PUBLIC UNIVERSITY OF NAVARRE DEPARTMENT OF HEALTH SCIENCES
DOCTORATE IN HEALTH SCIENCE
ROLE OF THE VENTILATORY CHEMOREFLEX AS RESPIRATORY ADAPTATION MECHANISM IN COMPETITIVE LEVEL SWIMMERS
Doctoral Thesis
Author: Alexis Arce Alvarez
Supervisor: Dr. Mikel Izquierdo Redín Co-supervisor: Dr. David Andrade Andrade
June 2022
https://doi.org/10.48035/Tesis/2454/44803 © Todos los derechos reservados
ACKNOWLEDGMENTS
A Francisca, mi ángel terrenal y Rafael, mi ángel celestial.
A mi amigo, mentor y guía David Andrade Andrade.
A Mikel Izquierdo Redín, por su confianza.
List of Publications
This doctoral thesis is a compendium of the following publications:
Research I. Andrade DC*, Arce-Alvarez A*, Parada F, Uribe S, Gordillo P, Dupre A, Ojeda C, Palumbo F, Castro G, Vasquez-Muñoz M, Del Rio R, Ramirez-Campillo R, Izquierdo M.
Acute effects of high-intensity interval training session and endurance exercise on pulmonary function and cardiorespiratory coupling. (2020). Physiological Reports, 8:e14455.
Research II. Beltrán AR*, Arce-Álvarez A*, Ramírez-Campillo R, Vásquez-Muñoz M, von Igel M, Ramírez MA, Del Rio R, Andrade DC. (2020). Baroreflex Modulation During Acute High-Altitude Exposure in Rats. Frontiers in Physiology, 11:1049.
Research III. Arce-Álvarez A, Veliz C, Vásquez-Muñoz M, von Igel M, Alvares C, Ramirez-Campillo R, Izquierdo M, Millet GP, Del Rio R, Andrade DC. (2021). Hypoxic Respiratory Chemoreflex Control in Young Trained Swimmers. Frontiers in Physiology, 12:632603.
Research IV. Alexis Arce-Álvarez; Camila Salazar; Carlos Cornejo; Valeria Paez; Manuel Vásquez-Muñoz; Katherine Stillner-Vilches; Catherine R. Jara; Rodrigo Ramirez-Campillo;
Mikel Izquierdo; David C. Andrade. Chemoreflex control as the cornerstone in immersion water sports: possible role on breath-hold. Front Physiol. 2022 Jun 6;13:894921.
RESUMEN
La presente tesis doctoral está basada en la publicación de 4 estudios que tienen como objetivo analizar el rol del quimiorreflejo periférico sobre el tiempo de apnea en atletas nadadores de nivel competitivo para lo cual se realizaron experimentos en población que va desde modelos animales, humanos físicamente activos y deportistas nadadores de nivel competitivo.
El objetivo del estudio 1 fue determinar los efectos agudos del ejercicio de entrenamiento interválico de alta intensidad (HIIT) y el ejercicio de endurance (EE) sobre la función pulmonar, el balance simpático/parasimpático y el acoplamiento cardiorrespiratorio (CRC) en sujetos sanos. Participaron ocho sujetos físicamente activos (corredores recreativos; cuatro hombres y cuatro mujeres): altura:
1,7 ± 0,1 m; masa corporal: 63,3 ± 5,7 kg; índice de masa corporal: 22,0 ± 2,4 kg/m2; edad: 23,9 ± 3,1 años). Usando un diseño cruzado de medidas repetidas los participantes fueron expuestos a EE (20 min al 80% de la frecuencia cardíaca (FC) máxima), HIIT (1 min de ejercicio al 90% de la FC máxima por 1 min de descanso, 10 veces) o condición de control (reposo). Se evaluó función pulmonar mediante espirometría (VC, PEF, PIF, FEV1, FEV1/VC, FEF 25, FEF 50, and FEF75), Electrocardiograma (ECG), presión arterial sistólica (SBP), diastólica (DBP) y presión arterial media (MAPB), saturación de oxígeno, CRC y la variabilidad de la frecuencia cardíaca (LFHRV, HFHRV, LF/HFHRV) antes y después de las intervenciones. Basalmente no hubo diferencias significativas entre EE, HIIT y control en los parámetros cardiovasculares y respiratorios. El EE y el HIIT aumentaron significativamente la SBP, la MABP y la FC (p < 0,05; pre v/s post). El ejercicio de resistencia y las sesiones de HIIT no indujeron cambios en FEV1, FEV1/VC, FEF 25, FEF o FEF 75 (p>0,05; pre vs post). Además, no se encontraron diferencias significativas en función pulmonar entre EE, HIIT y control (pre vs post). Una sesión aislada de EE no logró inducir cambios significativos en la relación LFHRV, HFHRV y LF/HFHRV (p<0,05; pre vs. post) (Figura 3). Sin embargo, la potencia espectral total se redujo significativamente después de la EE (p<0,05; pre vs. post). Por el contrario, HIIT induce cambios significativos en los datos espectrales de HRV. De hecho, LFHRV aumentó mientras que HFHRV se redujo con HIIT (todos p<0,05; pre vs post). En consecuencia, la relación LF/HFHRV
aumentó después del protocolo HIIT (p<0,05; pre vs. post). Además, la potencia espectral total de HRV se redujo con HIIT. El ejercicio de resistencia y el HIIT mostraron una disminución significativa en las variables del dominio del tiempo (SDNN; RMSSD, NN50, pNN50, p<.05; pre vs. post).
Además, SD1 y SD2 se redujeron significativamente después de EE y HIIT. El análisis de direccionalidad mostró que el acoplamiento entre la respiración y la frecuencia cardíaca (B → H) fue superior al que va de la frecuencia cardíaca a la respiración (H → B) en condiciones de control. El acoplamiento B → H pero no H → B aumentó significativamente después de EE (0,14 ± 0,03 vs 0,16
± 0,03 Hz, pre vs. post, respectivamente, p<0,05). No se encontró efecto del HIIT en el acoplamiento B → H ni en el acoplamiento H → B. Los principales hallazgos fueron: (a) ni EE ni HIIT cambian la función pulmonar; (b) HIIT y no EE produjeron cambios agudos de HRV evidenciados por una disminución de HFHRV y un aumento de LFHRV después del ejercicio; (c) HIIT pero no EE tiene un efecto perjudicial sobre el ajuste cardiovascular autonómico normal a un challenge de inclinación; y (d) EE pero no HIIT indujo un aumento agudo en CRC. En general, nuestros hallazgos sugieren que las respuestas cardiorrespiratorias agudas a EE y HIIT pueden diferir entre estas dos modalidades de ejercicio, lo que podría tener algunas implicaciones para las adaptaciones crónicas al ejercicio.
El objetivo del estudio 2 fue determinar el efecto de la exposición aguda a gran altitud sobre la modulación simpática/parasimpática y del control barorreflejo (BR) en ratas normales. Se utilizaron 12 ratas Sprague Dawley macho se asignaron aleatoriamente a grupos de nivel del mar (n = 7) y de gran altitud (n = 5) (3270 m sobre el nivel del mar). Las ratas fueron distribuidas aleatoriamente en grupo Nivel del Mar (n = 7) y grupo Gran Altitud (n = 5). Las ratas del nivel del mar fueron sometidas a cirugía de cateterismo y seles realizó un registro basal de presión arterial (BP) durante una hora.
Posteriormente, el experimento BR se realizó de la siguiente manera: se inyectaron 8 bolos de fenilefrina para aumentar la PA (i.v.) y después de 30 min de recuperación, se inyectaron 8 bolos de nitroprusiato de sodio para disminuir la PA (i.v.). La segunda serie de ratas (grupo Gran Altitud) ascendió a 3.270 m sobre el nivel del mar (Caspana, Antofagasta, Chile) en un laboratorio móvil y luego de 24 h se realizó la cirugía de cateterismo. Similar a la primera serie de animales (grupo Sea- Level), 8h después del procedimiento quirúrgico, se realizaron registros basales de BP (1 hora) y el experimento BR. Se midió presión arterial sistólica (SBP), presión arterial diastólica (DBP) presión arterial media (MABP), presión de pulso (PP), frecuencia cardiaca (HR). Se estimó el control autonómico usando una medición indirecta mediante el cálculo de variabilidad del ritmo cardiaco (HRV). Basalmente no hubo diferencias significativas entre la exposición al nivel del mar y a gran altitud en el peso corporal DBP, SBP, MABP, PP y HR. Después de la exposición aguda a gran altitud, las ratas mostraron un aumento del impulso simpático y una disminución de la modulación parasimpática del corazón. El componente LFHRV aumentó significativamente (p< 0,05) desde el nivel del mar (42,12 ± 7,44 nu) hasta gran altitud (60,55 ± 4,47 nu), mientras que el componente HFHRV se redujo significativamente (p< 0,05) desde el nivel del mar (39,37 ± 4,44 nu) hasta gran altitud (57,82
± 7,43 nu). Consecuentemente a relación LF/HFHRV aumentó significativamente (p<0,05) a gran altitud en comparación con el nivel del mar (1,66 ± 0,27 frente a 0,85 ± 0,23, respectivamente).
Después de la exposición aguda a gran altitud y después de la administración de Phe, las respuestas bradicárdicas disminuyeron significativamente (p<0,05). La curva sigmoidal del análisis BR mostró una bradicardia vagal BR disminuida. Además, la curvatura (0,04 ± 0,01 frente a 0,07 ± 0,01 mmHg/latidos/min) y la bradicardia máxima (50,70 ± 0,28 frente a 58,01 ± 0,81 latidos/min) se redujo significativamente (p<0,05) después de exposición aguda a gran altitud en comparación con el nivel del mar. La respuesta taquicárdica máxima a SNP, rango, pendiente, punto medio de BP, meseta inferior y meseta superior del análisis de BR no fueron significativamente diferentes entre los grupos a nivel del mar y a gran altitud. Los principales hallazgos del presente estudio fueron: (i) después de una exposición aguda a gran altitud hay un deterioro del control autonómico cardíaco; (ii) la exposición a Gran Altitud produce un deterioro del control barorreflejo cardiaco en ratas normales; y (iii) hay una activación reducida del impulso parasimpático durante la exposición aguda a gran altitud en ratas normales. Los presentes resultados sugieren que la exposición aguda a grandes altitudes produce un deterioro del control autonómico y un deterioro de la función barorrefleja, caracterizado principalmente por el descenso en la actividad parasimpática después de 24 h de exposición a grandes altitudes.
El objetivo del estudio 3 fue determinar la respuesta ventilatoria hipóxica (HVR), respuesta hipóxica cardiaca (CHR) y los ajustes cardiovasculares durante una apnea voluntaria máxima en nadadores jóvenes altamente entrenados. En el estudio participaron quince nadadores (ocho hombres y siete mujeres; edad, 20,93 ± 5,18 años; altura, 169,53 ± 10,44 cm; masa corporal, 71,5 ± 12,77 kg; índice de masa corporal (BMI), 24,7 ± 2,05 kg/m2), con 5-12 años de entrenamiento en natación y un volumen de entrenamiento semanal medio de 4h por día, cinco veces por semana y veintisiete controles (veintidós hombres y cinco mujeres; edad, 17,22 ± 2,42 años; altura, 169,52 ± 8,12 cm;
masa corporal, 63,85 ± 10,3 kg; BMI, 22,12 ± 2,49 kg/m2). Se realizó un estudio transversal descriptivo para determinar HVR y respuesta autonómica a hipoxia y apnea voluntaria máxima en nadadores jóvenes altamente entrenados en comparación con controles. La respuesta ventilatoria hipóxica se evaluó mediante un challenge hipóxico transitorio. Los participantes se sometieron a tres pruebas consecutivas que consistieron en cinco respiraciones de N2 al 100%. Posteriormente, los sujetos cambiaron de una posición supina a una sedente y se les solicito una apnea voluntaria máxima.
Para determinar la CHR, se utilizaron para el análisis 1 min de descanso en normoxia y 1 min de respuesta máxima de la HR en hipoxia. La variabilidad del ritmo cardiaco (HRV) se evaluó como una medida indirecta del equilibrio autonómico del corazón. A partir del registro de ECG, se obtuvieron series temporales del intervalo R-R y se utilizó un espectrograma variable de tiempo para obtener la densidad espectral (PSD) de HRV. Adicionalmente a los parámetros cardiovasculares se midió la función respiratoria mediante espirometría y se registró VC, PEF, PIF, FEV1, FEV1/VC, FEF 25, FEF 50 y FEF 75. Basalmente las variables demográficas, respiratorias, cardiovasculares y metabólicas no fueron diferentes entre los nadadores y los controles. La duración máxima de la apnea voluntaria fue mayor en los nadadores que en los controles (83,18 ± 41,43 vs. 55,77 ± 23,71 s respectivamente). La respuesta de FC durante la apnea fue mayor en los nadadores en comparación con los controles (HR: 71,99 ± 7,67 vs. 63,20 ± 10,07 lat/min). Además, la respuesta de HR máxima a la apnea voluntaria fue mayor en nadadores en comparación con los controles (∆HR: 2,94 ± 7,88 vs. 2,20 ± 7,86 1 lat/min). Durante una apnea voluntaria máxima, la LFHRV en los nadadores aumentó en comparación con la condición de reposo, sin embargo, los participantes del grupo control no mostraron cambios significativos en LFHRV, entre el reposo y la prueba de apnea. El ∆LFHRV fue mayor en los nadadores que en los controles (999.2 ± 1368 vs. 140.4 ± 1194 ∆AUC; p = 0.033). Con respecto al HFHRV durante la apnea, ambos grupos mostraron una disminución del impulso parasimpático. Ambos grupos mostraron un aumento en LF/HF desde el reposo hasta la apnea (nadadores: 0,23 ± 0,18 a 0,54 ± 0,44, p<0,001; control: 0,23 ± 0,22 a 0,37 ± 0,41, p=0,037). Con respecto a la respuesta ventilatoria y cardiaca a la hipoxia, en normoxia los nadadores mostraron un aumento menor en VE (0,11 ± 0,04 v/s a 0,19 ± 0,04 L*min*kg-1) desde la condición normóxica a la hipóxica que los controles (0,14 ± 0,04 v/s a 0,27 ± 0,06 a L/min/kg). La HVR, expresada como
∆VE/∆SpO2 fue significativamente menor en los nadadores en comparación con los participantes del grupo control (0,007 ± 0,001 v/s 0,016 ± 0,002 ∆VE/∆SpO2). HCR fue similar entre nadadores (0.27
± 0.51 ∆HR/∆SpO2) y controles (0,52 ± 1,04 ∆HR/∆SpO2). Los nadadores mostraron con respecto a los controles: (i) duración máxima de la apnea voluntaria más larga; (ii) marcada disminución de HVR; (iii) mayor respuesta cardíaca durante la prueba de apnea voluntaria máxima caracterizada por un desequilibrio autonómico general. Nuestros resultados sugieren fuertemente que la menor respuesta ventilatoria a la hipoxia (determinada a través de un challenge hipóxico) podría contribuir a una mayor duración de la apnea en los nadadores.
El objetivo del estudio 4 fue resumir la evidencia disponible relacionada con el control quimiorreflejo en los deportes acuáticos de inmersión. Los deportes acuáticos de inmersión implican apneas a largo plazo; por lo tanto, los atletas deben adaptarse fisiológicamente para mantener la oxigenación muscular, a pesar de no realizar ventilación pulmonar. La contención de la respiración (es decir, la apnea) es común en los deportes acuáticos e implica una disminución y un aumento de la PaO2 y la PaCO2, respectivamente, como las principales señales que desencadenan el final de la apnea. Los principales sensores fisiológicos de O2 son los cuerpos carotídeos, que son capaces de detectar gases arteriales y alteraciones metabólicas antes de llegar al cerebro, lo que ayuda a ajustar el sistema cardiorrespiratorio. Además, el principal sensor de H+/CO2 es el núcleo retrotrapezoide (RTN), que se encuentra a nivel del tronco encefálico; este mecanismo contribuye a detectar acidosis respiratoria y metabólica. Aunque estos sensores se han caracterizado en estados fisiopatológicos, la evidencia actual muestra un posible papel de estos mecanismos como sensores fisiológicos durante la apnea voluntaria. Se ha encontrado que los atletas buceadores y nadadores muestran tiempos de apnea más prolongados que los atletas de deportes terrestres, así como una disminución del control quimiorreflejo de O2 periférico y CO2 central. Sin embargo, aunque la quimiosensibilidad en reposo podría disminuir, recientemente encontramos una marcada simpatoexcitación durante la apnea voluntaria máxima en nadadores jóvenes, lo que podría activar el bazo (que es un órgano reservorio de sangre oxigenada). Por lo tanto, es posible que el quimiorreflejo, la función autonómica y los órganos de almacenamiento/suministro de oxígeno estén relacionados con la apnea en los deportes acuáticos de inmersión. En base a la información resumida proponemos un posible modelo mecanicista fisiológico que podría contribuir a proporcionar nuevas vías para comprender la fisiología respiratoria de los deportes acuáticos.
SUMMARY
This doctoral thesis is based on the publication of 4 studies that aim to analyze the role of the peripheral chemoreflex on apnea time in competitive swimming athletes, for which experiments were carried out on a population ranging from animal models, physically active humans and competitive swimmers.
The objective of Research 1 was to determine the acute effects of high-intensity interval training (HIIT) exercise and endurance exercise (EE) on lung function, sympathetic/parasympathetic balance, and cardiorespiratory coupling (CRC) in healthy subjects. Eight physically active subjects (recreational runners; four men and four women) participated: height: 1.7 ± 0.1 m; body mass: 63.3
± 5.7 kg; body mass index: 22.0 ± 2.4 kg/m2; age: 23.9 ± 3.1 years). Using a repeated measures crossover design, participants were exposed to EE (20 min at 80% of maximal heart rate (HR), HIIT (1 min of exercise at 90% of maximal HR for 1 min of rest, 10 times) or control condition (rest).
Pulmonary function was assessed by spirometry (VC, PEF, PIF, FEV1, FEV1/VC, FEF 25, FEF 50, and FEF75), Electrocardiogram (ECG), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MAPB), oxygen saturation, CRC, and heart rate variability (LFHRV, HFHRV, LF/HFHRV) before and after the interventions. At baseline, there were no significant differences between EE, HIIT, and control in cardiovascular and respiratory parameters. EE and HIIT significantly increased SBP, MABP, and HR (p < 0.05, pre vs post). Resistance exercise and HIIT sessions did not induce changes in FEV1, FEV1/CV, FEF 25, FEF, or FEF 75 (p>0.05; pre vs post).
Furthermore, no significant differences in lung function were found between EE, HIIT and control (pre vs post). An isolated EE session failed to induce significant changes in the LFHRV, HFHRV and LF/HFHRV ratio (p<0.05; pre vs. post) (Figure 3). However, total spectral power was significantly reduced after EE (p<0.05; pre vs. post). In contrast, HIIT induces significant changes in HRV spectral data. In fact, LFHRV increased while HFHRV decreased with HIIT (all p<0.05; pre vs post).
Consequently, the LF/HFHRV ratio increased after the HIIT protocol (p<0.05; pre vs. post).
Furthermore, the total spectral power of HRV was reduced with HIIT. Resistance exercise and HIIT showed a significant decrease in time domain variables (SDNN; RMSSD, NN50, pNN50, p<.05; pre vs. post). Furthermore, SD1 and SD2 were significantly reduced after EE and HIIT. Directionality analysis showed that the coupling between respiration and heart rate (B → H) was greater than that between heart rate and respiration (H → B) under control conditions. B → H but not H → B coupling increased significantly after EE (0.14 ± 0.03 vs. 0.16 ± 0.03 Hz, pre vs. post, respectively, p<0.05).
No effect of HIIT on B→H coupling or H→B coupling was found. The main findings were: (a) neither EE nor HIIT changed lung function; (b) HIIT and no EE produced acute HRV changes evidenced by a decrease in HFHRV and an increase in LFHRV after exercise; (c) HIIT but not EE has a detrimental effect on normal cardiovascular autonomic adjustment to an incline challenge; and (d) EE but not HIIT induced an acute increase in CRC. Overall, our findings suggest that acute cardiorespiratory responses to EE and HIIT may differ between these two exercise modalities, which could have some implications for chronic adaptations to exercise.
The aim of research 2 was to determine the effect of acute high-altitude exposure on sympathetic/parasympathetic modulation of baroreflex control (BR)in normal rats. Twelve male Sprague Dawley rats were randomly assigned to sea level (n = 7) and high altitude (n = 5) groups (3270 m above sea level). The rats were randomly distributed into the Sea Level group (n = 7) and the High-Altitude group (n = 5). Sea-level rats underwent catheterization surgery and baseline blood pressure (BP) recording for one hour. Subsequently, the BR experiment was performed as follows: 8 boluses of phenylephrine were injected to increase BP (i.v.) and after 30 min of recovery, 8 boluses of sodium nitroprusside were injected to decrease BP (i.v.). The second series of rats (High Altitude group) ascended to 3,270 m above sea level (Caspana, Antofagasta, Chile) in a mobile laboratory and after 24 h, catheterization surgery was performed. Similar to the first series of animals (Sea-Level group), 8h after the surgical procedure, baseline recordings of BP (1 hour) and the BR experiment were performed. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MABP), pulse pressure (PP), heart rate (HR) was measured. Autonomic control was estimated using an indirect measurement by calculating heart rate variability (HRV). Basally there were no significant differences between sea level and high-altitude exposure in body weight DBP, SBP, MABP, PP and HR. After acute exposure to high altitude, rats showed increased sympathetic drive and decreased parasympathetic modulation of the heart. The LFHRV component increased significantly (p<0.05) from sea level (42.12 ± 7.44 nu) to high altitude (60.55 ± 4.47 nu), while the HFHRV component decreased significantly (p<0.05) from sea level (39.37 ± 4.44 nu) to high altitude (57.82 ± 7.43 nu). Consequently, the LF/HFHRV ratio increased significantly (p<0.05) at high altitude compared to sea level (1.66 ± 0.27 vs. 0.85 ± 0.23, respectively). After acute exposure to high altitude and after Phe administration, bradycardic responses were significantly decreased (p<0.05).
The sigmoidal curve of the BR analysis showed diminished BR vagal bradycardia. In addition, curvature (0.04 ± 0.01 vs. 0.07 ± 0.01 mmHg/beats/min) and maximal bradycardia (50.70 ± 0.28 vs.
58.01 ± 0.81 beats/ min) was significantly (p<0.05) reduced after acute exposure at high altitude compared to sea level. Peak tachycardia response to SNP, range, slope, BP midpoint, lower plateau, and upper plateau of BR analysis were not significantly different between groups at sea level and high altitude. The main findings of the present study were: (i) after acute exposure to high altitude there is impaired cardiac autonomic control; (ii) High Altitude exposure produces impaired cardiac baroreflex control in normal rats; and (iii) there is reduced parasympathetic drive activation during acute high- altitude exposure in normal rats. The present results suggest that acute exposure to high altitudes leads to impaired autonomic control and impaired baroreflex function, mainly characterized by decreased parasympathetic activity after 24 h of exposure to high altitudes.
The aim of research 3 was to determine hypoxic ventilatory response (HVR), hypoxic cardiac response (HCR), and cardiovascular adjustments during maximal voluntary apnea in highly trained young swimmers. Fifteen swimmers participated in the research (eight men and seven women; age, 20.93 ± 5.18 years; height, 169.53 ± 10.44 cm; body mass, 71.5 ± 12.77 kg; mass index (BMI), 24.7
± 2.05 kg/m2), with 5-12 years of swimming training and a mean weekly training volume of 4h per day, five times per week and twenty-seven controls (twenty-two men and five women, age, 17.22 ± 2.42 years, height, 169.52 ± 8.12 cm, body mass, 63.85 ± 10.3 kg, BMI, 22.12 ± 2.49 kg/m2). A descriptive cross-sectional study was conducted to determine HVR and autonomic response to hypoxia and maximal voluntary apnea in highly trained young swimmers compared to controls.
Hypoxic ventilatory response was assessed by transient hypoxic challenge. Participants underwent three consecutive tests consisting of five breaths of 100% N2. Subsequently, subjects changed from a supine to a sitting position and were asked to hold maximal voluntary apnea. To determine CHR, 1 min of rest in normoxia and 1 min of maximal HR response in hypoxia were used for analysis. Heart rate variability (HRV) was assessed as an indirect measure of the autonomic balance of the heart.
From the ECG recording, time series of the R-R interval were obtained, and a time-varying spectrogram was used to obtain the spectral density (PSD) of HRV. In addition to cardiovascular parameters, respiratory function was measured by spirometry and TD, PEF, PIF, FEV1, FEV1/VC, FEF 25, FEF 50 and FEF 75 were recorded. At baseline, demographic, respiratory, cardiovascular and metabolic variables were not different between swimmers and controls. The maximum duration of voluntary apnea was longer in swimmers than in controls (83.18 ± 41.43 vs. 55.77 ± 23.71 s, respectively). HR response during apnea was higher in swimmers compared to controls (HR: 71.99
± 7.67 vs. 63.20 ± 10.07 beats/min). Furthermore, the maximal HR response to voluntary apnea was greater in swimmers compared to controls (∆HR: 2.94 ± 7.88 vs. 2.20 ± 7.86 1 beats/min). During a maximal voluntary apnea, the LFHRV in the swimmers increased compared to the resting condition, however, the control group participants did not show significant changes in LFHRV between the rest and the apnea test. ∆LFHRV was higher in swimmers than controls (999.2 ± 1368 vs. 140.4 ± 1194
∆AUC; p = 0.033). Regarding HFHRV during apnea, both groups showed decreased parasympathetic drive. Both groups showed an increase in LF/HF from rest to apnea (swimmers: 0.23 ± 0.18 to 0.54
± 0.44, p<0.001; control: 0.23 ± 0.22 to 0, 37 ± 0.41, p=0.037). Regarding the ventilatory and cardiac response to hypoxia, normoxic swimmers showed a smaller increase in VE (0.11 ± 0.04 v/s to 0.19 ± 0.04 L*min*kg-1) from normoxic to hypoxic condition than controls (0.14 ± 0.04 v/s at 0.27 ± 0.06 a L/min/kg). HVR, expressed as ∆VE/∆SpO2, was significantly lower in swimmers compared to control participants (0.007 ± 0.001 vs. 0.016 ± 0.002 ∆VE/∆SpO2). HCR was similar between swimmers (0.27 ± 0.51 ∆HR/∆SpO2) and controls (0.52 ± 1.04 ∆HR/∆SpO2). Swimmers showed with respect to controls: (i) longest maximum duration of voluntary apnea; (ii) marked decrease in HVR; (iii) increased cardiac response during maximal voluntary apnea testing characterized by a general autonomic imbalance. Our results strongly suggest that the lower ventilatory response to hypoxia (determined through a hypoxic challenge) could contribute to a longer duration of apnea in swimmers.
The aim of research 4 was to summarize the available evidence related to chemoreflex control in immersion water sports. Immersion water sports involve long-term apneas; therefore, athletes must physiologically adapt to maintain muscle oxygenation, despite not performing pulmonary ventilation.
Breath-holding (ie, apnea) is common in water sports and involves a decrease and increase in PaO2 and PaCO2, respectively, as the main signals that trigger the end of apnea. The main physiological sensors of O2 are the carotid bodies, which are capable of detecting arterial gases and metabolic alterations before reaching the brain, which helps to adjust the cardiorespiratory system. Furthermore, the main H+/CO2 sensor is the retrotrapezoidal nucleus (RTN), which is located at the level of the brainstem; this mechanism helps detect respiratory and metabolic acidosis. Although these sensors have been characterized in pathophysiological conditions, current evidence shows a possible role for these mechanisms as physiological sensors during voluntary apnea. Diving and swimming athletes have been found to show longer apnea times than land-sport athletes, as well as decreased chemoreflex control of peripheral O2 and central CO2. However, although resting chemosensitivity might decrease, we recently found marked sympathoexcitation during maximal voluntary apnea in young swimmers, which might activate the spleen (which is a reservoir organ for oxygenated blood).
Therefore, it is possible that the chemoreflex, autonomic function, and oxygen storage/supply organs are related to apnea in immersive water sports. Based on the summarized information we propose a possible physiological mechanistic model that could contribute to providing new ways to understand the respiratory physiology of aquatic sports.
CONTENTS Page
1. Introduction 21
2. Aims and hypothesis of the doctoral thesis 22
3. Methods 23
3.1 Research I 23
3.1.1 Subjects 23
3.1.2 Experimental procedure 23
3.1.3 Pulmonary function 24
3.1.4 Continuous recording of ventilation and electrocardiogram
24 3.1.5 Arterial blood pressure and oxygen saturation 25
3.1.6 Heart rate variability 25
3.1.7 Cardiorespiratory coupling 25
3.1.8 Statistical analysis 25
3.2 Research II 26
3.2.1 Ethical Approval and Animals 26
3.2.2 Experimental Procedure 26
3.2.3 Arterial Blood Pressure in Freely Moving Rats 26
3.2.4 Baroreflex Control 26
3.2.5 Dose-Responses Analysis to BR 27
3.2.6 Autonomic Control 27
3.2.7 Statistical Analysis 27
3.3 Research III 28
3.3.1 Ethics Statements 28
3.3.2 Subjects 28
3.3.3 Experimental Design 28
3.3.4 Hypoxic Ventilatory Response (HVR) and Maximum Voluntary Apnea Test 29
3.3.5 Cardiac Hypoxic Response (CHR) 29
3.3.6 Electrocardiogram (ECG) 30
3.3.7 Heart Rate Variability (HRV) 30
3.3.8 Resting Metabolic Rate 30
3.3.9 Pulmonary Function 31
3.3.10 Statistical Analysis 31
3.4 Research IV 32
4. Results 33
4.1 Research I 33
4.1.1 Baseline cardiorespiratory parameters and changes after an acute bout of EE
or HIIT exercise 33
4.1.2 Acute effects of EE and HIIT exercise bouts on pulmonary function 34 4.1.3 Acute effects of endurance and HIIT exercise bouts on heart rate variability 34 4.1.4 Acute effects of EE and HIIT exercise bouts on autonomic disturbances
induced by orthostatic sit-up test
38
4.1.5 Acute effects of EE and HIIT exercise bouts on cardiorespiratory coupling 42
4.2 Research II 43
4.2.1 Effect of High-Altitude exposure on baseline physiological variables 43 4.2.2 Effect of High-Altitude exposure on cardiac autonomic control at rest 43 4.2.3 Effect of High-Altitude exposure on cardiac baroreflex control 46 4.2.4 Effect of High-Altitude exposure on parasympathetic modulation of R-R
interval time series.
47
4.2.5 Effects of High-Altitude exposure on baroreflex control and parasympathetic modulation are not dependent of blood pressure stimulation
50
4.3 Research III 51
4.3.1 Baseline cardiorespiratory and pulmonary parameters 51 4.3.2 Maximum voluntary apnea time and cardiovascular response to hypoxia and
apnea 52
4.3.3 Autonomic control during maximum voluntary apnea test 53 4.3.4 Ventilatory and cardiac response to hypoxia
54
4.3.5 Hypoxic ventilatory response 57
4.3.6 Hypoxic cardiac response 57
5. Discussion 58
5.1 Research I 58
5.1.1 Effect of single bout of EE and HIIT on pulmonary function 58
5.1.2 Effect of EE and HIIT on HRV disturbances 58
5.1.3 Endurances exercise promotes cardiorespiratory coupling in healthy individuals
59
5.1.4 Limitations 60
5.2 Research II 61
5.2.1 Baroreflex control and High-Altitude environment 61 5.2.2 High-altitude exposure and autonomic control impairment 61
5.2.3 Limitations 62
5.3 Research III 64
5.3.1 Hypoxic ventilatory response and apnea duration in young highly trained swimmers
64
5.3.2 Autonomic control during apnea in young highly trained swimmers 65
5.4 Research IV 66
5.4.1 Peripheral and central chemoreflex control 66
5.4.1.1 Peripheral chemoreflex 66
5.4.1.2 Central chemoreflex 67
5.4.2 Central and peripheral interaction: a key point in autonomic response to voluntary apnea
67
5.4.3 Spleen-chemoreflex relationship in voluntary apnea 68 5.4.4 Chemoreflex responses and adaptations in immersion water sports 69
6. Conclusions 72
6.1 Research I 72
6.2 Research II 72
6.3 Research III 72
6.4 Research IV 72
7. References 73
7.1 Introduction 73
7.2 Research I 73
7.2 Research II 77
7.3 Research III 80
7.4 Research IV 84
8. Appendix (published studies) 89
8.1 Research I 89
8.2 Research II 103
8.3 Research III 113
8.4 Research IV 124
LIST OF TABLES AND FIGURES Page Table 1. Acute effect of endurance and high intensity Interval training (HIIT) on
cardiovascular and ventilatory variables (Research I)
33
Table 2. Acute effect of endurance and high intensity Interval training (HIIT) on heart rate variability (HRV) alterations (Research I)
36
Table 3. Acute effect of endurance and high intensity Interval training (HIIT) on heart rate variability (HRV) alterations following orthostatic sit-up test (Research I)
40
Table 4. Effect of high-altitude exposure on baseline physiological parameters (Research II)
43
Table 5. Effect of high-altitude exposure on heart rate variability parameters at rest (Research II)
45
Table 6. Effect of high-altitude exposure on baroreflex control (Research II) 47 Table 7. Effect of high-altitude exposure on heart rate variability parameters at sea level
and high-altitude during phenylephrine (Phe) and sodium nitroprusside (SNP) (Research II)
49
Table 8. Basal anthropometric, respiratory, and cardiovascular characteristics of swimmers compared to control participants at rest condition (Research III)
51
Table 9. Cardiovascular and respiratory responses to severe hypoxic challenge in swimmers and control participants (Research III)
56
Figure 1. Experimental design (Research I) 24
Figure 2. Timeline and design of the experiment (Research III) 29 Figure 3. Acute effect of high-intensity interval training and endurance exercise on
pulmonary function (Research I)
34
Figure 4. Acute effect of high-intensity exercise training and endurance exercise on heart rate variability alterations in healthy individuals (Research I)
35
Figure 5. Single bout of high-intensity exercise training elicits sympathoexcitation in healthy individuals (Research I)
39
Figure 6. Single bout of endurance exercise increases cardiorespiratory coupling in healthy individuals (Research I)
42
Figure 7. Effect of High-Altitude (3,270 m above sea level) on heart rate variability (HRV) alterations (Research II)
44
Figure 8. Effect of High-Altitude (3,270 m above sea level) on baroreflex (BR) control in freely moving rats (Research II)
48
Figure 9. Effect of High-Altitude (3,270 m above sea level) on heart rate variability (HRV) alteration in time-varying domain, following phenylephrine (Phe) administration in freely moving rats (Research II).
48
Figure 10. Dose-response curve of phenylephrine (Phe) and sodium nitroprusside (SNP) during Sea-Level and High-altitude (3,270 m above sea level) exposure (Research II).
50
Figure 11. Swimmers are able to maintain longer apnea time and a higher heart rate response during the apnea effort (Research III).
52
Figure 12. Autonomic control during the maximum voluntary apnea test in swimmers and control participants (Research III).
53
Figure 13. Hypoxic ventilatory (HVR) cardiac (HCR) responses in young trained swimmers. (Research III).
54
Figure 14: Mechanism of signal transduction and cell excitability of the peripheral and
central chemoreflex. (Research IV) 70
Figure 15: Hypothetical schematic representation model related to the influence of the central and peripheral chemoreflex on a breath hold. (Research IV)
71
LIST OF ABBREVIATIONS ApEn: Approximate entropy AUC: area under the curve BP: blood pressure
Bpm: beats per minute BR: baroreflex
CB: carotid body
CHR: cardiac hypoxic response CO2: carbon dioxide
CRC: cardiorespiratory coupling DBP: diastolic blood pressure
Dp/dt: first derivative of the blood pressure ECG: electrocardiogram
EE: endurance exercise
FECO2: expired fraction of end-tidal carbon dioxide FEF 25: forced expiratory flow at 25% of the vital capacity FEF 50: forced expiratory flow at 50% of the vital capacity FEF75: forced expiratory flow at 75% of the vital capacity FEO2: end-tidal oxygen
FEV1: forced expiratory volume in 1 second FiCO2: concentration of inspired carbon dioxide FiO2: concentration of inspired oxygen
FIO2: fraction inspired of O2
HFHRV: high frequency component of heart rate variability HIIT: high Intensity Interval Training
HR: heart rate
HRV: heart rate variability
HVR: hypoxic ventilatory response kHz: kilohertz
LFHRV: low frequency component of heart rate variability
MAPB: mean arterial blood pressure NADH: nicotinamide adenine dinucleotide n.u.: normalized units
N2: nitrogen
NN50: number of pairs of successive NN intervals that differ by more than 50 ms O2: oxygen
PaCO2: partial pressure of carbon dioxide PaO2: partial pressure of oxygen
PEF: peak expiratory flow PetCO2: end-tidal CO2 pressure PetO2: end-tidal oxygen pressure pH: potential hydrogen
Phe: phenylephrine PIF: peak inspiratory flow
PNN50: proportion of NN50 divided by the total number of NN intervals PP: pulse pressure
PSD: power spectral density REV: reserve expiratory volume RF: Respiratory frequency RMR: resting metabolic rate
RMSSD: root mean square of the successive differences between adjacent normal R-R intervals RQ: respiratory quotient
R-R: time between ECG peak R RTN: retrotrapezoid nucleus
RVLM: rostral ventrolateral medulla SampEn: Sample entropy
SBP: systolic blood pressure SD: standard deviation
SD1: short-term variability of NN intervals SD2: long-term variability of NN intervals
SDNN: standard deviation of the NN intervals SNP: sodium nitroprusside
SpO2: arterial saturation Te: expiratory time Ti: inspiratory time
Ttot: total time of one breath
TWIK: two pore domain potassium channel VC: vital capacity
VCO2: carbon dioxide production VE: minute ventilation
VLF: very low frequency component of HRV VO2: oxygen uptake
VT: tidal volume
1. INTRODUCTION
Water sports are subdivided into different modalities (i.e. artistic swimming, ornamental jumps, swimming in open or closed waters, water polo, among others), which combine very well coordinated movements along with long-term divers (Konstantinidou et al., 2017). Moreover, has been estimated that in artistic swimming, voluntary apnea during exercise (dynamic apnea) is approximately 21-s (Konstantinidou et al., 2017), which suggests that these athletes have significant cardiorespiratory control during these apnea episodes.
Apnea is defined as a cessation of ventilation, and this can be divided into obstructive apneas (decreased respiratory flow with movement of the respiratory musculature) and central apneas (cessation of ventilation, without movements of the respiratory musculature) (Toledo et al., 2017).
Both types of apneas have been widely studied in a pathological context (hypertension, heart failure, obstructive sleep apnea, etc.) (Toledo et al., 2017; Andrade et al., 2018; Rowley et al., 2006);
however, the evidence associated with voluntary static and dynamic apnea and how these apneas can be a physiological mechanism of adaptation in water sports is limited.
One of the more important factors implicated in all swimming disciplines that involve apnea events is the duration of thereof (Greco et al., 1996). It has been accepted that the important determining factors in the duration of apnea are the ability to store O2 in the body, tolerance to asphyxiation and metabolic rate (Konstantinidou et al., 2017). Indeed, it has been determined that higher motor efficiency and lower muscle energy expenditure during exercise, higher capacity to store O2 and higher anaerobic performance contribute significantly to improve the duration of dynamic apneas (Konstantinidou et al., 2017). However, there are mechanisms associated with cardiorespiratory neuronal control, which have been poorly explored in water sports. These mechanisms could contribute significantly to the duration of apnea and consequently on performance in sports that require a cessation of ventilation for a long period of time.
During an apnea event there is a decrease in pH, therefore, accumulation of H +, secondary to an increase in arterial pCO2, in addition to a progressive decrease in arterial pO2 (Andrade et al., 2018).
All this set of events stimulates both central and peripheral chemoreceptors (Andrade et al., 2018).
During an apnea, the increased activity of these chemoreceptors stimulates an increase in respiratory activity; therefore, apnea ends when breathing is involuntarily restored, induced by an increase in the activity of these chemoreceptors (Toledo et al., 2017) (Figure 1, 2). Considering this widely known physiological mechanism (Mansukhani et al., 2015; Kara et al., 2003), it is possible to suggest that athletes who have the ability to increase the duration of dynamic apneas may have a decreased chemoreflex (central or peripheral), which gives them greater resistance to hypercapnia-hypoxia events, therefore, increasing its competitive performance. Thus, the objective of this research is to determine the role of chemoreflex on voluntary apnea and aerobic performance in swimmers of competitive level.
2. AIMS AND HYPOTHESIS OF THE DOCTORAL THESIS
Hypothesis
Competitive swimmers’ athletes manifest a decrease in the chemoreflex function, giving them greater resistance to maximum breath-hold.
General Aim
To determine the role of the chemoreflex function on maximum voluntary breath-hold and aerobic performance in competitive swimmers’ athletes.
Specific Aims
1. To determine the acute effects of exercise on pulmonary function, autonomic modulation, and cardiorespiratory coupling (CRC) in control subjects.
2. To determine the effects of a hypoxic chemoreflex stimulation on the baroreflex function and autonomic modulation.
3. To determine the hypoxic chemoreflex ventilatory response and maximum voluntary breath-hold in competitive level swimmers and control subjects.
4. To summarize the more relevant evidence related to chemoreflex control and voluntary breath-hold in immersion water sports.
3. METHODS 3.1 Research I 3.1.1 Subjects
Eight physically active participants (recreational runners; four male and four female) were recruited in the present study: height: 1.7 ± 0.1 m; body mass: 63.3 ± 5.7 kg; body mass index (BMI): 22.0 ± 2.4 kg/m2; age: 23.9 ± 3.1 years). All females were measured in the same stage of menstrual cycle in different weeks. Exercise sessions were conducted between 13:00 and 17:00 hr. and subjects refrained from drinking alcohol, smoke, caffeine, or drugs that alter autonomic control 48 hr. before exercise session. To assess the effects of a single bout of HIIT and EE, as independent variables, on pulmonary function, cardiovascular responses, autonomic control, and cardiorespiratory coupling, the same subjects were randomly assigned to HIIT, EE, and control conditions. Accordingly, this was a mixed crossover, repeated-measures design. Participants were carefully informed about the experiment procedures, and the possible risks and benefits associated with their participation in the study. An appropriate signed informed consent document was obtained in accordance with the latest version of the Declaration of Helsinki. Separated by 7 days for men and accordingly to menstrual cycle of the female, participants completed an acute bout of HIIT (Francois & Little, 2015), EE (Corte de Araujo et al., 2012), or a control condition (20 min of resting standing on treadmill), on a treadmill (Power Jog, J100800 Cardio Sport running machine, North Charleston, SC).
3.1.2 Experimental procedure
None of the participants had any background (in the 6-month period preceding the study) in regular strength training or competitive sports activity. Exclusion criteria considered for enrollment were: (a) potential medical problems or a history of ankle, knee, or back injury; (b) any lower extremity reconstructive surgery in the past 2 years or unresolved musculoskeletal disorders; (c) autonomic control impairment at rest, estimated by HRV disturbances (low to high frequency ratio of HRV <2.3) (Nunan, Sandercock, & Brodie, 2010); and (d) history of chronic obstructive or restrictive pulmonary diseases and/or altered spirometry on the day of the pre exercise session (forced expiratory volume at first second (FEV1)/ vital capacity (VC) <70, FEV1 <80% of predicted value or VC <80% of predicted value). Participants were carefully familiarized with the tests procedures before the measurements were taken. Randomly the type of exercise of session was chosen (HIIT, EE, or control). All participants were subject at the same warm-up muscle actions prior to the exercises (Andrade et al., 2015). The warm-up consisted of running at 6–7 km/hr on a treadmill (Power Jog).
Tests were always administered in the same order (clinical spirometry, electrocardiogram, blood pressure, and sit-up test, Figure 1), time of day (between 13:00 and 17:00 hr), and by the same investigators. The day before each experimental condition, participants were instructed to (a) have a good night sleep (~8 hr) and (b) use the same athletic shoes and clothing during the protocols. HIIT consisted in 1 min of exercise at 90% of maximal heart rate (HRmax) and 1 min of rest, repeated 10 times (Francois & Little, 2015); EE involved 5 min to ramp-up to 80% of HRmax followed by 20 min at 80% HRmax (Corte de Araujo et al., 2012); and control involved 20 min of resting. HR during exercise was monitored with a telemetry device (Polar, V800, Finland). Prior to, and after each experimental condition, height, body mass, systolic (SBP) and diastolic (DBP) blood pressure, mean arterial blood pressure (MABP), pulse pressure (PP), HR, clinical spirometry (VC; peak expiratory flow [PEF]; peak inspiratory flow [PIF]; FEV1; FEV1/VC; forced expiratory flow at 25% [FEF 25], 50% [FEF 50] and 75% [FEF 75] of VC), and the orthostatic sit-up test measurements. were taken.
Height was measured using a wall-mounted stadiometer (HR-200, Tanita, Japan) recorded to the
nearest 0.1 cm. Body mass was measured to the nearest 0.1 kg using a digital scale (BF-350, Tanita, IL, USA). BMI was calculated as body mass/height2.
Figure 1. Experimental design. Physically active men (n=4) and women (n=4) performed 3 different randomly ordered protocols (HIIT, endurance exercise [EE] and control) in a treadmill. Before and after each experimental procedure individuals were assessed on: blood pressure, autonomic control, cardiorespiratory coupling, pulmonary function and orthostatic sit-up test. HRmax: maximal heart rate.
3.1.3 Pulmonary function
Pulmonary function was assessed according to the ATS/ERS Task Force consensus (Miller et al., 2005). Briefly, from the tidal volume, subjects were asked to perform a maximal inspiration (inspiratory reserve volume), return to tidal volume, and then perform a maximal expiration (expiratory reserve volume). We used the maximal expiratory curve to calculate VC, PEF, PIF, FEV1, FEV1/VC, FEF 25, FEF 50, and FEF 75. All recordings were performed with a clinical spirometry, calibrated according to the manufacturer instructions (Jaeger, Vyaire Medical Care).
3.1.4 Continuous recording of ventilation and electrocardiogram
In addition to pulmonary function assessments, prior to and after each experimental condition, ventilatory flow and a 2-lead ECG (10 min at rest pre and post exercise and during sit-up test) were recorded following the orthostatic sit-up test (20 min total time), similar to a previous study (Currie, Wong, Warburton, & Krassioukov, 2015). Briefly, subjects were asked to maintain a supine position (10 min) and then asked to change to an upright position as slowly and as smoothly as possible (10 min) (Currie et al., 2015). The heart rate (HR) response and R-R time series (5 min before orthostatic sit-up test and 5 min after orthostatic sit-up test) were used to estimate sympathoexcitation (see Autonomic control methods section). All recordings were sampled at 1 kHz with an analogic-digital recording system (ADInstruments). The HR and ventilator flow were analyzed with LabChart Pro 8.0 (ADInstruments).
3.1.5 Arterial blood pressure and oxygen saturation
Prior to, and after each experimental condition, SBP and DBP were determined. From SBP and DBP, MABP (1/3 of SBP + 2/3 of DBP) and PP (SBP-DBP) were calculated as previously described (Álvarez et al., 2013). Measurements were determined with a sphygmomanometer (Tenso Medical Instruments Co., Ltd.) and a stethoscope (Littmann Cardiology, 3M, Bracknell, UK) by the same experienced clinician. In addition, SpO2 was determined before and after each acute exercise session (BuleTooth PULSE OXIMETER, BK-P02, China).
3.1.6 Heart rate variability
Heart rate variability was used as an indirect measure of autonomic balance of the heart (Camm, Malik, & Bigger, 1996). From the ECG recordings, time series were obtained and autoregressive algorithm with Hann windowing of 50% overlap was used to obtain the power spectral density (PSD) of HRV. Cut-off frequencies were defined as: very low frequency: 0.00–0.04; low frequency (LFHRV): 0.04–0.15 Hz and high frequency (HFHRV) 0.15–0.45 Hz (Yuda et al., 2018). Additionally, we used the LF/HFHRV ratio as an indicator of autonomic balance of the heart. LFHRV and HFHRV were expressed as normalized units (n.u.) and raw dada. Analysis was performed within in a 10 min window using Kubios HRV Premium Software v3.1 (Kubios, Kuopio Finland). In addition, to estimate the overall autonomic disruption after the interventions, spectral non-stationary analysis was used (2 s resolution) during the orthostatic sit-up test. The LFHRV component was used as indicator of autonomic deregulation. Quantification was performed from the area under the curve (AUC), from continued non-stationary analysis. This analysis was performed with Kubios HRV Premium Software v3.1. Using the stationary and nonstationary analysis, time domain and nonlinear domain were plotted (Time domain: SDNN: standard deviation of the NN intervals; RMSSD: root mean square of the successive differences between adjacent normal R-R intervals; NN50: number of pairs of successive NN intervals that differ by more than 50 ms; PNN50: proportion of NN50 divided by the total number of NN intervals. Nonlinear domain: SD1: short-term variability of NN intervals; SD2: long-term variability of NN intervals; ApEn: Approximate entropy; SampEn: Sample entropy).
3.1.7 Cardiorespiratory coupling
The directionality and the magnitude of interactions between breathing and heart rate time series oscillations were quantified with the mutual information theory and phase models using a three-step protocol (Zhu et al., 2013). First, we evaluated the mutual information of the phases. After, we obtained the interaction functions of the oscillators by fitting the coupled oscillator model to the data, related to phase of the cycle. As a last step, we compared the joint probability of the empirical phases with that obtained from the simulated model, in order to validate the empirical oscillator model. The data were showed as power spectral density and coupling directionality (breath to heart and heart to breath). Analysis was performed using custom Matlab routines.
3.1.8 Statistical analysis
Data are expressed as mean ± standard deviation (SD). All data were subjected to normality (Shapiro–
Wilk) and homoscedasticity (Levene) testing. Data were evaluated using a 3 (control, EE, HIIT) × 2 (pre–post) analysis of variance (two-way ANOVA with repeated measures), followed by Holm–Sidak post hoc analysis according to the data structure. Nonparametric variables were evaluated using Kruskal– Wallis analysis followed by Dunn's post test. A p-value <.05 was considered statistically significant. All analyses were performed with GraphPad Prism 8.0 (La Jolla, CA).
3.2 Research II
3.2.1 Ethical Approval and Animals
Twelve male Sprague-Dawley rats were used in these experiments. All surgical procedures and protocols used, were in accordance with guidelines of the American Physiological Society and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Antofagasta Scientific Research Ethical Committee (CEIC-210/2019).
3.2.2 Experimental Procedure
Male Sprague-Dawley rats (n = 12) were housed in individual cages with a 12/12-h light/dark schedule and were allowed free access to food and water. The rats were randomly allocated into Sea- Level group (n = 7) and to High-Altitude group (n = 5). Sea-level rats were subjected to catheterization surgery according to the method of Li et al. (1999) and basal BP recording was preformed (1-hour). Afterward, BR experiment was performed as follows: 8 boluses of phenylephrine to increase BP were injected (i.v.) and after 30 min of recovery, 8 boluses of sodium nitroprusside to decrease BP were injected (i.v.). The second series of rats (High-Altitude group) ascended at 3,270 m above sea level (Caspana, Antofagasta, Chile) in a costume made mobile laboratory and after 24 h, the catheterization surgery was performed. Similar to the first animal series (Sea-Level group), 8 h after (Masson et al., 2014) the surgical procedure, basal recordings of BP (1-hour) and the BR experiment were performed. At Sea-Level the relative humidity was between 66 and 68% and the temperature between 19 and 21ºC, while at 3,270 m (High-Altitude), the relative humidity was between 21 and 25% and the temperature was 19 º (Chilean Meteorological Service).
3.2.3 Arterial Blood Pressure in Freely Moving Rats
Arterial BP measurement was performed in conscious freely moving rats. The carotid artery and jugular vein cannulations (PE-50 polyethylene tubing, Clay Adams, Parsippany, NJ, United States), were performed to measure BP and for drugs administration. The rats were anesthetized (i.p.) using ketamine (80 mg/kg; Fort Dodge Animal Health, United States) plus xylazine (12 mg/kg; Alcon, United States) (Li et al., 1999; Feng et al., 2015). A midline incision in the neck was performed to isolate a lateral branch of the carotid artery. A small incision was made and a 3 Fr polyurethane catheter was guided into the artery and was tunneled subcutaneously to the back of the neck and connected to a vascular access port. Eight hours before BP measurement, the rats were anesthetized (i.p.) using ketamine (80 mg/kg; Fort Dodge Animal Health, United States). plus xylazine (12 mg/kg;
Alcon, United States) for catheterization of the common carotid artery and jugular vein (Li et al., 1999; Feng et al., 2015). The BP was continuously recorded in a BIOPAC system (DA100C, BIOPAC system, United States) at a sampling rate of 1 KHz. From recordings we were able to estimate systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure (PP = SBP–DBP) and mean arterial blood pressure (MABP = 1/3 of SBP C 2/3 of DBP). In addition, the heart rate (HR) was derived from dP/dt signal obtained from the BP recordings (Del Rio et al., 2016; Andrade et al., 2017).
3.2.4 Baroreflex Control
The BR was evaluated by repeated bolus injections (0.1 ml) of graded doses of sodium nitroprusside (0.4, 0.8, 1.6, 3.2, 6.4, 12.8, and 25.6 mg/kg; Sigma-Aldrich, United States) and phenylephrine (0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 mg/kg; Sigma- Aldrich, United States). These drugs were used to induce a decrease or increase in BP, respectively. Sodium nitroprusside and phenylephrine injections were given in a random order and subsequent injections were not given until the recorded parameters
had returned to pre-injection levels. The cardiac BR function was analyzed using a logistic regression over the entire pressure range (Negrão et al., 1993; Michelini et al., 2003). Data was fit to the equation:
HR = A/[1Cexp{B(MAP-C)}]CD, where A is HR range; B is the slope coefficient; C is the pressure at the midpoint of the range (midpoint BP); and D is the minimum HR. The peak slope (maximum gain) was determined by the first derivative of the baroreflex curve and was calculated with the equation: Gain = A(1) A(2) [1/4], where A(1) is the range and A(2) is the average slope. The mean values for each curve parameter were used to derive composite curves for each group of rats.
3.2.5 Dose-Responses Analysis to BR
Stimulation To determine whether the effects of High-Altitude in BR control could be associated to differences in BP stimulus, we constructed a dose-response curve for SNP and Phe. We used 8 doses of SNP (concentration: 0.0512 mg/mL; at 0.1; 0.2; 0.4; 0.8; 1.6; 3.2; 6.4; and 12.8 mL/kg) and 8 doses of Phe (concentration: 0.1024 mg/mL; at 0.2; 0.4; 0.8; 1.6; 3.2; 6.4; 12.8; 25.6 mL/kg). The curve was constructed using the logarithm of different doses. The responses were estimated using the delta of MABP (1MABP) from previous baseline measurements.
3.2.6 Autonomic Control
Heart rate variability (HRV) was used as an indirect measurement of autonomic balance of the heart (Del Rio et al., 2016; Andrade et al., 2017). The first derivative of the BP (Dp/dt) signal was used to calculate the HR. Autoregressive algorithm, after Hann windowing with 50% overlap, was used to obtain power spectral density of HRV. Cut-off frequencies were defined as low frequency (LFHRV):
0.04–0.6 Hz and high frequency (HFHRV) 0.6–2.4 Hz (Andrade et al., 2017). Additionally, we used LF/HFHRV ratio as an indicator of autonomic balance of the heart. LFHRV and HFHRV were expressed as normalized units (n.u.). Analysis was performed within a 10 min window. This analysis was performed in LabChart 7.3.8 HRV module software (ADInstruments, Bella Vista, NSW, Australia). In addition, to estimate the autonomic contribution on BR function, spectral non-stationary analysis was used (2-s resolution). The HFHRV component (0.6–2.4 Hz) was used as an indicator of parasympathetic modulation. This analysis was performed with Kubios HRV Premium Software V 3.1 (Kubios, Finlandia).
3.2.7 Statistical Analysis
Data were expressed as mean ± standard error of the mean. All data were subjected to Shapiro-Wilk normality test. The unpaired t-test at two tails was employed to compare the differences between groups. p < 0.05 was considered statistically significant. Statistical analyses were performed by GraphPad Prism 8.0 (GraphPad software Inc., San Diego, CA, United States).
3.3 Research III 3.3.1 Ethics Statements
Protocols were approved by the Ethical Committee of the Universidad Mayor (Approval number
#169_2019) and were performed according to the standards set by the latest version of the Declaration of Helsinki. Participants were carefully informed about the experimental procedures, and the possible risks and benefits associated with their participation in the study. Thereafter, written informed assent and consent were obtained from the parents of under-age athletes and from adult athletes, respectively.
3.3.2 Subjects
Fifteen young trained regional- to national-level competitive swimmers [eight males and seven females; age, 20.93 ± 5.18 years; height, 169.53 ± 10.44 cm; body mass, 71.5 ± 12.77 kg; body mass index (BMI), 24.7 ± 2.05 kg/m2], with 5–12 years of swimming training, and a mean weekly training volume of ± 4 h per day, five times per week, participated in this study. Twenty-seven controls (22 males and five females; age, 17.22 ± 2.42 years; height, 169.52 ± 8.12 cm; body mass, 63.85 ± 10.3 kg; BMI, 22.12 ± 2.49 kg/m2) also volunteered to participate in this study. All females were assessed (by a female technician) during the early follicular phase of their menstrual cycle. Experiments were conducted between 08:00 and 17:00 h. Forty-eight hours before experiments participants, were asked to avoid consumption of alcohol, cigarettes, caffeine, or drugs that may alter autonomic control. None of the participants were taking any medication or had a personal or family history of any cardiac, ventilatory or endocrine disorder.
3.3.3 Experimental Design
A descriptive cross-sectional study was performed to determine HVR and autonomic response to hypoxia and maximum voluntary apnea in young highly trained swimmers compared to controls. The inclusion criteria were: (i) high-performance swimmers with less than 12 years of training; (ii) from national or university teams, active participants in national or international competitions; and a minimum of 20 h of training per week. Exclusion criteria were: (i) potential medical problems or history of cardiorespiratory diseases; (ii) any cardiovascular or respiratory surgery in the past 2 years;
(iii) autonomic control impairment at rest, estimated by heart rate variability (HRV) disturbances (low to high frequency ratio of HRV < 2.3) (Nunan et al., 2010); (iv) being in the course of an acute illness or consumption of any drug or pharmacological ergogenic aid and (v) history of chronic obstructive or restrictive pulmonary diseases and/or altered spirometry on the day of the testing session, including (vi) forced expiratory volume at first second (FEV1)/vital capacity (VC) < 70, (vii) FEV1 < 80% of predicted value, or (viii) VC < 80% of predicted value. On the first day, body mass, height, HR, clinical spirometry [VC; peak expiratory flow (PEF); FEV1; FEV1/VC; forced expiratory flow at 25% (FEF 25), 50% (FEF 50), and 75% (FEF 75) of VC] and resting metabolic ratio were measured.
Body mass was estimated to the nearest 0.1 kg using a digital scale (BF-350, Tanita, IL, United States). Height was measured using a wall-mounted stadiometer (HR-200, Tanita, Japan) and recorded to the nearest 0.1 cm. The BMI was calculated as kg/m2. On the second day, the participants were instrumented and positioned in the supine position, at an ambient temperature of ~22ºC.
Instrumentation includes: 3-leads electrocardiogram (ECG), core temperature regulator, oxygen saturation using a pulse oximeter measured on the index or thumb finger (SpO2) (BK-PO2, BIOBASE, China) and an orofacial mask (Hans Rudolph3, 3700A, Kansas City, MO, United States) connected to a gas mixing chamber to measure airflow and expired gases. From respiratory flow, tidal volume (VT) was calculated (FE141, ADInstruments Inc., New Zealand). The expired fraction of
end-tidal carbon dioxide (FECO2), end-tidal oxygen (FEO2) levels, and fraction inspired of O2 (FIO2) were measured with CO2 and O2 gas analyzers (ML206, ADInstruments Inc., New Zealand).
After instrumenting the subjects, they were given a 15-min rest period in supine position before the recording started. After this period, baseline parameters were recorded for 20 min. In addition, they were instrumented with eye mask and headphones to reduce external noise (MPA 101, Masprot, Chile) to blunt the effect of manipulation of gases on participants’ arousal. Ventilatory data, pulse oximeter, and gas exchanges were digitized using PowerLab Data Acquisition System (PowerLab, 16SP, ADInstruments Inc., New Zealand) and analyzed with LabChart 8.0 (ADInstruments Inc., New Zealand).
Figure 2. Timeline and design of the experiment. Fifteen young swimmers and twenty-seven control subjects were enrolled to participate in this study. Measurements were performed in two days.
At day 1, baseline parameters (height, weight, anthropometric, resting metabolic rate and clinical spirometry were performed. At day 2, respiratory, autonomic, cardiovascular parameters, hypoxic ventilatory response (HVR) and cardiac hypoxic response (CHR), and a maximum apnea duration test were estimated.
3.3.4 Hypoxic Ventilatory Response (HVR) and Maximum Voluntary Apnea Test
Hypoxic ventilatory response was evaluated by a poikilocapnic transient hypoxic challenge similar to the one previously described (Pfoh et al., 2016). Participants underwent three consecutive trials (each trial was separate by 5 min) that consisted of five-breaths of 100% N2. N2 was blended into a port on the mask through N2 tubing. After the application of N2, 15 min elapsed until ventilatory parameters returned to baseline levels. Afterward, the subjects changed from a supine to a sitting position, and they were instructed to perform a maximum voluntary apnea (after a maximum inspiration). This experiment was performed with a PowerLab Data Acquisition System (PowerLab, 16SP, ADInstruments Inc., New Zealand).
3.3.5 Cardiac Hypoxic Response (CHR)
Cardiac hypoxic response was assessed by the same stimulus of HVR, similar as in previous experiments (Richalet et al., 2012; Bourdillon et al., 2014). Briefly, three transient tests of five breaths at 100% N2 were applied with 5 min of rest between trials. The highest HR response from these trials was used. To determine the CHR, 1 min of rest in normoxia and 1 min of maximum HR response in
hypoxia were used for the analysis. Thus, CHR was calculated as follows: resting HR – maximum HR/SpO2 in normoxia – SpO2 in hypoxia (= 1HR/1SpO2) (Richalet et al., 2012; Bourdillon et al., 2014).
3.3.6 Electrocardiogram (ECG)
The 3-leads ECG was recorded using a bio-amplifier connected to a digital recording system (PowerLab, 16SP, ADInstruments Inc., New Zealand). The electrodes (3M, Saint Paul, MN, United States) were placed in second derivative (DII) from Einthoven triangle with participants in a supine position. The ECG was recorded continuously, along with breathing gases and ventilation in all experiments considering peripheral chemoreflex test and maximum voluntary apnea test. The sampling frequency was set at 4 kHz and was amplified x100. The ECG analysis was performed with LabChart software v.8 (ADInstruments Inc., New Zealand).
3.3.7 Heart Rate Variability (HRV)
The HRV was assessed as an indirect measure of autonomic balance of the heart (Camm, 1996). From the 3-lead ECG recording, time series were obtained from R-R interval and a time-varying spectrogram was used to obtain the power spectral density (PSD) of HRV (2 s resolution, Tarvainen et al., 2014). Cut-off frequencies were defined as very low frequency [Very low frequency (VLFHRV; 0.00–0.04 Hz), low frequency (LFHRV; 0.04–0.10 Hz), and high frequency (HFHRV;
0.10–0.40 Hz) (Yuda et al., 2018)]. Additionally, we used the LF/HFHRV ratio as an indicator of global autonomic balance of the heart. The LFHRV and HFHRV were expressed as normalized units (n.u.), calculated as follow: LF n.u. = LF power/(total power – VLF); and HF n.u. = HF power/(total power – VLF) (Camm, 1996); thereafter the area under curve (AUC) of the total responses was calculated, as previously described (Andrade et al., 2020). The baseline values and the maximum apnea event were analyzed using Kubios HRV Premium Software v3.1 (Kubios, Kuopio, Finland).
3.3.8 Resting Metabolic Rate
Resting metabolic rate (RMR) was measured by indirect calorimetry as previously described (Speakman and Selman, 2003). All participants were instructed to minimize movement after waking- up and to avoid vigorous exercise before the implementation of the calorimetry (Speakman and Selman, 2003; Compher et al., 2006; Nieman et al., 2006). Participants underwent RMR evaluation between 8:00 and 10:00 am. During RMR measurement participants breathed through an oronasal mask (7450 Series Silicone V2, Hans Rudolph, Kansas City, MO, United States) for expired gas collection and analysis (Quark CPET metabolic cart; COSMED, Rome, Italy). Every three measurements the metabolic cart was re-calibrated with a known calibration gas (O2 15%, CO2 5%, N2 balanced) (Nieman et al., 2013). The RMR measurement was performed in a specially conditioned room isolated from noise, at a temperature of 23ºC and 50% of humidity. Before the measurement, the participants rested for 30 min. The subjects were instrumented and placed in a supine position during the 40 min of measurements. From the total recording, the first 5-min were discarded as part of the acclimatization period. The calculation of respiratory quotient (RQ), protein, carbohydrates and lipids oxidation were obtained from the remaining 35 min. Protein, carbohydrates and lipids oxidation, were expressed as kcal/day and as % of total resting metabolic rate. The recording and analysis were performed with OMNIA, Cardiopulmonary Diagnostic Suite v 1.4 (Quark CPET metabolic cart; COSMED, Rome, Italy).
