Intercostal Muscles Oxygenation and Breathing Pattern during Exercise in Competitive Marathon Runners

ed to evaluate the association between the changes in ventilatory vari- ables (tidal volume (Vt), respiratory rate (RR) and lung ventilation ( . V E)) and deoxygenation of m.intescostales(DSmO 2-m.intercostales) during a maximal incremental exercise in 19 male high-level competitive marathon runners. The ventilatory variables and oxygen consumption ( . V O 2) were recorded breath-by-breath by exhaled gas analysis. A near-infrared spectroscopy device (MOXY ® ) located in the right-hemithorax allowed the recording of

respiratory rate (RR) and lung ventilation ( . V E)) and deoxygenation of m.intescostales(DSmO 2-m.intercostales) during a maximal incremental exercise in 19 male high-level competitive marathon runners. The ventilatory variables and oxygen consumption ( . V O 2) were recorded breath-by-breath by exhaled gas analysis. A near-infrared spectroscopy device (MOXY ® ) located in the right-hemithorax allowed the recording of SmO 2-m.intercostales. To explore changes in oxygen levels in muscles with high demand during exercise, a second MOXY ® records SmO 2-m.vastus laterallis. The triphasic model of exercise intensity was used for evaluating changes in SmO 2in both muscle groups. We found thatDSmO 2-m.intercostalescorrelated with . V O 2-peak(r = 0.65;p =0.002) and the increase of . V E (r = 0.78;p= 0.001), RR (r = 0.54;p =0.001), but not Vt (p =0.210). The interaction of factors (muscles exercise-phases) in SmO 2expressed as an arbitrary unit (a.u) was signi cant (p= 0.005). At VT1 there was no difference (p= 0.177), but SmO 2-m.intercostaleswas higher at VT2 (p< 0.001) and . V O 2-peak(p< 0.001). In high-level competitive marathon runners, them.intercostalesdeoxygenation during incremental exercise is directly associated with the aerobic capacity and increased lung ventilation and respiratory rate, but not tidal volume. Moreover, it shows less deoxygenation thanm.vastus laterallisat intensities above the aerobic ventilatory threshold. Keywords:exercise; runners; near-infrared spectroscopy; respiratory muscles; respiration 1. Introduction During the race, high-level competitive marathon runners use about 75% of the maxi- mum oxygen consumption ( . V O2-m¡x.) and keep lung ventilation ( . V E) near 80 L min 1 [1]. This requires the respiratory muscles recruitment (e.g.,m.diaphragma, m.intercostales, m.serratus (posteriorandanterior)), leading to an increase in . V O2distributed to these muscles ( . V O2-RM), which in trained men has been proven to reach up to 10% [2]. This elevated energy cost of breathing (COB) requires higher local nutrients and oxygen supply, an aspect that has been reported could diminish blood ow to locomotor muscles, causing an intolerance to effort and early end of exercise (metabolic re ex) [2–4]. Different factors are implicated in a high COB by exercise; one of them is

reach up to 10% [2]. This elevated energy cost of breathing (COB) requires higher local nutrients and oxygen supply, an aspect that has been reported could diminish blood ow to locomotor muscles, causing an intolerance to effort and early end of exercise (metabolic re ex) [2–4]. Different factors are implicated in a high COB by exercise; one of them is the breathing pattern adopted during physical exertion, understood as the relative contribution of Vt and RR to the increase in . V E. Few studies have evaluated how the relative changes in these Int. J. Environ. Res. Public Health2021,18, 8287.

Int. J. Environ. Res. Public Health2021,18, 8287 2 of 12 ventilatory variables during exercise could affect the oxygenation of respiratory muscles, a relevant aspect because of the wide variability observed according to the type (running or cycling), duration (short or long-exercise) and intensity (steady-state or incremental) of the physical exercise [5–7]. Evaluating the respiratory muscle's oxygenation level is dif cult due to the complex anatomical arrangement and the considerable variation in muscular recruitment with vary- ing degrees of ventilation. Nevertheless, a new non-invasive method has been proposed to determine the ratio between the supply-to-consumption of oxygen as re ected by muscle saturation (SmO2) evaluated in them.intercostales, which has great importance in high intensities of exercise; this can be determined by continuous near-infrared spectroscopy (NIRS, 630–850 nm) that measures the changes in oxygenated hemoglobin (O2Hb) and myoglobin (mHb) at a microvascular level [8–10]. In sports science, the changes of SmO2 (deoxygenation,DSmO2) during physical effort have been extensively studied in locomotor muscles, with far less attention to respiratory muscles [11–13]. To our knowledge, in high- level competitive marathon runners, it is unexplored how the breathing pattern adopted during physical exertion is associated with SmO2-m.intercostales, a novel aspect requiring attention due to the possible respiratory limitations to exercise that could be modulated by respiratory muscle training. Thus, the main goal of this study was to evaluate the association between breathing pattern and deoxygenation of them.intercostales(DSmO2-m.intercostales) during an incre- mental physical effort test in high-level competitive marathon runners. Secondarily, we aimed to determine the association between oxygen consumption and the relative increase of each ventilatory variable (lung ventilation ( . V E), respiratory rate (RR) and tidal volume (Vt)),DSmO2-m.intercostalesandDSmO2-m.vastus laterallis. Furthermore, we explore the possible limitation to exercise by comparing the changes betweenDSmO2-m.intercostales andDSmO2-m.vastus laterallisduring exercise protocol according to the triphasic exercise intensity model determined by the ventilatory thresholds (ventilatory threshold 1 (VT1) and ventilatory threshold 2 (VT2)). 2. Materials and Methods 2.1. Design of Study and Participants A cross-sectional observational study that assessed 19 healthy male high-level com- petitive marathon runners (age 22.9 1.9 years; height 173.1 4.2 cm; weight66.5 6.7 kg ;

laterallisduring exercise protocol according to the triphasic exercise intensity model determined by the ventilatory thresholds (ventilatory threshold 1 (VT1) and ventilatory threshold 2 (VT2)). 2. Materials and Methods 2.1. Design of Study and Participants A cross-sectional observational study that assessed 19 healthy male high-level com- petitive marathon runners (age 22.9 1.9 years; height 173.1 4.2 cm; weight66.5 6.7 kg ; body mass index (BMI) 22.2 2.8 kg m 2 ; peak oxygen consumption ( . V O 2-peak) 62.3 4.6 mL min 1 kg 1 ; training volume 125.1 31.4 km week 1 , best personal time obtained in a marathon (during the last 3 years) 157 23 min) without a history of systemic problems, such as respiratory, cardiovascular, metabolic, musculoskeletal or neoplastic diseases, or any infectious or in ammatory process, for at least two weeks before measure- ments (recruitment by convenience). The participants did not consume drugs, antioxidants or any nutritional support. The sample size calculation was done by the software G*Power ® 3.1 (Heinrich-Heine-University, Dusseldorf, Germany) using previous data concerning the association found between SmO2-m.intercostalesand . V O2in runners with similar charac- teristics to participants in this study (r = 0.64;p= 0.001), considering a signi cance level of 5%, power of 80% and a two-tail test, plus 10% of data losing. All the participants were informed of the purpose, protocol and procedures before informed consent was obtained from them. This study was approved by the ethics committee of the Ponti cia Universidad Catâlica de Chile (Institutional Review Board, protocol number 180305007, date of approval:5 June 2018). The study was carried out according to the Declaration of Helsinki for human experimentation. 2.2. Procedures The evaluations were done in the Exercise Physiology Laboratory of the Ponti cia Universidad Catâlica de Chile under controlled environmental conditions (room tempera- ture, 20 2 C; relative humidity, 40 2%) and xed schedule (9:00 to 14:00 h). All the

Int. J. Environ. Res. Public Health2021,18, 8287 3 of 12 participants were instructed to avoid physical activity 24 h before the day of measurement and not to engage in alcohol, caffeine or other stimulants and food intake for at least three hours before the tests. 2.3. Ergospirometry The aerobic capacity was assessed by determining the . V O 2-peak. This test was per- formed on a treadmill ergometer (HP Cosmos, Traunstein, Germany) in an incremental ex- ercise until voluntary exhaustion, despite verbal stimuli (respiratory quotient,1.20 0.05 ). The exercise protocol consisted of a 3-min rest, 5-min warm-up (8 km h 1 ) and subsequent increase of 2 km h 1 every 150 s, until all criteria for stopping the test were met. The treadmill slope throughout the test was xed at 2%. Heart rate, ngertip oxygen saturation and ventilatory variables ( . V E, RR, Vt) were continuously recorded. Ventilatory and spired gas composition data ( . V O2, . V CO2, . V E, RR and Vt) werebreath-by-breathobtained using an ergospirometer (MasterScreen CPX, Jaeger™, Traunstein, Germany) and expressed under standard temperature pressure dry air (STPD). 2.4. Triphasic Model of Exercise Intensity Determined by Ventilatory Thresholds To analyze the changes in the variables measured (SmO2, . V E, RR and Vt) according to the triphasic model, the ventilatory 1 (VT1) and 2 (VT2) and . V O 2-peak.were determined by two experienced and blinded evaluators using the visual method [14]. The VT1 was the initial departure from . V E linearity, the beginning of a systematic increase in the ventilatory equivalent of O2( . V E . VCO2 1) and the end-tidal pressure value of O2(Pet-O2). The VT2 was the secondary increase in . V E, . V E . VO2 1, a marked increase in the ventilatory equiva- lent of CO2( . V E . VCO2 1) and a decrease in the pressure value at the end of CO2expiration (Pet-CO2) during exercise, above VT1 [15]. In case of a discrepancy between the two evaluators, the opinion of an experienced third blinded evaluator was possible, accepting as the de nitive

VO2 1, a marked increase in the ventilatory equiva- lent of CO2( . V E . VCO2 1) and a decrease in the pressure value at the end of CO2expiration (Pet-CO2) during exercise, above VT1 [15]. In case of a discrepancy between the two evaluators, the opinion of an experienced third blinded evaluator was possible, accepting as the de nitive criterion that point at which at least two evaluators agreed [16]. However, none of the cases revealed differences between the two evaluators in this study. Regarding . V O 2-peak, the highest value of the last 30 s obtained during the incremental maximum effort test was considered, based on established criteria for the determination [17]. 2.5. Measurement of SmO2 During the protocol, muscle oxygen saturation (SmO2) was non-invasively evaluated using the monitor MOXY ® (Fortiori Design LLC, Hutchinson, MN, USA), which emits light waves close to the infrared range (near-infrared spectroscopy, NIRS (630–850 nm)) from diodes to the surrounding tissue and records total hemoglobin (THb) and myoglobin (mHb) concentrations at the microvascular level [9]. This device records the amount of light that returns to two detectors placed 12.5 and 25.0 mm from the source, thus locally recording SmO2through the interpretation of THb and mHb levels [18]. The SmO2data were recorded using the PeriPedal ® software (PeriPedal, Indianapolis, IN, USA) with a sampling frequency of 2 Hz [19]. The light penetration depth is half of the distance between the emitting source and the detector ( 12 mm) [19]. To determine the SmO2-m.intercostales, a MOXY ® device was located in the seventh intercostal space of the anterior axillary line of the right hemithorax [20]. A second MOXY ® device was placed 5 cm lateral to the midpoint of the imaginary line between the upper edge of the patella and the greater trochanter of the right femur [20]. The position of both MOXY ® devices was attached to the skin using the material suggested by the manufacturer, in addition to extra xation with a cohesive band on the surroundings of the measurement zone, avoiding excessive compression that could alter the SmO2record (similar to

the upper edge of the patella and the greater trochanter of the right femur [20]. The position of both MOXY ® devices was attached to the skin using the material suggested by the manufacturer, in addition to extra xation with a cohesive band on the surroundings of the measurement zone, avoiding excessive compression that could alter the SmO2record (similar to that used in our previous study) [20]. Figure illustrates the positions of the NIRS devices.

Int. J. Environ. Res. Public Health2021,18, 8287 4 of 12Int. J. Environ. Res. Public Health 2021, 18, x 4 of 12 with a cohesive band on the surroundings of the measurement zone, avoiding excessive compression that could alter the SmO 2 record (similar to that used in our previous study) [20]. Figure 1 illustrates the positions of the NIRS devices. Figure 1. Placement of the muscle oximetry NIRS devices in the thorax (m.intercostales) and leg (m.vastus laterallis). 2.6. Breathing Pattern To facilitate the interpretation of the breathing pattern of each participant (corresponding to changes in ventilatory variables (V 6E, RR and Vt) during the exercise protocol), a standardization of the obtained values was performed by transferring them to arbitrary relative units considering the number of times that each variable increased from rest (taken as the unit (1.0) reference value) to V 6O 2-peak phases (n° times = (ventilatory variable value at V 6O 2-peak phase–ventilatory variable value at rest phase) ventilatory variable value at rest phase −1 ). 2.7. Data Analysis The registration of the variables monitored by using the ergoespirometer and the SmO 2 devices was manually synchronized by two operators. Thus, each participant had an initial record of 90 s in the bipedal position, followed by 180 s corresponding to the resting phase. The values of ventilatory variables (V 6E, RR and Vt) and SmO 2 considered for calculations correspond to the average of the last 30 s of each phase recordings. The maximal deoxygenation (Δ) corresponds to (SmO 2 at rest-SmO2 at V 6O 2-peak) · (SmO2 at V 6O 2-peak) −1 (%). To compare the SmO2 in the muscle groups assessed during the exercise protocol, the data were expressed as an arbitrary unit (a.u), where the value of SmO 2 obtained at the rest phase was considered the maximum or 1, values obtained in other phases were expressed as a ratio (e.g., SmO 2-m.intercostales at VT1 (a.u) = SmO 2 at VT1 (%) · SmO 2 at rest −1 (%)). These methods of expressing muscle oxygenation are used because SmO 2 values present variability between muscle

value of SmO 2 obtained at the rest phase was considered the maximum or 1, values obtained in other phases were expressed as a ratio (e.g., SmO 2-m.intercostales at VT1 (a.u) = SmO 2 at VT1 (%) · SmO 2 at rest −1 (%)). These methods of expressing muscle oxygenation are used because SmO 2 values present variability between muscle groups mainly since their analysis includes both hemoglobin and myoglobin quantification (myoglobin level has been reported to correspond to between 30% to 50% of the signal at different muscles) [21], Figure 1. Placement of the muscle oximetry NIRS devices in the thorax (m.intercostales) and leg (m.vastus laterallis). 2.6. Breathing Pattern To facilitate the interpretation of the breathing pattern of each participant (correspond- ing to changes in ventilatory variables ( . V E, RR and Vt) during the exercise protocol), a standardization of the obtained values was performed by transferring them to arbitrary relative units considering the number of times that each variable increased from rest (taken as the unit (1.0) reference value) to . V O 2-peakphases (n times = (ventilatory variable value at . V O 2-peakphase–ventilatory variable value at rest phase) ventilatory variable value at rest phase 1 ). 2.7. Data Analysis The registration of the variables monitored by using the ergoespirometer and the SmO2devices was manually synchronized by two operators. Thus, each participant had an initial record of 90 s in the bipedal position, followed by 180 s corresponding to the resting phase. The values of ventilatory variables ( . V E, RR and Vt) and SmO2considered for calculations correspond to the average of the last 30 s of each phase recordings. The maximal deoxygenation (D) corresponds to (SmO2at rest-SmO2at . V O 2-peak) (SmO2 at . V O 2-peak) 1 (%). To compare the SmO2in the muscle groups assessed during the exercise protocol, the data were expressed as an arbitrary unit (a.u), where the value of SmO2obtained at the rest phase was considered the maximum or 1, values obtained in other phases were expressed as a ratio (e.g., SmO2-m.intercostalesat VT1 (a.u) = SmO2 at VT1 (%)

V O 2-peak) 1 (%). To compare the SmO2in the muscle groups assessed during the exercise protocol, the data were expressed as an arbitrary unit (a.u), where the value of SmO2obtained at the rest phase was considered the maximum or 1, values obtained in other phases were expressed as a ratio (e.g., SmO2-m.intercostalesat VT1 (a.u) = SmO2 at VT1 (%) SmO2at rest 1 (%)). These methods of expressing muscle oxygenation are used because SmO2values present variability between muscle groups mainly since their analysis includes both hemoglobin and myoglobin quanti cation (myoglobin level has been

Int. J. Environ. Res. Public Health2021,18, 8287 5 of 12 reported to correspond to between 30% to 50% of the signal at different muscles) [21], whose concentration is variable depending on the morphofunctional and structural characteristics (such as capillarization and skeletal muscle ber type composition) of the different muscle groups in each athlete. 2.8. Statistical Analysis Data normality was checked using the Shapiro–Wilk test. The association of . V O 2-peak, the relative increase of . V E, RR, Vt (breathing pattern) andDSmO2atm.intercostalesand m.vastus lateralliswere assessed using the Pearson correlation coef cient. A two-way ANOVA test allowed the comparison of the SmO2(a.u) levels between muscles assessed among different exercise protocol phases (VT1, VT2 and . V O 2-peak) when the interaction of the factors (muscles x exercise phases) was signi cant (p< 0.05). Subsequent multiple comparisons were analyzed using the Tukey post-hoc test. The level of signi cance for all analyses wasp< 0.05. The statistical software used was GraphPad Prism 7.0 (San Diego, CA, USA). 3. Results The values of SmO2(%) (m.intercostalesandm.vastus laterallis), total hemoglobin (THb) and ventilatory variables ( . VE, RR and Vt) are shown in Table. Table 1. Oxygen consumption, muscle oxygen saturation, total hemoglobin and ventilatory variables in the different phases of exercise (n = 19). Phases Variables Rest VT1 VT2 . VO 2-peak . VO 2(mL kg 1 min 1 ) 6.35 0.75 31.30 4.15 * 52.01 3.91 * 62.32 4.65 * SmO 2 m.intercostales(%) 74.6 10.7 65.1 12.6 * 52.9 11.4 * 35.2 12.8 * DSmO 2 m.intercostales(%) - - - 53.4 13.9 SmO 2 m.vastus lateralis(%) 63.2 10.9 48.7 9.6 * 36.1 10.2 * 17.1 7.5 * DSmO 2 m.vastus lateralis(%) - - - 72.4 13.5 THbm.intercostales(g dL 1 ) 12.4 0.5 12.4 0.5 12.4 0.5 12.3 0.6 THbm.vastus laterallis(g dL 1 ) 12.6 0.4 12.5 0.5 12.4 0.5 12.6 0.6 . VE (L min 1 ) 10.4 2.5 43.2 10.2 * 99.5 18.1 * 158.8 18.9 * RR (bpm) 13.1 1.7 25.3 6.7 * 40.7 7.6 * 59.3 8.4 * Vt (L) 0.81 0.20 1.75 0.41 * 2.47 0.43 * 2.71 0.30

This research investigates the relationship between breathing patterns and muscle oxygenation in marathon runners during exercise.