM; n = 12. Data were analyzed using two-way repeated-measure ANOVA with Tukey’s post hoc test and are presented as mean ± S.D.

Table 3.

Pulmonary gas exchange and cardiorespiratory responses to normoxia and hyperoxic hypercapnia at rest and during passive leg movement (LM) in men

	Normoxia		Hyperoxic hypercapnia		p value
	Rest	LM	Rest	LM	Main effect of LM	Main effect of hypercapnia	Interaction effect
Pulmonary gas exchange
SpO2 (%)	97 ± 2	97 ± 2	100 ± 1†	100 ± 0†	0.099	<0.001	0.364
PETO2 (mmHg)	85 ± 5	81 ± 5*	526 ± 10†	526 ± 9†	0.053	<0.001	0.051
PETCO2 (mmHg)	33 ± 3	35 ± 3*	45 ± 2†	45 ± 2†	0.001	<0.001	0.046
Ventilatory responses
V̇E (L·min−1)	11 ± 2	13 ± 3	31 ± 11†	33 ± 9†	0.086	<0.001	0.873
fB (breaths·min−1)	15 ± 4	18 ± 5*	19 ± 5†	20 ± 4	0.056	0.026	0.062
VT (L)	0.9 ± 0.2	0.8 ± 0.1	1.7 ± 0.4†	1.7 ± 0.3†	0.595	<0.001	0.065
Hemodynamic responses
MAP (mmHg)	101 ± 15	97 ± 18*	107 ± 17†	102 ± 15*†	0.020	0.002	0.992
HR (beats·min−1)	67 ± 9	77 ± 13*	69 ± 11	79 ± 9*	0.001	0.154	1.000
SV (mL)	78 ± 12	90 ± 14*	84 ± 18†	97 ± 18*†	<0.001	0.025	0.484
CO (L·min−1)	5.2 ± 0.7	6.6 ± 1.4*	5.7 ± 1.0†	7.1 ± 1.3*†	<0.001	0.021	0.934
LBF (L·min−1)	0.3 ± 0.1	1.6 ± 0.4*	0.3 ± 0.1	1.5 ± 0.4*†	<0.001	0.244	0.080
LVC (mL·min−1·mmHg)	2.8 ± 0.9	16.4 ± 5.8*	3.0 ± 0.8	14.4 ± 5.8*†	<0.001	0.063	0.023

S_pO₂: oxyhemoglobin saturation; P_ETO₂: end-tidal partial pressure of O₂; P_ETCO₂: end-tidal partial pressure of CO₂; V̇_E: minute ventilation; f_B: breathing frequency; V_T: tidal volume; MAP: mean arterial pressure; HR: heart rate; SV: stroke volume; CO: cardiac output; LBF: leg blood flow; LVC: leg vascular conductance.

^*p < 0.05 vs. rest;

^†p < 0.05 vs. normoxia. Pulmonary gas exchange and ventilatory responses are 60-s average at rest and during LM, while hemodynamic responses are 60-s average at rest and the maximal response during LM; n = 12. Data were analyzed using two-way repeated-measure ANOVA with Tukey’s post hoc test and are presented as mean ± S.D.

Table 4.

Pulmonary gas exchange and cardiorespiratory responses to normoxia and hyperoxic hypercapnia at rest and during passive leg movement (LM) in women

	Normoxia		Hyperoxic hypercapnia		p value
	Rest	LM	Rest	LM	Main effect of LM	Main effect of hypercapnia	Interaction effect
Pulmonary gas exchange
SpO2 (%)	97 ± 1	97 ± 1	100 ± 0†	100 ± 1†	0.748	<0.001	0.596
PETO2 (mmHg)	85 ± 3	83 ± 5	519 ± 20†	522 ± 15†	0.633	<0.001	0.098
PETCO2 (mmHg)	32 ± 4	33 ± 1	43 ± 2†	43 ± 2†	0.732	<0.001	0.394
Ventilatory responses
V̇E (L·min−1)	9 ± 2	11 ± 2	24 ± 12†	28 ± 9*†	0.010	<0.001	0.282
fB (breaths·min−1)	15 ± 5	17 ± 4*	19 ± 8†	21 ± 6†	0.049	0.009	0.420
VT (L)	0.7 ± 0.2	0.6 ± 0.1	1.3 ± 0.2†	1.4 ± 0.3†	0.388	<0.001	0.094
Hemodynamic responses
MAP (mmHg)	100 ± 17	95 ± 23*	101 ± 17	102 ± 18†	0.172	0.024	0.029
HR (beats·min−1)	66 ± 10	75 ± 15*	72 ± 14†	81 ± 15*†	<0.001	0.014	0.886
SV (mL)	70 ± 14	73 ± 16	76 ± 15	78 ± 16	0.262	0.053	0.760
CO (L·min−1)	4.6 ± 1.1	5.0 ± 1.1*	5.5 ± 1.3†	5.9 ± 1.3*†	0.004	0.002	0.797
LBF (L·min−1)	0.5 ± 0.4	1.1 ± 0.5*	0.5 ± 0.4	1.3 ± 0.6*†	<0.001	0.011	0.076
LVC (mL·min−1·mmHg)	4.1 ± 2.8	11.1 ± 5.3*	4.6 ± 2.6	12.1 ± 5.7*	<0.001	0.122	0.380

S_pO₂: oxyhemoglobin saturation; P_ETO₂: end-tidal partial pressure of O₂; P_ETCO₂: end-tidal partial pressure of CO₂; V̇_E: minute ventilation; f_B: breathing frequency; V_T: tidal volume; MAP: mean arterial pressure; HR: heart rate; SV: stroke volume; CO: cardiac output; LBF: leg blood flow; LVC: leg vascular conductance.

^*p < 0.05 vs. rest;

^†p < 0.05 vs. normoxia. Pulmonary gas exchange and ventilatory responses are 60-s average at rest and during LM, while hemodynamic responses are 60-s average at rest and the maximal response during LM; n = 12. Data were analyzed using two-way repeated-measure ANOVA with Tukey’s post hoc test and are presented as mean ± S.D.

Individual and interactive hemodynamic effects of the MMR and the O₂-CR

Men (n = 12).

The resting responses (60-s averages) and the maximal responses to LM under normoxia and normocapnic hypoxia are shown in Table 1. Figure 2 illustrates the separate and combined hemodynamic effects triggered by the activation of the MMR and the O₂-CR. During the individual activation of the MMR (i.e., ∆Norm_LM-Norm_Rest), HR, SV, CO, LBF, and LVC were significantly increased. During the individual activation of the O₂-CR (i.e., ∆Hypo_Rest-Norm_Rest), HR and CO were significantly increased. When both reflexes were activated concurrently (i.e., ∆Hypo_LM-Norm_Rest), HR, SV, CO, LBF, and LVC were significantly increased. In terms of the mode of interaction between the MMR and the O₂-CR (i.e., ∆Hypo_LM-Norm_Rest vs. ∆Norm_LM-Norm_Rest + ∆Hypo_Rest-Norm_Rest), the LBF and LVC responses were significantly smaller during the coactivation of the two reflexes compared with the summation of the responses elicited by each reflex alone, reflecting a hypo-additive interaction. The MAP, HR, SV, and CO responses to the coactivation of the MMR and O₂-CR were not different from the summated responses, indicating an additive interaction.

Figure 2. Hemodynamic changes during the individual and the concurrent activation of the muscle mechanoreflex (MMR) and the hypoxia-induced chemoreflex (O₂-CR) in men.

MMR & O₂-CR: observed changes during coactivation of the MMR and O₂-CR; MMR + O₂-CR: sum of the changes elicited by each reflex alone; MAP: mean arterial pressure; HR: heart rate; SV: stroke volume; CO: cardiac output; LBF: leg blood flow; LVC: leg vascular conductance. *Significantly different from zero, p < 0.05; †significantly different between MMR & O₂-CR and MMR + O₂-CR, p < 0.05. Individual subject data are denoted by ◇ and solid lines; n = 12. Data were analyzed using a priori planned comparisons with the Holm-Bonferroni correction and are presented as mean ± S.D.

Women (n = 12).

The resting responses (60-s averages) and the maximal responses to LM under normoxia and normocapnic hypoxia are shown in Table 2. Figure 3 illustrates the separate and combined hemodynamic effects triggered by the activation of the MMR and the O₂-CR. During the individual activation of the MMR, MAP was significantly decreased while HR, CO, LBF, and LVC were significantly increased. During the individual activation of the O₂-CR, HR and CO were significantly increased but SV was decreased (p = 0.002). When both reflexes were activated concurrently, HR, CO, LBF, and LVC were significantly increased. In terms of the mode of interaction between the MMR and the O₂-CR, the SV, CO, LBF, and LVC responses were significantly greater during the reflex coactivation compared with the sum of the responses elicited by each reflex alone, reflecting a hyper-additive interaction. The MAP and HR responses to the coactivation of the MMR and O₂-CR were not different from the summated responses, indicating an additive interaction.

Figure 3. Hemodynamic changes during the individual and the concurrent activation of the muscle mechanoreflex (MMR) and the hypoxia-induced chemoreflex (O₂-CR) in women.

MMR & O₂-CR: observed changes during coactivation of the MMR and O₂-CR; MMR + O₂-CR: sum of the changes elicited by each reflex alone; MAP: mean arterial pressure; HR: heart rate; SV: stroke volume; CO: cardiac output; LBF: leg blood flow; LVC: leg vascular conductance. *Significantly different from zero, p < 0.05; †significantly different between MMR & O₂-CR and MMR + O₂-CR, p < 0.05. Individual subject data are denoted by ◇ and solid lines; n = 12. Data were analyzed using a priori planned comparisons with the Holm-Bonferroni correction and are presented as mean ± S.D.

Individual and interactive hemodynamic effects of the MMR and the CO₂-CR

Men (n = 12).

The resting responses (60-s averages) and the maximal responses to LM under normoxia and hyperoxic hypercapnia are shown in Table 3. Figure 4 illustrates the separate and combined hemodynamic effects evoked by the activation of the MMR and the CO₂-CR. During the individual activation of the CO₂-CR (i.e., ∆Hyper_Rest-Norm_Rest), MAP and CO were significantly increased. When both reflexes were activated simultaneously (i.e., ∆Hyper_LM-Norm_Rest), HR, SV, CO, LBF, and LVC were significantly increased. Regarding the interaction mode of the MMR and CO₂-CR (i.e., ∆Hyper_LM-Norm_Rest vs. ∆Norm_LM-Norm_Rest + ∆Hyper_Rest-Norm_Rest), the LVC response was significantly lower during the coactivation of the two reflexes compared with the sum of the responses induced by each reflex alone, reflecting a hypo-additive interaction. The MAP, HR, SV, CO, and LBF responses to the coactivation of the MMR and CO₂-CR were not different from the summated responses, indicating an additive interaction.

Figure 4. Hemodynamic changes during the individual and the concurrent activation of the muscle mechanoreflex (MMR) and the hypercapnia-induced chemoreflex (CO₂-CR) in men.

MMR & CO₂-CR: observed changes during coactivation of the MMR and CO₂-CR; MMR + CO₂-CR: sum of the changes elicited by each reflex alone; MAP: mean arterial pressure; HR: heart rate; SV: stroke volume; CO: cardiac output; LBF: leg blood flow; LVC: leg vascular conductance. *Significantly different from zero, p < 0.05; †significantly different between MMR & CO₂-CR and MMR + CO₂-CR, p < 0.05. Individual subject data are denoted by ◇ and solid lines; n = 12. Data were analyzed using a priori planned comparisons with the Holm-Bonferroni correction and are presented as mean ± S.D.

Women (n = 12).

The resting responses (60-s averages) and the maximal responses to LM under normoxia and hyperoxic hypercapnia are shown in Table 4. Figure 5 illustrates the separate and combined hemodynamic effects evoked by the activation of the MMR and the CO₂-CR. During the individual activation of the CO₂-CR, HR and CO were significantly increased. When both reflexes were activated simultaneously, HR, SV, CO, LBF, and LVC were significantly increased. Regarding the interaction mode of the MMR and CO₂-CR, the MAP response was significantly greater during the reflex coactivation compared with the sum of the responses induced by each reflex alone, reflecting a hyper-additive interaction. The HR, SV, CO, LBF, and LVC responses to the coactivation of the MMR and CO₂-CR were not different from the summated responses, indicating an additive interaction.

Figure 5. Hemodynamic changes during the individual and the concurrent activation of the muscle mechanoreflex (MMR) and the hypercapnia-induced chemoreflex (CO₂-CR) in women.

MMR & CO₂-CR, observed changes during coactivation of the MMR and CO₂-CR; MMR + CO₂-CR, sum of the changes elicited by each reflex alone; MAP: mean arterial pressure; HR: heart rate; SV: stroke volume; CO: cardiac output; LBF: leg blood flow; LVC: leg vascular conductance. *Significantly different from zero, p < 0.05; †significantly different between MMR & CO₂-CR and MMR + CO₂-CR, p < 0.05. Individual subject data are denoted by ◇ and solid lines; n = 12. Data were analyzed using a priori planned comparisons with the Holm-Bonferroni correction and are presented as mean ± S.D.

DISCUSSION

The purpose of this study was to examine the interaction and hemodynamic consequence of the MMR and CR. LM and hypoxia, or hypercapnia, were used to activate the MMR and the O₂-CR, or CO₂-CR, individually and simultaneously. In men, the interaction between the MMR and the CR, regardless of being activated by hypoxia or hypercapnia, was found to be additive for MAP and central hemodynamics, and, as indicated by the restriction in LVC, hyper-additive in terms of sympathetic vasoconstriction. In women, while additive for MAP and HR, the MMR:O₂-CR interaction was, potentially due to an augmented sympathetic vasodilation, hyper-additive for the central and peripheral hemodynamic responses. However, when the CR was activated by hypercapnia in women, the reflex interaction effect was hyper-additive for MAP and additive for central and peripheral hemodynamics. These findings suggest that the interaction between the MMR and the CR exerts a profound influence on the autonomic cardiovascular control, with the hemodynamic consequences differing between women and men. Of particular functional relevance is that the MMR:CR interaction with hypoxia restricts locomotor muscle perfusion in men, but facilitates the muscle perfusion in women.

Hemodynamic responses to MMR and CR activation

LM-induced activation of the MMR significantly increased central and peripheral hemodynamics in both sexes (Figs. 2–5, black bars). While the MMR accounts for most of the changes in CO and a considerable portion of the LBF response (Trinity et al., 2010), it should be recognized that a part of the hyperemic response to LM is mediated by endothelial nitric oxide-dependent local vasodilation (Mortensen et al., 2012; Trinity et al., 2012). Regardless, the observed increases in CO are in line with previous investigations utilizing passive muscle stretch to activate the MMR and reflective of reflex-mediated changes in the sympathetic and parasympathetic innervation of the heart (Gladwell & Coote, 2002; Gladwell et al., 2005; Cui et al., 2006; Drew et al., 2017; Venturelli et al., 2017). Furthermore, in agreement with earlier studies (Somers et al., 1989; Somers et al., 1991; Xie et al., 2001; Cooper et al., 2005; Steinback et al., 2009), activation of the CR via hypoxia, or hypercapnia, augmented central, but not peripheral, hemodynamics in both sexes (Figs. 2–5, white bars). While an excitation of the sympathetic nervous system in response to hypoxia and hypercapnia may explain the increases in CO, the lack of a subsequent impact on peripheral hemodynamics is likely due to the systemic vasodilation associated with arterial hypoxemia and hypercapnia (Saito et al., 1988; Dinenno, 2016). Intriguingly, with no effect in women, men also exhibited an increase in MAP when the CR was activated by hyperoxic hypercapnia, but not when activated by normocapnic hypoxia. While this effect could, in part, be due to vasoconstriction resulting from exposure to hyperoxia (Ranadive et al., 2014), the reasons for the absence of this effect in women remain unknown and could reflect yet another sex difference associated with the exposure to hypercapnia (Sayegh et al., 2022). Taken together, both MMR and CR activation evoked significant cardiovascular changes in men and women.

Hemodynamic responses to MMR:CR coactivation

Coactivation of both reflexes increased HR, CO, LBF and LVC in both sexes (Figs. 2–5, gray bars). Furthermore, with no change in MAP in men, the MMR:CR coactivation abolished the decrease in MAP observed in women during normoxic LM (i.e., MMR activation alone). However, the mode of interaction between the MMR and the CR and the potential for a sex difference cannot be addressed by these observations. To evaluate whether the observed hemodynamic responses are a simple addition of those evoked by the MMR and the CR alone, or the result of significant interactions between the two reflexes, we compared the hemodynamic responses during the coactivation with the mathematical sum of the responses evoked by each reflex.

Mode of the MMR:CR interaction

Based on voluntary knee-extension exercise, our recent study demonstrated the exercise pressor reflex:O₂-CR interaction to be hypo-additive for LBF and LVC and hyper-additive for MAP and HR, leading to the conclusion that this reflex interaction results in a synergistic effect on global sympathetic outflow (Wan et al., 2020). Interestingly, the present study documented that, in men, the MMR:O₂-CR interaction is also hypo-additive for LBF and LVC (Fig. 2E & F), suggesting that the potentiated sympathetic vasoconstriction resulting from the coactivation of the exercise pressor reflex and O₂-CR (Wan et al., 2020), is, at least partly, attributed to the MMR:O₂-CR interaction. However, in contrast to the hyper-additive effect of the exercise pressor reflex:O₂-CR interaction on MAP and HR (Wan et al., 2020), the consequence of the MMR:O₂-CR interaction was just additive in terms of these variables (Fig. 2A & B). A potential explanation for this inconsistency could be a different influence of the arterial baroreflex. Specifically, the exercise pressor reflex (comprising the MMR and the muscle metaboreflex) and central command, both of which were activated during the prior study utilizing voluntary exercise, shift the arterial baroreflex to operate at a higher MAP and HR and away from the centering point (Raven et al., 2006; Hureau et al., 2018). By contrast, LM (i.e., passive exercise) in the present study did not engage central command or the muscle metaboreflex and should therefore be characterized by a limited resetting of the arterial baroreflex. Thus, although the MMR:O₂-CR interaction could increase sympathetic outflow, the resulting impact on MAP and HR might have been buffered by the arterial baroreflex, presumably through an increased parasympathetic drive to the resting heart (Mitchell et al., 1989; White & Raven, 2014; Doherty et al., 2018).

In women, the MMR:O₂-CR interaction was, similar to men, additive in terms of MAP and HR (Fig.3A & B). However, in contrast to men, the MMR:O₂-CR coactivation exerted a hyper-additive effect on SV, CO, and the peripheral hemodynamics (Fig. 3C–F), indicating an augmented vasodilation. While the exact mechanisms remain unclear, a greater β-adrenergic vasodilation in young women and the subsequent reduction in afterload may contribute to this sex-related disparity. In fact, there is compelling evidence of an upregulated β-adrenergic responsiveness of the peripheral vasculature to sympathetic outflow in premenopausal women (Kneale et al., 2000; Hart et al., 2011) and, as such, the vasodilatory response to hypoxic exercise is higher in women compared to men (Casey et al., 2014). Moreover, Jacob and colleagues (2021) recently reported that acute hypoxia increases the vasoconstrictor response to sympathoexcitatory stressors (e.g., cold pressor test) in men, but attenuates the response in women, a finding which further substantiates the current conclusion of the sex difference in the MMR:O₂-CR interaction. Importantly, considering the dominant β-adrenergic vasodilation in women, which likely explains these sex-specific consequences of the reflex interaction, the current findings support the hypothesis of a potentiated sympathoexcitation during the MMR:O₂-CR coactivation in humans.

The MMR:CO₂-CR interaction in men was additive for most circulatory variables, but hypo-additive for LVC (Fig. 4), which suggests, in keeping with the consequence of the MMR:O₂-CR coactivation, a potentiated sympathetic vasoconstriction. In women, however, the MMR:CO₂-CR interaction was hyper-additive for MAP (Fig. 5A), but additive for all central and peripheral hemodynamic variables (Fig. 5B–F). In comparison with the additive effect on MAP and the hyper-additive effect on central and peripheral hemodynamics during the MMR:O₂-CR coactivation, it is not clear why the women (but not the men) exhibited disparate interactive effects with CR activation via hypercapnia vs. hypoxia. These discrepancies potentially result from a combination of multiple factors, including that baroreflex buffering of the CR-mediated sympathetic outflow is more effective in hypoxia compared to hypercapnia (Somers et al., 1991) and that the effect of hypoxia on altering baroreflex sensitivity differs from that in hypercapnia (Cooper et al., 2005; Steinback et al., 2009). In fact, using gas conditions similar to those in the present study, Sayegh et al. (2022) recently reported that the increase in muscle sympathetic nerve activity in response to hyperoxic hypercapnia at rest is greater in women vs. men, but not different between the sexes when exposed to normocapnic hypoxia. In addition, because of the enhanced β-adrenergic activity, premenopausal women display a different hemodynamic consequence in response to increasing muscle sympathetic nerve activity, i.e., a blunted neurovascular transduction, when compared with men (Hogarth et al., 2007; Hart et al., 2009; Usselman et al., 2015; Briant et al., 2016; Robinson et al., 2019). Collectively, the current CR type- and sex-related differences in the interaction mode may be ascribed to the combined influence of these factors on altering the relationship between baroreflex sensitivity and neurovascular transduction (Hissen et al., 2019) and the subsequent consequence for the hemodynamic manifestation of neurocirculatory control.

Limitations

Although the present study was intended to activate the MMR and the CR to examine the hemodynamic effect of their interaction, the resulting impact on SV and CO could have also triggered the cardiopulmonary baroreflex, a secondary consequence which might have influenced our findings. However, given that the consequences for SV and CO were additive during the MMR:O₂-CR coactivation in men and during the MMR:CO₂-CR coactivation in both sexes, the observed hyper- or hypo-additive effect on the peripheral hemodynamics was most likely due to the interaction of the MMR and CR, and not due to the engagement of the cardiopulmonary baroreflex. The circumstance may have been different in women where the MMR:O₂-CR coactivation resulted in a hyper-additive effect on SV and CO, i.e., SV and CO were augmented, which could have further activated the cardiopulmonary baroreflex. Hence, the resulting baroreflex-mediated sympathetic inhibition might have counteracted the sympathoexcitatory effect of the MMR:O₂-CR interaction and could have led to an underestimation of the interaction effect on the peripheral vasculature in women. Finally, sympathetic outflow was not directly measured in this study and all conclusions related to sympathetic nervous system activity were derived from hemodynamic changes. It is, in this context, crucial to emphasize that both evoked parasympathetic alterations and blunted neurovascular transduction in women could have resulted in a disassociation between sympathetic outflow and hemodynamic responses.

CONCLUSION

The interaction of the MMR and CR has a pronounced influence on the autonomic cardiovascular control, with the hemodynamic consequences differing between men and women. Of particular interest for future cardiovascular studies at altitude are the findings that the MMR:O₂-CR interaction restricts peripheral hemodynamics in young men, but, likely due to the greater β-adrenergic vasodilation characterizing premenopausal women, facilitates central and peripheral hemodynamics in their female counterparts. Despite this sex difference, the current data point to a synergistic effect of the MMR:O₂-CR coactivation upon sympathetic nervous system activity.

KEY POINTS.

• The cardiovascular response resulting from the activation of the muscle mechanoreflex (MMR), or the chemoreflex (CR), was previously shown to be different between women and men; this study focused on the hemodynamic consequence of the interaction of these two sympathoexcitatory reflexes.

• MMR and CR were activated by passive leg movement and exposure to hypoxia (O₂-CR), or hypercapnia (CO₂-CR), respectively.

• Individual and interactive reflex effects on central and peripheral hemodynamics were quantified in healthy young women and men.

• In men, the MMR:O₂-CR and MMR:CO₂-CR interactions restricted peripheral hemodynamics, likely by potentiating sympathetic vasoconstriction.

• In women, the MMR:O₂-CR interaction facilitated central and peripheral hemodynamics, likely by potentiating sympathetic vasodilation; however, the MMR:CO₂-CR interaction was simply additive for the central and peripheral hemodynamics.

• The interaction between the MMR and the CR exerts a profound influence on the autonomic control of cardiovascular function in humans, with the hemodynamic consequences differing between women and men.

Biography

Hsuan-Yu Wan completed his VMD (National Chung Hsing University, Taiwan) and PhD in Exercise Physiology (Indiana University- Bloomington, USA), and currently is a postdoctoral fellow with Professor Markus Amann at the University of Utah Vascular Research Laboratory. Hsuan-Yu is interested in the reflex regulation of cardiovascular and ventilatory responses to exercise under environmental stress in health and disease. His long-term research goal is to better understand the mechanisms for the neural integration of autonomic and cardiorespiratory control, and associated development of muscle fatigue in exercising humans.