Eric Hermand

Aurélien Pichon

François J Lhuissier

Jean-Paul Richalet
email: richalet@univ-paris13.fr

Periodic breathing in healthy humans at exercise in hypoxia. Running head: Intrinsic periodic breathing in humans

Keywords:

Periodic breathing is frequent in heart failure or ventilatory disorders during sleep, and common during sleep at high altitude, but has been rarely studied in wakefulness and during exercise. A retrospective analysis of ventilation from hypoxia exercise tests was realized in 82 healthy subjects, separated in two groups with either high or low ventilatory response to hypoxia at exercise (HVRe). A FFT spectral analysis of the breath-by-breath ventilation (E) signal, O 2 saturation and end-tidal PCO 2 evidenced a periodic pattern with a period of 11.1 to 12.0 seconds. Peak power of E spectrum was higher in the high HVRe group (P<0.001). A prospective study (25 subjects) was performed to evaluate the influence of cardiorespiratory factors on the amplitude and period of oscillations in various conditions of exercise (20 to 40% maximal aerobic power) and hypoxia (0 to 4,000m altitude). E period was shorter at exercise (vs rest, P<0.001) and hypoxia (vs normoxia, P<0.001) and inversely related with cardiac output and E (P<0.001). E peak power was higher at exercise (P<0.001) and hypoxia (P<0.001) and was positively related with cardiac output and E (P<0.001). E peak power in hypoxia was positively related with the ventilatory response to CO 2 (HCVR). This novel observation suggests that healthy subjects demonstrate a spontaneous periodic breathing, not clearly observable at rest and in normoxia, but triggered by hypoxic exercise. The periodic pattern is enhanced in subjects with high HVRe and high HCVR, suggesting that oxygen and CO 2 play a synergistic role in the modulation of these oscillations. Clinical Trial reg.

Introduction

The control of ventilation involves three main components: the respiratory centers, responsible for the genesis of the respiratory rhythm, the effector system (the ventilatory muscles) and the receptors (mainly the chemoreceptors) that inform the centers of the status of the whole system. Central chemoreceptors are sensitive to the level of arterial PCO 2 with gain adjustments from peripheral chemoreceptors [START_REF] Blain | Peripheral chemoreceptors determine the respiratory sensitivity of central chemoreceptors to CO2: Peripheral-central chemoreceptor interdependence[END_REF][START_REF] Lahiri | CO2/H(+) sensing: peripheral and central chemoreception[END_REF], although these interactions have been debated in humans [START_REF] Duffin | Cross-Talk opposing view: peripheral and central chemoreflexes have additive effects on ventilation in humans[END_REF], while the response to hypoxia is mainly due to peripheral chemoreceptors (carotid bodies) [START_REF] Kumar | Peripheral Chemoreceptors: Function and Plasticity of the Carotid Body [Online[END_REF][START_REF] Lahiri | Oxygen sensing in the body[END_REF]. All these components are involved in a closed loop system of control that produces, in most usual circumstances, a relatively stable level of ventilation adapted to the needs of the organism. However, in some cases, the system shows a marked degree of instability with periodic breathing or interruption of ventilation. Sleep apneas or hypopneas might be of obstructive or central origin and are accompanied by oscillations in the level of end tidal PCO 2 and arterial O 2 saturation [START_REF] Bålfors | Impairment of cerebral perfusion during obstructive sleep apneas[END_REF][START_REF] Xie | Influence of arterial O2 on the susceptibility to posthyperventilation apnea during sleep[END_REF]. Indeed, patients with cardiac failure may show a periodic breathing or Cheyne-Stokes pattern of ventilation at rest [START_REF] Brack | Cheyne-Stokes Respiration in Patients with Heart Failure: Prevalence, Causes, Consequences and Treatments[END_REF][START_REF] Pinna | Cardiorespiratory interactions during periodic breathing in awake chronic heart failure patients[END_REF][START_REF] Ponikowski | Oscillatory breathing patterns during wakefulness in patients with chronic heart failure: clinical implications and role of augmented peripheral chemosensitivity[END_REF][START_REF] Schulz | working group Kreislauf und Schlaf of the German Sleep Society (DGSM). Sleep apnoea in heart failure[END_REF] and during exercise [START_REF] Dhakal | Exercise oscillatory ventilation in heart failure[END_REF][START_REF] Ben-Dov | Evidence that circulatory oscillations accompany ventilatory oscillations during exercise in patients with heart failure[END_REF]. Normal subjects may also show a periodic breathing pattern during sleep at high altitude [START_REF] Ainslie | Breathing and sleep at high altitude[END_REF][START_REF] Berssenbrugge | Mechanisms of hypoxia-induced periodic breathing during sleep in humans[END_REF][START_REF] Küpper | Cheyne stokes breathing at high altitude: a helpful response or a troublemaker?[END_REF][START_REF] Lipsitz | Heart rate and respiratory rhythm dynamics on ascent to high altitude[END_REF][START_REF] Masuyama | Periodic breathing at high altitude and ventilatory responses to O2 and CO2[END_REF]. The factors affecting breathing instability have been extensively discussed [START_REF] Cherniack | Mathematical models of periodic breathing and their usefulness in understanding cardiovascular and respiratory disorders[END_REF][START_REF] Khoo | Determinants of ventilatory instability and variability[END_REF]. However, studies made in awake subjects at high altitude are very scarce [START_REF] Brusil | Methods for identifying respiratory oscillations disclose altitude effects[END_REF][START_REF] Chadha | Periodic breathing triggered by hypoxia in normal awake adults. Modification by naloxone[END_REF][START_REF] Fan | Effects of acetazolamide on cerebrovascular function and breathing stability at 5,050 m[END_REF][START_REF] Mateika | Peripheral chemoreflex responsiveness is increased at elevated levels of carbon dioxide after episodic hypoxia in awake humans[END_REF][START_REF] Waggener | Strength and cycle time of highaltitude ventilatory patterns in unacclimatized humans[END_REF], and only one observation of periodic breathing during exercise at high altitude, in field conditions, has been reported [START_REF] Garde | Periodic breathing during ascent to extreme altitude quantified by spectral analysis of the respiratory volume signal[END_REF].

The instability of the ventilation control system in patients is related to three main factors: the low reserve of O 2 and CO 2 in the lungs and tissues, the delay of signal transmission between the lungs and the central/peripheral chemoreceptors, and a high gain of the chemoreceptor response to O 2 or CO 2 or of the central response to input signals [START_REF] Cherniack | Mathematical models of periodic breathing and their usefulness in understanding cardiovascular and respiratory disorders[END_REF][START_REF] Pack | Mechanisms of ventilatory periodicities[END_REF]. A physiological or environmental stress can reveal the unstable nature of the ventilatory control system. This can be initiated by a mechanical (obstruction of airways), chemical (rapid variation of O 2 or CO 2 pressures) or central (sigh) constraint [START_REF] Dempsey | Pathophysiology of sleep apnea[END_REF].

When ventilation was recorded breath-by-breath, a periodic breathing was observed in healthy subjects coming to our outpatient mountain medicine consultation at Avicenne hospital in Bobigny (France). The ventilation signal showed a periodic breathing pattern, which increased when the subject started to exercise in hypoxic conditions and was subsequently maintained during normoxic exercise. During this consultation, subjects perform a hypoxia exercise test, which allows a good prediction of their risk factors for severe high altitude illnesses [START_REF] Canouï-Poitrine | Risk prediction score for severe high altitude illness: a cohort study[END_REF][START_REF] Richalet | Physiological risk factors for severe high-altitude illness: a prospective cohort study[END_REF]. Therefore, our objective was to confirm this observation of periodic breathing in healthy subjects in a retrospective study led in 82 subjects who performed this hypoxia exercise test. We hypothesized that subjects with a high ventilatory response to hypoxia might show a more pronounced periodic pattern of ventilation, due to a higher gain of the chemoreceptor feedback loop. Then, in a prospective study conducted in 25 subjects, we determined the influence of cardiorespiratory parameters (ventilation, cardiac output) on the observed periodic pattern, at different levels of exercise intensity and hypoxic stress. We also conducted a CO 2 rebreathing test on each subject to assess the potential role of central chemoreceptors [START_REF] Duffin | Measuring the respiratory chemoreflexes in humans[END_REF][START_REF] Read | A clinical method for assessing the ventilatory response to carbon dioxide[END_REF] on the genesis of ventilation oscillations.

If the hypothesis were confirmed, periodic breathing would not be considered as a rare phenomenon observed in some patients or in normal subjects in hypoxic conditions, but as a common characteristic of the breathing pattern in humans, exacerbated in some pathological or physiological conditions. The observation of this phenomenon in high-risk patients may help the early detection of patients susceptible to develop apneic syndromes.

Subjects and methods

Retrospective study

Subjects.

Among the population coming to the outpatient consultation of mountain medicine at Avicenne hospital in 2012, 82 subjects (38 females and 44 males) were retrospectively randomly selected and separated in two groups of 41 high and 41 low responders to hypoxia according to the median value of the hypoxic ventilatory response to hypoxia at exercise (HVRe > or < 0.84 L.min -1 .kg -1 ) derived from the hypoxic exercise test (inspired fraction of O 2 : 0.115, exercise intensity of 30% of maximal aerobic power), as previously described [START_REF] Canouï-Poitrine | Risk prediction score for severe high altitude illness: a cohort study[END_REF][START_REF] Lhuissier | Ventilatory and cardiac responses to hypoxia at submaximal exercise are independent of altitude and exercise intensity[END_REF][START_REF] Lhuissier | Ageing and cardiorespiratory response to hypoxia[END_REF][START_REF] Read | A clinical method for assessing the ventilatory response to carbon dioxide[END_REF].

Characteristics of the two groups are shown in table 1.

Procedure.

The hypoxic exercise test consists in 4 successive phases of 3 to 4 minutes each with the following sequence: rest in normoxia (RN), rest in hypoxia (RH), exercise in hypoxia (EH) and exercise in normoxia (EN) (Figure 1). Minute ventilation (E , L.min -1 ) was measured through a metabograph (Vmax Encore, SensorMedics, Yorba Linda, CA). Tidal volume (VT), total respiratory cycle time (Ttot) and inspiratory time (Ti) were derived from the ventilation signal.

Pulse O 2 saturation (SpO 2 , %) was measured by transcutaneous oximetry (Nellcor N-595, Nellcor, Pleasanton, CA) on a pre-warmed ear lobe. End tidal PCO 2 (PETCO 2 ) was measured by infrared thermopile (Vmax Encore, SensorMedics, Yorba Linda, CA). As previously defined, ventilatory response to hypoxia at exercise was measured as the ratio of increase in E over the decrease in SpO2 between normoxic and hypoxic conditions at exercise [START_REF] Bourdillon | Cerebral oxygenation during the Richalet hypoxia sensitivity test and cycling time-trial performance in severe hypoxia[END_REF][START_REF] Canouï-Poitrine | Risk prediction score for severe high altitude illness: a cohort study[END_REF][START_REF] Lhuissier | Ventilatory and cardiac responses to hypoxia at submaximal exercise are independent of altitude and exercise intensity[END_REF][START_REF] Lhuissier | Ageing and cardiorespiratory response to hypoxia[END_REF][START_REF] Richalet | Physiological risk factors for severe high-altitude illness: a prospective cohort study[END_REF].

During the whole test, E, SpO 2 and PETCO2 were recorded breath-by-breath (Figure 1). Data were transferred to a computer for further variability analysis. A Fast Fourier Transform (FFT) was then applied to the ventilation signal in sequences of 128 seconds of a steady state interval in each phase of the test. This method allowed us to detect the presence of peaks in the frequency domain of the ventilation signal (Figure 2). Two main parameters were derived from the FFT: the frequency in hertz (or period in seconds) of the larger peak and its power estimated as the area under the peak at ± 0.02 Hz around the peak (in L 2 .min -2 , % 2 and mmHg 2 , respectively for E, SpO 2 and PETCO 2 spectra). This method allowed us to precisely quantify the presence of oscillations in the signals that are not observable in the standard protocol routinely used in the hypoxic exercise test where the signals are averaged every 20 seconds [START_REF] Richalet | Physiological risk factors for severe high-altitude illness: a prospective cohort study[END_REF].

Prospective study

In order to determine the main factors likely to influence ventilatory oscillations, we designed two separate protocols: one was centered on the effect of intensity of power output at exercise (group Intensity), the second on the effect of the level of hypoxia (group Altitude).

Subjects.

For both protocols, healthy and non-smoking male subjects volunteered and were given exhaustive information about the successive tests. All were in good physical condition, with a medium level of regular physical activities (from 2 to 10 hours per week). They showed no evidence of cardiovascular or pulmonary disease. Subjects' characteristics are presented in table 2.

Procedure.

All subjects were first asked to perform a standard ramp test protocol on a cycloergometer to determine their maximal aerobic power (MAP, Table 2): after a 3 minute warm-up at 60 watts, power output was increased by 30-watt steps every two minutes until exhaustion.

Group Intensity: six tests were randomly performed with a minimum of 2-day interval. After one minute rest for material habituation and cardiorespiratory parameters stabilization, subjects were first asked to keep a resting sitting position on the ergometer for 6 min, and then to pedal for 6 min at around 65 rpm pedaling cadence at an exercise intensity of 20, 30 and 40% of MAP.

The whole test was executed either in normoxia or in normobaric hypoxic condition simulating an altitude of 4,000 m.

Group Altitude: four tests were randomly conducted, at different simulated altitudes: sea level, 2,000m, 3,000m and 4,000m, with fractions of inspired oxygen of respectively 0.209, 0.168, 0.145 and 0.127. A 6-min rest / 6-min exercise test was performed and exercise intensity was set at 30% of MAP.

The ventilatory response to CO 2 (HCVR) was determined using a modified Read's rebreathing method [START_REF] Mohan | Measuring central-chemoreflex sensitivity in man: rebreathing and steady-state methods compared[END_REF]. After breathing room air through a mouthpiece device to establish a baseline, the valve was switched to a 10-liter rebreathing bag containing a mixture of 93% O 2 and 7% CO 2 . After two deep breaths in order to speed up the gas mixing in the lungs, the subject was asked to spontaneously breath in the bag until ventilation reached 60 L.min -1 . Collected data was then compiled in a E-PETCO 2 graph to calculate HCVR from the slope of the linear regression between E and PETCO 2 .

The same data acquisition and analysis as in the retrospective study was performed in the prospective study. In addition, instantaneous cardiac output was measured using a transthoracic impedance technique (Physioflow®, Manatec Biomedical, France) throughout the different phases.

The protocols were approved by the Ile-de-France Ethics Committee (CPP-IDF2) and an individual written informed consent has been collected from all subjects.

Statistical analysis.

Results are presented as mean ± standard deviation. In the retrospective study, a two-way analysis of variance was performed to evaluate the difference in period and peak power of E, SpO 2 and PETCO 2 spectra between groups (low and high HVRe) and between conditions (rest, exercise, hypoxia, normoxia). A Bonferroni post-hoc test was then used when applicable. In the prospective study, regarding the expected difference in mean period (3 seconds), the standard deviation (2.4 seconds), a level of significance at 0.05% and a statistical power at 80%, the minimum number of subjects to be included was 7 for each protocol. Since spectra peak power showed a high standard deviation and a non-normal distribution, we performed a logarithmic transformation of raw data, and calculated a minimum number of subjects to include of 10. A two-way analysis of variance was then performed to evaluate the difference in period and power of the E, SpO 2 and PETCO 2 spectra between conditions (rest and exercise, hypoxia and normoxia). A multivariate regression was done to establish potential correlations between period and peak power of E, SpO 2 and PETCO 2 spectra on one hand, and cardiorespiratory parameters on the other hand: E, SpO 2 , PETCO 2 , Ttot, Ti, VT, heart rate (HR) and cardiac output (c).

Results

Retrospective study

Typical recordings of a hypoxic exercise test with breath-by-breath output are shown in The corresponding frequency spectrum of the ventilation signal during the four phases of the hypoxic exercise test in a given subject (high HVRe) is presented in figure 2 as an example. Note that a peak was observable in all phases but particularly sharp in exercise in hypoxia (EH). Mean values of period of oscillation of E and SpO 2 signals, as well as power of the corresponding peak in the four phases of the test are presented in figure 3. Two-way ANOVA showed that E, SpO 2 and PETCO 2 periods were lower at exercise (P<0.001) and similar in both groups. E and SpO 2 peak powers were higher at exercise (P<0.001) and higher in the high HVRe group (P<0.001). Detailed differences are showed in figure 3 for E and SpO 2 (PETCO 2 not shown).

For the whole population, the average period was around 12.5 s at rest and significantly decreased to 11.1 s at exercise (P<0.001), without difference between the two HVRe groups. The mean period of the E signal (11.1±1.8 s) was slightly shorter than the period for SpO 2 (12.0±1.8 s, P<0.001) and PETCO 2 (11.5±1.8 s, P<0.001). The differences are small but highly significant (paired comparison).

Prospective study

The same breath-by-breath recording methods were used in the prospective study. Varying altitude and exercise intensities allowed identifying some of the factors influencing period and peak power. Following data are presented by combining the results of the Intensity and Altitude groups.

As expected, E increased with exercise and from normoxia to hypoxia, SpO 2 decreased with hypoxia and with exercise (in hypoxia) and PETCO 2 decreased with hypoxia and increased with exercise (Figure 4, left column).

As in the retrospective study, a peak in E, SpO 2 and PETCO 2 spectra was clearly noticeable, especially during exercise in the hypoxic phase. Mean values for period and peak power are presented in figure 4. The period of oscillations during exercise was shorter than in rest, for all altitudes (P<0.001), decreased with increasing exercise intensity (P<0.001) and was not influenced by altitude. Peak power of E oscillations increased with altitude (P<0.001) and from rest to exercise (P<0.001) but was not influenced by the level of exercise intensity. A similar trend was shown in the SpO 2 and PETCO 2 signals, although the variations were less marked (Figure 4). Multivariate regressions evidenced significant correlations during exercise between E period and E (P<0.001) or c (P<0.001) and hypoxia (P<0.01), as well as between E peak power and E (P<0.001) or c (P<0.001) and hypoxia (P<0.001). Regressions for E period and E peak power, for each condition (normoxia and hypoxia) are shown in figure 5. E period was negatively related to the intensity of E and c in hypoxic conditions. E peak power was positively related to E and c both in normoxic and hypoxic conditions (Figure 5). For a given level of E (or c), E peak power and period were greater in hypoxia than in normoxia (P<0.001). Similar correlations were found for the PETCO 2 peak power with E and c (results not shown). No significant correlation was found for the SpO 2 signal.

Interestingly, relating E period with Ttot, we found a very significant correlation both at rest and exercise (Figure 6). Two-factor regression analysis of E period as a function of Ttot and exercise showed a significant correlation with Ttot (P<0.001) and with exercise (P<0.05).

When pooling the individual values of oscillation periods at exercise in hypoxic conditions, the mean period of the E signal was 10.9 ± 1.3 s, slightly shorter than the SpO 2 period (11.6 ± 1.5 s, P<0.001) and PETCO 2 period (11.6 ± 1.3 s, P<0.001), similarly to what was found in the retrospective study.

Relating E peak power to HCVR, we found a significant correlation in hypoxic (3,000 m) conditions but not in normoxia (Figure 7, left). Thus the intensity of E oscillations is higher in subjects with a higher ventilatory response to CO 2 , but only in hypoxic conditions. A similar relationship was found for PETCO 2 peak power (Figure 7, right) but not for SpO 2 (results not shown). No correlation was found between E, SpO 2 or PETCO 2 period and HCVR (results not shown).

Discussion

To our knowledge, this is the first description of a spontaneous oscillation of ventilation in awake normal subjects at rest and during exercise in normoxic and hypoxic conditions. We observed a period of oscillations between 8 and 16 seconds, meaning that spontaneous ventilation oscillates between a maximum and a minimum every 3 to 5 breaths at rest and 4 to 7 breaths during exercise. No such observation has been made in previous studies of ventilation in hypoxic exercise conditions, probably because it requires a breath-by-breath recording of the ventilation signal and in most cases a spectral analysis to evidence the periodicity of the signal.

Both retrospective and prospective study show that the period is shorter at exercise when cardiac output and ventilation are higher. In addition, the oscillation is more pronounced in subjects with a higher sensitivity to hypoxia of their peripheral chemoreceptors. These findings provide novel evidence that support the theory of the closed loop system of control of ventilation, involving the sensitivity of the central and peripheral chemoreceptors and the time delay of the O 2 /CO 2 signals between the receptors and the respiratory control centers [START_REF] Fowler | Periodic breathing at high altitude[END_REF][START_REF] Jubran | Effect of isocapnic hypoxia on variational activity of breathing[END_REF][START_REF] Lipsitz | Heart rate and respiratory rhythm dynamics on ascent to high altitude[END_REF]. The lung to carotid body circulation time may also affect the oscillation period [START_REF] Ward | Inspiratory-expiratory responses to alternate-breath oscillation of PACO2 and PAO2[END_REF].

Exercise may shorten the delay through the increased blood velocity and therefore decrease the period of oscillation. The high gain of the feedback control system in high responders to hypoxia may increase the instability of the system and favor the appearance of oscillations. This spontaneous periodic breathing was probably uncovered by the brisk stimulation of the closed loop control system by imposing a hypoxic exercise to the subject, probably inducing rapid variations in the O 2 and CO 2 signals.

The combination of exercise and hypoxia may trigger the instability of the system since exercise by itself induces a further drop in O 2 arterial pressure and saturation in hypoxic conditions [START_REF] Woorons | Moderate exercise in hypoxia induces a greater arterial desaturation in trained than untrained men[END_REF]. This is confirmed in the present study by the fact that, at the same level of ventilation during exercise, oscillations of ventilation are much greater in hypoxia than in normoxia (Figure 5, lower left panel), supporting the hypothesis that hypoxia, through peripheral chemoreceptors, may be a main source of the system instability. This periodic breathing is not readily observable at rest in normoxia in awake subjects, probably because of the chaotic pattern of spontaneous ventilation and the influence of the cortical control [START_REF] Wysocki | Chaotic dynamics of ventilatory flow in humans[END_REF].

Our results indicate that E peak power rises with ventilatory response to CO 2 in hypoxia.

This suggests that the well-established synergistic effect of hypoxia and hypercapnia on the control of ventilation [START_REF] Duffin | Measuring the respiratory chemoreflexes in humans[END_REF][START_REF] Mohan | The effect of hypoxia on the ventilatory response to carbon dioxide in man[END_REF] is involved in the genesis of ventilatory oscillations.

Oscillations were found in the ventilation signal as well as in SpO 2 and PETCO 2 , which are indirect markers of the two main physiological variables controlling ventilation, i.e. arterial PO 2 and PCO 2 . The fact that these three signals were obtained through completely independent techniques reinforces the validity of the observed periodic pattern. The slight but highly significant difference in the period for E, SpO 2 and PETCO 2 is surprising, but could be in fact explained by either a non-linearity of the control system involving O 2 and CO 2 signals, or the existence of the lung O 2 /CO 2 stores which would act as a low pass filter reducing the oscillation frequency of the SpO 2 and PETCO 2 signals.

The mean period of E oscillation (11 s) observed at exercise in the present study is shorter than the one observed at rest at high altitude (20s at 3050m) [START_REF] Brusil | Methods for identifying respiratory oscillations disclose altitude effects[END_REF], during sleep at high altitude (15s at 8000m) [START_REF] Fowler | Periodic breathing at high altitude[END_REF] or in patients suffering from cardiac failure (around 1 minute) [START_REF] Dempsey | Pathophysiology of sleep apnea[END_REF][START_REF] Pinna | Periodic breathing in heart failure patients: testing the hypothesis of instability of the chemoreflex loop[END_REF][START_REF] Ponikowski | Oscillatory breathing patterns during wakefulness in patients with chronic heart failure: clinical implications and role of augmented peripheral chemosensitivity[END_REF] or obstructive sleep apneas (> 38 s) [START_REF] Dempsey | Pathophysiology of sleep apnea[END_REF][START_REF] Ryan | Periodicity of obstructive sleep apnea in patients with and without heart failure[END_REF]. It stands in the large range of oscillations (5 to 100 s) observed in uncontrolled conditions in subjects climbing at high altitude [START_REF] Garde | Periodic breathing during ascent to extreme altitude quantified by spectral analysis of the respiratory volume signal[END_REF]. It is clearly out of the range of the periods observed in the present hypoxia exercise test for breathing frequency (3 to 5 s), heart rate (0.4 to 1.0 s) or pedaling rate (1s). It is interesting to note that, in the present study, an increase in cardiac output (as expected during exercise) exacerbated the oscillations whereas in patients with cardiac failure, the predominant hypothesis to explain the Cheyne-Stokes breathing pattern is an increase in time delay due to low cardiac output [START_REF] Cherniack | Mathematical models of periodic breathing and their usefulness in understanding cardiovascular and respiratory disorders[END_REF][START_REF] Fowler | The role of the central chemoreceptor in causing periodic breathing[END_REF]. A phase shift between the various physiological signals involved in the control loop might contribute to the instability of the system, as seen with heart rate and blood pressure, with arterial O 2 saturation, or tidal volume in normal subjects during sleep in hypoxia [START_REF] Berssenbrugge | Mechanisms of hypoxia-induced periodic breathing during sleep in humans[END_REF][START_REF] Lipsitz | Heart rate and respiratory rhythm dynamics on ascent to high altitude[END_REF] or in patients with cardiac failure [START_REF] Binggeli | Spontanous periodic breathing is associated with sympathetic hyperreactivity and baroreceptor dysfunction in hypertension[END_REF][START_REF] Pinna | Periodic breathing in heart failure patients: testing the hypothesis of instability of the chemoreflex loop[END_REF]. The observation that exercise appears to increase ventilation oscillations suggests that whatever effect enhances oscillations is strong enough to offset the stabilizing effect of the reduced phase shift. In obstructive sleep apneas, an initial apnea may trigger oscillations in an already unstable system, and airway obstructions (permanent or intermittent) may promote hypoxia and make the system more unstable.

In conclusion, a spontaneous periodic breathing pattern with a period of around 11 seconds at exercise has been evidenced in normal subjects, uncovering the intrinsic unstable structure of the ventilation control loop. The intensity of these oscillations depends on the ventilatory response to hypoxia, suggesting that peripheral chemoreceptors play a role in the genesis or modulation of this phenomenon. Further studies will be necessary to determine the respective influence of O 2 and CO 2 signals, sensitivity of the central and peripheral chemoreceptors, and intensity of cardiac output on the period and power of this observed phenomenon. This observation may have an impact on the comprehension of central or obstructive apneas and periodic breathing in diseases and on early detection of high-risk subjects with a hypoxic exercise test.

Figure 1 .

 1 Figure 1. In the subject with a high HVRe (lower panel), oscillations of E, especially in the hypoxic exercise phase, are clearly shown. Note that the oscillations are also observable on the SpO 2 and PETCO 2 signals. Oscillations are less apparent in the subject with low HVRe (upper panel).

Figure 1 .

 1 Figure 1. Breath-by-breath output of minute ventilation (E), pulse O 2 saturation (SpO 2 ) and end tidal PCO 2 (PETCO 2 ) during a hypoxia exercise test. Lower panel: subject with a high HVRe (0.93 L.min -1 .kg -1 ). Upper panel: subject with a low HVRe (0.60 L.min -1 .kg -1 ).

Figure 2 .

 2 Figure 2. Example of spectral analysis of minute ventilation by Fast Fourier Transform in the four phases of a hypoxia exercise test. RN: rest in normoxia, RH: rest in hypoxia, EH: exercise in hypoxia, EN: exercise in normoxia. PSD: Power Spectral Density. Note that the frequency of the main peak in hypoxic exercise conditions (EH) is around 0.08 Hz, which corresponds to a period of 12.5 seconds.

Figure 3 .

 3 Figure 3. E and SpO 2 period and power of the main peak obtained from the spectral analysis of minute ventilation during the various phases of a hypoxia exercise test. RN: rest in normoxia, RH: rest in hypoxia, EH: exercise in hypoxia, EN: exercise in normoxia. Mean ± SD. Condition vs RN: *, P<0.05; **, P<0.01; ***, P<0.001. High HVRe vs Low HVRe: +, P<0.05 ; ++, P<0.01.

Figure 4 .

 4 Figure 4. Mean values of E, SpO 2 and PETCO 2 and corresponding periods and peak powers extracted from the prospective study. Mean ± SD. Condition vs Rest: +, P<0.05; ++, P<0.01; +++, P<0.001. Condition vs Sea level: *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 5 .

 5 Figure 5. Linear regressions between E period and peak power, and E (left column) and Cardiac Output (right column) during exercise, in normoxic and hypoxic conditions, extracted from the prospective study.

Figure 6 .

 6 Figure 6. Linear regressions between E period and total respiratory cycle time (Ttot) at rest and exercise, extracted from the prospective study.

Figure 7 .

 7 Figure 7. Linear regressions between E (left) and PETCO 2 (right) peak powers during exercise and hypercapnic ventilatory response (HCVR), extracted from the prospective study.

Table 1 .

 1 Characteristics of the subjects following their hypoxic ventilatory response at exercise (HVRe).

		n	Sex (female)	Age (yrs)	Body weight (kg)	Height (cm)	HVRe (L.min -1 .kg -1 )
	High HVRe	41	18	47 ± 14	71 ± 9	171 ± 7	1.26 ± 0.40
	Low HVRe	41	20	49 ± 16	66 ± 13	167 ± 9	0.55 ± 0.15
	P High vs Low		ns	ns	ns	ns	<0.001
	Mean ± SD

Table 2 .

 2 Characteristics of the subjects of the Intensity and Altitude studies.

		n	Age (years)	Height (cm)	Weight (kg)	Maximal aerobic power (MAP) (Watts)
	Group Intensity	12 28.6 ± 6.7	175.1 ± 7.4	75.1 ± 14.7	270.0 ± 67.0
	Group Altitude	13 27.2 ± 6.5	173.8 ± 9.0	69.4 ± 12.4	235.8 ± 47.9
	Mean ± SD


Eric Hermand
email: eric.hermand17@gmail.com

François J Lhuissier

Julie Larribaut

Aurélien Pichon

Jean-Paul Richalet

Ventilatory oscillations at exercise: effects of hyperoxia, hypercapnia and acetazolamide

Keywords:

Introduction

In most usual circumstances, human ventilation adapts to the demand of the organism around a steady state value. However, its control is known to be challenged under various specific conditions. For example, patients suffering from chronic heart failure (CHF) or apnea syndrome (central, obstructive or mixed) show breathing pattern marked by large oscillations with a period of around 1 minute at rest, awake or asleep, and during exercise. Oscillations are also present in healthy subjects at high altitude during sleep [START_REF] Berssenbrugge | Mechanisms of hypoxia-induced periodic breathing during sleep in humans[END_REF][START_REF] Ainslie | Breathing and sleep at high altitude[END_REF], in wakeful state [START_REF] Waggener | Strength and cycle time of high-altitude ventilatory patterns in unacclimatized humans[END_REF][START_REF] Fan | Effects of acetazolamide on cerebrovascular function and breathing stability at 5,050 m[END_REF] and in patients suffering from chronic mountain sickness [START_REF] Richalet | Acetazolamide: a treatment for chronic mountain sickness[END_REF]. Several factors are involved in breathing instability. Reduced blood flow and exacerbated chemosensitivity are common in CHF at rest [START_REF] Pinna | Cardiorespiratory interactions during periodic breathing in awake chronic heart failure patients[END_REF] and during exercise [START_REF] Agostoni | Mechanisms of Periodic Breathing During Exercise in Patients With Chronic Heart Failure[END_REF][START_REF] Dhakal | Exercise oscillatory ventilation in heart failure[END_REF]. Obstructive sleep apneas (OSA) are associated with anatomical factors inducing airway obstruction and decreased central respiratory drive to genioglossus muscle during sleep [START_REF] Dempsey | Pathophysiology of sleep apnea[END_REF]. These factors may disturb the respiratory control loop and impair the ability of the system to effectively control ventilation.

In awake subjects, only one observation of ventilatory oscillations during exercise has been reported at high altitude [START_REF] Garde | Periodic breathing during ascent to extreme altitude quantified by spectral analysis of the respiratory volume signal[END_REF]. We recently described this phenomenon, with a period of around 11 seconds, in subjects exercising in mild hypoxia [START_REF] Hermand | Periodic breathing in healthy humans at exercise in hypoxia[END_REF]. The analysis of cardiorespiratory parameters has highlighted some factors that may have an impact on the period and amplitude of ventilatory oscillations: unlike CHF patients whose severity of periodic breathing is related to reduced cardiac output, the amplitude of oscillations in healthy subjects was positively correlated to cardiac output and ventilation during exercise in hypoxia, while their period was shorter during exercise as compared to rest [START_REF] Hermand | Periodic breathing in healthy humans at exercise in hypoxia[END_REF].

Peripheral and central chemoreceptors respectively inform respiratory centers about O 2 and CO 2 status. The response to hypoxia is mainly due to peripheral chemoreceptors [START_REF] Lahiri | Oxygen sensing in the body[END_REF][START_REF] Kumar | Peripheral Chemoreceptors: Function and Plasticity of the Carotid Body[END_REF] while the level of arterial or interstitial partial pressure of CO 2 (PCO 2 ) is detected by central chemoreceptors, with gain adjustments from peripheral chemoreceptors [START_REF] Lahiri | CO2/H(+) sensing: peripheral and central chemoreception[END_REF][START_REF] Blain | Peripheral chemoreceptors determine the respiratory sensitivity of central chemoreceptors to CO2: Peripheral-central chemoreceptor interdependence[END_REF], although these interactions have been debated in humans [START_REF] Duffin | Cross-Talk opposing view: peripheral and central chemoreflexes have additive effects on ventilation in humans[END_REF]. We suggested that peripheral chemoreceptors play an important role in the genesis of ventilatory oscillations, more pronounced in subjects with a higher hypoxic ventilatory response, thus revealing the unstable nature of the ventilatory control system under physiological and environmental stresses. The degree of ventilatory instability is linked to the loop gain, which is the product of the controller gain, i.e. the ventilatory response to hypoxia and/or hypercapnia, and the plant gain, i.e. the effect of a ventilation variation on arterial PCO 2 (PaCO 2 ) and/or arterial PO 2 (PaO 2 ). The higher the gain, the more unstable the system [START_REF] Cherniack | Mathematical models of periodic breathing and their usefulness in understanding cardiovascular and respiratory disorders[END_REF][START_REF] Burgess | New insights from the measurement of loop gain in obstructive sleep apnoea[END_REF]. Therefore, as in CHF, a greater sensitivity of central and peripheral respiratory drive to CO 2 and O 2 , through greater hypoxic and hypercapnic ventilatory responses [START_REF] Solin | Peripheral and central ventilatory responses in central sleep apnea with and without congestive heart failure[END_REF][START_REF] Giannoni | Combined increased chemosensitivity to hypoxia and hypercapnia as a prognosticator in heart failure[END_REF][START_REF] Maestri | Assessment of the peripheral ventilatory response to CO2 in heart failure patients: reliability of the single-breath test[END_REF] would lead to more pronounced oscillations of ventilation [START_REF] Francis | Quantitative general theory for periodic breathing in chronic heart failure and its clinical implications[END_REF][START_REF] Basner | Loop gain: can it help close the heart failure-periodic breathing loop?[END_REF].

Various interventions are available to modulate this breathing instability. Occasional CO 2 inhalation during sleep considerably diminishes apnea occurrences in Cheynes-Stokes respiration (CSR) by keeping CO 2 arterial pressure above apneic threshold [START_REF] Andreas | Treatment of Cheyne-Stokes respiration with nasal oxygen and carbon dioxide[END_REF][START_REF] Lorenzi-Filho | Effects of inhaled carbon dioxide and oxygen on cheyne-stokes respiration in patients with heart failure[END_REF]. Hyperoxia has more conflicting effects: it reduces the hypoxemia involved in the mechanisms of periodic breathing, and therefore improves heart condition, physical performance and apnea/hypopnea index in CSR patients [START_REF] Andreas | Improvement of exercise capacity with treatment of Cheyne-Stokes respiration in patients with congestive heart failure[END_REF][START_REF] Franklin | Reversal of central sleep apnea with oxygen[END_REF], but does not abolish ventilatory oscillations in OSA [START_REF] Gold | A shift from central and mixed sleep apnea to obstructive sleep apnea resulting from low-flow oxygen[END_REF] and CHF patients [START_REF] Ponikowski | Oscillatory breathing patterns during wakefulness in patients with chronic heart failure: clinical implications and role of augmented peripheral chemosensitivity[END_REF]. Conversely, at high altitude, O 2 therapy significantly reduces sleep apneas and periodic breathing, and improves sleep quality in normal subjects [START_REF] Mcelroy | Nocturnal O2 enrichment of room air at high altitude increases daytime O2 saturation without changing control of ventilation[END_REF][START_REF] Moraga | Periodic breathing and oxygen supplementation in Chilean miners at high altitude (4200m)[END_REF].

Pharmacological interventions are also available for breathing disorders. Carbonic anhydrase inhibitors are proven to be effective treatment for acute [START_REF] Swenson | Renal carbonic anhydrase inhibition reduces high altitude sleep periodic breathing[END_REF] and chronic [START_REF] Richalet | Acetazolamide: a treatment for chronic mountain sickness[END_REF] mountain sickness, as well as for ventilatory disorders in healthy subjects at high altitude [START_REF] Swenson | Renal carbonic anhydrase inhibition reduces high altitude sleep periodic breathing[END_REF][START_REF] Fischer | Theophylline and acetazolamide reduce sleep-disordered breathing at high altitude[END_REF][START_REF] Ainslie | Breathing and sleep at high altitude[END_REF]. They are also experimentally used to treat periodic breathing in CSR, CHF and OSA patients [START_REF] Edwards | Acetazolamide improves loop gain but not the other physiological traits causing obstructive sleep apnoea[END_REF][START_REF] Javaheri | Acetazolamide attenuates hunter-cheyne-stokes breathing but augments the hypercapnic ventilatory response in patients with heart failure[END_REF][START_REF] Apostolo | Acetazolamide and Inhaled Carbon Dioxide Reduce Periodic Breathing During Exercise in Patients with Chronic Heart Failure[END_REF]. Their complex action is still to be fully understood, and involves several contradictory mechanisms, associating central stimulation and reduction of peripheral chemoreceptors activity [START_REF] Swenson | Carbonic anhydrase inhibitors and ventilation: a complex interplay of stimulation and suppression[END_REF][START_REF] Teppema | Multifaceted clinical effects of acetazolamide: will the underlying mechanisms please stand up?[END_REF]. Through a greater stimulation of central chemoreceptors by CO 2 , acetazolamide (ACZ) elevates ventilation level and O 2 saturation in patients suffering from apneas and in healthy subjects at high altitude, and considerably reduces occurrence and length of apnea episodes. ACZ also increases ventilatory response to CO 2 in patients with chronic mountain sickness [START_REF] Rivera-Ch | Effect of acetazolamide on ventilatory response in subjects with chronic mountain sickness[END_REF]. A similar effect on ventilatory oscillations was observed in awake CHF patients at rest and during exercise [START_REF] Fontana | Effect of acetazolamide on chemosensitivity, Cheyne-Stokes respiration, and response to effort in patients with heart failure[END_REF][START_REF] Apostolo | Acetazolamide and Inhaled Carbon Dioxide Reduce Periodic Breathing During Exercise in Patients with Chronic Heart Failure[END_REF]. Similarly, the role of ACZ-induced changes in controller gain and plant gain are still debated [START_REF] Edwards | Acetazolamide improves loop gain but not the other physiological traits causing obstructive sleep apnoea[END_REF][START_REF] Burgess | New insights from the measurement of loop gain in obstructive sleep apnoea[END_REF].

Until now, the phenomenon of ventilatory oscillations was well described mostly in patients and in sojourners at high altitude during sleep.

Recently, a similar pattern was unveiled in normal subjects during exercise in hypoxia [START_REF] Hermand | Periodic breathing in healthy humans at exercise in hypoxia[END_REF]. Since the period of oscillations observed in this preliminary study is much shorter than in patients, and since the intensity of oscillations is associated with high cardiac output (exercise) contrary to pathological conditions (CHF, CSR), this strongly suggests that the involved mechanisms are different. Therefore, the objective of the present study is to unravel the mechanisms by which the ventilatory control system becomes unstable and generates oscillations in awake subjects at exercise.

In order to assess the respective role of central and peripheral chemoreceptors, respectively sensitive to variations of CO 2 and O 2 , we propose to submit our model of ventilatory oscillations in healthy subjects to various external constraints such as hyperoxia, hypercapnia and pharmacological treatment by ACZ.

Subjects and methods

Subjects.

Twenty-six healthy and non-smoking male subjects volunteered for the study and were given complete information about the successive tests.

All were in good physical condition, with a medium to high level of regular physical activity (from 2 to 10 hours per week). They showed no evidence of cardiovascular or pulmonary disease. Subjects' characteristics are presented in table 1. The protocol was approved by the Ile-de-France Ethics Committee (CPP-IDF2) and an individual written informed consent has been collected from all subjects. The study was registered as Clinical Trial reg. n°: NCT02201875.

Procedure.

All subjects were first asked to perform a standard ramp test protocol on a cycloergometer to determine their maximal aerobic power (MAP, Table 1): after a 3-minute warm-up at 60 watts, power output was increased by 30-watt steps every two minutes until exhaustion.

For all studies, minute ventilation ( E, L.min -1 ) was measured through a metabograph (Vmax Encore, SensorMedics, Yorba Linda, CA). Tidal volume (VT, L), total respiratory cycle time (Ttot, s) and inspiratory time (Ti, s) were derived from the ventilation signal. Pulse O 2 saturation (SpO 2 , %) was measured by transcutaneous oximetry (Nellcor N-595, Nellcor, Pleasanton, CA) on a pre-warmed ear lobe. End tidal PCO 2 (PETCO 2 , mmHg) was measured by infrared thermopile (Vmax Encore, SensorMedics, Yorba Linda, CA). During the whole test, E, SpO 2 and PETCO 2 were recorded breath-by-breath (Fig. 1). Data were transferred to a computer for further variability analysis. A Fast Fourier Transform (FFT) was then applied to the breath-by-breath ventilation signal, extracted from the raw data, in sequences of 128 points (one point per second) of a steady state interval at the end of each phase of the test. This method allowed us to detect the presence of peaks in the frequency domain of the ventilation signal (Fig. 2) [START_REF] Hermand | Periodic breathing in healthy humans at exercise in hypoxia[END_REF]. Two main parameters were derived from the FFT: the frequency in hertz (or period in seconds) of the larger peak and its power estimated as the area under the peak at ± 0.02 Hz around the peak (in L 2 .min -2 , % 2 and mmHg 2 , respectively for E, SpO 2 and PETCO 2 spectra). Thus, a high peak power translates into greater ventilatory oscillations. This method allowed us to precisely quantify the presence of oscillations in the signals that are not observable in the standard protocol routinely used in the hypoxic exercise test where the signals are averaged every 20 seconds [START_REF] Richalet | Physiological risk factors for severe high-altitude illness: a prospective cohort study[END_REF][START_REF] Lhuissier | Ventilatory and cardiac responses to hypoxia at submaximal exercise are independent of altitude and exercise intensity[END_REF][START_REF] Canouï-Poitrine | Risk prediction score for severe high altitude illness: a cohort study[END_REF][START_REF] Bourdillon | Cerebral oxygenation during the Richalet hypoxia sensitivity test and cycling time-trial performance in severe hypoxia[END_REF]. Cardiac output ( c, L.min -1 ) was measured using a non-invasive impedance cardiograph device (PhysioFlow PF-05, Manatec biomedical, Paris, France) [START_REF] Charloux | A new impedance cardiograph device for the non-invasive evaluation of cardiac output at rest and during exercise: comparison with the "direct" Fick method[END_REF][START_REF] Richard | Noninvasive cardiac output evaluation during a maximal progressive exercise test, using a new impedance cardiograph device[END_REF].

The ventilatory response to CO 2 (HCVR) was determined using a modified Read's rebreathing method [START_REF] Read | A clinical method for assessing the ventilatory response to carbon dioxide[END_REF]. After breathing room air through a mouthpiece device to establish a baseline, the valve was switched to a 10-liter rebreathing bag containing a mixture of 93% O 2 and 7% CO 2 . After two deep breaths in order to speed up the gas mixing in the lungs, the subject was asked to spontaneously breathe in the bag until ventilation reached 60 L.min -1 . Collected data were then compiled in a E -PETCO 2 graph to calculate HCVR from the slope of the linear regression between E and PETCO 2 (see inlets in Fig. 1). HCVR was measured once in each subject for the hyperoxia and hypercapnic hyperoxia studies and twice (under placebo and ACZ) in the ACZ study.

Hyperoxia and hypercapnic hyperoxia studies

Four tests were randomly conducted, in normoxia and hyperoxia (O 2 100%), then, at least one week apart, in normoxia and hypercapnic hyperoxia (O 2 93%, CO 2 7%) in 13 subjects. After one minute rest for material habituation and cardiorespiratory parameters stabilization, subjects were first asked to keep a resting sitting position on the ergometer for 6 min, and then to pedal for 6 min at around 65 rpm pedaling cadence at an exercise intensity of 30 % of MAP.

ACZ study

In a double-blind, placebo-controlled, cross-over study, 13 subjects were asked to take either a placebo or a 250 mg ACZ pill, on morning breakfast and at midday lunch, on the day before and on tests days. Tests were performed in the afternoon following the last intake. One week at least separated the two testing days. Under each condition, subjects underwent two randomized tests: one in normoxia and one in hypoxia (normobaric simulated altitude of 3000 m). As in the hypercapnic hyperoxia studies, a 6-min rest / 6-min exercise test was performed and exercise intensity was set at 30% of MAP.

Statistical analysis.

Results are presented as mean ± standard deviation. Regarding the expected difference in mean period (3 seconds), the standard deviation (2.4 seconds), a level of significance at 0.05% and a statistical power at 80%, the minimum number of subjects to be included was 7 for each protocol. Since spectra peak power showed a high standard deviation and a non-normal distribution, we performed a logarithmic transformation of raw data, and calculated a minimum number of subjects of 10 to include. Normality of data on each condition (rest/exercise, normoxia/hypoxia/hyperoxia/hypercapnia, placebo/ACZ) were verified by a Shapiro-Wilk normality test. In the hyperoxia and hypercapnic hyperoxia studies, two-way analyses of variance (ANOVA) with repeated measures were done (rest/exercise, normoxia/hyperoxia or normoxia/hypercapnic hyperoxia). The same subjects participated in both hyperoxic and hypercapnic hyperoxic studies. Therefore, we performed a two-way analysis of variance (rest/exercise, hyperoxia/hypercapnic hyperoxia) with repeated measures to point out the specific role of hypercapnia on cardiorespiratory parameters. In the ACZ study, a three-way analysis of variance with repeated measures was performed to evaluate the difference in period and peak power of E, SpO 2 and PETCO 2 spectra between conditions (rest/exercise, normoxia/hypoxia, placebo/ACZ). A posthoc paired Student's test was then used when applicable. As exercise is associated with a concomitant variation of multiple variables, we performed a multivariate regression analysis in order to evaluate the independent influence of each cardiorespiratory variable on period and peak power of E, SpO 2 and PETCO 2 spectra. This approach, for example, will allow us to assess the specific influence of E and/or c on peak power and period of E.

Results

Values of measured cardiorespiratory variables are presented in Table 2.

Effect of hyperoxia (Fig. 3) Effect of exercise vs rest. In the two-way ANOVA, E and PETCO 2 increased with exercise (P < 0.001), SpO 2 being unchanged. E period was shorter (P < 0.001) and its peak power higher (P < 0.001). PETCO 2 period decreased (P < 0.001) and its peak power increased (P < 0.05). SpO 2 period was shorter (P < 0.001).

Effect of hyperoxia vs normoxia.

In the two-way ANOVA, E was not different in hyperoxia, whereas SpO 2 increased (P < 0.001) and P ETCO 2 decreased (P < 0.05). E period tended to be slightly shorter (P = 0.07), as well as PETCO 2 period. PETCO 2 peak power decreased (P < 0.01).

The post-hoc analysis showed that, at exercise, E and PETCO 2 periods were shorter in hyperoxia (vs normoxia, P < 0.01). E peak power remained unchanged, whereas PETCO 2 peak power decreased significantly (P < 0.05).

Effect of hypercapnic hyperoxia (Fig. 3) Effect of exercise vs rest. In the two-way ANOVA, E and PETCO 2 increased with exercise (P < 0.001). E and PETCO 2 periods were shorter (P < 0.01 and P < 0.001 respectively) and their peak power greater (P < 0.001).

Effect of hypercapnic hyperoxia vs normoxia. In the two-way ANOVA, E, SpO 2 and PETCO 2 increased (P < 0.001) in hypercapnic hyperoxia.

E period was shorter and its peak power was greater (P < 0.05 and P < 0.001 respectively). PETCO 2 period was shorter (P < 0.01) and its peak power was smaller (P < 0.001).

The post-hoc analysis showed that, at exercise, SpO 2 and PETCO 2 periods decreased in hypercapnic hyperoxia (vs normoxia, P < 0.05). E peak power was greater (vs normoxia, P < 0.01) whereas PETCO 2 peak power was lower (vs normoxia, P < 0.01).

Effect of hypercapnia: hypercapnic hyperoxia vs. hyperoxia (Fig. 3) A two-way ANOVA evidenced an increase in E and PETCO 2 in hypercapnic hyperoxia (vs hyperoxia, P < 0.001). As expected, SpO 2 was not significantly different between conditions. The period of ventilatory oscillations tended to be shorter in hypercapnic hyperoxia (vs hyperoxia, P = 0.054). E peak power was greater in hypercapnic hyperoxia (vs hyperoxia, P < 0.001). PETCO 2 period was not modified by hypercapnia while PETCO 2 peak power decreased (P < 0.001).

Effect of ACZ (Fig. 4)

One subject had to stop the experimentation due to side effects of ACZ administration (headaches, nausea), so that 12 subjects performed the experiment.

Typical complete breath-by-breath recordings in hypoxia under placebo (upper panel) and ACZ (lower panel) are presented in Fig. 1. Oscillations of E during exercise are clearly less noticeable under ACZ condition, which is confirmed by a lower peak power in the corresponding frequency spectrum of the ventilation signal (Fig. 2,right).

Effect of exercise vs rest.

During exercise, E and PETCO 2 increased (P < 0.001) and SpO 2 decreased (P < 0.001). E, SpO 2 and PETCO 2 periods were shorter (P < 0.001). E and PETCO 2 peak powers were higher (P < 0.001 and P < 0.01 respectively).

Effect of hypoxia vs normoxia. In hypoxia, SpO 2 and PETCO 2 were lower (P < 0.001 and P < 0.01 respectively), and E tended to be higher (P = 0.053). E, SpO 2 and PETCO 2 periods were not significantly different while E and PETCO 2 peak power increased (P < 0.001 and P < 0.01 respectively).

Effect of ACZ vs placebo.

ACZ increased E (P < 0.01) and lowered PETCO 2 (P < 0.001). As expected, SpO 2 did not change in normoxia. When considering only hypoxic condition, ACZ increased SpO 2 (P < 0.05). E, SpO 2 and P ETCO 2 periods were not modified. E and PETCO 2 peak powers were lower under ACZ (P < 0.001). For all subjects, the average period was around 12.4 s at rest and significantly decreased to 10.4 s at exercise (P < 0.001), without any difference between the placebo and the ACZ groups.

Cardiorespiratory variables affecting period and peak power

In a first univariate approach, regressions for E period and E peak power, for each condition (normoxia, hyperoxia, hypercapnic hyperoxia, placebo and ACZ), with c or E were assessed (Figs 5 and 6 respectively). In all conditions, E period was negatively related to E and c (upper panels), while E peak power was positively correlated to E and c (lower panels). It is noticeable that ACZ administration significantly decreased (vs placebo) the slope of the regression between E peak power and c as well as E (P < 0.05): the higher c or E, the greater the effect of ACZ (Figs 5 and 6, lower left panels). In addition, for a given level of c, E peak power was always higher in hypercapnia, the regression slopes being similar (Fig. 5, lower right panel). HCVR was higher under ACZ (vs placebo, P < 0.01, table 2). However, there was no significant correlation between HCVR and the corresponding E peak power, under placebo and ACZ (Fig. 7), nor between HCVR placebo-ACZ difference and the corresponding E peak power placebo-ACZ difference (P = 0.18). Although the linear relationship between E peak power and HCVR did not reach significance, Fig. 7 clearly shows that for a given level of HCVR, ACZ decreases E peak power.

As several variables, such as c, E and Ttot, may vary simultaneously in response to exercise or environmental constraints, we performed a multivariate regression analysis to assess the specific role of each of these variables on ventilatory oscillations. When pooling all values obtained at rest and exercise, this analysis evidenced Ttot as the most significant factor explaining changes in E period (P < 0.001), as illustrated in Fig. 8.

Concerning E peak power, E (P < 0.001) and Ttot (P < 0.02), but not c (P = 0.094), were found as explaining variables.

To summarize the main results of these three studies:

1. Hyperoxia reduced the period of E and PETCO 2 oscillations and decreased PETCO 2 peak power at exercise.

2. Hypercapnia increased E peak power and decreased E period, while it reduced the amplitude of PETCO 2 oscillations.

3. ACZ blunted E and PETCO 2 oscillations but did not alter corresponding periods.

4. Exercise increased amplitudes and shortened periods of E and PETCO 2 oscillations in all conditions.

Discussion

To our knowledge, this is the first study on ventilatory oscillations performed on healthy subjects under physiological (exercise) and environmental stress (hypoxia/hyperoxia/hypercapnia) and under pharmacological challenge (ACZ). Our previous work evidenced for the first time ventilatory oscillations in hypoxia at exercise in normal subjects, and the importance of peripheral chemoreceptors in their genesis [START_REF] Hermand | Periodic breathing in healthy humans at exercise in hypoxia[END_REF]. The present study reveals further factors that may modulate these oscillations and proposes some mechanistic hypotheses. Hyperoxia, which silences peripheral chemoreceptors [START_REF] Sinski | Deactivation of carotid body chemoreceptors by hyperoxia decreases blood pressure in hypertensive patients[END_REF], did not modify E peak power and therefore E oscillations in healthy subjects, in accordance with some but not all studies in patients with mixed sleep apnea [START_REF] Brack | Cheyne-Stokes Respiration in Patients with Heart Failure: Prevalence, Causes, Consequences and Treatments[END_REF]. We evidenced a very significant increase in E peak power in hypercapnia, conversely to what was found in central and obstructive apneas, where CO 2 inhalation drastically reduced ventilatory oscillations [START_REF] Hudgel | Alteration in obstructive apnea pattern induced by changes in oxygen-and carbon-dioxide-inspired concentrations[END_REF][START_REF] Brack | Cheyne-Stokes Respiration in Patients with Heart Failure: Prevalence, Causes, Consequences and Treatments[END_REF]. The effect of ACZ treatment in healthy subjects in hypoxia at exercise was similar to what was observed in subjects at high altitude [START_REF] Ainslie | Breathing and sleep at high altitude[END_REF] and CSR-OSA patients [START_REF] Lalande | The effects of sildenafil and acetazolamide on breathing efficiency and ventilatory control during hypoxic exercise[END_REF][START_REF] Brack | Cheyne-Stokes Respiration in Patients with Heart Failure: Prevalence, Causes, Consequences and Treatments[END_REF]: average minute ventilation was augmented and PETCO 2 decreased, whereas ventilatory oscillations were considerably reduced at exercise in hypoxia. This confirms the blunting of loop gain by ACZ observed in OSA patients [START_REF] Edwards | Acetazolamide improves loop gain but not the other physiological traits causing obstructive sleep apnoea[END_REF]. It has been reported that ACZ could impair respiratory muscle and therefore modify the breathing pattern during exercise [START_REF] Gonzales | Effect of acetazolamide on respiratory muscle fatigue in humans[END_REF]. However, a much lower dose of ACZ was used in the present study and exercise intensity was limited to 30% of MAP. Moreover, as VT and Ti were not modified by ACZ in rest or exercise conditions (Table 2), we therefore assume that ACZ did not impact breathing pattern.

Mechanisms.

These results provide new insights on mechanisms involved in ventilatory oscillations in healthy subjects (Fig. 9). The activity of the respiratory central pattern generator could be modulated by an oscillator, the activity of which would depend on various chemical stimuli. This oscillator would promote the instability of the system. Given that this instability seems to be directly related to the intensity of E and c, it is not surprising to observe the destabilizing effect of exercise, hypoxia and hypercapnia, which are known to increase E and c. ACZ has contrasting effects: it inhibits the effect of hypoxia [START_REF] Ainslie | Breathing and sleep at high altitude[END_REF] and enhances the effect of hypercapnia on ventilation [START_REF] Vovk | Changes in chemoreflex characteristics following acute carbonic anhydrase inhibition in humans at rest[END_REF]. In the present study, ACZ also blunts the relation between ventilatory oscillations and E or c (Fig. 5 and9). Hyperoxia deactivates peripheral chemoreceptors [START_REF] Sinski | Deactivation of carotid body chemoreceptors by hyperoxia decreases blood pressure in hypertensive patients[END_REF], and, to our knowledge, has no action on central chemoreceptors in humans. Although hyperoxia did not significantly decrease E peak power, as we could expect regarding the inhibition of peripheral chemoreceptors, there is a tendency for a decrease in E and SpO 2 peak power at exercise and a significant decrease of PETCO 2 peak power. Therefore, the hyperoxia-induced inhibition of carotid bodies would not be strong enough, alone, to modulate ventilatory oscillations.

CO 2 stimulates central chemoreceptors, but is also, in a lesser extent, a determinant factor in peripheral chemoreflex activation in hypoxia [START_REF] Mohan | The effect of hypoxia on the ventilatory response to carbon dioxide in man[END_REF]. Therefore, the noticeable increase in ventilatory oscillations in hypercapnia could be mainly attributed to the activation of central chemoreceptors, without completely ruling out a potential peripheral involvement of CO 2 in hyperoxia.

Acetazolamide stimulates central chemoreceptors but reduces peripheral chemoreceptors activity [START_REF] Ainslie | Breathing and sleep at high altitude[END_REF]. However, its mechanisms of action have been extensively debated [START_REF] Teppema | Multifaceted clinical effects of acetazolamide: will the underlying mechanisms please stand up?[END_REF]. Putting the decrease in ventilatory oscillations under ACZ in perspective with the increase of these oscillations in hypoxia when peripheral chemoreceptors are stimulated, confirms that peripheral chemoreceptors play a key role in the genesis of ventilatory oscillations. It validates the hypothesis we put forward in our preliminary study showing that oscillations are enhanced in subjects with high HVR [START_REF] Hermand | Periodic breathing in healthy humans at exercise in hypoxia[END_REF]. However, ventilatory oscillations are also greater under a hyperoxic hypercapnic stress, when peripheral chemoreceptors are silenced, thus implying that central chemoreceptors are also a major actor of breathing instability. This suggests that ventilatory oscillations depend on a subtle and synergic action of both central and peripheral chemoreceptors, through arterial O 2 and CO 2 pressure.

Moreover, we observed that, in hypercapnic hyperoxia, PETCO 2 and E exhibited dichotomous oscillations properties: PETCO 2 peak power was paradoxically lower in hypercapnia whereas E peak power was higher. CO 2 stores being close to saturation in hypercapnia, despite a higher ventilation to flush it out, the measured expired CO 2 level is then not subjected to oscillations as observed for E, as a full CO 2 store will damp systemic CO 2 variations.

ACZ augments the controller gain (HCVR) but blunts the enhancing effects of controller gain on ventilatory oscillations (Fig. 7). This paradoxical effect of ACZ, reducing ventilatory oscillations (i.e. loop gain) in spite of the rise of controller gain, means a very significant reduction of plant gain, evoked in Javaheri's work [START_REF] Javaheri | Acetazolamide attenuates hunter-cheyne-stokes breathing but augments the hypercapnic ventilatory response in patients with heart failure[END_REF]. While we were not able to determine the value of the plant gain, we nevertheless observed a noticeable decrease in PETCO 2 under ACZ in all conditions (Fig. 4), therefore inducing a lower CO 2 reserve and a lower plant gain [START_REF] White | Pathogenesis of Obstructive and Central Sleep Apnea[END_REF][START_REF] Javaheri | Acetazolamide attenuates hunter-cheyne-stokes breathing but augments the hypercapnic ventilatory response in patients with heart failure[END_REF].

Mechanisms leading to reduced ventilatory oscillations under ACZ are still debated. This may involve a third arm, the mixing gain, depending, among other factors, on cerebral blood flow which might increase the CO 2 washout of the cerebral spinal fluid and therefore blunt ventilatory oscillations [START_REF] Younes | Chemical control stability in patients with obstructive sleep apnea[END_REF][START_REF] Dempsey | The ventilatory responsiveness to CO(2) below eupnoea as a determinant of ventilatory stability in sleep[END_REF][START_REF] Skow | The effects of head-up and head-down tilt on central respiratory chemoreflex loop gain tested by hyperoxic rebreathing[END_REF]. However, both exercise [START_REF] Ogoh | Cerebral blood flow during exercise: mechanisms of regulation[END_REF] and ACZ [START_REF] Okazawa | Effects of Acetazolamide on Cerebral Blood Flow, Blood Volume, and Oxygen Metabolism: A Positron Emission Tomography Study With Healthy Volunteers[END_REF] are known to increase cerebral blood flow but have opposite effects on breathing instability (present study).

The multivariate analysis showing that the intensity of oscillations is related to E and Ttot, but insignificantly to c, gives a clue to the apparent contradiction between our observations of greater ventilatory oscillations with increasing c (fig. 5) and the current hypothesis of low c-induced periodic breathing in CHF patients [START_REF] Agostoni | Mechanisms of Periodic Breathing During Exercise in Patients With Chronic Heart Failure[END_REF].

The tight relationship between E period and Ttot observed in our previous work is confirmed (Fig. 8): E period is strongly linked to the duration of the respiratory cycle, independently of other cardiorespiratory factors such as E and c. This suggests that Ttot is a determinant factor of the intrinsic oscillator. This correlation also pointed out a theoretical limit of E oscillation period: when Ttot tends towards zero (breathing frequency tending to infinity), E oscillation period tends to approximately 7 seconds. This tight relationship between E period and Ttot also suggests the involvement of an internal oscillator that would modulate the activity of the central pattern generator, responsible for the generation of respiratory rhythm in mammals [START_REF] Forster | Contributions of the pre-Bötzinger complex and the Kölliker-fuse nuclei to respiratory rhythm and pattern generation in awake and sleeping goats[END_REF].

In conclusion, these findings confirm the existence of a mechanism that modulates ventilation and induces breathing instability, especially during exercise in hypoxic and hypercapnic conditions. This instability, unraveled in healthy awake subjects after a brisk challenge, depends on a complex regulation system involving O 2 and CO 2 sensing where both peripheral (high HVR and effect of ACZ) and central (hypercapnia and tight relationship between period and Ttot) chemoreflex seem to play a key role. We expect this study to contribute to the knowledge of factors involved in sleep apneas of central and obstructive origins. Exercise vs Rest: #, P < 0.05; ##, P < 0.01; ###, P < 0.001. Hypercapnic hyperoxia or hyperoxia or hypoxia vs normoxia: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Hypercapnic hyperoxia vs hyperoxia: +, P < 0.05, ++, P < 0.01, +++, P < 0.001. ACZ vs placebo: †, P < 0.05, † †, P < 0.01, † † †, P < 0.001.

CAPTIONS

Figure 1 .

 1 Figure 1. Breath-by-breath ventilation recordings in subject n° 4, under placebo (upper panel) and acetazolamide (lower panel) treatment. Inlets: ventilatory response to CO 2 (upper right: placebo, lower right: acetazolamide).

Figure 2 .

 2 Figure 2. Power spectral density of the ventilation signal at exercise. Left panel: normoxia, hyperoxia and hypercapnic hyperoxia (subject n°4).Right panel: placebo and ACZ (subject n°4) in hypoxia.

Figure 3 .

 3 Figure3. Mean (± SD) values of E, SpO 2 and PETCO 2 and corresponding periods and peak powers in normoxia, hyperoxia and hypercapnic hyperoxia. Exercise vs Rest: #, P < 0.05; ##, P < 0.01; ###, P < 0.001. Hypercapnic hyperoxia or hyperoxia vs normoxia: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Hypercapnic hyperoxia vs hyperoxia: +, P < 0.05, ++, P < 0.01, +++, P < 0.001.

Figure 4 .

 4 Figure 4. Mean (± SD) values of E, SpO 2 and PETCO 2 and corresponding periods and peak powers in normoxia (Nx) and hypoxia (Hx), under placebo or acetazolamide treatment. Exercise vs Rest: #, P < 0.05; ##, P < 0.01; ###, P < 0.001. Hypoxia vs normoxia: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Acetazolamide vs placebo: †, P < 0.05, † †, P < 0.01, † † †, P < 0.001.

Figure 5 .

 5 Figure 5. Ventilation period (upper panels) and peak power (lower panels) as a function of cardiac output ( c). Left panels: ACZ and placebo. Right panels: normoxia, hyperoxia and hypercapnic hyperoxia. Rest and exercise values have been pooled in all conditions. Normoxia and hypoxia values have been pooled in the ACZ vs placebo study. Slopes are significantly different between placebo and ACZ for the E peak power vs c correlation (P < 0.05).

Figure 6 .

 6 Figure 6. Ventilation period (upper panels) and peak power (lower panels) as a function of ventilation ( E). Left panels: ACZ and placebo. Right panels: normoxia, hyperoxia and hypercapnic hyperoxia. Rest and exercise values have been pooled in all conditions. Normoxia and hypoxia values have been pooled in the ACZ vs placebo study. Slopes are significantly different between placebo and ACZ for the E peak power vs c correlation (P < 0.05).

Figure 7 .

 7 Figure 7. E peak power as a function of HCVR for each subject under placebo and ACZ (large symbols: mean values ± SD, black dot, Placebo;white dot, ACZ). ACZ increases HCVR and reduces E peak power, but these relationships do not reach significance (Placebo: r² = 0.123, P = 0.26; ACZ: r² = 0.273, P = 0.08). However, it is clear that ACZ blunts the effect of HCVR on ventilatory oscillations (arrow).

Figure 8 .

 8 Figure 8. Linear regressions between E period and total respiratory cycle time (Ttot) in the ACZ study. All values (rest / exercise, normoxia / hypoxia, placebo / ACZ) have been pooled.

Figure 9 .

 9 Figure9. Schematic diagram of potential mechanisms involved in the genesis of ventilatory oscillations in normal subjects at exercise. Breathing instability is directly related to the intensity of E and c. Exercise, hypoxia and hypercapnia increase E and c, and therefore increase ventilatory oscillations. ACZ inhibits the effect of hypoxia and enhances the effect of hypercapnia on ventilation, and blunts the relation between E or c, and ventilatory oscillations. The effects of ACZ on this regulation system are still debated (see discussion).

Table 2 .

 2 Mean values of cardiorespiratory parameters for the hyperoxia (Hox), hypercapnic hyperoxia (Hox Hcap) and acetazolamide (ACZ) studies. Rest Exer. Rest Exer. Rest Exer. Rest Exer. Rest Exer. Rest Exer. Hx: hypoxia; Exer.: Exercise. E: minute ventilation; SpO 2 : pulse O 2 saturation; PETCO 2 : end-tidal PCO 2 ; c: cardiac output; Ti: inspiration time; Ttot: total respiration cycle time; VT: tidal volume; HCVR: hypercapnic ventilatory response. Mean ± SD.

			E (L.min -1 )	SpO 2 (%)	PETCO 2 (mmHg)	c (L.min -1 )	Ti (s)	Ttot (s)	VT (L)	HCVR (L.min -1 .
	Rest 11.4 ± 1.9 Exer. Study Nx 33.9 ± 4.2 ### 12.5 32.9 Hox ± 2.5 ± 4.7 Hyperoxia ###	96.2 ± 1.2 98.0 ± 0.8	96.0 ± 1.4 98.0 ± 0.7	38.7 ± 1.7 36.8 ± 2.1	45.8 ± 2.3 ### 45.4 ± 3.4 ###	5.53 ± 1.07 5.34 ± 0.79	10.33 ± 1.81 ### 9.78 ± 1.42 ###	1.84 ± 0.62 1.42 ± 0.37	1.40 ± 0.43 1.34 ± 0.44	5.04 ± 1.56 4.75 ± 1.65	3.16 ± 0.92 ## 3.28 ± 0.94 ##	0.92 ± 0.20 0.93 ± 0.21	1.74 ± 0.41 ### 1.73 ± 0.34 ###	mmHg -1 )
					***	***	***			*	**
			11.9	34.2	96.6	96.3	38.9	45.7	5.66	10.22	1.75	1.39	4.65	3.08	0.90	1.70	2.78 ± 0.86
		Nx	± 2.0	± 5.6	± 1.4	± 1.6	± 2.2	± 2.7	± 1.14	± 1.47	± 0.51	± 0.43	± 1.38	± 0.90	± 0.24	± 0.38
	Study Hox Hcap	Hypercapnic hyperoxia	35.5 ± 8.5 ***	### 72.9 ±13.9 ### ***	97.9 ± 0.5 **	# 97.9 ± 0.5 **	55.4 ± 2.7 ***	### 66.3 ± 3.4 ### ***	5.25 ± 0.93	### 10.76 ± 1.62 ### *	1.37 ± 0.29 **	## 1.04 ± 0.23 ### ***	3.36 ± 0.79 ***	### 2.29 ± 0.50 ### ***	1.91 ± 0.42 ***	### 2.59 ± 0.37 ### ***
			+++	+++			+++	+++		+		++	++	+++	+++	+++
			11.6	31.5	96.3	96.1	40.5	46.9	6.18	11.19	1.88	1.54	4.58	3.34	0.87	1.71
		Nx	± 1.2	± 4.4	± 1.6	± 1.3	± 3.1	± 3.1	± 1.06	± 1.60	± 0.81	± 0.54	± 1.54	± 0.99	± 0.28	± 0.38
				###				###		###		#		###		###
		Placebo	12,3	33.9	91.8	86.8	40.2	44.8	6.93	12.45	1.77	1.46	4.23	3.12	0.85	1.72	2.46 ± 0.53
		Hx	± 1.5	± 4.2 ###	± 1.9	± 1.5 ###	± 2.9	± 1.8 ###	± 1.17	± 2.35 ###	± 0.61	± 0.53	± 1.07	± 0.90 ###	± 0.21	± 0.38 ###
				*	***	***		*	*	*
	Study ACZ	Nx	12.3 ± 1.8	35.3 ± 4.7 ###	96.3 ± 1.4	96.0 ± 1.1	34.4 ± 3.5	40.3 ±4.6 ###	6.93 ± 1.17	12.45 ± 2.35 ###	1.88 ± 0.61	1.46 ± 0.53 #	4.73 ± 2.20	3.19 ± 0.91 ##	0.93 ± 0.30	1.82 ± 0.41 ###
		ACZ	12.8 ± 1.5	† † 36.3 ± 5.5	92.5 ± 1.3	88.3 ± 2.5	† † † 34.1 ± 3.8	† † † 37.9 ± 4.5	6.52 ± 1.42	12.08 ± 2.36	1.89 ± 0.86	1.42 ± 0.44	4.64 ± 1.58	3.19 ± 0.82	0.96 ± 0.27	1.87 ± 0.34	3.78 ± 1.67 † †
		Hx		###		###		###		###		##		###		###
					***	***		**	**	***
						†	† † †	† † †
	Nx: normoxia;