CO 2 -Enriched Air Inhalation Modulates the Ventilatory and Metabolic Responses of Endurance Runners During Incremental Running in Hypobaric Hypoxia

Abstract

Cao, Yinhang, Naoto Fujii, Tomomi Fujimoto, Yin-Feng Lai, Takeshi Ogawa, Tsutomu Hiroyama, Yasushi Enomoto, and Takeshi Nishiyasu. CO₂-enriched air inhalation modulates the ventilatory and metabolic responses of endurance runners during incremental running in hypobaric hypoxia. High Alt Med Biol. 23:125–134, 2022.

Aim:

We measured the effects of breathing CO₂-enriched air on ventilatory and metabolic responses during incremental running exercise under moderately hypobairc hypoxic (HH) conditions.

Materials and Methods:

Ten young male endurance runners [61.4 ± 6.0 ml/(min·kg)] performed incremental running tests under three conditions: (1) normobaric normoxia (NN), (2) HH (2,500 m), and (3) HH with 5% CO₂ inhalation (HH+CO₂). The test under NN was always performed first, and then, the two remaining tests were completed in random and counterbalanced order.

Results:

End-tidal CO₂ partial pressure (55 ± 3 vs. 35 ± 1 mmHg), peak ventilation (163 ± 14 vs. 152 ± 12 l/min), and peak oxygen uptake [52.3 ± 5.5 vs. 50.5 ± 4.9 ml/(min·kg)] were all higher in the HH+CO₂ than HH trial (all p < 0.01), respectively. However, the duration of the incremental test did not differ between HH+CO₂ and HH trials.

Conclusion:

These data suggest that chemoreflex activation by breathing CO₂-enriched air stimulates breathing and aerobic metabolism during maximal intensity exercise without affecting exercise performance in male endurance runners under a moderately hypobaric hypoxic environment.

Introduction

Peak oxygen uptake ( ${\dot{V O}}_{2 p e a k}$ ) and exercise performance are lower at high altitude than at sea level due to the reduction in inspired oxygen (Stenberg et al., 1966; Lawler et al., 1988; Martin and O'Kroy, 1993; Ferretti et al., 1997). In addition, it has been shown that ventilatory responses to hypoxia are important determinants of ${\dot{V O}}_{2 p e a k}$ and exercise performance at high altitude (Gavin et al., 1998; Chapman, 2013). This is because increasing ventilatory responses in hypoxia can increase alveolar O₂ pressure, and thus arterial O₂ saturation, thereby increasing oxygen delivery to the peripheral tissues (e.g., locomotor muscles) (Lawler et al., 1988; Gavin et al., 1998; Calbet et al., 2003; Chapman, 2013). For instance, we previously observed that individuals exhibiting smaller reductions in ${\dot{V O}}_{2 p e a k}$ under moderately hypobaric hypoxic conditions (e.g., 2,500 m) also exhibit greater increases in peak ventilation (Ogawa et al., 2007). We therefore postulated that any intervention that can enhance the ventilatory response to hypoxia would increase aerobic energy provision and improve exercise performance at high altitude.

It is well known that elevating carbon dioxide (CO₂) can enhance ventilatory responses by stimulating respiratory chemoreflexes. This is because CO₂ that diffuses into the interstitial fluid in the brain and periphery reacts with the water to form carbonic acid (H₂CO₃), which then dissociates into bicarbonate (HCO₃⁻) and hydrogen ion (H⁺). The increased H⁺ then activates both central chemoreceptors in the medulla and peripheral chemoreceptors in the carotid bodies, ultimately increasing ventilation (Dempsey et al., 1984; Duffin, 2005). In line with this, breathing CO₂-enriched air increases peak ventilation during maximal exercise at sea level (Babb, 1997; Kato et al., 2005; Fan et al., 2012). On the contrary, the effect of breathing CO₂-enriched air on peak ventilation during maximal exercise at high altitude remains unclear, as the results from earlier studies are inconsistent (Subudhi et al., 2011; Fan and Kayser, 2013; Doutreleau et al., 2017).

Furthermore, the three abovementioned studies were all conducted under normobaric or hypobaric hypoxic conditions equivalent to 4,000–5,000 m. No study has yet investigated the effect of breathing CO₂-enriched air on peak ventilation under moderately hypobaric hypoxic conditions (e.g., 2,500 m), which is an altitude frequently used by athletes for “live high-train low” model of altitude training (Chapman et al., 2014).

It should be noted that higher peak ventilation during high-intensity exercise increases the work of breathing, which can induce a redistribution of blood flow from the locomotor to respiratory muscles (Harms et al., 1997; Sheel et al., 2001) or exacerbate the perception of dyspnea and respiratory muscle fatigue under normobaric normoxic conditions (Harms et al., 2000; Yates, 2019). These factors may result in an early termination of exercise (Dempsey et al., 2008b; Romer and Dempsey, 2014). Also, arterial oxygen saturation is already near maximal at sea level; hence, increases in ventilation do not mediate a substantial increase in arterial oxygen saturation. Consequently, the increases in peak ventilation and peak aerobic energy production associated with breathing CO₂-enriched air, if they occur, do not necessarily translate into improved exercise performance under those conditions.

However, this may not occur under hypobaric hypoxic conditions. Breathing helium–oxygen (He-O₂), which has a lower gas density than normobaric air, can reduce airflow resistance and increase peak ventilation, ${\dot{V O}}_{2 p e a k}$ , and exercise performance under normobaric hypoxia (4,800 m) (Esposito and Ferretti, 1997). The reduced air density at high altitude (i.e., hypobaric hypoxia [HH]) may also alleviate the work of breathing related to higher peak ventilation. However, the air density in a moderately hypobaric hypoxic environment equivalent to 2,500 m is still greater than that with He-O₂ (∼25% vs. 80% reduction in air density relative to normal air). In addition, increases in ventilation can greatly increase arterial oxygen saturation since it is reduced under HH relative to normobaric normoxia (NN). It therefore remains unclear whether inhalation of CO₂-enriched air mediates increases in peak ventilation and ${\dot{V O}}_{2 p e a k}$ at moderate HH or improves exercise performance.

To address this question, the present study was designed as a mechanistic study to test the hypothesis that breathing CO₂-enriched air increases peak ventilation by stimulating chemoreflexes, thereby increasing arterial O₂ saturation and oxygen delivery to locomotor muscles, ultimately improving ${\dot{V O}}_{2 p e a k}$ and exercise performance in male endurance runners during incremental running exercise under moderately HH conditions equivalent to 2,500 m.

Materials and Methods

Ethics approval

This study was approved by the Human Subjects Committee of the University of Tsukuba (No. 30-121) and adhered to the guidelines in the Declaration of Helsinki, except for registration in a database. All participants provided written informed consent before participating in the study.

Participants

Ten healthy young male endurance runners participated in this study. Participant characteristics and pulmonary function values are presented in Table 1. All participants were lowlanders with no previous exposure to hypoxic conditions equal to or above 1,000 m for >6 months before participating in the present study. All participants were members of the university track and field team. They were all free from cardiopulmonary disease and were not cigarette smokers.

Table 1.

Participant Characteristics and Pulmonary Function

Age (years)	22 ± 1
Height (m)	1.74 ± 0.03
Weight (kg)	60.4 ± 2.9
FVC (l)	4.76 ± 0.46
FEV₁ (l)	4.16 ± 0.28
FEV/FVC (%)	88 ± 4
PEFR (l/s)	9.5 ± 0.6
MEF_25–75 (l/s)	4.8 ± 0.7
MVV (l/min)	176 ± 11

Values are mean ± SD.

FEV₁, forced expired volume in 1 second; FVC, forced vital capacity; MEF_25–75, maximal expiratory flow between 25% and 75% of forced vital capacity; MVV, maximal voluntary ventilation; PEFR, peak expiratory flow rate; SD, standard deviation.

Experimental procedure

Participants visited the laboratory before the main experiment and were familiarized with the experimental protocols and equipment. Thereafter, experimental sessions were initiated, in which the participants completed exhaustive incremental running tests in a custom-made environmental chamber (Shimazu, Kyoto, Japan) under three conditions: (1) NN, (2) HH, and (3) HH with CO₂-enriched air (5% CO₂ [25.7 mmHg], 21% O₂ [107.7 mmHg], and balance N₂) (HH+CO₂). The custom-made environment chamber can simulate hypobaric hypoxic conditions up to 8,000 m above sea level. In addition, the temperature in the chamber can be controlled, and the room air can be continuously ventilated to minimize any increase in CO₂ inside the chamber. The three trials were separated by 4–7 days, with the last two trials completed in a random and counterbalanced order. The 5% CO₂-enriched air, which can increase peak ventilation under normobaric hypoxia (Doutreleau et al., 2017), was adopted in the present study. The inspired O₂ concentration and O₂ tension were maintained at their same respective levels in the HH and HH+CO₂ trials by adjusting the N₂ concentration. The NN trial was always performed first, which enabled us to estimate the total amount of gas that would be inhaled during the HH and HH+CO₂ trials.

In all three trials, the participants breathed through the same circuit connected to a large prefilled reservoir held in three to four bags (total 2,100–2,800 l) and were unaware of which gas they were breathing. The gas in the prefilled reservoir was humidified. In the HH+CO₂ trial, CO₂-enriched air inhalation was initiated at 3 minutes into the 6-minute rest period and was continued until the end of the exercise. Under hypobaric hypoxic conditions, the chamber was gradually decompressed at a rate of ∼130 m/min (∼20 minutes) to achieve a barometric pressure equivalent to that at 2,500 m (560 mmHg). Each exhaustive incremental running test began within 20 minutes after completing the decompression.

Pulmonary function assessment

Pulmonary function was assessed under NN condition using a portable spirometer (AS-507; Minato Medical Science, Osaka, Japan) as previously described (Crapo et al., 1995).

Maximal incremental running tests

On experimental days, all participants performed self-selected 10-minute warm-up exercises (e.g., stretching and jogging) outside the laboratory. The structure of the warm-up was matched for all three conditions. They then entered the environmental chamber, where the room temperature was regulated to 20.2°C ± 0.3°C. The room air was continuously ventilated to minimize any increase of CO₂ inside the chamber. After a 6-minute rest period, participants initiated a 2-minute warm-up at a speed of 180 m/min on the treadmill, followed by a 1-minute rest. An exhaustive incremental running test then commenced at a speed of 220 m/min. The running speed was increased by 20 m/min every 2 minutes until 280 m/min was reached, after which the running speed was increased by 10 m/min every 1 minute until volitional fatigue. Participants were verbally encouraged throughout the running test. Near the end of the test, expiratory gases were directly assessed using the Douglas bag method.

We elected to use the Douglas bag method as the measurement error can be large if data were assessed by a mass spectrometer (ARCO-2000; ARCO SYSTEM, Chiba, Japan), especially at maximal intensity exercise. All participants met at least two of the following three criteria for ${\dot{V O}}_{2 p e a k}$ (Rice et al., 2000): (1) $\dot{V O}$ ₂ reached a plateau and did not increase further, despite increases in running speed (<150 ml); (2) peak heart rate reached >90% of the age predicted value; and (3) the respiratory exchange ratio was >1.1.

Measurements

Participants breathed through a face mask attached to a two-way nonrebreathing valve (Hans Rudolph, Shawnee, KS) with the expiration side connected to a Douglas bag (200–250 l) via a smooth bore hose. A pneumotachograph transducer (PN-230; ARCO SYSTEM, Chiba, Japan) and a gas sampling tube with a sampling rate of <0.5 l/min were attached between the mask and the two-way valve, and the expired volume and gases were analyzed using a mass spectrometer (ARCO-2000; ARCO SYSTEM). The temperature of expired gas proximal to the pneumotachograph transducer was measured to express volume parameters (i.e., minute ventilation) under body temperature, pressure, and saturated conditions, whereas metabolic parameters (i.e., $\dot{V O}$ ₂, CO₂ output) under standard temperature and pressure, and dry conditions. The flowmeter was calibrated by a syringe with a known volume of 3 l and reference gases at known concentrations immediately before starting incremental running tests under each condition (NN, HH, or HH+CO₂ condition). Continuous breath-by-breath variables of minute ventilation, tidal volume, breathing frequency, $\dot{V O}$ ₂, CO₂ output, respiratory exchange ratio, ventilatory equivalents for O₂ and CO₂, end-tidal O₂, and CO₂ partial pressures were determined during rest and submaximal exercise periods using the mass spectrometer mentioned above. Immediately after the incremental running tests, the gas in the Douglas bag was mixed and analyzed using the same gas sampling tube, and then passed slowly through a dry gas meter (DC-5A; Shinagawa, Tokyo, Japan) to obtain its volume under each condition.

This information was used to assess peak ventilation, ${\dot{V O}}_{2 p e a k}$ , and peak CO₂ output. Arterial O₂ saturation was estimated with a forehead pulse oximeter (N-595; Nellcor, Hayward, CA). Heart rate was recorded every 5 seconds using a heart rate monitor (RS400; POLAR, Finland). Ratings of perceived exertion (Borg 6–20) and dyspnea (Borg 0–10) were also recorded at each running stage (Borg, 1982). All respiratory variables and arterial O₂ saturation were continuously recorded at a sampling rate of 1,000 Hz and stored in a computer via a data acquisition system (PowerLab 16/30; ADInstruments).

Estimated respiratory muscle oxygen uptake

Respiratory muscle oxygen uptake ( $\dot{V O}$ _2RM) at maximal exercise was estimated using a second-order regression equation as described previously (Wilhite et al., 2013): [ $\dot{V O}$ _2RM (ml/min) = −9.396 + 1.015 × minute ventilation +0.0121 × minute ventilation²]. The second-order regression equation mentioned above was developed based on the data provided by Aaron et al. (1992).

Data analysis

During rest and the first four stages of the exercise (220–280 m/min), data averaged over the last 1 minute of each stage were used for analysis. Data collected during the last 1 minute of the last stage that participants completed were defined as the peak value. If a participant did not complete the 1-minute stage, data at this speed were not included in the data analysis. The incremental test time was determined as the time from the start of the exercise to the point where the participant could no longer continue to run. In addition, the percent differences in ${\dot{V O}}_{2 p e a k}$ , peak ventilation, and arterial O₂ saturation between the HH and HH+CO₂ trials were calculated to determine the extent to which peak ventilation and arterial O₂ saturation contribute to the difference in ${\dot{V O}}_{2 p e a k}$ .

The relationships between the percent change in ${\dot{V O}}_{2 p e a k}$ and peak ventilation as well as between the percent change in ${\dot{V O}}_{2 p e a k}$ and arterial O₂ saturation from the HH to the HH+CO₂ trial were examined in correlation analyses.

Statistical analyses

Data are presented as mean ± standard deviation (SD). Two-way repeated-measures analysis of variance (ANOVA) was used to compare the submaximal exercise data; the two factors were trial (NN, HH, and HH+CO₂) and treadmill speed (220–280 m/min). One-way repeated-measures ANOVA was used to compare values recorded at maximal exercise; the factor was trial (NN, HH, and HH+CO₂). When main effects or interactions were detected, post hoc multiple comparisons were made using the Holm–Bonferroni method. Values of p < 0.05 were considered statistically significant. Pearson product moment correlations were used to analyze the relationships between the percent changes in ${\dot{V O}}_{2 p e a k}$ , peak ventilation, and arterial O₂ saturation from the HH to the HH+CO₂ trial. The SPSS 25 statistical software package for Windows (IBM, Armonk, NY) was used for all statistical analyses.

Results

Respiratory and cardiovascular responses at maximal exercise

As designed, breathing CO₂-enriched air increased end-tidal CO₂ partial pressure compared with breathing room air (55 ± 3 vs. 35 ± 1 mmHg, p < 0.01, Fig. 1).

FIG. 1.

End-tidal CO₂ partial pressure during maximal exercise. Dotted lines represent each participant's data. Data are mean ± SD. *p < 0.05 versus normobaric normoxia; ^†p < 0.05 hypobaric hypoxia+CO₂ versus hypobaric hypoxia. SD, standard deviation.

In the HH+CO₂ trial, peak ventilation was higher than in the HH trial (163 ± 14 vs. 152 ± 12 l/min) (Fig. 2A), and this effect was mediated by an increment in tidal volume compared with HH alone (Fig. 2B) (all p < 0.01). Breathing frequency did not differ between HH and HH+CO₂ trials (Fig. 2C). Moreover, at maximal exercise, ${\dot{V O}}_{2 p e a k}$ [52.3 ± 5.5 vs. 50.5 ± 4.9 ml/(min·kg)] (Fig. 3A), arterial O₂ saturation (79 ± 2 vs. 75 ± 3%) (Fig. 3B), and end-tidal O₂ partial pressure (89 ± 2 vs. 83 ± 3 mmHg) (Fig. 3C) were all higher in the HH+CO₂ than the HH trial (all p < 0.01). Thus, the hypoxia-induced reduction in ${\dot{V O}}_{2 p e a k}$ was smaller when the participants breathed CO₂-enriched air than when they breathed normal air (15 ± 3 vs. 18 ± 3%, p < 0.01).

FIG. 2.

Peak ventilation (A), tidal volume (B), and breathing frequency (C) during maximal exercise. Dotted lines represent each participant's data. Data are mean ± SD. *p < 0.05 versus normobaric normoxia; ^†p < 0.05 hypobaric hypoxia+CO₂ versus hypobaric hypoxia.

FIG. 3.

Peak oxygen uptake ( ${\dot{V O}}_{2 p e a k}$ ) (A), arterial O₂ saturation (B), and end-tidal O₂ partial pressure (C) during maximal exercise. Dotted lines represent each participant's data. Data are mean ± SD. *p < 0.05 versus normobaric normoxia; ^†p < 0.05 hypobaric hypoxia+CO₂ versus hypobaric hypoxia.

In addition, the percent increase in ${\dot{V O}}_{2 p e a k}$ with CO₂-enriched air inhalation in the hypobaric hypoxic environment correlated positively with both peak ventilation (r = 0.76, p = 0.02) and arterial O₂ saturation (r = 0.82, p < 0.01) (Fig. 4).

FIG. 4.

Relationships between the percent increase in peak oxygen uptake ( ${\dot{V O}}_{2 p e a k}$ ) and the percent increase in peak ventilation (A) or percent increase in arterial O₂ saturation (B) with CO₂-enriched air inhalation under hypobaric hypoxic conditions.

Ventilatory equivalents for O₂ (p < 0.01) and CO₂ (p < 0.01) were higher, whereas the respiratory exchange ratio was lower at maximal exercise in the HH+CO₂ trial than in the HH trial (Table 2). Peak CO₂ output (p = 0.10) and peak heart rate (p = 0.73) did not differ between the HH and HH+CO₂ trials (Table 2).

Table 2.

Variables Obtained During Maximal Exercise

	Normobaric normoxia	Hypobaric hypoxia	Hypobaric hypoxia+CO₂
Peak CO₂ output (l/min)	4.24 ± 0.35	3.66 ± 0.25^*	3.59 ± 0.28^*
Ventilatory equivalents for O₂	39 ± 2	49 ± 3^*	50 ± 3^*^,†
Ventilatory equivalents for CO₂	34 ± 2	41 ± 2^*	45 ± 3^*^,†
Respiratory exchange ratio	1.14 ± 0.02	1.19 ± 0.04^*	1.13 ± 0.04^†
Peak heart rate (beats/min)	196 ± 10	186 ± 10^*	184 ± 7^*
Rating of perceived exertion	19 ± 1	19 ± 1	19 ± 1
Dyspnea	9 ± 1	9 ± 1	10 ± 1^*^,†
$\dot{V O}$ _2RM (ml/min)	386 ± 55	413 ± 59^*	465 ± 77^*^,†

Values are mean ± SD.

p < 0.05 versus normobaric normoxia.

†

p < 0.05 hypobaric hypoxia+CO₂ versus hypobaric hypoxia.

$\dot{V O}$ _2RM, estimated respiratory muscle O₂ consumption.

Perceptions of dyspnea and exertion at maximal exercise

Perception of dyspnea at maximal exercise was higher in the HH+CO₂ trial than in the HH trial (p = 0.02, Table 2). There was no difference in the rating of perceived exertion between the HH and HH+CO₂ trials (p = 0.89, Table 2).

Incremental test time

Incremental test time did not differ between the HH and HH+CO₂ trials (p = 0.25, Fig. 5).

FIG. 5.

Total exercise time. Data are mean ± SD. *p < 0.05 versus normobaric normoxia. Dotted lines represent each participant's data.

Estimated respiratory muscle oxygen uptake at maximal exercise

In parallel with the increase in peak ventilation from the HH to the HH+CO₂ trials (Δ11 ± 5 l/min), the estimated $\dot{V O}$ _2RM was also higher in the HH+CO₂ than in the HH trial (p < 0.01, Table 2). As a result, an estimated 50 ± 15% (Δ53 ± 24 ml/min) of the 109 ± 39 ml/min increase in whole-body ${\dot{V O}}_{2 p e a k}$ is accounted for by CO₂-enriched air inhalation.

Respiratory and cardiovascular responses during rest and submaximal exercise

In resting subjects, minute ventilation, tidal volume, arterial O₂ saturation, end-tidal O₂ and CO₂ partial pressures, and ventilatory equivalents for O₂ and CO₂ were all higher, whereas CO₂ output and respiratory exchange ratio were lower in the HH+CO₂ than in the HH trial (Table 3, all p < 0.05). In addition, minute ventilation, tidal volume, $\dot{V O}$ ₂, arterial O₂ saturation, end-tidal O₂ and CO₂ partial pressures, and ventilatory equivalents for O₂ and CO₂ were all higher in the HH+CO₂ than in the HH trial at running speeds of 220–280 m/min (Table 3, all p < 0.05). Respiratory exchange ratio was lower in the HH+CO₂ than in the HH trial at running speeds of 280 m/min (Table 3, p < 0.05). CO₂ output and heart rate did not differ between the HH and HH+CO₂ trials at running speeds of 220–280 m/min (Table 3).

Table 3.

Variables Obtained During Rest and Submaximal Exercise

	Trial	Rest	220 (m/min)	240 (m/min)	260 (m/min)	280 (m/min)
End-tidal CO₂ partial pressure (mmHg)	Normobaric normoxia	36.5 ± 1.6	43.2 ± 2.7	44.5 ± 2.3	44.6 ± 2.9	43.4 ± 2.6
	Hypobaric hypoxia	37.4 ± 1.7	39.6 ± 1.3^*	39.4 ± 1.6^*	38.4 ± 1.8^*	36.9 ± 1.5^*
	Hypobaric hypoxia+CO₂	43.9 ± 1.2^*^,†	51.1 ± 2.7^*^,†	52.8 ± 3.0^*^,†	53.6 ± 2.6^*^,†	54.0 ± 2.6^*^,†
End-tidal O₂ partial pressure (mmHg)	Normobaric normoxia	114.4 ± 3.7	106.5 ± 4.9	107.5 ± 4.1	109.0 ± 4.5	111.8 ± 3.5
	Hypobaric hypoxia	72.6 ± 3.8^*	73.5 ± 3.3^*	75.7 ± 3.5^*	77.8 ± 3.7^*	80.8 ± 3.6^*
	Hypobaric hypoxia+CO₂	89.7 ± 2.9^*^,†	85.1 ± 3.3^*^,†	85.8 ± 2.6^*^,†	86.3 ± 2.0^*^,†	87.4 ± 1.9^*^,†
Minute ventilation (l/min)	Normobaric normoxia	18.7 ± 3.0	64.7 ± 7.2	73.2 ± 6.8	82.0 ± 8.7	94.7 ± 9.3
	Hypobaric hypoxia	18.7 ± 2.6	73.7 ± 6.9^*	85.4 ± 8.5^*	97.8 ± 9.9^*	111.4 ± 9.7^*
	Hypobaric hypoxia+CO₂	26.6 ± 2.8^*^,†	96.8 ± 13.0^*^,†	109.0 ± 14.1^*^,†	120.9 ± 12.2^*^,†	130.3 ± 13.9^*^,†
Tidal volume (l)	Normobaric normoxia	0.83 ± 0.12	1.51 ± 0.14	1.61 ± 0.10	1.73 ± 0.12	1.79 ± 0.16
	Hypobaric hypoxia	0.85 ± 0.18	1.58 ± 0.20	1.67 ± 0.15	1.78 ± 0.20	1.82 ± 0.20
	Hypobaric hypoxia+CO₂	1.09 ± 0.10^*^,†	1.97 ± 0.17^*^,†	2.12 ± 0.21^*^,†	2.13 ± 0.23^*^,†	2.14 ± 0.25^*^,†
Breathing frequency (breaths/min)	Normobaric normoxia	23 ± 3	43 ± 6	46 ± 5	48 ± 6	53 ± 5
	Hypobaric hypoxia	23 ± 3	47 ± 6^*	51 ± 6^*	56 ± 7^*	62 ± 11^*
	Hypobaric hypoxia+CO₂	25 ± 3	49 ± 6^*	52 ± 8^*	57 ± 9^*	62 ± 11^*
${\dot{V O}}_{2}$ (l/min)	Normobaric normoxia	0.48 ± 0.05	2.38 ± 0.19	2.62 ± 0.16	2.80 ± 0.14	3.04 ± 0.20
	Hypobaric hypoxia	0.49 ± 0.08	2.22 ± 0.12^*	2.42 ± 0.12^*	2.56 ± 0.15^*	2.66 ± 0.16^*
	Hypobaric hypoxia+CO₂	0.49 ± 0.07	2.32 ± 0.13^†	2.51 ± 0.16^†	2.71 ± 0.20^†	2.87 ± 0.22^*^,†
CO₂ output (l/min)	Normobaric normoxia	0.42 ± 0.06	2.08 ± 0.22	2.44 ± 0.21	2.71 ± 0.21	3.12 ± 0.27
	Hypobaric hypoxia	0.43 ± 0.09	2.13 ± 0.17	2.47 ± 0.22	2.80 ± 0.33	3.11 ± 0.36
	Hypobaric hypoxia+CO₂	0.35 ± 0.04^*^,†	1.99 ± 0.16	2.41 ± 0.19	2.70 ± 0.17	3.04 ± 0.21
Respiratory exchange ratio	Normobaric normoxia	0.87 ± 0.05	0.87 ± 0.06	0.93 ± 0.06	0.97 ± 0.06	1.01 ± 0.05
	Hypobaric hypoxia	0.87 ± 0.08	0.96 ± 0.06^*	1.02 ± 0.08^*	1.10 ± 0.15^*	1.18 ± 0.18^*
	Hypobaric hypoxia+CO₂	0.72 ± 0.05^*^,†	0.96 ± 0.07^*	1.00 ± 0.07^*	1.06 ± 0.07^*	1.08 ± 0.06^*^,†
	Normobaric normoxia	39 ± 4	27 ± 3	28 ± 3	29 ± 3	31 ± 3
Ventilatory equivalents for O₂	Hypobaric hypoxia	39 ± 4	33 ± 3^*	35 ± 3^*	38 ± 4^*	42 ± 5^*
	Hypobaric hypoxia+CO₂	55 ± 7^*^,†	42 ± 5^*^,†	44 ± 6^*^,†	45 ± 6^*^,†	46 ± 6^*^,†
	Normobaric normoxia	45 ± 4	31 ± 2	30 ± 2	30 ± 2	31 ± 2
Ventilatory equivalents for CO₂	Hypobaric hypoxia	44 ± 5	35 ± 3^*	35 ± 3^*	35 ± 3^*	36 ± 3^*
Ventilatory equivalents for CO₂	Hypobaric hypoxia+CO₂	76 ± 8^*^,†	49 ± 5^*^,†	45 ± 5^*^,†	45 ± 5^*^,†	43 ± 4^*^,†
Heart rate (beats/min)	Normobaric normoxia	77 ± 10	142 ± 14	156 ± 14	166 ± 15	174 ± 16
	Hypobaric hypoxia	77 ± 8	149 ± 13	161 ± 10	169 ± 12	177 ± 12
	Hypobaric hypoxia+CO₂	77 ± 5	149 ± 10	158 ± 13	169 ± 11	177 ± 9
Arterial O₂ saturation (%)	Normobaric normoxia	99 ± 1	93 ± 3	92 ± 5	93 ± 3	93 ± 2
	Hypobaric hypoxia	92 ± 1^*	82 ± 4^*	80 ± 4^*	79 ± 3^*	79 ± 2^*
	Hypobaric hypoxia+CO₂	97 ± 2^†	87 ± 4^*^,†	85 ± 4^*^,†	83 ± 3^*^,†	82 ± 2^*^,†
Dyspnea	Normobaric normoxia	NA	1 ± 1	2 ± 1	3 ± 1	4 ± 1
	Hypobaric hypoxia	NA	2 ± 1	3 ± 1	4 ± 2	5 ± 2
	Hypobaric hypoxia+CO₂	NA	2 ± 1^*	3 ± 2^*	5 ± 2^*^,†	7 ± 2^*^,†
Rating of perceived exertion	Normobaric normoxia	NA	9 ± 1	10 ± 2	12 ± 2	13 ± 2
	Hypobaric hypoxia	NA	9 ± 2	10 ± 2	12 ± 2	13 ± 3
	Hypobaric hypoxia+CO₂	NA	10 ± 2^*	11 ± 2	13 ± 3^†	15 ± 3^*^,†

Values are mean ± SD.

p < 0.05 versus normobaric normoxia.

†

p < 0.05 hypobaric hypoxia+CO₂ versus hypobaric hypoxia.

NA, not available; ${\dot{V O}}_{2}$ , oxygen uptake.

Perceptions of dyspnea and exertion during submaximal exercise

Perceptions of dyspnea and exertion at running speeds of 260 and 280 m/min were also higher in the HH+CO₂ than in the HH trial (all p < 0.05, Table 3).

Discussion

The major findings of the present study are as follows: (1) breathing 5% CO₂-enriched air increases end-tidal CO₂ partial pressure, peak ventilation, and ${\dot{V O}}_{2 p e a k}$ under moderately hypobaric hypoxic conditions equivalent to 2,500 m above sea level; and (2) the duration of the incremental test under hypobaric hypoxic conditions was not affected by breathing CO₂-enriched air. Taken together, these findings suggest that chemoreflex activation by breathing CO₂-enriched air increases peak ventilation. This thereby increases aerobic energy supply without affecting exercise performance under moderately hypobaric hypoxic conditions in male competitive endurance runners.

Ventilatory response

Consistent with an earlier study on the effects of normobaric hypoxia (4,000 m) (Doutreleau et al., 2017), we found that breathing CO₂-enriched air increased peak ventilation during maximal exercise (Fig. 2A). This may be the result of H⁺-mediated central and peripheral chemoreceptor activation related to the increased CO₂, which combines with water to form H₂CO₃ and then ionizes to HCO₃⁻ and H⁺ (Dempsey et al., 1984; Duffin, 2005). However, in contrast to our findings and those of Doutreleau et al. (2017), two earlier studies reported no increase in peak ventilation with end-tidal CO₂ partial pressure clamped at 45–50 mmHg under HH or normobaric hypoxia (4,875 or 5,000 m) (Subudhi et al., 2011; Fan and Kayser, 2013).

A possible explanation for the disparate findings is differences in the level of end-tidal CO₂ partial pressures. The end-tidal CO₂ partial pressures in the present study and the study by Doutreleau et al. (2017) were 55–60 mmHg, which are higher than in the other two studies (Subudhi et al., 2011; Fan and Kayser, 2013). This suggests that end-tidal CO₂ partial pressures greater than 50 mmHg may be necessary to increase peak ventilation in a moderately hypobaric hypoxic environment. Alternatively, the different levels of hypoxia may underlie the disparate results. The level of hypoxia achieved in the two earlier studies (4,875–5,000 m) was greater than in the present study and the study by Doutreleau et al. (2017) (4,000 m).

In addition, it has been reported that another respiratory neural mechanism independent of chemoreceptor feedback, defined as short-term modulation, is also involved in the regulation of exercise ventilatory response to maintain normal blood gas homeostasis (Wood et al., 2008). Furthermore, Dempsey and Smith (1994) proposed that enhanced ventilatory response during high-intensity exercise may be, in part, due to the increased descending activity from the locomotor-linked central nervous system. Thus, the role of short-term modulation and feedforward mechanisms in increasing peak ventilation cannot be ruled out in the present study.

During maximal or near-maximal exercise under normobaric normoxic conditions, the ventilatory responses of some endurance athletes may be restricted because of mechanical limitations classically termed expiratory flow limitation (Johnson et al., 1992; Dominelli et al., 2012). However, we previously observed that expiratory flow limitation does not affect ventilatory responses during incremental running in a moderate hypobaric hypoxic environment (2,500 m) (Cao et al., 2019). Thus, it does not appear that expiratory flow limitation, if there was any, affected ventilatory responses in the present study.

Metabolic response and exercise performance

As we hypothesized, there was an increase in ${\dot{V O}}_{2 p e a k}$ following the elevation in peak ventilation that accompanied breathing CO₂-enriched air under HH (Fig. 3A). This may reflect an increase in alveolar O₂ partial pressure associated with the elevated ventilation, which would increase the gradient for O₂ diffusion across the alveolar–capillary membrane (Esposito and Ferretti, 1997; Gavin et al., 1998; Calbet et al., 2003), ultimately increasing arterial O₂ saturation and the amount of O₂ transport to body tissues and organs. In line with this, our results demonstrated that arterial O₂ saturation during maximal exercise was higher in the HH+CO₂ trial than in the HH trial (Fig. 3B). Furthermore, the percent increment in ${\dot{V O}}_{2 p e a k}$ with CO₂-enriched air inhalation under HH correlated positively with peak ventilation (r = 0.76, p < 0.05) (Fig. 4A) and explained 58% of the increase in ${\dot{V O}}_{2 p e a k}$ based on the R² values.

Despite the elevated provision of aerobic energy in the HH+CO₂ trial, as reflected by the higher ${\dot{V O}}_{2 p e a k}$ , the incremental test time remained unchanged (Fig. 5). There are five possible explanations for this phenomenon. First, oxygen consumption by respiratory muscles increases as peak ventilation increases (Dominelli et al., 2015). Our results suggest that of the estimated $\dot{V O}$ _2RM, ∼50% of the increment in ${\dot{V O}}_{2 p e a k}$ associated with CO₂-enriched air inhalation was utilized by the respiratory muscles. Accumulation of metabolites in the respiratory muscles during high-intensity exercise can compromise leg blood flow via a sympathetically mediated vasoconstrictor reflex (Dempsey et al., 2006), which may limit $\dot{V O}$ ₂ by locomotor muscles. As a result, only a fraction of the increased oxygen taken up was utilized by the locomotor muscles, and this was perhaps insufficient to improve exercise performance in the present study. Second, perception of dyspnea was higher during maximal exercise when breathing CO₂-enriched air than when breathing normal air (Table 2). The increased perception of dyspnea could inhibit descending commands from the central nervous system (Fan and Kayser, 2016), which may inhibit exercise performance (Dempsey et al., 2008a; Wells and Norris, 2009; Ramsook et al., 2017).

This notion is supported by our results showing that the peak heart rate in the HH+CO₂ trial was slightly lower than that in the HH trial (Table 2). Third, the lower pH associated with CO₂-enriched air inhalation may shift the hemoglobin O₂ dissociation curve to the right, decreasing arterial O₂ saturation for a given arterial O₂ partial pressure (Doutreleau et al., 2017). Thus, the Bohr effect may prevent elevations in arterial O₂ saturation and O₂ content. Fourth, $\dot{V O}$ ₂ at all stages of the incremental running test was higher in the HH+CO₂ trial than in the HH trial (Table 3), suggesting that the running economy was decreased with breathing CO₂-enriched air. The impaired running economy might partly underlie unchanged exercise performance. Fifth, CO₂-enriched air inhalation has been shown to reduce limb muscle contractility by decreasing intracellular pH, which may in turn inhibit exercise performance (Vianna et al., 1990; Mador et al., 1997). Clamping pH through bicarbonate infusion (Peronnet et al., 2007; Volianitis et al., 2011) while breathing CO₂-enriched air may be a way to test this possibility in the future.

Previous studies reported that increased work of breathing with higher minute ventilation may result in an early termination of exercise under normobaric normoxic conditions (Dempsey et al., 2008b; Romer and Dempsey, 2014). However, our results demonstrated that breathing CO₂-enriched air increased peak ventilation and ${\dot{V O}}_{2 p e a k}$ without affecting exercise performance. This may be due to the reduced air density under moderate hypobaric hypoxic conditions used in the present study, which reduces the airflow resistance and thus alleviates the burden of respiratory muscles, ultimately offsetting the negative effects of increased work of breathing on exercise performance.

Furthermore, Esposito and Ferretti (1997) reported that breathing He-O₂ increased peak ventilation, ${\dot{V O}}_{2 p e a k}$ , and exercise performance at normobaric hypoxia (4,800 m) (Esposito and Ferretti, 1997). It should be noted that the percent increment in ${\dot{V O}}_{2 p e a k}$ (4% vs. 14%) and peak ventilation (7% vs. 31%) was lower in the present study than in the study by Esposito and Ferretti (1997). This appears to be due to the fact that the reduction in air density in the moderately hypobaric hypoxic environment used in the present study is smaller than with He-O₂ (∼25% vs. 80% reduction in air density relative to normal air). It may be that a greater reduction in air density is required to observe a clear performance-enhancing effect, but this remains to be tested.

Variables during submaximal exercise

Consistent with earlier studies (Subudhi et al., 2011; Doutreleau et al., 2017), we found that breathing CO₂-enriched air increased end-tidal CO₂ and O₂ partial pressures, minute ventilation, and arterial O₂ saturation, whereas it had no effect on heart rate or CO₂ output during maximal and submaximal intensity exercise (Table 3). Thus, breathing CO₂-enriched air appears to modulate ventilatory and metabolic responses independently of exercise intensity. Because breathing CO₂-enriched air increased the submaximal arterial O₂ saturation, it may enhance prolonged submaximal exercise performance (e.g., duration of exercise) under hypobaric hypoxic conditions, but this needs to be directly assessed in the future.

Limitations

As the calculation of estimated $\dot{V O}$ _2RM was designed under normobaric conditions only (Aaron et al., 1992), the estimated $\dot{V O}$ _2RM assessed under hypobaric conditions (i.e., HH vs. HH+CO₂ trial) may not be accurate. Future studies directly assessing $\dot{V O}$ _2RM under moderate hypobaric hypoxic conditions are required to precisely estimate $\dot{V O}$ _2RM under these conditions. Second, we did not measure blood gases, cardiac output, alveolar-arterial O₂ differences, arterial O₂ content, cerebral blood flow, or anaerobic metabolism. Consequently, we do not know how these variables were affected by breathing CO₂-enriched air. Third, we cannot rule out an order effect, as the NN trial was always performed first. Fourth, the hypobaric isocapnic condition was not used. Thus, the influence of reduction in end-tidal CO₂ pressure from NN to HH was not assessed.

Fifth, only young males were included in the present study, which limits the generalizability of our findings. In comparison with males, females have smaller lungs and conducting airways (Sheel et al., 2009). Consequently, expiratory flow limitation is a factor limiting exercise ventilation in females more often than in males (Guenette et al., 2007). Therefore, the increase in peak ventilation with breathing CO₂-enriched air may be smaller in females than in males. This warrants future investigation.

Conclusion

Chemoreflex activation by breathing CO₂-enriched air was shown to augment ventilatory and metabolic responses, but the incremental exercise performance by competitive endurance runners remained unchanged under moderately hypobaric hypoxic conditions (2,500 m above sea level).

Footnotes

Acknowledgments

We sincerely thank the participants for their time and efforts to participate in this study.

Authors' Contributions

C.Y., N.F., F.T., T.O., and T.N. conceived and designed the experiments. C.Y., F. T., and L.Y. contributed to data collection. C.Y. and N.F. performed the data analysis. C.Y., N.F., F.T., L.Y., T.O., T.H., Y.E., and T.N. interpreted the experimental results. C.Y. prepared the figures. C.Y. drafted the article. C.Y., N.F., F.T., L.Y., T.O., T.H., Y.E., and T.N. edited and revised the article. All authors approved the final version of the article, and all experiments took place at the Institute of Health and Sport Sciences located at the University of Tsukuba.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan and the “Human High Performance (HHP) Research Project,” at the University of Tsukuba, and supported by Shanghai Key Lab of Human Performance at Shanghai University of Sport (No. 11DZ2261100).

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