Chronic moderate hypercapnia suppresses ventilatory responses to acute CO₂ challenges

Abstract

Chronic hypercapnia (CH) is a hallmark of chronic lung disease, and CH increases the risk for acute-on-chronic exacerbations leading to greater hypoxemia/hypercapnia and poor health outcomes. However, the role of hypercapnia per se (duration and severity) in determining an individual’s ability to tolerate further hypercapnic exacerbations is unknown. Our primary objective herein was to test the hypothesis that mild-to-moderate CH (arterial $\text{P}{\text{CO}}_2$ ∼50–70 mmHg) increases susceptibility to pathophysiological responses to severe acute CO₂ challenges. Three groups (GR) of adult female goats were studied during 14 days of exposure to room air (GR 1; control) or 6% inspired CO₂ (GR 2; mild CH), or 7 days of 6% inspired CO₂ followed by 7 days of 8% inspired CO₂ (GR 3; moderate CH). Consistent with previous reports, there were no changes in physiological parameters in GR 1 (RA control), but mild CH (GR 2) increased steady-state ventilation and transiently suppressed CO₂/[H⁺] chemosensitivity. Further increasing InCO₂ from 6% to 8% (GR 3) transiently increased ventilation and arterial [H⁺]. Similar to mild CH, moderate CH increased ventilation to levels greater than predicted. However, in contrast to mild CH, acute ventilatory chemosensitivity was suppressed throughout the duration of moderate CH, and the arterial − mixed expired CO₂ gradient became negative. These data suggest that moderate CH limits physiological responses to acute severe exacerbations and provide evidence of recruitment of extrapulmonary systems (i.e., gastric CO₂ elimination) during times of moderate-severe hypercapnia.

NEW & NOTEWORTHY Moderate levels of chronic hypercapnia (CH; ∼70 mmHg) in healthy adult female goats elicited similar steady-state physiological adaptations compared with mild CH (∼55 mmHg). However, unlike mild CH, moderate CH chronically suppressed acute CO₂/[H⁺] chemosensitivity and reversed the arterial to mixed expired CO₂ gradient. These findings suggest that moderate CH suppresses vital mechanisms of ventilatory control and recruits additional physiological systems (i.e., gastric CO₂ release) to help buffer excess CO₂.

INTRODUCTION

CO₂ retention (hypercapnia) results from inadequate alveolar ventilation relative to metabolic rate. Chronic hypercapnia (CH) is present in several clinical conditions and disease states, including chronic lung disease, central and peripheral neuromuscular diseases (1, 2), obesity hypoventilation syndrome (3), sleep disordered breathing (4), and sedative use. Among these various etiologies, hypercapnia is most prevalent in chronic lung diseases such as chronic obstructive pulmonary disease (COPD) (5–7) and is a significant global health problem (8, 9). COPD can present with or without CH depending on the stage of disease progression and underlying etiology. Hypercapnia is clinically defined as arterial PCO₂ ($\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$) > 45 mmHg, and persistent hypercapnia has been identified as an independent risk factor for increased morbidity and mortality in patients with COPD (7, 10, 11). As lung function progressively deteriorates, patients with advanced stage COPD experience increases in $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ levels that often exceed 70 mmHg. However, data are lacking that determine the role of CH per se in its effects on ventilatory control mechanisms, disease progression, and associated comorbidities.

In addition to progressive worsening of lung function, COPD is further complicated by acute-on-chronic exacerbations, which result from acute stressors to the respiratory system. Acute exacerbations are clinically defined as “a sustained (24–48 h) increase in cough, sputum production, and/or dyspnea” (12). During an exacerbation, acid-base and blood gas homeostasis is further disrupted beyond the previous steady state, resulting in worsening hypoxemia and hypercapnia. Frequent occurrence of exacerbations leads to secondary complications ranging from functional limitations in day-to-day activities to increased rates of mortality (13–15). It has been reported that hypercapnia, among other comorbidities of COPD such as hypoxemia, age, sex, etc., is an independent risk factor for more frequent, severe exacerbations that result in pathophysiological responses (i.e., respiratory failure, stroke, myocardial infarction) (5, 13, 16, 17). Indeed, hypercapnia at the time of hospital admission is correlated to coagulation dysfunction, increased susceptibility to respiratory infection, and other negative outcomes (7, 11, 18, 19). Furthermore, repeated incidence of severe exacerbations is strongly correlated with longer hospitalizations, decreased quality of life, socioeconomic burdens, and high mortality (20–22). Despite clear indications of the negative consequences associated with hypercapnia, there appears to be a dichotomy of patients that retain CO₂ between those that tolerate acute exacerbations in hypercapnia and others that are susceptible to more negative physiological consequences during acute and chronic hypercapnia (clinically known as a “frequent exacerbator”) (22–24). Individuals characterized as a “frequent exacerbator” not only experience more exacerbations per year, but the acute-on-chronic events of a “frequent exacerbator” phenotype are classified as more moderate-to-severe events compared with nonfrequent or infrequent phenotypes (23, 24). Yet, the underlying feature that distinguishes an individual from becoming a nonfrequent versus frequent exacerbator is not well understood.

We recently found that chronic exposure to mild hypercapnia (inspired CO₂ of 6%; $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ ∼ 55 mmHg) in otherwise healthy goats has multiple physiological effects, including increased ventilation (V̇_I), acid-base shifts, electrolyte imbalance, modest hypertension, and significant impairments in cognitive function (25, 26). Moreover, we found that during mild CH, acute challenges with greater levels of inspired CO₂ (InCO₂) to 7% and 8% showed no obvious pathophysiological responses suggesting that acute exacerbations could be tolerated (25, 26). In contrast, humans with more progressive disease states where $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ can exceed 70 mmHg, experience more frequent, severe exacerbations that result in pathophysiological responses (i.e., CO₂ narcosis, respiratory failure, stroke, and myocardial infarction) (5, 16, 27). It is well established that multiple factors, including hypercapnia, influence the progressive deterioration of health in patients with COPD. However, it is unclear if CH per se and the duration or severity of hypercapnia, in the absence of additional complications (i.e., hypoxemia, decreased lung function), are determinants of the phenotype of “frequent exacerbation” and subsequent pathophysiological responses during exacerbations. Accordingly, the primary aim of the present study was to test the hypothesis that moderate CH increases the susceptibility to pathophysiological responses to more severe acute CO₂ challenges.

A secondary aim herein was to further explore whether exposure to mild-to-moderate CH changes measurements that are associated with recruitment of extrapulmonary mechanisms to assist countering excess levels of CO₂. This objective was based on 1) the previous finding of a decrease in the difference between arterial and mixed expired CO₂, suggesting an additional source of CO₂ was added to expired air from the alveoli (25, 26); and 2) recent theories that suggest that during times of respiratory acidosis, the gastroesophageal system, via a controlled mechanism within sites of central chemosensitivity, eliminates CO₂ through the esophagus and thus affects the arterial − mixed expired CO₂ gradient (28).

MATERIALS AND METHODS

Ethical Approval

The Medical College of Wisconsin (MCW) Institutional Animal Care and Use Committee (IACUC) has reviewed and approved all experimental protocols. MCW has remained accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AALAC #000129) and remains in compliance with the Public Health Services Policy on Humane Care and Use of Laboratory Animals (PHS Policy), provisions of the Animal Welfare Act and all other Federal statutes and regulations.

Study Population and Conditions

Three groups each with seven female, adult (2+ yr) goats weighing ∼40–50 kg were used in this study. Goats used in this study were obtained from outbred herds and included various strains (i.e., Alpine, Saanen, and Toggenburg). The use of female goats is relevant as clinical studies suggest that women are disproportionately affected by COPD compared with male counterparts. Women experience a greater rise in mortality, worse prognostic outcomes, increased risk for severe and frequent exacerbations, and a greater loss of pulmonary function (29–32). Each goat was randomly assigned to an experimental group. Group 1 (GR 1) was surgical room air controls and exposed to 14 days of room air. Group 2 (GR 2) was exposed to 14 days of 6% InCO₂. Group 3 (GR 3) was exposed to 7 days of 6% InCO₂ followed by 7 days of 8% InCO₂. Goats were reared and transported under conditions specified by the United States Department of Agriculture. All goats were chronically housed in environmental chambers as previously outlined (26), where temperature and humidity were maintained within normal limits. Photoperiods for all animals were fixed between 0600 h and 1800 h. Excluding periods of study, food, and water were provided ad libitum.

Surgery

Surgical methods for subcutaneous elevation of carotid arteries and diaphragm electromyogram (EMG) implementation were conducted as described previously (25, 26). Briefly, goats were anaesthetized with ketamine (15 mg/kg iv) for intubation, and then mechanically ventilated with 2%–3% isoflurane in 100% O₂. For elevation of carotid arteries, each artery was isolated from the vagus nerve, superficially elevated above the muscle, and then sutured in place underneath the skin. For EMG implantation, wire EMG electrodes were woven into the diaphragm, and externalized through the overlaying skin. In the postoperative period, goats were administered flunixin meglumine (2 mg/kg im) for analgesia daily for 3 days and ceftiofur sodium (4 mg/kg im) to minimize infections daily for 7 days. Following a 14-day recovery period, carotid arteries were catheterized via an indwelling catheter and flushed daily with heparinized saline (0.1% heparin in saline) followed by heparin.

Physiological Experiment

For physiological studies, a custom-fitted mask was fastened to the snout of the goat, and a one-way breathing valve was attached to the mask. A pneumotach was connected to the inspiratory port of the valve, and the expiratory port was connected to a Tissot gasometer. Inspiratory flow was digitally measured and recorded with LabChart software (ADInstruments, Colorado Springs, CO). Expired air was collected in a Tissot gasometer for measurement of mixed expired gas composition via a gas analyzer (OxiGraf, Sunnyvale, CA). Tissot collections were used for assessment of fractional concentration of expired O₂ ($\text{F}{\text{E}}_{{\text{O}}_2}$) and CO₂ ($\text{F}{\text{E}}_{\text{C}{\text{O}}_2}$) and used to calculate oxygen consumption (V̇o₂), CO₂ excretion (V̇co₂), and mixed expired oxygen ($\text{P}{\text{E}}_{{\text{O}}_2}$) and CO₂ ($\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$). The catheter chronically inserted in a carotid artery allowed for sampling arterial blood (every 10–20 min) and for continuous measurement of heart rate and blood pressure throughout each study. All measurements were obtained during the awake period. Arterial blood gases and pH were measured with a Siemens blood gas analyzer (Rapid Lab 248, Bayer Health Care, Leverkusen, Germany).

Experimental Procedure

All goats were allowed 2 wk for surgical recovery, during which they were acclimatized to the environmental chamber and trained with the data collection system. Following recovery, daily physiological measurements of ventilatory, cardiovascular, acid/base, metabolic, and cognitive function were measured under room air conditions during the awake state to establish a baseline. Animals were randomly assigned into three experimental groups: group 1 (GR 1) was exposed to room air for 14 days, and group 2 (GR 2) was exposed to 6% InCO₂ for 14 days. Group 3 (GR 3) was exposed to 6% InCO₂ for 7 days followed by 8% InCO₂ for an additional 7 days. Assessments of ventilatory, renal (blood gas and pH), metabolic rate, acute CO₂ sensitivity, and cardiovascular function (heart rate and blood pressure) were measured every 2–3 days during room air control period conditions and throughout the 14-day period experimental period. At the completion of the 14-day period, all goats were euthanized by initial deep anesthesia with a ketamine/xylazine solution (24:1; iv) followed by phenobarbital sodium and phenytoin sodium (FatalPLUS, 97.5 mg/kg iv). A graphical illustration of the experimental protocol is presented in Fig. 1.

Figure 1.

Experimental protocol. Three groups (n = 7/group) of female, adult (+2 yr) goats were randomly assigned to an experimental group. Each group was exposed to room air (RA) for ∼7 days to establish a baseline (termed: “RA Control Period”). Group 1 (GR 1) was surgical room air control and exposed to 14 days of room air. Group 2 (GR 2) was exposed to 14 days of 6% inspired CO₂ (InCO₂). Group 3 (GR 3) was exposed to 7 days of 6% InCO₂ followed by 7 days of 8% InCO₂. Steady-state (magenta circles) variables (i.e., V̇_I, V_T, f, $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$, [H⁺], etc.) were measured throughout the RA control period and experimental periods. Acute CO₂ chemosensitivity (green circles) was assessed following the completion of each 20-min period of steady-state measurements except on the first day of raising InCO₂ to allow for acclimation to the new steady state of increased InCO₂. f, breathing frequency; V̇_I, ventilation; V_T, tidal volume.

Acute Chemoreflex

The acute CO₂/[H⁺] chemoreflex was measured repeatedly during all phases of the study by acutely increasing InCO₂ above the chronic, steady-state level of InCO₂, which is consistent with previous studies (25, 26). When goats were living at room air conditions, InCO₂ was acutely increased to 3%, 5%, and 7% for 5 min each. When goats were chronically inhaling in 6% InCO₂, the InCO₂ was increased for 5 min each to 7% and 8%. For goats chronically inhaling 8% InCO₂, the InCO₂ was increased to 9% and 10% for 5 min each. Between minutes 3 and 5 of each level of acutely elevated InCO₂, arterial blood was sampled to assess arterial CO₂ and [H⁺]. The change in ventilation relative to the changes in arterial CO₂ (ΔV̇_I/Δ$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$) and [H⁺] (ΔV̇_I/Δ[H⁺]) were calculated and used as an index of the acute ventilatory CO₂/[H⁺] chemoreflex. Measurements of acute [H⁺] chemosensitivity and the level of arterial [H⁺] were used in the calculation of predicted steady-state ventilation [(ΔV̇_I/Δ [H⁺]) × (arterial [H⁺] – SS[H⁺]) + SS V̇_I].¹

Cognitive Function

Assessment of cognitive function was completed by a daily visual discrimination test as previously reported (25, 33) and because goats have been shown to excel at this type of cognitive test (34–36) Each day, goats were presented with two shapes (X and O) on the outside Plexiglas wall of the environmental chamber. Before the beginning of each visual discrimination test, a shape was randomly designated as the correct shape. Presentation of both shapes on the exterior of the goat’s environmental chamber prompted the goat to select a shape with its snout. Each day, on selection of the correct shape by the goat’s snout, the goat was rewarded with a food-related reward and the number of correct choices was recorded out of 10 total attempts.

Physiological Data Acquisition and Statistical Analysis

Ventilatory measures (V̇_I, f, and V_T) were analyzed (LabChart Software) and binned at 5-min intervals. Additional physiological variables were averaged across each study. Due to occasional nonfunctional catheters, days of study were binned as follows: days 1, 2, 5–7, 8, 9–10, and 12–14. To isolate the effects of varying levels of CH, per se, a two-way RM mixed-model ANOVA [time and group (GR 2 and GR 3) as factors] and appropriate post hoc tests for each physiological variable were used to identify significant effects across time and group (GraphPad Prism 8 software). Statistical significance for cognitive function was determined by a two-way RM mixed-model ANOVA (time and group as factors) and appropriate post hoc test to identify significant effects across time and between groups (GraphPad Prism 8 software). Significance of the V̇_I/[H⁺] and V̇_I/$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ relationship was analyzed with an analysis of covariance (ANCOVA). Statistically significant difference was determined as P < 0.05.

RESULTS

Ventilatory Adaptation, Timing, and Drive

No significant changes were found across all ventilatory measurements in 14-day room air control animals (GR 1; Table 1). Consistent with previous reports (25, 26), 6% InCO₂ initially increased minute ventilation (V̇_I) to ∼340% of the room air control period in GR 2 and 3 (P < 0.001), but V̇_I decreased (P = 0.008) thereafter to ∼263% of the room air control period (Fig. 2A). There was a significant interaction between time and CO₂ exposure (group) for V̇_I [F(8,91) = 3.578, P = 0.001]. Increasing InCO₂ to 8% after day 7 in GR 3 further increased V̇_I from 22.74 ± 3.44 L/min to 33.90 ± 2.20 L/min (P < 0.001), which was greater (P = 0.028) than V̇_I in GR 2 goats at the same time point. Following this increase in ventilation in GR 3 on day 1 of 8% InCO₂, V̇_I did not significantly differ from GR 2 goats throughout the remaining 14 days of CO₂ exposure. For breathing frequency [f; breaths/min (BPM)], there was a significant main effect of time [F(8,91) = 15.33, P < 0.001] but not group [F(1,12) = 0.01751, P = 0.896]. Frequency increased in GR 2 during day 1 6% InCO₂ from 20.23 ± 2.16 BPM to 35.02 ± 4.25 BPM (P < 0.001) but did not change significantly thereafter (Table 2). Similarly, f increased in GR 3 on day 1 of 6% InCO₂ (P < 0.001) but decreased significantly (P = 0.048) by 7 days. When InCO₂ was raised from 6% to 8% in GR 3, there was a secondary increase (P = 0.021) in f from 6 to 7 days. Furthermore, f did not significantly differ from 1 day of 8% InCO₂ during the remaining days of 8% InCO₂ (Table 3). There was a significant main effect of time [F(8, 91) = 23.69, P < 0.001] but not group [F(1, 12) = 0.062, P = 0.896] for tidal volume (V_T). In GR 2 and GR 3, V_T increased (P < 0.001) on day 1 of 6% InCO₂ and remained at or near that level thereafter irrespective of any change in InCO₂ (Tables 2 and 3). Despite decreases in both inspiratory time (T_I; P < 0.05; Table 2) and expiratory time (T_E; P < 0.001; Tables 2 and 3) and increases in ventilatory drive (V_T/T_I; P < 0.001; Tables 2 and 3) throughout CO₂ exposure, there was no significant additional effect of 8% InCO₂ on these measures throughout the study. For T_I and T_E, there was a significant main effect of time [F(8,91) = 4.690, P < 0.001; F(8,91) = 40.45, P < 0.001], but no significant effect of group [F(1,12) = 0.007, P = 0.931; F(1,12) = 0.1956, P = 0.666]. V_T/T_I demonstrated a significant interaction between time and group [F(1,12) = 0.1956, P = 0.004] but post hoc analysis determined there were no significant differences between GR 2 and GR 3.

Table 1.

Physiological parameters during 14 days of room air exposure in goats

	Control	Day 1	Day 2	Days 5–7	Day 8	Days 9–10	Days 12–14	P Value
V̇I, L/min	9.34 ± 0.32	8.71 ± 0.68	8.36 ± 0.40	8.40 ± 0.59	8.65 ± 0.39	9.08 ± 0.68	9.49 ± 0.75	ns
f, BPM	19.34 ± 0.51	19.69 ± 1.22	18.51 ± 0.81	18.54 ± 0.72	18.18 ± 1.02	18.31 ± 0.68	18.82 ± 0.98	ns
VT, L	0.49 ± 0.02	0.45 ± 0.01	0.46 ± 0.03	0.47 ± 0.02	0.47 ± 0.03	0.49 ± 0.03	0.51 ± 0.04	ns
TI, s	1.24 ± 0.04	1.25 ± 0.06	1.27 ± 0.06	1.24 ± 0.04	1.26 ± 0.03	1.28 ± 0.04	1.29 ± 0.06	ns
TE, s	1.90 ± 0.07	1.88 ± 0.14	2.04 ± 0.11	2.10 ± 0.13	1.99 ± 0.15	2.13 ± 0.15	1.98 ± 0.15	ns
VT/TI, L/s	0.40 ± 0.02	0.36 ± 0.02	0.37 ± 0.03	0.36 ± 0.02	0.39 ± 0.02	0.38 ± 0.01	0.40 ± 0.03	ns
$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$, mmHg	39.04 ± 1.55	39.24 ± 1.74	41.35 ± 1.00	42.42 ± 0.83	40.01 ± 1.17	40.60 ± 1.21	42.04 ± 1.21	ns
$\text{P}{\text{a}}_{{\text{O}}_2}$, mmHg	89.54 ± 3.38	84.89 ± 4.86	85.12 ± 4.30	90.38 ± 8.88	83.08 ± 4.06	86.29 ± 2.53	83.46 ± 2.57	ns
[${\text{HCO}}_3^{-}$], mEq/L	24.33 ± 1.11	24.83 ± 1.68	25.65 ± 0.98	24.55 ± 1.37	24.69 ± 0.73	24.90 ± 0.86	26.33 ± 1.05	ns
[H+], nmol/L	38.40 ± 1.10	38.15 ± 1.91	38.47 ± 0.89	41.64 ± 2.24	38.25 ± 1.64	39.27 ± 0.72	38.19 ± 0.99	ns
V̇o2, L/min	0.24 ± 0.02	0.21 ± 0.03	0.20 ± 0.01	0.21 ± 0.01	0.23 ± 0.02	0.21 ± 0.02	0.22 ± 0.02	ns
V̇co2, L/min	0.20 ± 0.02	0.17 ± 0.02	0.17 ± 0.02	0.17 ± 0.01	0.20 ± 0.02	0.18 ± 0.02	0.19 ± 0.02	ns
RER (V̇co2/V̇o2)	0.82 ± 0.03	0.80 ± 0.03	0.83 ± 0.04	0.80 ± 0.03	0.85 ± 0.03	0.84 ± 0.03	0.87 ± 0.04	ns
V̇I/V̇o2	39.76 ± 2.70	43.80 ± 3.54	42.55 ± 2.99	41.21 ± 2.53	40.77 ± 3.89	43.98 ± 3.73	43.40 ± 1.99	ns
$\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$, mmHg	23.00 ± 1.20	24.40 ± 2.27	27.14 ± 5.23	28.58 ± 4.96	23.22 ± 1.51	27.80 ± 4.41	27.19 ± 4.23	ns
ΔP($\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ − $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$), mmHg	17.83 ± 1.72	17.92 ± 1.70	20.38 ± 1.12	19.94 ± 0.95	17.36 ± 1.57	18.87 ± 1.15	20.10 ± 1.02	ns
$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ slope, L/min/mmHg	2.17 ± 0.26	–	1.75 ± 0.25	1.68 ± 0.28	–	1.94 ± 0.39	1.97 ± 0.26	ns
[H+] slope, L/min/nmol/L	2.68 ± 0.29	–	2.31 ± 0.13	2.45 ± 0.49	–	2.46 ± 0.44	2.81 ± 0.52	ns

Across 14 days of room air exposure, there were no significant changes in physiological parameters measured as determined by a one-way RM ANOVA. Values expressed as means ± SE. n ≥ 6. BPM, breaths per minute; $\text{P}{\text{a}}_{{\text{O}}_2}$, arterial partial pressure of oxygen; $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$, mixed expired CO₂; RER, respiratory exchange ratio; f, breathing frequency; T_E, expiratory time; T_I, inspiratory time; V̇co₂, CO₂ excretion; V̇_I, ventilation; V̇o₂, oxygen consumption; V_T, tidal volume.

Figure 2.

Mild-moderate hypercapnia leads to transient increases in ventilation and [H⁺]. Minute ventilation (L/min, A), $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ (mmHg, B), and arterial [H⁺] (nmol/L_, C) adaptations during 14 days inhaling 6% InCO₂ (GR 2; closed, blue circles) or 7 days inhaling 6% InCO₂ (GR 3; open, red squares) followed by 7 days at 8% InCO₂ (GR 3; closed, red squares) are shown. Increasing InCO₂ to 8% initially augmented V̇_I compared with time-matched GR 2 goats (A). On day 2 of 8% InCO₂ (days 9 and 10 overall), $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ (B) and arterial [H⁺] (C) were greater in GR 3 goats vs. GR 2 goats. #Significance between GR 2 and GR 3. ‡Significance between days in GR 3. Significance was determined by two-way mixed-model ANOVA Holm-Sidak post hoc analysis. Data are presented as individual values (gray: GR 2: closed circles; GR 3: open-closed squares) with means ± SE (GR 2: blue error bars and GR 3: red error bars). n = 7/group. GR, group; InCO₂, inspired CO₂.

Table 2.

Physiological parameters during 14 days of 6% InCO₂ exposure in goats

	Control	Day 1	Day 2	Days 3–5	Days 6–7	Day 8	Days 9–10	Days 11–12	Days 13–14	P Value
V̇I, L/min	9.42 ± 0.79	32.38 ± 3.10*	25.41 ± 1.20*	25.31 ± 0.81*	23.99 ± 1.09*	24.11 ± 1.38*	23.06 ± 1.23*	27.14 ± 1.70*	28.08 ± 2.17*	<0.001
f, BPM	20.23 ± 2.16	35.02 ± 4.25*	32.38 ± 3.32*	30.46 ± 3.95*	31.36 ± 4.34*	30.44 ± 3.69*	32.08 ± 5.24*	36.50 ± 8.92*	34.00 ± 5.85*	<0.001
VT, L	0.50 ± 0.05	0.97 ± 0.08*	0.82 ± 0.07*	0.90 ± 0.09*	0.82 ± 0.07*	0.85 ± 0.09*	0.79 ± 0.11*	0.84 ± 0.13*	0.91 ± 0.10*	<0.001
TI, s	1.23 ± 0.09	1.08 ± 0.06*	1.05 ± 0.05*	1.13 ± 0.07	1.12 ± 0.06	1.15 ± 0.08	1.08 ± 0.09	1.06 ± 0.11*	1.07 ± 0.07*	<0.05
TE, s	1.97 ± 0.23	0.78 ± 0.12*	0.90 ± 0.10*	0.99 ± 0.13*	0.95 ± 0.12*	0.97 ± 0.12*	0.96 ± 0.16*	0.82 ± 0.21*	0.90 ± 0.14*	<0.001
VT/TI, L/s	0.40 ± 0.03	0.90 ± 0.06*	0.79 ± 0.05*	0.79 ± 0.05*	0.73 ± 0.04*	0.73 ± 0.05*	0.71 ± 0.05*	0.78 ± 0.06*	0.84 ± 0.06*	<0.001
$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$, mmHg	38.68 ± 1.54	48.97 ± 2.59*	52.57 ± 1.45*	–	54.31 ± 1.37*	54.78 ± 1.53*	53.09 ± 1.49*	–	54.51 ± 1.49*	<0.001
$\text{P}{\text{a}}_{{\text{O}}_2}$, mmHg	92.21 ± 2.98	111.91 ± 2.34*	112.02 ± 2.70*	–	111.95 ± 3.83*	110.70 ± 3.88*	109.61 ± 2.77*	–	110.80 ± 4.22*	<0.001
[${\text{HCO}}_3^{-}$], mEq/L	23.11 ± 1.33	25.23 ± 1.50	27.75 ± 1.11*	–	29.82 ± 0.90*	30.81 ± 1.46*	30.19 ± 1.04*	–	29.50 ± 0.83*	<0.001
[H+], nmol/L	40.09 ± 0.96	46.40 ± 1.30*	45.27 ± 0.73*	–	43.49 ± 0.99	42.58 ± 1.05	41.95 ± 0.52	–	44.05 ± 0.29*	<0.05
V̇o2, L/min	0.26 ± 0.03	0.30 ± 0.03	0.34 ± 0.04	0.36 ± 0.03	0.30 ± 0.02	0.31 ± 0.02	0.29 ± 0.03	0.30 ± 0.04	0.31 ± 0.05	ns
V̇co2, L/min	0.21 ± 0.03	0.16 ± 0.02	0.20 ± 0.02	0.21 ± 0.02	0.19 ± 0.01	0.19 ± 0.01	0.18 ± 0.03	0.17 ± 0.02	0.18 ± 0.02	ns
RER (V̇co2/V̇o2)	0.80 ± 0.01	0.55 ± 0.05*	0.58 ± 0.02*	0.61 ± 0.03*	0.63 ± 0.03*	0.64 ± 0.03*	0.61 ± 0.06*	0.59 ± 0.10*	0.60 ± 0.03*	<0.05
V̇I/V̇o2	37.99 ± 3.01	112.49 ± 13.56*	79.17 ± 8.25*	75.03 ± 7.72*	81.61 ± 7.95*	80.81 ± 7.28*	85.70 ± 8.39*	93.02 ± 22.96	97.70 ± 9.36*	<0.05
$\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$, mmHg	22.55 ± 1.26	45.60 ± 2.12*	51.68 ± 0.87*	52.64 ± 0.90*	52.13 ± 0.59*	52.39 ± 0.49*	51.38 ± 1.26*	51.22 ± 0.79*	51.19 ± 0.75*	<0.001
ΔP($\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ − $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$), mmHg	16.61 ± 2.19	3.81 ± 4.06*	1.49 ± 1.38*	3.12 ± 1.72*	2.65 ± 1.23*	2.64 ± 1.36*	3.12 ± 2.52*	5.10 ± 1.95*	3.81 ± 1.72*	<0.001
$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ slope, L/min/mmHg	1.76 ± 0.21	–	1.11 ± 0.18	–	1.56 ± 0.22	–	1.35 ± 0.24	–	1.72 ± 0.25	ns
[H+] slope, L/min/nmol/L	2.29 ± 0.17	–	1.83 ± 0.09	–	2.29 ± 0.17	–	1.83 ± 0.09	–	2.49 ± 0.14	ns

Means ± SE values for 14 days of 6% InCO₂ goats are shown. n ≥ 6. *Significance vs. GR 2 RA control period values as determined by two-way RM mixed-model ANOVA. BPM, breaths per minute; InCO₂, inspired CO₂; $\text{P}{\text{a}}_{{\text{O}}_2}$, arterial partial pressure of oxygen; $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$, mixed expired CO₂; RER, respiratory exchange ratio; f, breathing frequency; T_E, expiratory time; T_I, inspiratory time; V̇co₂, CO₂ excretion; V̇_I, ventilation; V̇o₂, oxygen consumption; V_T, tidal volume.

Table 3.

Physiological parameters during 7 days of 6% InCO₂ followed by 7 days of 8% InCO₂ exposure in goats

	Control	Day 1	Day 2	Days 3–5	Days 6–7	Day 8	Days 9–10	Days 11–12	Days 13–14	P Value
V̇I, L/min	8.71 ± 0.55	28.65 ± 2.90*	23.83 ± 3.05*	25.50 ± 3.16*	22.74 ± 3.44*	33.90 ± 2.20*	29.65 ± 2.79*	29.29 ± 3.02*	30.68 ± 2.95*	<0.001
f, BPM	21.16 ± 1.76	33.10 ± 1.71*	29.35 ± 1.34*	30.59 ± 1.81*	26.37 ± 1.12	33.03 ± 1.56*	33.67 ± 1.99*	31.92 ± 2.11*	34.60 ± 2.16*	<0.05
VT, L	0.43 ± 0.04	0.91 ± 0.15*	0.84 ± 0.14*	0.86 ± 0.13*	0.87 ± 0.13*	1.05 ± 0.09*	0.90 ± 0.11*	0.94 ± 0.11*	0.92 ± 0.11*	<0.001
TI, s	1.17 ± 0.06	1.07 ± 0.04	1.09 ± 0.04	1.10 ± 0.04	1.18 ± 0.03	1.10 ± 0.03	1.06 ± 0.03	1.10 ± 0.04	1.08 ± 0.04	ns
TE, s	1.86 ± 0.20	0.79 ± 0.07*	0.98 ± 0.06*	0.92 ± 0.10*	1.13 ± 0.09*	0.75 ± 0.07*	0.78 ± 0.09*	0.85 ± 0.08*	0.73 ± 0.08*	<0.001
VT/TI, L/s	0.37 ± 0.02	0.83 ± 0.10*	0.75 ± 0.10*	0.77 ± 0.09*	0.72 ± 0.09*	0.95 ± 0.06*	0.85 ± 0.08*	0.84 ± 0.08*	0.84 ± 0.08*	<0.001
$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$, mmHg	37.57 ± 1.13	49.30 ± 2.01*	50.69 ± 1.63*	–	52.30 ± 1.67*	58.20 ± 1.61*	60.58 ± 2.38*	–	60.12 ± 2.20*	<0.001
$\text{P}{\text{a}}_{{\text{O}}_2}$, mmHg	99.34 ± 1.88	114.52 ± 4.00*	114.98 ± 2.60*	–	117.22 ± 2.74*	117.51 ± 1.97*	116.36 ± 2.44*	–	112.92 ± 1.79*	<0.001
[${\text{HCO}}_3^{-}$], mEq/L	23.81 ± 1.28	25.76 ± 1.38*	28.55 ± 1.71*	–	30.14 ± 1.38*	29.55 ± 1.76*	31.82 ± 1.64*	–	31.95 ± 1.35*	<0.001
[H+], nmol/L	37.92 ± 0.87	45.93 ± 1.62*	42.73 ± 1.30*	–	41.52 ± 0.85*	47.48 ± 1.63*	45.55 ± 0.87*	–	45.01 ± 1.36*	<0.05
V̇o2, L/min	0.24 ± 0.02	0.36 ± 0.07*	0.34 ± 0.04	0.32 ± 0.03	0.34 ± 0.05	0.37 ± 0.04*	0.38 ± 0.05*	0.36 ± 0.04*	0.34 ± 0.04	<0.05
V̇co2, L/min	0.20 ± 0.01	0.20 ± 0.02	0.22 ± 0.01	0.21 ± 0.00	0.22 ± 0.02	0.21 ± 0.01	0.24 ± 0.03	0.22 ± 0.02	0.22 ± 0.03	ns
RER (V̇co2/V̇o2)	0.84 ± 0.03	0.60 ± 0.03*	0.70 ± 0.05	0.69 ± 0.05	0.68 ± 0.04	0.59 ± 0.05*	0.64 ± 0.04*	0.63 ± 0.04*	0.66 ± 0.04*	<0.05
V̇I/V̇o2	36.80 ± 1.98	87.45 ± 11.54*	71.32 ± 2.87*	79.68 ± 2.71*	66.88 ± 2.84*	95.89 ± 8.53*	81.56 ± 6.95*	82.94 ± 5.48*	93.76 ±7.46*	<0.05
$\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$, mmHg	23.56 ± 0.70	47.58 ± 2.98*	53.33 ± 0.56*	52.87 ± 0.76*	53.93 ± 0.48*	62.39 ± 2.42*	65.96 ± 0.69*	65.84 ± 0.73*	65.80 ± 0.87*	<0.001
ΔP($\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ − $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$), mmHg	14.02 ± 0.88	1.71 ± 2.35*	−2.64 ± 1.47*	−1.09 ± 1.76*	−1.63 ± 1.26*	−4.18 ± 1.72*	−5.39 ± 2.12*	−4.39 ± 1.96*	−7.46 ± 2.28*	<0.001
$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ slope, L/min/mmHg	1.87 ± 0.21	–	0.85 ± 0.20*	–	1.70 ± 0.28	–	0.86 ± 0.26*	–	0.83 ± 0.28*	<0.05
[H+] slope, L/min/nmol/L	2.53 ± 0.21	–	1.57 ± 0.46	–	3.28 ± 0.46	–	0.86 ± 0.19*	–	1.30 ± 0.38*	<0.001

Means ± SE values for 7 days of 6% InCO₂ followed by 7 days of 8% InCO₂ goats are shown. n ≥ 6. *Significance versus GR 3 RA control period values as determined by two-way RM mixed-model ANOVA. BPM, breaths per minute; InCO₂, inspired CO₂; $\text{P}{\text{a}}_{{\text{O}}_2}$, arterial partial pressure of oxygen; $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$, mixed expired CO₂; RER, respiratory exchange ratio; f, breathing frequency; T_E, expiratory time; T_I, inspiratory time; V̇co₂, CO₂ excretion; V̇_I, ventilation; V̇o₂, oxygen consumption; V_T, tidal volume.

Arterial Blood Gases and Acid-Base Buffering

As shown previously (25, 26), we found no changes in blood gas and acid-base measurements in room air control animals (GR 1; Table 1). There was a significant interaction between time and group for $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ [F(6,65) = 6.877; P < 0.001]. The effects of 7 days of 6% InCO₂ in GR 2 and GR 3 were consistent with previous reports (25, 26). On day 1 of 6% InCO₂, $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ was increased 10–12 mmHg from baseline in GR 2 and GR 3 (P < 0.001; Fig. 2B). Thereafter, $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ increased to ∼15 mmHg above baseline by 7 days of 6% InCO₂ (P = 0.006). Further increasing InCO₂ from 6% to 8% in GR 3 increased $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ from 52.30 ± 1.67 mmHg to 58.20 ± 1.61 mmHg (P < 0.001), which was also higher than GR 2 animals (which remained at 6% InCO₂) on days 2 and 3 of 8% exposure (P = 0.045). No significant interactions between time and group were identified for $\text{P}{\text{a}}_{{\text{O}}_2}$ [time: F(6, 65) = 25.08, P < 0.001; group: F(1, 11) = 2.106, P = 0.174]. $\text{P}{\text{a}}_{{\text{O}}_2}$ increased from baseline values in both GR 2 (P < 0.001) and GR 3 (P < 0.001) initially on day 1 of 6% InCO₂, and $\text{P}{\text{a}}_{{\text{O}}_2}$ in GR 2 or GR 3 did not change thereafter nor differ between GR 2 and GR 3 (Tables 2 and 3).

For [${\text{HCO}}_3^{-}$] there was no significant interaction between time [F(6,65) = 15.23, P < 0.001] and group [F(1, 11) = 0.064, P = 0.804]. In GR 2 and GR 3, arterial [${\text{HCO}}_3^{-}$] during exposure to 6% InCO₂, reached a peak by day 2 (P < 0.001), where it remained elevated thereafter with no additional significant effect during 8% InCO₂ (Tables 2 and 3). There was a significant interaction between time and group for [H⁺] [F(6,65) = 5.919, P < 0.001]. As expected, there was an increase in arterial [H⁺] in both GR 2 (P < 0.001) and GR 3 (P < 0.001) after 1 day of 6% InCO₂ but arterial [H⁺] decreased thereafter from 46.40 ± 1.30 nmol/L to 41.95 ± 0.52 nmol/L in GR 2 (P = 0.046; Fig. 2C) with a similar decrease from 45.93 ± 1.62 nmol/L to 41.53 ± 0.85 nmol/L in GR 3 (P = 0.036; Fig. 2C). In GR 3, when InCO₂ was increased from 6% to 8%, there was an additional increase in [H⁺] compared with GR 3 at 7 days of 6% InCO₂ values (P < 0.001), that was higher than 9–10 days of 6% in GR 2 (P = 0.021). However, by 7 days of 8% InCO₂, arterial [H⁺] did not significantly differ from GR 2 animals at the same time point (day 14). Overall, these data suggest that despite the additional acid load experienced during the increase in InCO₂ from 6% to 8%, the goats can adequately buffer this load despite the maintenance of arterial $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ well above normal (+15 mmHg).

Ventilatory Responses to Acute Severe Hypercapnia

Ventilation/$P{a}_{C{O}_2}$ and ventilation/[H⁺] relationships.

In Fig. 3, the slopes of the V̇_I $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ and V̇_I [H⁺] relationships represent the degree of acute sensitivity of the ventilatory system during acute hypercapnic challenges. The first points along each line are measured ventilation and CO₂/[H⁺] during the steady state and the additional points are V̇_I and CO₂/[H⁺] measured at each level of InCO₂ during an acute hypercapnic challenge. In Fig. 3, A–D, the black line is the relationship of ventilation to $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ or [H⁺] at room air. In Fig. 3, A and B, the gray and blue lines are the relationship for GR 2 at 7 and 14 days of 6% InCO₂, respectively. In Fig. 3, C and D, the dashed and solid red lines are the relationship for GR 3 at 7 days of 6% InCO2 and 7 days of 8% InCO₂, respectively.

Figure 3.

8% InCO₂ alters slope of the ventilation/$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ and ventilation/[H⁺] relationship. The first point of each relationship reflects measurements of V̇_I (L/min) and $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ (mmHg) or [H⁺] (nmol/L) obtained during the “steady state.” The additional 2–3 points along the line are measurements of V̇_I and $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ or [H⁺] during the acute InCO₂ challenges. A regression line was fitted to each line and represents the sensitivity of the ventilatory system to acute changes in $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ or [H⁺]. For GR 2 (A), the V̇_I/$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ relationship was significantly shifted to the right of the room air control period at 7 days of 6% InCO₂, but not at 14 days of 6%, with no change in the slope of the relationship at either time point. In GR 2, the V̇_I/[H⁺] relationship was significantly shifted leftward at 7 and 14 days of 6% in GR 2 (B). For GR 3 (C), the V̇_I/$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ relationship was significantly shifted to the right at 7 days of 6%. However, at 7 days of 8% InCO₂, the slope of the relationship was not significantly rightward shifted, but the slope was decreased relative to the RA control period. The V̇_I/[H⁺] relationship in GR 3 (D) at 7 days of 6% did not change from the RA control period but at 8 days of 8%, the relationship was shifted upward. Moreover, at 7 days of 8%, the slope of the V̇_I/[H⁺] relationship was decreased. Significance of the shifts in V̇_I/$\text{P}{\text{A}}_{\text{C}{\text{O}}_2}$ and V̇_I/[H⁺] relationships was determined by an ANCOVA. Data are presented as means ± SE. N ≥ 6/group. GR, group; InCO₂, inspired CO₂; RA, room air; V̇_I, ventilation.

In GR 2, the V̇_I/$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ (Fig. 3A) relationship at 7 days of 6% is significantly shifted to the right of the RA control period (P = 0.008), but by 14 days, the shift of the relationship does not significantly differ from the RA control period (P = 0.156). As shown previously, GR 2 (Fig. 3B) demonstrated a leftward shift (P < 0.001) of the V̇_I/[H⁺] relationship at 7 and 14 days of 6% InCO₂. In GR 3 (Fig. 3C), the V̇_I/$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ is also rightward shifted (P = 0.035) compared with the RA control period at 7 days of 6%. By 7 days of 8% InCO₂, the line is shifted to the right but does not reach significance (P = 0.065). GR 3 (Fig. 3D) did not demonstrate a leftward shift in the V̇_I/[H⁺] relationship at 7 days of 6% but did have a significant upward shift (P = 0.010) in the relationship at 7 days of 8%. In GR 3 at 7 days of 8%, there was a decrease in the V̇_I/[H⁺] relationship that was not evident during any duration of exposure to 6% InCO₂ in GR 2 or GR 3.

$P{a}_{C{O}_2}$ and arterial [H⁺] slopes.

$\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ and arterial [H⁺] slopes did not change during chronic room air exposure (Table 1). There was a significant interaction between time and group for $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ [F(4,37) = 3.232, P = 0.022] and [H⁺] [F(4,37) = 6.596, P < 0.001] slopes. Similar to previous studies (25, 26), GR 2 and GR 3 demonstrated a transient suppression in the $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ and [H⁺] slopes within 2 days of 6% InCO₂ (Fig. 4 and Tables 2 and 3). However, statistical significance was reached in GR 3 for only $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ slopes (P = 0.004). Consistent with previous reports (25, 26), there was no significant difference in $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ or [H⁺] slopes at 7 days of 6% in GR 2 and GR 3, or thereafter in GR 2 (Fig. 4 and Tables 2 and 3). In GR 3, at 2 days of 8%, there was a significant suppression in both $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ (P = 0.002) and [H⁺] (P < 0.001) slopes. Unlike chronic exposure to 6% reported here and previously (25, 26), $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ (P < 0.001) and [H⁺] (P = 0.003) slopes remained chronically decreased relative to room air in GR 3. Moreover, in GR 3 at 7 days of 8%, the [H⁺] slope was significantly decreased compared with GR 2 (Fig. 4B; P = 0.049). The sustained decrease in CO₂ and [H⁺] slopes during 8% InCO₂ indicates that acute chemosensitivity is chronically suppressed.

Figure 4.

Eight percent InCO₂ chronically suppresses acute CO₂/[H⁺] chemosensitivity. Previous reports have shown that 6% InCO₂ transiently suppresses acute CO₂/[H⁺] sensitivity. During the first 7 days of 6% InCO₂, GR 3, but not GR 2, demonstrated a significant decrease in the $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ slope (A). GR 2 did not exhibit a significant change in the acute $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ throughout the remaining 7 days of 6% InCO₂ A). However, in GR 3, when InCO₂ was chronically raised from 6% to 8% InCO₂, there was a significant, sustained suppression in the $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ slopes at days 2 and 7 (A). Similar to the $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ slopes, GR 2 did not demonstrate a significant change in acute [H⁺] slopes across 14 days of 6% InCO₂ (B). Conversely, in GR 3, [H⁺] slopes were significantly and chronically suppressed at 2 and 7 days of 8% InCO₂. Moreover, at 7 days of 8% InCO₂ (days 11–14 overall) GR 3 had a significantly lower [H⁺] slope than the GR 2 [H⁺] slope (B). #Significance between GR 2 and GR 3. *Significance versus the RA control period. Significance was determined by two-way mixed-model ANOVA Holm–Sidak post hoc analysis. Data are presented as individual values (gray: GR 2: closed circles; GR 3: open-closed squares) with means ± SE (GR 2: blue error bars and GR 3: red error bars). n ≥ 6/group. GR, group; InCO₂, inspired CO₂; RA, room air.

Predicted Ventilation Versus Actual Ventilation

Predicted ventilation was determined through the following equation: [(ΔV̇_I/Δ [H⁺]) × (arterial [H⁺] – SS[H⁺]) + SS V̇_I]. There was a significant interaction between time and condition (predicted vs. actual) in both GR 2 [F(4, 39) = 7.387, P < 0.001] and GR 3 [F(4, 44) = 3.134, P = 0.023]. Consistent with previous studies (25, 26), increasing InCO₂ resulted in measured ventilation levels that exceeded levels of ventilation predicted by the animals’ ventilatory sensitivity to [H⁺] (Fig. 5; P < 0.05). However, on 7 days of 6% InCO₂ in GR 3 (Fig. 5B), there was no significant difference observed between actual and predicted ventilation. Increasing InCO₂ to 8% had no additional effect on the relationship between actual and predicted ventilation. As previously concluded by our laboratory, actual ventilation exceeding ventilation predicted by acute [H⁺] chemosensitivity strongly suggests additional stimuli beyond the traditional chemical stimuli to breath are contributing to measured ventilation. Although previous studies have identified potential neurochemical substrates that can attribute to measured ventilation exceeded predicted, none to date have fully explained this phenomenon. The exact mechanism driving actual ventilation has yet to be elucidated.

Figure 5.

Actual ventilation exceeds predicted ventilation during 6% and 8% InCO₂. Predicted ventilation was calculated by the following equation: [(ΔV̇_I/Δ [H⁺]) × (arterial [H⁺] – SS[H⁺]) + SS V̇_I]. During both 6% (A) and 8% (B) InCO₂, actual ventilation was greater than ventilation predicted by the acute chemoreflex, suggesting additional stimuli beyond acute chemosensitivity to [H⁺] contributes to ventilation (26). #Significance between actual and predicted ventilation. Significance was determined by two-way mixed-model ANOVA and Holm–Sidak post hoc analysis. n ≥ 6/group. V̇_I, ventilation.

Metabolic Rate and Extrapulmonary CO₂ Buffering

Changes in metabolic rate were determined through measurements of oxygen consumption (V̇o₂), CO₂ production (V̇co₂), and the respiratory exchange ratio (RER). For V̇o₂, there was a significant main effect of time [F(8,91) = 3.205, P = 0.003] but not group [F(1,12) = 0.4406, P = 0.519]. V̇co₂ revealed no significant main effect of time [F(8, 91) = 0.7707, P = 0.629] or group [F(1, 12) = 1.603, P = 0.229]. There was a significant main effect of time [F(8, 91) = 8.154, P < 0.001] but not group [F(1,12) = 2.072, P = 0.175] for RER. Similar to previous studies (25, 26), exposure to 6% InCO₂ increased V̇o₂ (P = 0.007; Table 3) but did not alter V̇co₂ across day or condition (Tables 2 and 3). Thus, due to the respective changes in V̇o₂ and V̇co₂, there was a significant decline in RER in both GR 2 and GR 3 (Tables 2 and 3; P < 0.050). Increasing InCO₂ from 6% to 8% resulted in an increase in V̇o₂ that was greater than RA control period values across all days at 8% InCO₂ (P < 0.05), but these values were not different between GR 2 and GR 3. Raising InCO₂ to 8% did not significantly affect V̇co₂. Convection requirement (V̇_I/V̇o₂) was also measured throughout the study in all groups. There was a significant main effect of time [F(8, 91) = 20.71, P < 0.001] but not group [F(1,12) = 0.397, P = 0.5407]. Both GR 2 and GR 3 demonstrated an increase in V̇_I/V̇o₂ relative to each groups RA control period values, but no significant differences between groups were determined (Tables 2 and 3, P < 0.05). As expected, there were no changes in V̇o₂, V̇co₂, RER, or V̇_I/V̇o₂ in GR 1 across time (Table 1).

Mixed expired CO₂ ($\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$) revealed a significant interaction between time and group [F(8, 90) = 14.16, P < 0.001]. In GR 2 and GR 3, $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ increased by ∼23 and 24 mmHg, respectively, following initial exposure to 6% InCO₂ (P < 0.001; Fig. 6A). By day 2, $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ increased further from day 1 values to 51.68 ± 0.87 mmHg in GR 2 (P < 0.010) and 53.33 ± 0.56 mmHg in GR 3 (P = 0.008). In GR 2, $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ remained elevated throughout the 14-day exposure to 6% InCO₂. However, in GR 3 when increasing InCO₂ from 6% to 8%, $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ increased further above the steady state established during exposure to 6% (P < 0.001) where it remained elevated relative to previous exposure to 6% and was significantly greater than GR 2 (6% InCO₂) values across the 7 days of 8% InCO₂ (P < 0.001). Given that $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ (Fig. 2) and $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ (Fig. 6A) were increased with elevated InCO₂, we calculated the arterial to mixed expired CO₂ difference (Fig. 6B) to determine if there was a reversal of the gradient as previously reported (25, 26). There was a significant main effect of both time [F(8, 80) = 25.36, P < 0.001] and group [F(1, 11) = 9.230, P = 0.011] for the CO₂ gradient. When chronically exposed to 6% InCO₂ for 14 days (GR 2), the $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$–$\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ difference decreased relative to room air control period values (P < 0.001) and remained at or greater than 0 throughout exposure to 6%. However, in GR 3, when InCO₂ was raised to 8%, the increase in $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ exceeded increases in $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ which resulted in a negative P(arterial − mixed expired)CO₂ difference. During 8%, the $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ − $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ difference during day 2 (GR 2 days 9 and 10; P = 0.029), days 3 and 5 (GR 2 days 11 and 12; P = 0.016), and days 6 and 7 (GR 2 days 13 and 14; P = 0.014) was significantly decreased compared with time matched values of GR 2 (Fig. 6B). The reversed gradient of $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ to $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ suggests that additional mechanisms beyond the pulmonary system contributes to CO₂ buffering/elimination and ultimately increased expired CO₂ concentrations (28).

Figure 6.

Extrapulmonary CO₂ buffering likely occurs during 7 days of 8% InCO₂. In GR 2 and GR 3, 6% InCO₂ significantly increased mixed-expired CO₂ ($\text{P}{\text{E}}_{\text{C}{O}_2}$, A) compared with the RA control period values. When raising InCO₂ from 6% to 8% in GR 3, $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ was significantly increased compared with time-matched GR 2 values and with GR 3 values obtained during 6–7 days at 6% (A). The arterial − mixed expired CO₂ difference [ΔP(arterial − mixed expired)CO₂; B] decreased during 14 days of 6% InCO₂ (blue, closed circles) and 7 days of 6% and 7 day of 8% InCO₂ (open-closed, red squares) compared with RA control period values. By 2 days of 8% InCO₂, the ΔP(arterial − mixed expired)CO₂ was significantly lower than time-matched GR 2 values (B). #Significance between GR 2 and GR 3. Significance was determined by two-way mixed-model ANOVA and Holm–Sidak post hoc analysis. Data are presented as individual values (gray: GR 2: closed circles; GR 3: open-closed squares) with means ± SE (GR 2: blue error bars and GR 3: red error bars). n ≥ 6/group. GR, group; InCO₂, inspired CO₂; RA, room air.

Cognitive Function

We have previously shown that CH significantly reduced cognitive function as measured by the number of correct choices during an established visual discrimination test (25, 26). GR 1 animals demonstrated ∼8 correct choices per assessment during the “control” period. GR 1 animals consistently maintained this measure of cognitive function throughout the 2 wk. For GR 2 and GR 3 comparisons, there was a main effect of time [F(14,98) = 15.40, P < 0.001] but not group [F(1, 7) = 1.256, P = 0.299]. Similar to previous data (25, 26), GR 2 animals exposed to 6% InCO₂ resulted in a reduction of number of correct choices by ∼50% (P < 0.001) which persisted throughout 14 days. Increasing InCO₂ to 8% in GR 3 did not have an additional effect on cognitive function, and the number of correct choices remained at ∼5 out of 10 attempts each day.

Diaphragm activation during acute CO₂ challenges in mild and moderate hypercapnia.

Statistical analysis revealed a significant main effect of time [F(4, 23) = 3.346, P = 0.269] but not group [F(1, 6) = 0.0001, P = 0.990] on diaphragm activation during acute CO₂ challenges. In GR 2 and GR 3, chronic mild (6%) hypercapnia did not significantly change diaphragm activation during acute challenges (Supplemental Fig. S1; all Supplemental Material is available at https://doi.org/10.6084/m9.figshare.20263800.v1). However, in GR 3 during 8% InCO₂, there was a trend for diminished diaphragm activation during acute hypercapnic challenges at 2 days (P = 0.085) and 7 days (P = 0.053). The reduced stimulation of the diaphragm is consistent with the reduced $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ and [H⁺] slope data and may partially explain the loss of CO₂/[H⁺] responsiveness reported throughout the exposure to 8% InCO₂ in GR 3. However, due to EMG failure in some goats, diaphragm measurements were only obtained in four goats per group and thus additional studies are needed to fully determine the effect of moderate CH on diaphragmatic activity.

DISCUSSION

Chronic hypercapnia (CH) is a hallmark of chronic respiratory disease and several clinical conditions, and more severe levels of hypercapnia are correlated with poor clinical outcomes (7, 11, 12). However, CH can often coexist with hypoxemia. Thus, the contributions of the CH (and the level of CH) per se on respiratory dysfunction, particularly during acute exacerbations of CH, are unknown. The goal of the present study was to gain insight into the physiological consequences of mild-to-moderate CH in otherwise healthy female adult goats and determine if higher levels of CH increase the susceptibility to pathophysiological responses to acute CO₂ challenges. Here we showed that as expected, raising inspired InCO₂ to 6% (mild CH) stimulates ventilation and increases arterial CO₂ and [H⁺], where over 1-wk steady-state ventilation plateaued and remained above the level predicted within a few days without affecting acute chemosensitivity. Further increasing InCO₂ to 8% (moderate CH) resulted in a secondary, transient increase in ventilation accompanied by a further transient increase in $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ and acidosis, which was adequately buffered for [H⁺] thereafter. These data suggest that healthy animals adequately adapt to moderate levels of CH. However, exposure up to 7 days of moderate CH shifted the relationships of ventilation to $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ and [H⁺] reducing the slopes of these responses, indicative of a blunting of the acute chemoreflexes. During challenges of severe 9%–10% InCO₂ ventilation failed to increase in proportion to increases in CO₂/[H⁺] and even was reduced in some animals. These data obtained during acute CO₂ challenges suggests that there may indeed be an “upper limit,” even in healthy animals, to withstand secondary, more severe hypercapnic challenges, and perhaps leave them susceptible to pathophysiological respiratory dysfunction during acute exacerbations of CH.

Moderate Hypercapnia Chronically Suppresses Acute CO₂/[H⁺] Chemosensitivity

Consistently, we have reported in previous studies and herein that mild CH only transiently suppresses acute CO₂/[H⁺] chemosensitivity. However, in the present study we have found that moderate levels of CH lead to a sustained suppression of the acute CO₂/[H⁺] chemoreflex. To our knowledge, this is the first study that examined how varying levels of hypercapnia, per se, can alter the fundamental responses to sustained and acute-on-chronic hypercapnic challenges. Our data may also further explain the consistent variability reported in human studies, as several studies have reported varying data on acute chemoreflex adaptation during CH (37–41). Yet, these previous studies failed to examine an increasing severity of hypercapnia within a single study. Data from the present study suggest that the severity of hypercapnia rather than just presence of hypercapnia within experimental and clinical conditions must be considered when determining the possible (mal)adaptations that may occur.

The mechanism(s) underlying chronically diminished acute CO₂/[H⁺] sensitivity during moderate CH in GR 3 has not been elucidated; however, there are several attractive hypotheses that should be further examined. A possible explanation for the sustained reduction in CO2/[H⁺] slopes observed during moderate to severe levels of hypercapnia may be due to the curvilinear shape of the CO₂ response curve. Within normal physiological ranges the relationship between $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ and ventilation is linear, where V̇_I increases ∼2–5 L/min per every 1 mmHg increase of $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ (33). However, at the extreme ends of the $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ range experienced under pathological conditions ($\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ < 35 mmHg and $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ > 80 mmHg), the curve begins to flatten resulting in diminished ventilatory responses. In the present study, when GR 3 animals were acutely exposed to 10% InCO₂, $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ levels well exceeded 70 mmHg. Such levels are the beginning threshold in humans for the onset of CO₂ narcosis and subsequentially acute-on-chronic respiratory failure (42, 43). Clinically, consequences of acute-on-chronic respiratory failure have been primarily attributed to worsening hypoxemia, with hypercapnia being secondary to changes in $\text{P}{\text{a}}_{{\text{O}}_2}$ (44). However, herein we have clearly demonstrated that regardless of oxygenation state, when hypercapnia reaches a moderate-severe level, chronically, it can elicit limitations within respiratory control such as diminished respiratory responses observed during acute-on-chronic respiratory failure.

It should also be appreciated that hypercapnia can greatly alter the effectiveness of the respiratory system as a whole. It has been suggested that patients with severe COPD and thus $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ levels that exceed 70 mmHg experience a clinical phenomenon of “submissive hypercapnia.” Whereas, similar to permissive hypercapnia that is induced by clinicians to allow for $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ to increase, submissive hypercapnia is the decision of one’s respiratory controller to succumb to the present hypercapnia through limiting increases in ventilation and thus preserving energy requirements (45). In addition, prolonged exposure to severe levels of hypercapnia can greatly impair diaphragm and other respiratory muscle activity (46–49). In fact, mice chronically exposed to 10% demonstrated delayed diaphragmatic contraction and relaxation in addition to the size and composition of diaphragmatic fibers (48). Finally, in vivo and in vitro studies have shown that continuous severe levels of hypercapnia (8%–10% $\text{F}{\text{I}}_{\text{C}{\text{O}}_2}$) can lead to blunted cellular responsiveness to acute changes in CO₂/[H⁺] in sites of central chemosensitivity (50) and long-term depression of phrenic motor nerves (51, 52). When taken into consideration with the data presented herein, these aforementioned mechanisms could result in the blunted acute chemoreflex observed over 7 days of 8% InCO₂ in our goats. However, further studies are needed to fully examine the underlying mechanisms attributed to the chronically suppressed CO₂/[H⁺] slopes reported herein.

Integrated Responses Occur in Mild and Mild-to-Moderate Chronic Hypercapnia

Our results also demonstrate that as the ventilatory system may reach its limits, additional physiological systems may participate in buffering excess CO₂. Consistent with previous studies (25, 26), we have shown that mild and, as demonstrated herein, moderate CH does not significantly alter V̇co₂. As suggested by others, it is likely that the lack of change in CO₂ production may be attributed to increases in CO₂ storage throughout the body, such bone, muscle, and other tissues (26, 53–55). The rise in $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ during moderate CH is consistent with previous reports of chronic mild hypercapnia. During mild CH the rise of $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ never exceeded the levels of arterial resulting in a significant, but non-negative arterial − mixed expired CO₂ gradient. During moderate CH rise in $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ exceeds the increases in $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$. The disproportionate increases in mixed expired and arterial CO₂ during moderate led to a negative CO₂ gradient [ΔP(arterial − mixed expired)CO₂] that was only evident during moderate CH in the current study. A reversal of the CO₂ gradient has been reported across several different conditions and species (56–59). In initial studies that demonstrated a negative arterial − mixed expired CO₂ difference, there were conflicting views on whether the reversed gradient was due to an unknown mechanism (57, 60) or attributed to inaccurate measurement/technical errors (61, 62). However, a study by Dean et al. (28) proposed a potential mechanism to support findings of a negative $\text{P}{\text{a}}_{\text{C}{\text{O}}_2}$ − $\text{P}{\text{E}}_{\text{C}{\text{O}}_2}$ difference during respiratory acidosis. The theory suggests that in times of respiratory acidosis, excess arterial CO₂ that reaches the gastric system is buffered in the stomach and excreted by route of the esophagus. The additional CO₂ from the gastric system is added to the expired alveolar CO₂ and thus results in expired CO₂ exceeding that of arterial demonstrated as a negative P(arterial − mixed expired)co₂ (28). Herein, we found that during mild CH the arterial − mixed expired difference was near or greater than zero. However, when InCO₂ was raised to 8%, the difference was consistently negative. These data suggest gastric contribution to CO₂ elimination during mild CH is, at most, minimal. Conversely, during moderate CH, there is appreciable evidence of the gastric CO₂ ventilation, as indicated by the reversed gradient. However, further studies are needed to conclude there is a gastric contribution to CO₂ elimination.

As importantly highlighted in the study by Dean et al., acknowledgment of gastric ventilation in times of respiratory acidosis brings into focus two fundamental questions of central chemosensitivity: 1) what is the need for multiple sites of chemoreception? and 2) what functions do the numerous chemoreceptors possess? (28, 63, 64) The data herein, along with the gastric ventilation theory and phenomenon of submissive hypercapnia, suggest that multiple sites of chemoreception are needed, particularly in severe disease states. Conceivably, under normal “steady-state” conditions, primary chemoreceptive sites are able to meet the body’s metabolic demand and can readily response to changes in pH and thus, secondary chemosensitive sites remain relatively quiescent. However, in more demanding states (i.e., diseases), more predominate sites of chemosensitivity may dampen to preserve energy expenditure, thus recruiting the secondary chemosensitive sites to help regulate pH.

Finally, consistent with previous studies in our laboratory, we have shown that chronic mild hypercapnia has a deleterious effect on cognitive function (25, 26). Interestingly, increasing to more moderate levels of CH does not have any additional effect on shape discrimination tests. The lack of change in cognitive function at more severe levels of hypercapnia is intriguing; however, it suggests that impairments in cognitive function in clinical setting may not be dependent on the severity of hypercapnia.

Summary

The overall goal of the present study was to determine if increasing severity of chronic hypercapnia limits the fundamental responses to acute-on-chronic hypercapnic challenges. The data presented herein demonstrate that, indeed, there appears to be an “upper limit” to the ventilatory response to hypercapnic challenges and this limit is reached following at least 1 wk of recurring bouts of severe CO₂ challenges. To our knowledge, the present data suggest that increasing severity of hypercapnia, in isolation of other common comorbidities associated with chronic lung disease, can elicit unique effects on the respiratory system’s ability to adapt to acute-on-chronic hypercapnic challenges and ultimately lead to greater susceptibility to pathophysiological responses during exacerbations of disease states. This observation is critical to individuals with chronic respiratory diseases as it implies that the level of hypercapnia can set the threshold for an individual’s ability to tolerate acute perturbations. Our data add to the ongoing argument that CO₂ retention worsens prognosis. Moreover, findings presented herein emphasize the need for consideration of severity of hypercapnia, per se in patients as it may influence clinical outcomes including tolerance to exacerbations, quality of life, and overall mortality. Furthermore, future studies must address the underlying mechanisms that set the hypercapnic-induced thresholds, in hopes that clinical interventions can be used to prevent further deterioration of an individual’s health.

SUPPLEMENTAL DATA

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.20263800.v1.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant F31HL159908 and Department of Veterans Affairs Grant BX003284.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.J.B., N.J.B., M.R.H., and H.V.F. conceived and designed research; K.J.B., S.E.N., N.J.B., and L.P. performed experiments; K.J.B., S.E.N., and N.J.B. analyzed data; K.J.B., N.J.B., M.R.H., and H.V.F. interpreted results of experiments; K.J.B. prepared figures; K.J.B. drafted manuscript; K.J.B., S.E.N., N.J.B., M.R.H., and H.V.F. edited and revised manuscript; K.J.B., S.E.N., N.J.B., M.R.H., L.P., and H.V.F. approved final version of manuscript.