Education, Faculty of
Kinesiology, School of
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UBCV
Payne, Owen
Master of Science - MSc
University of British Columbia
The magnitude of exercise-induced diaphragmatic fatigue (EIDF) is similar in males irrespective of aerobic fitness. When compared to aerobically fit males, aerobically fit females appear less susceptible to EIDF. We hypothesized that chronic endurance training and regular exposure to high levels of exercise hyperpnea may confer EIDF resistance in aerobically fit females when compared to healthy females with average aerobic fitness. Healthy females (25 ± 4 years) performed graded cycle exercise to exhaustion to characterize aerobic fitness and were assigned into a highly-aerobically fit (Hi-Fit) or averagely-aerobically fit (Avg-Fit) group (Hi-Fit: n = 9, V̇O₂max = 56.1 ± 3.4 mL/kg/min; Avg-Fit: n = 9, V̇O₂max = 35.7 ± 4.9, p < 0.001). Participants then completed high intensity (~90% V̇O₂max) cycling exercise (Hi-Fit: 257 ± 29 W, Avg-Fit: 135 ±10 W, p < 0.001) to exhaustion. Changes in transdiaphragmatic twitch pressure (Pdi,tw), evoked by cervical magnetic stimulation (CMS), were used to assess EIDF between baseline and 5-, 30-, and 60-minutes after exercise cessation. Time to exhaustion (TTE) was similar between groups (Hi-Fit: 546 ± 178 seconds; Avg-Fit: 559 ±175 seconds, p = 0.876). The reduction in Pdi,tw relative to baseline at 5-, 30-, and 60-minutes after exercise cessation was 82.5 ± 12.0, 93.9 ± 9.8, 97.5 ± 5.6% and 87.8 ± 8.5, 97.7 ± 6.9, 101.8 ± 5.0%, in the Hi-Fit and Avg-Fit groups, respectively. There was no main effect of fitness on the exercise-induced fall in Pdi,tw or during the subsequent recovery (p = 0.186). We conclude that aerobic fitness does not affect the relative magnitude of EIDF in females. Similar to what has been observed in aerobically fit males, chronic endurance training and its concomitant hyperpnea do not appear to provide additional EIDF protection in females.
The Effect of Aerobic Fitness on Exercise-Induced Diaphragmatic Fatigue in Females by Owen Payne B.Sc., Queen’s University, 2021 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Kinesiology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2023 © Owen Payne, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: The Effect of Aerobic Fitness on Exercise-Induced Diaphragmatic Fatigue in Females submitted by Owen Payne in partial fulfilment of the requirements for the degree of Master of Science in Kinesiology Examining Committee: Dr. William Sheel, Professor, Kinesiology, UBC Supervisor Dr. Donald McKenzie, Professor Emeritus, Medicine, UBC Supervisory Committee Member Dr. Jeremy Road, Professor Emeritus, Medicine, UBC Supervisory Committee Member iii Abstract The magnitude of exercise-induced diaphragmatic fatigue (EIDF) is similar in males irrespective of aerobic fitness. When compared to aerobically fit males, aerobically fit females appear less susceptible to EIDF. We hypothesized that chronic endurance training and regular exposure to high levels of exercise hyperpnea may confer EIDF resistance in aerobically fit females when compared to healthy females with average aerobic fitness. Healthy females (25  4 years) performed graded cycle exercise to exhaustion to characterize aerobic fitness and were assigned into a highly-aerobically fit (Hi-Fit) or averagely-aerobically fit (Avg-Fit) group (Hi-Fit: n = 9, V̇O2max = 56.1  3.4 mL/kg/min; Avg-Fit: n = 9, V̇O2max = 35.7  4.9, p < 0.001). Participants then completed high intensity (~90% V̇O2max) cycling exercise (Hi-Fit: 257  29 W, Avg-Fit: 135  10 W, p < 0.001) to exhaustion. Changes in transdiaphragmatic twitch pressure (Pdi,tw), evoked by cervical magnetic stimulation (CMS), were used to assess EIDF between baseline and 5-, 30-, and 60-minutes after exercise cessation. Time to exhaustion (TTE) was similar between groups (Hi-Fit: 546  178 seconds; Avg-Fit: 559  175 seconds, p = 0.876). The reduction in Pdi,tw relative to baseline at 5-, 30-, and 60-minutes after exercise cessation was 82.5  12.0, 93.9  9.8, 97.5  5.6% and 87.8  8.5, 97.7  6.9, 101.8  5.0%, in the Hi-Fit and Avg-Fit groups, respectively. There was no main effect of fitness on the exercise-induced fall in Pdi,tw or during the subsequent recovery (p = 0.186). We conclude that aerobic fitness does not affect the relative magnitude of EIDF in females. Similar to what has been observed in aerobically fit males, chronic endurance training and its concomitant hyperpnea do not appear to provide additional EIDF protection in females. iv Lay Summary The diaphragm, like other skeletal muscles, fatigues following heavy use. Fatigue of the diaphragm may limit whole-body exercise capacity. Females tend to have a less fatigable diaphragm than males. Previous studies which have identified that females have a less fatigable diaphragm have used females that are very aerobically fit. The purpose of this thesis is to determine if diaphragmatic fatigue is affected by aerobic fitness in females. The results suggest that aerobic fitness is not a major factor affecting the development of diaphragmatic fatigue in females. v Preface I, Owen Payne designed the experiment discussed in this thesis under the guidance of Doctors William Sheel, Jeremy Road, Don McKenzie, and Jamie Burr. I recruited participants, scheduled experimental dates, and performed data collection with assistance from Katelyn Cotterell, Jules Clark, and Mick Leahy. Data analysis was performed by me; interpretation of the data was aided by all the members of the Health and Integrative Physiology Laboratory. The experiment contained in this thesis was approved by the University of British Columbia’s Research Ethics Board (H22-00157). vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi List of Abbreviations .................................................................................................................. xii Acknowledgements .................................................................................................................... xiv Dedication .....................................................................................................................................xv Chapter 1: Introduction ................................................................................................................1 1.1 Review of Literature ....................................................................................................... 2 1.1.1 Defining Fatigue ......................................................................................................... 2 1.1.1.1 Peripheral Sources of Performance Fatigability ................................................. 3 1.1.1.2 Metabolic Contributions to Performance Fatigability ........................................ 4 1.1.1.3 Central Sources of Performance Fatigability ...................................................... 6 1.1.2 Factors Contributing to the Development of EIDF..................................................... 7 1.1.3 Consequences of Diaphragmatic Fatigue.................................................................. 11 1.1.4 Assessment of Diaphragmatic Fatigue...................................................................... 12 1.1.5 Aerobic Fitness, The Respiratory System, and Diaphragmatic Fatigue ................... 15 1.1.5.1 The Static Respiratory System .......................................................................... 15 1.1.5.2 Ventilatory Cost of High Aerobic Fitness ........................................................ 16 1.1.5.3 Aerobic Fitness and Diaphragmatic Fatigue ..................................................... 17 vii 1.1.6 The Effect of Sex on the Respiratory System and Diaphragmatic Fatigue .............. 18 1.1.6.1 The Effect of Sex on Respiratory Structure ...................................................... 18 1.1.6.2 Sex Differences and the Mechanical Cost of Hyperpnea ................................. 20 1.1.6.3 Sex Differences and Diaphragmatic Fatigue .................................................... 21 1.2 Summary and Rationale ................................................................................................ 23 1.3 Purpose .......................................................................................................................... 24 1.4 Hypothesis..................................................................................................................... 24 Chapter 2: Methodology..............................................................................................................25 2.1 Ethical Approval ........................................................................................................... 25 2.2 Participants .................................................................................................................... 25 2.3 Experimental Overview ................................................................................................ 25 2.4 Pulmonary Function ...................................................................................................... 26 2.5 Maximal Ramp Cycle Test ........................................................................................... 26 2.6 Constant Load Exercise Test Familiarization ............................................................... 27 2.7 Constant Load Exercise Test to Exhaustion ................................................................. 27 2.8 Metabolic and Cardiorespiratory Measurement............................................................ 29 2.9 Pulmonary Pressure Measurement ................................................................................ 29 2.10 Surface Electromygraphy of the Diaphragm ................................................................ 30 2.11 Cervical Magnetic Stimulation ..................................................................................... 30 2.12 Cervical Magnetic Stimulation Ramp ........................................................................... 31 2.13 Diaphragmatic Fatigue Assessment .............................................................................. 31 2.14 Data Sampling and Recording ...................................................................................... 34 2.15 Data Analysis ................................................................................................................ 34 viii 2.16 Statistical Analysis ........................................................................................................ 34 Chapter 3: Results........................................................................................................................36 3.1 Subjects ......................................................................................................................... 36 3.2 Maximal Cycle Exercise ............................................................................................... 36 3.3 Constant Load Exercise Test to Exhaustion ................................................................. 38 3.4 Diaphragmatic Function................................................................................................ 43 3.4.1 Supramaximal Stimulation........................................................................................ 43 3.4.2 Diaphragmatic Fatigue .............................................................................................. 43 3.5 Breathing Mechanics .................................................................................................... 51 Chapter 4: Discussion ..................................................................................................................58 4.1 Main Findings ............................................................................................................... 58 4.2 EIDF and Aerobic Fitness ............................................................................................. 59 4.3 Ventilatory Response and Mechanical Cost ................................................................. 61 4.4 Aerobic Fitness and Respiratory Mechanics................................................................. 63 4.5 Diaphragmatic Fatigue and Absolute Diaphragmatic Load .......................................... 65 4.6 Diaphragmatic Intramuscular Adaptions ...................................................................... 67 4.7 Methodological Considerations .................................................................................... 70 4.8 Unresolved Questions and Future Directions ............................................................... 72 Chapter 5: Conclusion .................................................................................................................74 Bibliography .................................................................................................................................75 Appendices ....................................................................................................................................93 Appendix A Forms and Questionnaires .................................................................................... 93 A.1 Consent Form ............................................................................................................ 93 ix A.2 Medical History Form ............................................................................................. 102 A.3 Data Collection Form .............................................................................................. 103 A.4 Par-Q+ ..................................................................................................................... 105 A.5 Physical Activity and Sedentary Behaviour Questionnaire .................................... 109 x List of Tables Table 3.1 Descriptive Characteristics of the Participants ............................................................. 37 Table 3.2 Maximal Exercise Values ............................................................................................. 38 Table 3.3 Representative Participants ........................................................................................... 41 Table 3.4 Cardiorespiratory Data During the Constant Load Exercise Test to Exhaustion ......... 42 Table 3.5 Absolute and Relative Pdi,tw .......................................................................................... 46 Table 3.6 Potentiated Twitch Control Measures........................................................................... 57 xi List of Figures Figure 2.1 Exercise Prescription Illustration ................................................................................ 28 Figure 2.2 Twitch Potentiation Protocol ....................................................................................... 32 Figure 2.3 The Response of the Diaphragm to Phrenic Nerve Stimulation.................................. 33 Figure 3.1 Ratio of V̇O2 During the Constant Load Exercise Test to V̇O2max .............................. 40 Figure 3.2 Illustration of Potentiated Twitches............................................................................. 44 Figure 3.3 CMS Recruitment Curves............................................................................................ 45 Figure 3.4 Relative and Absolute Pdi,tw ......................................................................................... 47 Figure 3.5 Relationship Between EIDF and Aerobic Fitness ....................................................... 48 Figure 3.6 Relationship between EIDF and Exercise Time .......................................................... 49 Figure 3.7 Relationship Between EIDF and Respiratory Mechanics ........................................... 50 Figure 3.8 WB versus V̇E for All Participants During the Constant Load Exercise Test .............. 52 Figure 3.9 WB and Components of WB During the Constant Load Exercise Test ....................... 53 Figure 3.10 Rate of Pressure Produced During the Constant Load Exercise Test ....................... 54 Figure 3.11 Cumulative Mechanical Load at Exercise Cessation ................................................ 55 Figure 3.12 Relationship between Cumulative Diaphragmatic Work, the Rate of Diaphragmatic Pressure Production, and Time ..................................................................................................... 56 xii List of Abbreviations ADP Adenosine Diphosphate ANOVA Analysis of Variance ATP Adenosine Triphosphate Avg-Fit Averagely-Aerobically Fit Group in the Present Thesis BMI Body Mass Index CMS Cervical Magnetic Stimulation DLCO Diffusion Capacity of Carbon Monoxide EFL Expiratory Flow Limitation EIDF Exercise-Induced Diaphragmatic Fatigue EMG Electromyography fB Breathing Frequency FEF25-75% Forced Expiratory Flow Between 25 and 75% of FVC FEV1 Fraction of Expired Volume in 1-second FVC Full Vital Capacity Hi-Fit Highly-Aerobically Fit Group in the Present Thesis HR Heart Rate LHD Left Hemi-Diaphragm M-wave Compound Muscle Action Potential Peso Esophageal Pressure Pdi Transdiaphragmatic Pressure Pdi,tw Transdiaphragmatic Twitch Pressure PETCO2 Pressure of End-Tidal CO2 xiii PTPeso Pressure Time Product of the Esophagus PTPdi Pressure Time Product of the Diaphragm RER Respiratory Exchange Ratio RHD Right Hemi-Diaphragm SD Standard Deviation TLC Total Lung Capacity TTE Time to Exhaustion VA Alveolar Volume VT Tidal Volume V̇A Alveolar Ventilation V̇E Minute Ventilation V̇Emax Maximum Minute Ventilation V̇CO2 Carbon Dioxide Production V̇O2 Oxygen Uptake V̇O2max Maximum Oxygen Uptake V̇O2rm Respiratory Muscle Oxygen Uptake WB Work of Breathing xiv Acknowledgements This project would not have been possible without the willingness of Dr. Sheel to take me, a very inexperienced undergraduate student, on as a Master’s student. For your willingness then and your continued support throughout the entire process, I am profoundly grateful. To my committee members Dr. McKenzie and Dr. Road, your expertise on this topic has been instrumental in the completion of the project. I appreciate your thoughtful questions and your kind words of encouragement. Thank you. While not a part of the project when we first started, I would like to thank Dr. Burr. The contributions that you made to this process cannot be understated. Your enthusiasm for science is infectious. To all the students who spent time in Chan Gunn and Med Block C, past and present, it was an honour to get to spend my days working with such an excellent group of people. I appreciate the support and guidance that every single person gave to me. I look forward to one day getting to work with you all again. xv Dedication For Christina, Greg, and Shannon. Provinces apart, together in heart. 1 Chapter 1: Introduction High-intensity exercise to exhaustion (>80-85% V̇O2max) has been demonstrated to temporarily reduce the pressure producing capacity of the diaphragm, this reduction has been termed exercise-induced diaphragmatic fatigue (EIDF) (Johnson et al., 1993) The onset of EIDF is temporal (Archiza et al., 2018) and is likely caused by the combined effects of exercise hyperpnea (Babcock et al., 1995), uptake of circulating metabolites (Fregosi & Dempsey, 1986), and blood flow competition between the respiratory and locomotor muscles (Romer & Polkey, 2008). While EIDF does not limit alveolar ventilation (V̇A) during exercise, it is believed to contribute to exercise intolerance due to its association with activation of the respiratory muscle metaboreflex (Sheel et al., 2018) and the sensation of dyspnea (Boyle et al., 2020). Sex has been identified as a possible determinant of EIDF. Females, despite greater respiratory work for a given minute ventilation (V̇E) during intense exercise (Dominelli et al., 2015), appear to have a reduced magnitude and incidence of EIDF (Guenette et al., 2010; Ramsook et al., 2022; Welch et al., 2018a). Sex differences in skeletal muscle fatigability (Hunter, 2015) and accessory inspiratory muscle activity during exercise (Guenette et al., 2010; Mitchell et al., 2018) may contribute to the lower incidence of EIDF in females. Aerobic fitness may also be associated with EIDF. For example, during intense exercise highly-aerobically fit individuals are faced with ventilatory demands which encroach on the capacity of the respiratory system incurring a large mechanical cost (Johnson et al., 1992), suggesting a greater susceptibility to EIDF. Contrarily, regular exposure to intense exercise and its concomitant hyperpnea may result in EIDF resistance via training induced adaptations to the diaphragm. The lone study to investigate the relationship between aerobic fitness and EIDF was done by Babcock et al. (1996) where they observed no difference in EIDF between two groups stratified by aerobic 2 fitness. However, the population investigated in this study was almost exclusively male (20 males, 4 females). Presently, no study has investigated if aerobic fitness affects the incidence of EIDF in females. There is reason to believe that aerobic fitness may protect against EIDF in females. The first studies demonstrating sex differences in EIDF showed that, aerobically fit females (V̇O2max > 50 mL/kg/min) experience less EIDF than males of similar fitness (Guenette et al., 2010; Welch et al., 2018a). It is possible that the observed difference is not only due to sex, but an interaction between sex and fitness; EIDF may be mitigated in highly-aerobically fit females but sex differences in EIDF are contradictory: one study reported no sex difference (Reinhard et al., 2023) while another reported a reduced incidence in females (Ramsook et al., 2022). Therefore, the purpose of this thesis is to determine if aerobic fitness affects EIDF in females. Knowledge of the relationship between EIDF and aerobic fitness in females will advance the current understanding of the factors that affect EIDF and will improve the interpretation of the sex differences in EIDF observed previously. 1.1 Review of Literature 1.1.1 Defining Fatigue Fatigue can be defined as a disabling symptom which limits cognitive and physical performance due to underlying physiological and psychological factors (Enoka & Duchateau, 2016). Within the definition of fatigue there are two attributes which determine the onset and magnitude fatigue: perceived fatigability and performance fatigability, (Enoka & Duchateau, 2016). Perceived fatigability is defined as the changes in sensations that regulate the ability of an individual to perform a task (Enoka & Duchateau, 2016). Performance fatigability, also referred to as muscle fatigue, can be defined as an acute exercise-induced reduction in the force 3 generating capacity of the muscles involved in exercise performance (Enoka & Duchateau, 2016; Hunter, 2018). Distinct from neuromuscular injury and weakness, performance fatigability is reversible with relatively brief rest (NHLBI, 1990). Performance fatigability is not a phenomenon localized within one aspect of the neuromuscular system. Instead, various sites throughout the neuromuscular system can be responsible for performance fatigability. Sites of performance fatigability can be divided into central and peripheral sources. Central sources of performance fatigability refer to mechanisms prior to the neuromuscular junction (Gandevia, 2001). Whereas peripheral sources of performance fatigability occur at or distal to the neuromuscular junction and encompass mechanisms responsible for contractile impairment, despite no reduction in neural drive (Gandevia, 2001). For the purposes of this thesis, diaphragmatic fatigue refers to performance fatigability of the diaphragm, and EIDF refers to performance fatigability of the diaphragm caused by whole-body exercise. 1.1.1.1 Peripheral Sources of Performance Fatigability The predominate reason that causes a muscle to lose contractile function, depends on the type of task being performed (Bigland-Ritchie et al., 1995). There are two unique types of performance fatigability in the peripheral muscle, high-frequency performance fatigability and low-frequency performance fatigability. High-frequency performance fatigability occurs after high-frequency stimulation of a motor unit and is rapidly reversed when stimulation frequency is reduced (Jones, 1996). Alterations in sarcolemma excitability is the likely causative factor of high-frequency performance fatigability (Jones, 1996). Sarcolemma excitability is the ability of the muscle 4 membrane to respond to and propagate an action potential from a motor neuron (Rodriguez-Falces & Place, 2018). Action potentials propagate from the sarcolemma and into the muscle cell via the t-tubules, where the electric potential causes Ca2+ release from the sarcoplasmic reticulum (Allen et al., 2008). For example, external high-frequency muscle stimulation in vivo has indicated that changes in sarcolemma excitability, related to intramyocellular efflux of K+, can cause performance fatigability (Allen et al., 2008). Impaired sarcolemma excitability may contribute in part to performance fatigability following high-intensity exercise in humans, indicated by the observation that the total rectified area of the compound muscle action potential (M-wave) in a fatigued muscle is commonly reduced, but the relative importance is unclear (Kent-Braun et al., 2012). Low-frequency performance fatigability is elicited by much lower stimulation frequencies and is characterized by a reduction in the force generating capacity of a muscle which recovers over a much longer time course, hours to days (Jones, 1996). Low-frequency performance fatigability can be mediated by stressed induced changes in the physical property of the sarcomere (Jones, 1996). Stress induced changes are most apparent following eccentric and isometric loading (Jones, 1996). Another mechanism which has been shown to cause low-frequency performance fatigability is the accumulation of intramuscular metabolites. 1.1.1.2 Metabolic Contributions to Performance Fatigability As previously stated, performance fatigability is task specific (Bigland-Ritchie et al., 1995). Factors which determine task specificity of performance fatigability are the type of contraction, specific muscles performing the contraction, and intensity of the contraction (Bigland-Ritchie et al., 1995). Because of the discrepancy in intensity, the predominate mechanisms that cause performance fatigability during short-duration high-intensity exercise are 5 distinct from the mechanisms of prolonged submaximal exercise (Fitts, 1994). Prolonged-submaximal exercise causes performance fatigability primarily through depletion of intramuscular glycogen stores (Green, 1997); this is not the case in short-duration high-intensity exercise (Green, 1997). Short-duration high-intensity dynamic exercise requires greater contractile forces and peak energy requirements (Fitts, 1994). To meet the force demand, high-threshold motor units composed of type II muscle fibres are recruited (Green, 1997). Type II muscle fibres rely heavily on anaerobic metabolism. Furthermore, when aerobic ATP supply cannot match ATP demand within a muscle fibre, its reliance on anaerobic ATP supply increases (Allen et al., 2008). Inadequate aerobic ATP supply is of particular importance when oxygen supply to the working muscle is reduced, such as during ischemia (Kent-Braun et al., 2012). Because high-intensity short-duration exercise elevates rates of ATP hydrolysis and relies heavily on anaerobic metabolism, concentrations of intramuscular ADP, Pi, and H+ increase (Fitts, 1994). Elevated concentrations of ADP, Pi, and H+ exert significant effects on contractile function, which gradually dissipates following exercise cessation as metabolite concentrations return to baseline (Jones, 1996). Both excitation-contraction coupling (Kent-Braun et al., 2012) and actin-myosin interactions (Green, 1997) are directly impaired by intramuscular metabolite accumulation. Excitation-contraction coupling is the process which connects sarcolemma excitation to the development of muscle tension (Sandow, 1952). Excitation-contraction coupling includes the transition from a t-tubule action potential to Ca2+ release from the sarcoplasmic reticulum, the subsequent binding of Ca2+ to troponin, and the uptake of Ca2+ into the sarcoplasmic reticulum (Kent-Braun et al., 2012). Fatigued muscle cells experience a reduction in sarcoplasmic reticulum Ca2+ release despite normal t-tubular charge (Györke, 1993). Pi is proposed to limit 6 available intramyocellular Ca2+ through two mechanisms, impairment of Ca2+ release from the sarcoplasmic reticulum and the formation of a precipitate with Ca2+ (Allen et al., 2008). Another contributing factor to impaired excitation-contraction coupling is an elevation in concentrations of ADP and Pi, which inhibit the sarcoplasmic reticulum Ca2+ pump, reducing Ca2+ uptake (Kent-Braun et al., 2012). Furthermore, elevated Pi and reduced pH impair myofibrillar Ca2+ sensitivity, this further inhibits force production (Allen et al., 2008). Impaired myofibrillar Ca2+ sensitivity is particularly important when Ca2+ release is impeded (Allen et al., 2008; Kent-Braun et al., 2012). The cross-bridge cycle is the force producing mechanism of the neuromuscular system (Fitts, 2008). Myosin heads, energized through ATP hydrolysis, connect to actin filaments causing the sarcomere to shorten or resist lengthening, therefore producing force. Initially, myosin binds weakly to actin before transitioning to the strongly bound state (Allen et al., 2008). Elevated concentrations of Pi within the muscle fibre inhibit the transition from taking place, reducing the force generating capacity of the fibre (Allen et al., 2008). H+ also impairs the transition from the weakly to strongly bound state, but through a different mechanism (Kent-Braun et al., 2012). As a result, there are less cross-bridges in the strongly bound state and the force generating capacity of the muscle fibre is reduced. Additionally, elevations in H+ and ADP impair shortening velocity of the muscle fibre (Kent-Braun et al., 2012). Metabolite accumulation within active skeletal muscle inhibits multiple steps required for muscular contraction making it an important causative factor in performance fatigability. 1.1.1.3 Central Sources of Performance Fatigability Central sources of performance fatigability are characterized by a progressive reduction in the voluntary activation of a muscle during exercise (Gandevia, 2001). Observations that a 7 supramaximal stimulation of a fatigued muscle causes an increase in force production during a maximum voluntary contraction of the fatigued muscle suggest that there is a central contribution to performance fatigability (Bellemare & Bigland-Ritchie, 1987). The described technique, called twitch-interpolation, is the primary method used to assess central contributions to performance fatigability (Gandevia, 2001). Possible sites of performance fatigability have been identified in supraspinal and spinal locations, however the relative importance of these sites is unclear (Gandevia, 2001). Central contributions to performance fatigability will not be examined in the remainder of this thesis. 1.1.2 Factors Contributing to the Development of EIDF The development of performance fatigability is dependent on the balance between muscular energetic demands and supply (Monod & Scherrer, 1965). When the energetic demands of the diaphragm exceed supply progressive development of diaphragmatic fatigue will occur (Roussos et al., 1979). To properly discuss the energetic demands of the respiratory muscles the measure work of breathing (WB) must be defined. “WB is the mechanical work necessary to achieve a given V̇E with a given breathing pattern” (Dominelli et al., 2019). WB is largely determined by its elastic and resistive components (Otis et al., 1950) The elastic component refers to the work needed to overcome elastic resistance in the lung and chest wall (Roussos & Campbell, 1986). The resistive component refers to the work needed to overcome airway resistance to flow and the nonelastic deformation of tissue (Roussos & Campbell, 1986). Both inspiration and expiration have elastic and resistive components (Roussos & Campbell, 1986). At rest in the average human, blood gas homeostasis can be maintained with a V̇E of ~7.5 L/min (West & Luks, 2020). The WB required to maintain adequate V̇A is minimal at rest (Otis et 8 al., 1950). As V̇E increases, WB increases exponentially (Otis et al., 1950). V̇E can increase twenty-times greater than resting levels at peak exercise (West & Luks, 2020); so the hyperpnea of high-intensity exercise bares a significant mechanical cost (Dominelli et al., 2015). Furthermore, because WB is linearly related with respiratory muscle oxygen uptake (V̇O2rm) the substantial mechanical cost of exercise hyperpnea is directly reflected in the metabolic demands of the respiratory muscles (Dominelli et al., 2015). The elevation of WB seen during high-intensity dynamic exercise in humans is directly related to the development of EIDF (Archiza et al., 2018). Specifically, the magnitude of EIDF is proportional to the cumulative WB during high-intensity exercise (Archiza et al., 2018). When WB is manipulated experimentally the relationship between WB and EIDF is further illustrated. Babcock et al. (2002), reduced the WB during high-intensity dynamic exercise, using a positive assist ventilator. Following exercise with positive assist ventilation EIDF was eliminated which was attributed to the reduction in WB (Babcock et al., 2002). However, the mechanical demand on the diaphragm cannot solely explain the development of EIDF. Babcock et al. (1995), had participants mimic their ventilatory pattern during high-intensity dynamic exercise while at rest, to examine if exercise hyperpnea alone was adequate to cause EIDF. Even though tidal volume (VT), breathing frequency (fB), diaphragmatic pressure production, and the tension-time index were not significantly different between rest and exercise, participants did not develop diaphragmatic fatigue at rest (Babcock et al., 1995). Interestingly, in participants who completed a ‘supramimic’ protocol, where breath-by-breath diaphragmatic pressure targets were set at 50% greater than those achieved during exercise, diaphragmatic fatigue occurred (Babcock et al., 1995). High intensity dynamic exercise must place an additional stress on the diaphragm (Babcock et al., 1995). 9 The metabolic environment that occurs during high-intensity whole-body exercise may be partially responsible for the additional fatiguing stress. During light and moderate exercise, ATP production can be met almost exclusively through oxidative phosphorylation, limiting the accumulation of metabolites that interfere with contractile function, allowing exercise to be sustained for extended periods of time (Green, 1997). High-intensity exercise occurs once critical power is surpassed (Jones et al., 2010). During high intensity exercise a significant proportion of ATP supply occurs anaerobically, generating metabolites that impair muscular function, therefore limiting exercise tolerance (Green, 1997; Jones et al., 2010; Babcock et al., 1995), proposed that circulating metabolites, including H+ and Pi, which transiently increase during high-intensity exercise, could accumulate in the diaphragm causing EIDF. For example, it has been demonstrated that in rats elevated lactate uptake and accumulation within the diaphragm does occur following exercise (Fregosi & Dempsey, 1986). Whole-body dynamic exercise may also limit the energetic supply to the diaphragm (Babcock et al., 1995). For example, a reduction in blood supply to the diaphragm would decrease aerobic ATP synthesis, increasing metabolites implicated in performance fatigability. Indeed, evidence has shown that experimentally increasing phrenic artery blood flow, partially reverses diaphragmatic fatigue (Supinski et al., 1988). Currently, it is believed that during high-intensity exercise cardiac output is not able to supply both respiratory and active locomotor muscles adequately (Sheel et al., 2018). As a result blood flow competition occurs between the respiratory muscles and locomotor muscles which limits oxygen supply and causes metabolite accumulation within the diaphragm, precipitating EIDF (Sheel et al., 2018). Evidence for blood flow competition between the respiratory muscles and active locomotor muscles during dynamic exercise has been clearly demonstrated. Experimentally reducing the WB using a positive assist 10 ventilator increases blood flow to active locomotor muscles, whereas increasing the WB using resistors, reduces the blood flow to active locomotor muscles (Harms et al., 1997). Dominelli et al. (2017), extended on the findings of Harms et al. (1997) by demonstrating that during exercise respiratory muscle blood flow decreased during positive assist ventilation and increased during resistive breathing, and that these changes corresponded with an increase and decrease in locomotor blood flow, respectively (Dominelli et al., 2017). Blood flow competition is proposed to stem from a sympathetically mediated metaboreflex, arising from type III and IV afferents located in the respiratory muscles (Dempsey et al., 2006). High respiratory muscle work would cause sympathetic restraint of locomotor muscle vascular beds reducing locomotor blood flow (Dempsey et al., 2006). Consequently, during exercise an increase in WB causes the active locomotor muscles to become more fatigable (Dempsey et al., 2006). Blood flow competition is postulated to be bidirectional, meaning that a metaboreflex arising from active locomotor muscles will increase sympathetic restraint within the respiratory muscles, reducing available blood supply to the diaphragm, producing EIDF (Sheel et al., 2018). Further clues behind the factors contributing to EIDF can be gathered by examining the exercise modalities which cause it. EIDF consistently occurs following exercise to exhaustion at intensities >85% V̇O2max (Archiza et al., 2018; Babcock et al., 1995, 1996b; Guenette et al., 2010; Johnson et al., 1993). During exercise to exhaustion at a high intensity both the mechanical demands placed on the respiratory muscles and blood flow competition with locomotor muscles are likely contributing factors in the development of EIDF (Johnson et al., 1993). Furthermore, the duration of high-intensity exercise is a key determinant of the degree of EIDF; the degree of EIDF is progressively reduced as exercise duration is reduced (Archiza et al., 2018). A relevant example to illustrate the importance of exercise duration on the development of EIDF is 11 incremental exercise to exhaustion. Incremental exercise to exhaustion does not consistently elicit EIDF (Levine & Henson, 1988; Romer et al., 2007; Verin et al., 2004). It is possible that during incremental exercise the duration of high-intensity exercise which occurs immediately prior to exhaustion is too short to cause EIDF. 1.1.3 Consequences of Diaphragmatic Fatigue Under normal conditions, in healthy humans, EIDF does not cause respiratory failure. EIDF studies which have measured the pressure of end-tidal carbon dioxide (PETCO2) and oxygen saturation indicate that while EIDF occurred blood gas homeostasis was maintained, indicating that there was no alveolar hypoventilation (Romer & Polkey, 2008). While EIDF does not appear to cause inadequate V̇A, it could still affect exercise tolerance, but through different means. But, it is difficult to isolate the effect of EIDF on exercise tolerance, because EIDF occurs progressively throughout exhaustive exercise (Archiza et al., 2018). Experimental designs which induce diaphragmatic fatigue prior to exercise, using a resisted breathing task or isocapnic hyperventilation, attempt to isolate the effects of diaphragmatic fatigue on exercise tolerance. By comparing time to exhaustion when the diaphragm is pre-fatigued versus a control, the effect of diaphragmatic fatigue can be elucidated. Pre-fatigue studies have had mixed results, with some demonstrating a significant decrease in performance (Mador & Acevedo, 1991; Martin et al., 1982), while others indicate no change (Sliwinski et al., 1996; Spengler et al., 2000). A criticism of many pre-fatigue studies is that diaphragmatic fatigue was measured using voluntary techniques (Romer & Polkey, 2008). However, a recent investigation by Welch et al. (2018b) included an involuntary measurement of diaphragmatic fatigue, along with sex-based comparisons. Prior to exercise, participants performed pressure-threshold loading to elicit diaphragmatic fatigue, which was then assessed 12 using magnetic stimulation of the phrenic nerves. Participants immediately exercised to exhaustion on a cycle ergometer at a predetermined work rate (Welch et al., 2018a). When the same participants did not engage in pressure-threshold loading prior to exercise, they were able to bike for significantly longer at an identical workload. Furthermore, this study demonstrated that the reductions in performance were similar between males and females (Welch et al., 2018a). While diaphragmatic fatigue reduces exercise tolerance, the mechanisms behind the reduction are less clear. One possible mechanism is the effect of diaphragmatic fatigue on blood flow redistribution (Romer & Polkey, 2008). As mentioned, competition between respiratory and locomotor muscles is believed to be mediated by a sympathetic metaboreflex arising from type III and IV afferents within the respective muscular bed (Dempsey et al., 2006; Sheel et al., 2018). An increase in fatigue causing metabolites within the diaphragm could exacerbate blood flow competition through greater stimulation of local III and IV afferents, causing increased sympathetic restriction of locomotor muscles (Romer & Polkey, 2008). EIDF could also increase sensations of dyspnea precipitating a faster time to task failure (Romer & Polkey, 2008). Pre-fatiguing the diaphragm does result in greater feelings of breathlessness (Welch et al., 2018a). It has been suggested that an increase in accessory inspiratory muscle contribution, in response to reduced diaphragm contractile function, causes increased afferent feedback to the central nervous system exacerbating dyspnea contributing to exercise intolerance (Romer & Polkey, 2008). 1.1.4 Assessment of Diaphragmatic Fatigue The potential of the respiratory muscles to experience performance fatigability has been investigated for over fifty years. Early studies, using either voluntary hyperpnea (Shephard, 13 1967; Tenney & Reese, 1968) or inspiratory loading (Roussos et al., 1979) demonstrated that when ventilatory demand was raised to a critical point breathing could not be indefinitely maintained. However, these investigations did not attempt to quantify the degree of diaphragmatic fatigue. As mentioned previously, performance fatigability is defined as an acute exercise-induced reduction in the force generating capacity of the muscles involved in exercise performance (Hunter, 2018). Therefore, to accurately assess the force generating capacity of a muscle an identical contractile stimulus must be applied to the muscle before and after exercise. Maximal inspiratory maneuvers have been used in an attempt to elicit an identical contractile stimulus through voluntary activation (Bye et al., 1984; Coast et al., 1990; Loke et al., 1982; Roussos & Macklem, 1977). However, voluntary maneuvers are limited in their ability to accurately assess EIDF because they can be influenced by factors such as motivation. Consequently, voluntary activation of the diaphragm cannot be used to validly assess EIDF, instead methods which initiate an involuntary contraction of the diaphragm are needed. The use of phrenic nerve stimulation before and after fatiguing bouts of respiratory muscle work provides a tool to objectively assess EIDF (Bellemare & Bigland-Ritchie, 1987). Importantly, to reliably assess diaphragmatic fatigue using phrenic nerve stimulation, stimulation must be supramaximal, the diaphragm must be potentiated, and lung volume must be standardized (Laveneziana et al., 2019). Levine & Henson (1988) were the first to use transcutaneous electrical phrenic nerve stimulation to provide an involuntary measurement of EIDF. A drawback of transcutaneous electrical stimulation is that it is painful to experience (Similowski et al., 1989). However, 14 magnetic stimulation of the phrenic nerves is painless, making its use more attractive in clinical and research settings (Similowski et al., 1989). Due to the anatomical position of the diaphragm within the thoracoabdominal cavity, measurement of its contractile function is methodologically challenging relative to muscles involved in the movement of limbs. By placing catheters above and below the diaphragm, in the esophagus and stomach, respectively, it is possible to measure diaphragmatic pressure (Dominelli & Sheel, 2012). The absolute difference between the pressures measured using the esophageal and gastric balloon catheters is transdiaphragmatic pressure (Pdi). The pairing of bilateral phrenic nerve stimulation with balloon catheter derived measurements of Pdi is an ideal strategy to assess diaphragmatic fatigue. In response to a single stimulation of the phrenic nerves, the diaphragm rapidly contracts and relaxes, this pattern of contraction is called a diaphragmatic twitch. Diaphragmatic twitches are observed as a rapid increase in Pdi followed by an rapid decrease to its baseline value (ATS/ERS, 2002). The increase in Pdi in response to phrenic nerve stimulation is referred to as Pdi,tw. A reduction in Pdi,tw following fatiguing exercise has been used as the primary determinant of EIDF in modern studies (Archiza et al., 2018b; Babcock et al., 1995, 1996b; Guenette et al., 2010; Johnson et al., 1993). The diaphragmatic M-wave produced during stimulation of the phrenic nerves is another measure which can be used in the assessment of diaphragmatic fatigue. The M-wave of the diaphragm is recorded as an EMG signal collected by surface electrodes placed along the 6th-8th intercostal spaces (Welch et al., 2017) or internally using an esophageal electrode placed at the level of the diaphragm (ATS/ERS, 2002). The M-wave represents the synchronous electrical activity of motor units depolarized by the stimulus (Rodriguez-Falces & Place, 2018). Changes in the diaphragmatic M-wave following fatiguing exercise indicate that diaphragmatic fatigue is 15 related to changes in neuromuscular propagation (Enoka & Stuart, 1992). The M-wave is also useful as a control measure because it can confirm that both hemidiaphragms are activated with magnetic stimulation (Welch et al., 2017). The M-wave acts as an invaluable measurement in diaphragmatic fatigue investigations. 1.1.5 Aerobic Fitness, The Respiratory System, and Diaphragmatic Fatigue 1.1.5.1 The Static Respiratory System The respiratory system has been described as ‘overbuilt’; its capacity exceeds the demands placed on it, even during maximal exercise. For example, in young healthy males the respiratory system is unlikely to impose a limitation to dynamic exercise performance (Dempsey et al., 2020). As a result of the ‘overbuilt’ notion, the impact of the respiratory system on oxygen delivery and exercise performance has been largely ignored with focus instead placed on the capacity of the cardiovascular system and the metabolic characteristics of skeletal muscle (Dempsey et al., 2020). Unlike the cardiovascular system and skeletal muscle which exhibit adaptive changes in response to chronic exercise training, the structure of the respiratory system remains largely unmalleable (Dempsey et al., 2020). Young endurance trained athletes do not have significantly different pulmonary function when compared to their sedentary peers (Hagberg et al., 1988). Despite a lack of training induced changes in respiratory structure and function, highly-aerobically fit humans ventilate significantly more at maximal exercise placing an increased demand on the respiratory system (Johnson et al., 1992). Therefore, the adaptive changes in oxygen transport and locomotor muscle metabolism produce a demand which encroaches on respiratory capacity. 16 1.1.5.2 Ventilatory Cost of High Aerobic Fitness The WB is a useful tool to describe the ventilatory demand that highly-aerobically fit athletes experience during high-intensity exercise. No difference in WB is seen between aerobically fit and normal humans at a given V̇E (Milic-Emili et al., 1962). However, during high-intensity exercise, aerobically fit athletes are likely to experience a greater WB when compared to their less fit counterparts (Johnson et al., 1992). Aerobically fit individuals can exercise at greater absolute intensities necessitating a greater V̇E. Because WB increases exponentially as V̇E increases, highly-aerobically fit humans experience a disproportionately greater WB during high-intensity dynamic exercise (Guenette & Sheel, 2007). Expiratory flow limitation (EFL) also contributes to an elevation in the WB in many aerobically fit humans during high-intensity exercise and is a clear example of the capacity of the respiratory system being insufficient to support the demands placed on it during exercise. EFL occurs when an increase in expiratory muscle effort does not lead to an increase in expiratory flow due to partial collapse of airways (Hyatt, 1983). EFL is primarily determined by ventilatory demand, which comprises V̇E and breathing pattern, and ventilatory capacity, which comprises lung volume and maximal expiratory flow (Molgat-Seon et al., 2022). The ventilatory demands that highly-aerobically fit humans experience during high-intensity exercise should make them particularly susceptible to EFL (Guenette & Sheel, 2007). The development of EFL during exercise is associated with an increase in end-expiratory lung volume called dynamic hyperinflation (which can also occur without EFL) (Aliverti, 2008). While dynamic hyperinflation allows expiratory flow rate to increase further, WB is increased due to decreased lung compliance at higher lung volumes (Pellegrino et al., 1993). Furthermore, hyperinflation results in the inspiratory muscles contracting from a shorter length, reducing their efficiency, 17 increasing the probability of the development of performance fatigability (Roussos et al., 1979; Tzelepis et al., 1988). The high demands placed on the respiratory musculature result in a greater metabolic requirement of hyperpnea during maximum exercise in aerobically fit individuals. At maximum exercise V̇O2rm accounts for 15-16% of whole-body V̇O2 in highly-aerobically fit individuals, and only 10% of V̇O2 in healthy individuals of average fitness (Aaron et al., 1992). As a result, a greater proportion of cardiac output could be directed towards the respiratory muscles in aerobically fit individuals when compared to healthy individuals of average fitness. Therefore, a smaller proportion of total cardiac output would be portioned towards the locomotor muscles, implicating the capacity of the respiratory system as a limiting factor in exercise tolerance (Harms et al., 1997). 1.1.5.3 Aerobic Fitness and Diaphragmatic Fatigue The greater mechanical and metabolic requirements of the respiratory muscles in highly-aerobically fit individuals during high-intensity exercise would suggest that EIDF would be more common in this population. But unlike the rest of the structures in the respiratory system, there is evidence that the respiratory muscles exhibit adaptation to chronic endurance training. For example, in response to intense endurance training diaphragmatic proteins associated with an enhanced aerobic capacity increased in a population of mice (Sollanek et al., 2017). Other studies in rodents have shown greater oxidative enzyme activity (Green et al., 1989; Metzger & Fitts, 1986; Powers et al., 1994) and reduced capillary diffusion distance within the diaphragm following chronic endurance training (Green et al., 1989; Powers et al., 1992, 1994). The aerobic capacity of the diaphragm may increase with exercise training in other mammals, including humans. 18 Babcock et al. (1996), tested the hypothesis that aerobically fit individuals would be less susceptible to EIDF following high-intensity exercise to exhaustion when compared to healthy less-aerobically fit individuals. They did not observe any differences in EIDF between groups, despite the aerobically fit group ventilating significantly more. Interestingly, the absolute contribution of the diaphragm to total inspiratory pressure generation was not different between the groups in the final 40-50% of exercise. It is possible that the higher ventilatory demand of the highly fit group was achieved through greater activation of accessory respiratory muscles, reducing the demand placed on the diaphragm therefore reducing EIDF. It is also possible that histologic and metabolic adaptations occur within the diaphragm following regular endurance training, which increase fatigue resistance in highly-aerobically fit individuals. Regardless of the cause, the ratio of demand to capacity of the diaphragm remains unchanged in highly-aerobically fit individuals during high-intensity dynamic exercise. Unfortunately, results from this study do not indicate statistical equivalence between groups, it simply demonstrated that the group differences were not significant. Furthermore, most participants were male; important sex-based differences may result in different findings in a female population. 1.1.6 The Effect of Sex on the Respiratory System and Diaphragmatic Fatigue 1.1.6.1 The Effect of Sex on Respiratory Structure For clarity, sex and gender must be defined. Sex refers to a set of biological attributes in humans and animals and is often categorized using the terms male and female (CIHR, 2021). Gender refers to the socially constructed, roles, behaviours, and expressions. Most often using the terms man and woman to differentiate (CIHR, 2021). Differences in the respiratory system related to sex only, will be discussed in this thesis. 19 On average males have larger lungs than their female counterparts (Crapo et al., 1982). Part of the discrepancy in lung size can be attributed to group differences in standing height which is closely related to lung volume (Boren et al., 1966). Since males on average are taller than females, they will tend to have larger lungs. However, when matched for standing and sitting height, females have smaller lungs than males, implying an effect of sex on lung size which is not due to dimorphism in height (Schwartz et al., 1988; Thurlbeck, 1982). There is no significant difference between alveolar dimensions or alveoli per unit area between the sexes (Thurlbeck, 1982). But because males tend to have larger lungs than females, male lungs contain a greater number of alveoli (Thurlbeck, 1982). Differences in ribcage geometry have also been identified. Males have relatively shorter and broader ribcages and the bases of their lungs are larger than their apices forming a ‘pyramidal’ shape (Torres-Tamayo et al., 2018). The shape of the female lung is described as ‘prismatic’ due to the lung bases and apices being similarly sized (Torres-Tamayo et al., 2018). Sex-differences are also present in the calibre of airways. To properly discuss airway differences, the term ‘dysanapsis’ must be defined. Dysanapsis refers to the interindividual variation in the size of airways relative to lung volume (Mead, 1980). Individuals with the same sized lungs do not necessarily have the same sized airways (Green et al., 1974). The concept of dysanapsis is relevant to sex because females have been demonstrated to have proportionally smaller airways than males; females exhibit a smaller dysanapsis ratio (Sheel et al., 2016). An analysis of indices sensitive to lung size against indices sensitive to airway calibre revealed that males had airways 17% larger in diameter than females (Mead, 1980). A similar result was observed when airways were measured using acoustic reflectance (Martin et al., 1987). The most direct between sex comparison of airway and lung size was investigated in a cohort of former 20 smokers (Sheel et al., 2009). Both the trachea and large conducting airways were smaller in females than males when matched for lung size, providing clear evidence of sex-based dysanapsis. Unfortunately, computed tomography, the technique used in the previous study, exposes participants to radiation, disqualifying it from use in healthy populations. However, a retrospective analysis of data collected using computed tomography in a cohort of healthy individuals revealed that when matched for height females had smaller large conducting airways (1st -5th generation) than males (Dominelli et al., 2018). Recent evidence gathered using a bronchoscopy procedure further demonstrated that when matched for height healthy females have smaller 4th -6th generation airways than healthy males (Peters et al., 2021). 1.1.6.2 Sex Differences and the Mechanical Cost of Hyperpnea While the observed differences in respiratory system morphology have little impact on the WB at rest, they appear to cause significant effects when the demand on the respiratory system is increased during exercise (Molgat-Seon et al., 2018). Sex differences can exert significant effects on the WB. Of particular importance is the influence of airway geometry on the resistive WB. Poiseuille’s law dictates that a small decrease in radius exponentially increases resistance; smaller airway size predisposes females to a greater resistive WB (Dominelli et al., 2015). When V̇E rises above 50-70 L/min females due exhibit a greater WB (Sheel et al., 2016) and this has been demonstrated in healthy normal individuals (Dominelli et al., 2015), highly-aerobically fit individuals, (Guenette et al., 2007) and across age (Molgat-Seon, Peters, et al., 2018). As suggested by the smaller size of the female airway, the observed sex difference in WB is related to a greater resistive component, not the elastic component (Guenette et al., 2009; Molgat-Seon et al., 2019). Until recently no direct evidence 21 connecting airway size to WB has been observed. Peters et al. (2021), demonstrated that smaller airways in females result in a greater resistive WB during exercise when compared to males. Another factor which may predispose females to a higher WB is the development of EFL. Females have lower maximal expiratory flow rates than height matched males, which can likely be attributed to their proportionally smaller lungs and airways (McClaran et al., 1998). It is possible that females are more predisposed to EFL. But recent evidence indicates that young healthy females do not experience a greater incidence of EFL than young healthy males during peak exercise (Molgat-Seon et al., 2022). However, when the development of EFL was compared between endurance trained females and males, females experienced a higher incidence of EFL (90% of females vs 43% of males) (Guenette et al., 2007). Sex-based differences in EFL may only be evident in highly-aerobically fit populations. The observed difference in WB between males and females during exercise, suggests that females have a higher V̇O2rm at a given V̇E (Topin et al., 2003). Females do exhibit a higher absolute V̇O2rm and WB at a given V̇E above 55 L/min (Dominelli et al., 2015). Furthermore, V̇O2rm was a significantly greater fraction of V̇O2 in females, at >75% maximum V̇E (Dominelli et al., 2015). A greater proportion of total cardiac output may be partitioned to the respiratory muscles in females for a given increase in WB. 1.1.6.3 Sex Differences and Diaphragmatic Fatigue The observed sex-based differences in respiratory system structure and the effect of these differences on function, specifically WB, suggest that females may be more predisposed to EIDF. Guenette et al. (2010), tested the hypothesis that females would be more predisposed to EIDF than males in a cohort of endurance trained individuals with high aerobic fitness. Results from this study demonstrated that fewer females developed diaphragmatic fatigue and among those 22 who did, the magnitude was significantly less in females, contradicting their hypothesis (Guenette et al., 2010). Supporting the findings of Guenette et al. (2010), Welch et al. (2018), also demonstrated a reduced incidence of EIDF in females when compared to males. There have been a few proposed explanations for the apparent sex difference in EIDF. First, it has been observed that there are sex differences in respiratory mechanics. Guenette et al. (2010) observed that females had a lower diaphragmatic contribution to total inspiratory pressure generation suggesting a greater contribution of accessory inspiratory muscles, which may reduce the susceptibility of the diaphragm to performance fatigability. Furthermore, EMG data has demonstrated that females activate accessory inspiratory muscles to a greater degree than males during dynamic exercise (Mitchell et al., 2018). Second, differences in neuromuscular function between males and females may explain why females are more resistant to EIDF. In general females, when compared to males, are more resistant to performance fatigability during isometric and slow-velocity dynamic contractions (Hunter, 2015). Numerous sex differences may contribute to these observations including muscle mass, muscle perfusion, fibre type composition, and muscle metabolism (Hunter, 2014). Unfortunately, no investigations have examined sex differences in diaphragm morphology, histology, and metabolism. Accordingly, sex-based differences that underpin greater performance fatiguability resistance in females cannot be assumed to be responsible for the observed differences in EIDF. However, evidence from pressure-threshold loading studies, where the diaphragmatic contribution to inspiratory pressure generation is controlled for, indicates that the diaphragm of females has a greater endurance capacity than males. At the same relative diaphragmatic load, females exhibited a significantly longer time to exhaustion (Welch et al., 2018b). Furthermore, when matched for absolute diaphragmatic load females experienced 23 similar degrees of diaphragmatic fatigue, despite working at a relatively greater load (Geary et al., 2019). Another possible reason for the observed sex differences in EIDF could be related to the fitness of the female participants. Both Guenette et al. (2010) and Welch et al. (2018) used a cohort of highly-aerobically fit females (V̇O2max > 50 mL/kg/min) when they demonstrated a sex difference in EIDF. In close agreement, Guenette et al. (2010) and Welch et al. (2018) demonstrated a 13.0% and 13.5% reduction in Pdi,tw following exercise completion. Studies that have used cohorts of averagely fit females have demonstrated conflicting results. Reinhard et al. (2023), in a cohort of more moderately fit females (V̇O2max = 45.4 mL/kg/min), demonstrated 21% reduction in Pdi,tw following exercise cessation and no sex difference in EIDF. However, Ramsook et al. (2022) demonstrated no sex difference in EIDF and a more modest post-exercise reduction in Pdi,tw (14.8%) in an investigation on averagely fit females (V̇O2max = 40.1 mL/kg/min). While aerobic fitness does not appear to affect EIDF in males (Babcock et al., 1996), it is unknown if aerobic fitness effects EIDF in females. It is possible that the observed sex-based differences in EIDF may be due to an interaction between sex and aerobic fitness. 1.2 Summary and Rationale It is counterintuitive that the diaphragm, a muscle which has worked continuously since birth, is susceptible to performance fatigability. While resting function poses no challenge to the capacity of the diaphragm, the exponential increase in mechanical work which accompanies exercise hyperpnea places a significant demand on the principal muscle of respiration. High mechanical demand combined with impaired energetic supply, stemming from competition with locomotor muscles, is the perfect storm to induce performance fatigability. Careful assessment of diaphragm contractile function using involuntary techniques with precise measures has revealed 24 the incidence and magnitude of diaphragmatic fatigue in different populations. In the current review, attention was paid to the effect of aerobic fitness and sex on diaphragmatic fatigue. Highly-aerobically fit humans, despite disproportionately higher demands on the diaphragm during high-intensity exercise, experience the same degree of EIDF as their averagely-aerobically fit counterparts. Females, despite well-established morphological and functional differences in pulmonary capacity exhibit a diaphragm which is resistant to diaphragmatic fatigue when compared to males. What is unclear is if the effect of aerobic fitness and sex exhibit a significant interaction. Studies addressing this gap in knowledge are warranted to better understand the effect of sex and aerobic fitness on the occurrence of diaphragmatic fatigue. 1.3 Purpose The purpose of this thesis was to determine the effect of aerobic fitness on EIDF in females. 1.4 Hypothesis Highly-aerobically fit females will experience a lower incidence and relative magnitude of EIDF vs averagely-aerobically fit females. 25 Chapter 2: Methodology 2.1 Ethical Approval This study was approved by the Clinical Research Ethics Board at the University of British Columbia (H22-00157). 2.2 Participants Thirty-three females were recruited to participate in the study. Participants were divided into two predetermined groups based on aerobic fitness, Avg-Fit (V̇O2max < 40 mL/kg/min) and Hi-Fit (V̇O2max > 50 mL/kg/min). A V̇O2max < 40 mL/kg/min was chosen for the Avg-Fit group as it is slightly greater than the age predicted value (V̇O2max = 37.9 mL/kg/min) for females 20-29 years of age (Myers et al., 2017). A V̇O2max > 50 mL/kg/min was chosen for the Hi-Fit group to provide a minimum of a 10 mL/kg/min separation between groups. All participants were healthy, non-smoking/vaping, and had normal pulmonary function. Participants were randomly tested throughout their menstrual cycle and were not excluded if using oral contraceptives. Ten participants were ineligible for day 2 because their aerobic fitness fell between fitness groups (V̇O2max = 41-49 mL/kg/min) and two participants dropped out of the study following day 1. Of the participants that completed day 2, three did not experience a plateau in Pdi,tw with increasing CMS intensity and were excluded from the analysis. In total eighteen participants (Hi-Fit: n = 9, Avg-Fit: n = 9) were included in the data analysis. 2.3 Experimental Overview Testing took place on two days separated by at least 48 hours. Before both experimental visits participants were asked to avoid food and caffeine for at least 2 hours, exercise for at least 12 hours, and alcoholic beverages for at least 24 hours prior to all testing sessions. On the first day, after providing written informed consent, participants completed screening questionnaires, 26 anthropometric measures and resting pulmonary function testing. Participants then performed an incremental exercise test on a cycle ergometer to volitional exhaustion. Following the incremental test to exhaustion participants performed a 5-minute constant load exercise familiarization bout. On the second day, participants performed a constant load exercise test to exhaustion. Diaphragm contractile function was assessed before and after the constant load test to exhaustion using CMS. 2.4 Pulmonary Function Spirometry, plethysmography, and single-breath carbon monoxide diffusing capacity were all performed using a commercially available system (Vmax Autobox V62, CareFusion, USA). Single-breath diffusion capacity tests were adjusted for hemoglobin concentration, which was measured by a digital hemoglobinometer (Hb201+, HemoCue, Sweden). All procedures were performed in accordance with American Thoracic Society standardization and were compared to age and sex-related reference values (Hankinson et al., 1999; Quanjer et al., 1993; Stanojevic et al., 2017). 2.5 Maximal Ramp Cycle Test All exercise tests were performed on an upright electronically braked cycle ergometer (Velotron, RacerMate, Seattle, WA). Following a self-selected warmup, the test began at a work rate of 20 W, increased by 1 W every 2 seconds. The test was completed when the participant could no longer maintain 60 RPM despite verbal encouragement, or until volitional exhaustion. The maximal ramp cycle test was used to characterize the aerobic fitness of participants and to determine maximal exercise values. 27 2.6 Constant Load Exercise Test Familiarization An additional exercise test was performed approximately 20 minutes following the cessation of the ramp exercise test. Participants performed 5 minutes of exercise at a workload slightly greater than their respiratory compensation point. The respiratory compensation point was identified visually using a V-slope method from data collected during the maximal ramp cycle test (Beaver et al., 1986). This constant load exercise bout was designed to familiarize the participants with the intensity of the constant load exercise test used on the second day and to determine if exercise prescription was accurate. 2.7 Constant Load Exercise Test to Exhaustion Participants performed the constant load exercise test to exhaustion test at a workload determined to be slightly greater than their respiratory compensation point. Participants were encouraged to maintain their cadence around 90 RPM and were able to visually track their RPM on a display. The test was completed when the participant could no longer maintain a cadence over 60 RPM despite verbal encouragement, or until volitional exhaustion. During the entire test participants were blinded to their exercise time. 28 Figure 2.1 Exercise Prescription Illustration (Top Panels) Intensity was chosen when V̇E began rising out of proportion with V̇CO2 during the incremental ramp test. (Middle Panels) Chosen intensity corresponds to a drop in PETCO2 during the incremental ramp test. Arrows indicate intensity chosen. (Bottom Panels) Time to exhaustion was similar on Day 2. Abbreviations: V̇E, minute ventilation; V̇CO2, carbon dioxide output; PETCO2, end-tidal carbon dioxide. 29 2.8 Metabolic and Cardiorespiratory Measurement During all exercise tests participants were instrumented with a mouthpiece connected to a two-way nonrebreathing valve (model 2700B, Hans-Rudolph, Kansas City, MO). Expired air was collected in a mixing chamber. Measurement of mixed expired gases was performed by a calibrated oxygen analyzer (17518A, Vacumed, Ventura, CA) and calibrated carbon dioxide analyzer (17515A, Vacumed, Ventura, CA) connected to ports on the mixing chamber. Inspired and expired flow was measured using calibrated pneumotachographs (model 3813, Hans Rudolph, Kansas City, MO, USA) volume was obtained by integration of the respective flow channels. The expired pneumotachograph was heated. PETCO2 was measured from a port on the mouthpiece attached to a separate CO2 gas analyzer (17515A, Vacumed, Ventura, CA). Heart rate was measured using a heart rate telemetry chest strap (T31, Polar, Kempele, Finland). 2.9 Pulmonary Pressure Measurement Viscous lidocaine (Lidodan 2% Viscous, ODAN, Montreal, Canada) was applied to the nares and upper pharynx to minimize discomfort during balloon placement. an esophageal and gastric balloon catheter were inserted trans-nasally into the stomach. Once in the stomach participants performed a Valsalva maneuver to expel residual air from the catheter. The esophageal and gastric balloon catheters were filled with 1 mL and 2 mL of air, respectively. The esophageal catheter was then slowly raised out of the stomach while the participant performed sharp inspiratory sniffs. The balloon was determined to be in the esophagus when there was a negative deflection in inspiratory pressure during a sniff. To ensure the entire balloon was within the esophagus the balloon was raised another 10 cm. Balloon position was further adjusted so that end-expiratory esophageal pressure (Peso) was between -3 and -6 cmH2O in a sitting position 30 during eupneic breathing (Henke et al., 1988). Each balloon catheter was connected to a pressure transducer (DP45-34, Validyne, California) and demodulator (CD16, Validyne, California). Pressure transducers were calibrated using a digital manometer (2021P, Digitron, Torquay, UK). 2.10 Surface Electromyography of the Diaphragm Surface electrodes (H59P, Kendall-LTP, MA, USA) were placed in a bipolar arrangement at the 6th and 8th intercostal spaces along the anterior-axillary line to measure electrical activity of the right and left costal diaphragm (Welch et al., 2017). A ground electrode was placed on the acromion process of the scapula. All signals were amplified (x200) and band pass filtered (0.1Uz to 2kHz; Model P511, Grass Instruments; Warwick, RI, USA). M-wave onset latency, duration, peak-to-peak amplitude, and total rectified area were later analyzed using custom software (MatLab 2022b, MathWorks, Natick, MA). 2.11 Cervical Magnetic Stimulation Diaphragmatic contractile function was assessed using CMS in accordance with previously described techniques (Similowski et al., 1989) and all stimulations occurred at end-expiratory lung volume. Participants were seated in a chair with their neck slightly flexed. A 90-mm circular coil (P/N 3192-00; peak electric field strength = 530 V/m, peak magnetic field strength = 2.0 T, inductance = 23.5 μH) attached to a magnetic stimulator (Magstim 200-2, Magstim; Wales, UK) was applied to the 7th cervical vertebrae, activating both phrenic nerves. To determine the optimal stimulation location, CMS (70% of peak power output) was applied beginning at this vertebra and was moved between the 3rd to 8th cervical vertebrae in ~1 cm increments until the largest Pdi,tw is observed. The site that elicited the largest Pdi,tw was marked with ink and was used for all subsequent stimulations. 31 2.12 Cervical Magnetic Stimulation Ramp Maximum stimulation of the phrenic nerves was determined by a plateau in Pdi,tw despite an increase in stimulator intensity. CMS was applied with power output increasing in a ramp fashion (60%, 70%, 80%, 90%, 95%, and 100% of the 2 Tesla max stimulator output). At each intensity three stimulations were performed with a minimum of 30 seconds in between stimulations to avoid potentiation of the diaphragm. If the increase in Pdi,tw between the maximal and highest submaximal intensity was less than 5% and lower than the Pdi,tw coefficient of variation for that individual during the CMS ramp, we assumed maximal stimulation of the phrenic nerves. 2.13 Diaphragmatic Fatigue Assessment A potentiated twitch protocol was used for the assessment of EIDF. The potentiated twitch protocol was completed prior to exercise (baseline), and at 5, 30, and 60 minutes after exercise completion. At each time point seven potentiated twitches were performed. The first two were discarded and the final five were used for analysis. Each potentiated twitch began with a maximal inspiratory effort, lasting approximately 5 seconds, against an occluded mouthpiece at functional residual capacity. Following the maximal inspiratory effort, at the end of the second tidal expiration, CMS was applied at maximum power (100% stimulator output) to elicit a diaphragmatic twitch. As used previously by others, for individual participants EIDF was considered to be present if there is a >15% decrement from baseline Pdi,tw (Kufel et al. 2002, Guenette et al. 2010). A 15% decrease in Pdi,tw was approximately three times greater than the observed coefficient of variation of Pdi,tw in the present study (Section 3.4.2). 32 Figure 2.2 Twitch Potentiation Protocol Raw traces of respiratory pressures and diaphragmatic EMG measurements during the potentiated twitch protocol. Lighter shading indicates the first maximal inspiratory pressure maneuver, darker shading indicates the first diaphragmatic twitch. Abbreviations: Peso, esophageal pressure; Pga, gastric pressure; Pdi, diaphragmatic pressure; EMGRHD, right hemidiaphragm EMG; EMGLHD, left hemidiaphragm EMG. 33 Figure 2.3 The Response of the Diaphragm to Phrenic Nerve Stimulation Example of phrenic nerve stimulation from a single participant. The EMG signal is of the right hemi-diaphragm Abbreviations: Pdi, diaphragmatic pressure; Pdi,tw, transdiaphragmatic twitch pressure; M-wave, compound muscle action potential; EMG, electromyography. 34 2.14 Data Sampling and Recording Electromyography signals were collected at 10,000 Hz and all other data collected was sampled at 200 Hz (PowerLab 16/35, ADInstruments, Colorado Springs, CO) and stored on a computer for later analysis (LabChart 8, ADInstruments, Colorado Springs, CO). 2.15 Data Analysis Approximately ten to fifteen consecutive breaths were averaged at increments throughout the constant load test to exhaustion, with these averaged breaths WB was calculated using the tidal Peso-volume loop method. WB also included the portion of the elastic WB which falls outside the Peso-volume loop (Dominelli & Sheel, 2012). WB was then multiplied by fB and expressed as joules per minute. Additionally, diaphragmatic pressure-time product (PTPdi) and esophageal pressure-time product (PTPeso) were determined by integrating Pdi and Peso, respectively, over time during inspiration. These values were then multiplied by fB. The ratio PTPdi to PTPeso was used to determine the fraction of inspiratory pressure generated by the diaphragm. Additionally, at each increment, V̇O2, V̇CO2, V̇E, respiratory exchange ratio (RER), PETCO2, HR, fB, and VT were averaged to characterize the metabolic and cardiorespiratory response to exercise. 2.16 Statistical Analysis An independent samples t-test was used to compare anthropometric, pulmonary function, and maximum exercise values between fitness groups. In each fitness group a repeated measures ANOVA was used to confirm if the phrenic nerves were maximally stimulated. If there was a significant main effect of stimulator intensity pairwise t-tests were performed between Pdi,tw at 100% of stimulator output and lower stimulator outputs. Aerobic fitness differences in Pdi,tw, cardiorespiratory variables, and respiratory mechanics were assessed using a simple mixed ANOVA (aerobic fitness by time). If a significant effect of fitness was found pairwise t-tests 35 were performed to assess fitness differences at each time point. Twitch control characteristics (M-wave characteristics, contraction time and ½ relaxation time) were assessed using a separate repeated measures ANOVA for each group. If there was a significant main effect of time, pairwise t-tests were performed to compare baseline to subsequent time points. The relationship between EIDF and various parameters was further assessed via correlation. An independent samples t-test was used to compare aerobic fitness differences in the constant load exercise test to exhaustion time and cumulative respiratory mechanics during exercise. Significance was set at p < 0.05. Values are expressed as mean ± SD). 36 Chapter 3: Results 3.1 Subjects Demographics, anthropometric characteristics, and pulmonary function measures are detailed in Table 3.1. The Hi-Fit group was on average slightly older, taller, and heavier than the Avg-Fit group, however these differences did not reach statistical significance (p = 0.143, p = 0.124, p = 0.180, respectively). Body mass index (BMI) was not significantly different between groups (p = 0.680). Hi-Fit participants had a significantly larger FVC (p = 0.032), FEV1 (p = 0.017), and PEF (p < 0.001). Hi-Fit subjects self-reported a significantly greater amount of time spent performing aerobic exercise per week (Hi-Fit: 458 ± 166 vs Avg-Fit: 186 ± 128 min/week, p = 0.001). 3.2 Maximal Cycle Exercise Peak cardiorespiratory and metabolic values, corresponding to the final 30 second average of ramp cycle exercise test are presented in Table 3.2. Hi-Fit individuals reached a significantly greater maximum work rate (p < 0.001). Both absolute and percent predicted V̇O2max were significantly greater in in the Hi-Fit group (both, p < 0.001). The Hi-Fit group achieved a significantly higher V̇E (p < 0.001), this was due to a greater VT (p = 0.005), as fB was not different between groups (p = 0.785). No fitness difference was observed in PETCO2 (p = 0.223) and HR (p = 0.535). 37 Table 3.1 Descriptive Characteristics of the Participants Avg-Fit (n = 9) Hi-Fit (n = 9) Age, years 23 ± 3 (21 – 29) 26 ± 1 (18 – 31) Height, cm 163 ± 7 (155 – 173) 169 ± 8 (157 – 185) Mass, kg 61.7 ± 8.1 (51.4 – 76.8) 64.9 ± 6.1 (54.0 – 77.6) BMI, kg/m2 23.2 ± 1.9 (20.1 – 26.0) 22.9 ± 1.7 (19.8 – 25.9) FVC, L 4.0 ± 0.6 (2.9 – 4.9) 4.7 ± 0.6* (4.1 – 5.9) FVC, % predicted 104 ± 10 (79 – 116) 114 ± 8* (103 – 126) FEV1, L 3.3 ± 0.4 (2.8 – 3.8) 3.8 ± 0.4* (3.4 – 4.8) FEV1, % predicted 101 ± 7 (85 – 107) 111 ± 5* (102 – 119) FEV1/FVC, % 84 ± 5 (77 – 94) 82 ± 5 (75 – 90) FEV1/FVC, % predicted 98 ± 5 (91 – 109) 97 ± 5 (86 – 104) FEF25-75%, L/sec 3.6 ± 0.5 (2.7 – 4.4) 4.1 ± 0.5 (3.1 – 5.0) FEF25-75%, % predicted 98 ± 12 (74 – 123) 109 ± 11 (80 – 120) PEF, L/sec 6.9 ± 0.9 (5.8 – 8.2) 9.1 ± 1.3* (7.4 – 11.4) PEF, % predicted 100 ± 9 (91 – 121) 125 ± 13* (103 – 142) TLC, L 5.1 ± 0.9 (4.1 – 7.0) 5.6 ± 0.9 (4.6 – 7.5) TLC, % predicted 102 ± 10 (86 – 126) 105 ± 7 (95 – 117) VA, L 4.6 ± 0.8 (3.5 – 6.3) 5.3 ± 0.8 (4.1 – 6.9) VA, % predicted 97 ± 10 (76 – 115) 102 ± 12 (82 – 125) DLCO, mL/min/mmHg 23.8 ± 2.9 (16.9 – 30.0) 25.8 ± 7.1 (17.9 – 31.4) DLCO, % predicted 100 ± 13 (83 – 124) 103 ± 18 (76 – 133) DLCO/VA, mL/min/mmHg 4.9 ± 0.4 (4.3 – 5.5) 4.5 ± 0.7 (3.4 – 5.2) DLCO/VA, % predicted 104 ± 10 (90 – 116) 100 ± 14 (76 – 120) Hemoglobin, g/dL 13.4 ± 1.4 (11.3 – 15.1) 14.1 ± 0.9 (12.3 – 15.1) Values are means ± SD. Abbreviations: BMI, body mass index; FVC, forced vital capacity; FEV1, forced expired volume in 1 second; PEF, peak expiratory flow; FEF25-75%, forced expiratory flow between 25 and 75% of FVC; TLC, total lung capacity; VA, alveolar volume; DLCO, diffusing capacity of the lungs for carbon monoxide. Spirometry predicted equations are from Hankinson et al. (1999), TLC predicted equation is from Quanjer et al. (1993), VA and DLCO is from Stanojevic et al. (2017). * = significant difference between fitness groups (p < 0.05). 38 Table 3.2 Maximal Exercise Values Avg-Fit (n = 9) Hi-Fit (n = 9) HR, beats/min 185 ± 10 (163 – 200) 188 ± 7 (177 – 197) V̇O2max, mL/kg/min 35.7 ± 4.9 (25.3 – 40.6) 56.1 ± 3.4* (51.9 – 61.5) V̇O2max, % predicted 91 ± 20 (61 – 120) 148 ± 10* (130 – 171) V̇O2max, L/min 2.2 ± 0.4 (1.5 – 3.1) 3.6 ± 0.3* (3.3 – 4.2) V̇CO2, L/min 2.7 ± 0.4 (1.9 – 3.4) 4.1 ± 0.2* (3.8 – 4.5) RER 1.23 ± 0.07 (1.12 – 1.30) 1.13 ± 0.05* (1.08 – 1.22) V̇E, L/min 93 ± 14 (69 – 111) 125 ± 16* (95 – 144) fb, breaths/min 55 ± 12 (41 – 70) 57 ± 12 (35 – 77) VT, L 1.7 ± 0.3 (1.2 – 2.1) 2.3 ± 0.5* (1.8 – 3.2) PETCO2, mmHg 27.1 ± 4.7 (20.8 – 35.4) 29.8 ± 3.7 (23.1 – 34.9) Work rate, W 190 ± 30 (146 – 248) 317 ± 26* (282 – 368) Values are means ± SD. Abbreviations: HR, heart rate; V̇O2, oxygen uptake; V̇CO2, carbon dioxide production; RER, respiratory exchange ratio; V̇E, minute ventilation; fb, breathing frequency; VT, tidal volume; PETCO2, end-tidal pressure of carbon dioxide. V̇O2 predicted equation is from (Myers et al., 2017). * = significant difference between fitness groups (p < 0.05). 3.3 Constant Load Exercise Test to Exhaustion Values from the constant load exercise test to exhaustion are presented in Table 3.4, and displayed in increments corresponding to 20, 40, 60, 80, and 100 percent of exercise duration. The Hi-Fit group exercised at a significantly greater absolute workload (257  29 W) compared to the Avg-Fit group (135  10 W) during the test (p < 0.001). Constant load exercise test duration was similar between fitness groups (Hi-Fit: 546.1  177.8 versus Avg-Fit: 559.3  175.0 seconds, p = 0.876). As shown in Figure 3.1, V̇O2 over the duration of the constant load exercise test was similar between fitness groups when expressed as a percentage of V̇O2max; there was no main effect of fitness (p = 0.764) or fitness by time interaction (p = 0.320). There was no main effect of fitness on HR (p = 0.391) or fitness by time interaction (p = 0.382). There was a significant main effect of time on V̇E, fB, HR, V̇O2, and V̇CO2, which increased on average throughout exercise (all, p < 0.001). A significant main effect of time was 39 observed for PETCO2 (p < 0.001) which decreased during exercise. Furthermore, there was a main effect of time on both VT (p < 0.001) and RER (p = 0.004) which rose rapidly initially but steadily decreased once peak values were reached. There was a significant main effect of fitness observed in V̇O2 (p < 0.001) and V̇CO2 (p < 0.001) as values were consistently greater in the Hi-Fit group. A fitness by time interaction was observed for V̇O2 (p = 0.045) with a slightly greater increase over time in the Hi-Fit group, but not for V̇CO2 (p = 0.222). There was no main effect of fitness on RER (p = 0.314) or fitness by time interaction (p = 0.457). We observed a main effect of fitness on V̇E (p < 0.001), but no fitness by time interaction (p = 0.185). There was no main effect of fitness on fB (p = 0.686) or fitness by time interaction (p = 0.810). There was a significant main effect of fitness on VT (p = 0.006) and no fitness by time interaction (p = 0.200), with Hi-Fit individuals displaying greater values throughout exercise. There was a main effect of fitness on V̇E/V̇O2 (p = 0.002) and V̇E/V̇CO2 (p = 0.001), with Avg-Fit displaying higher values throughout exercise. There was no fitness by time interaction for V̇E/V̇O2 (p = 0.462) or V̇E/V̇CO2 (p = 0.213). A significant main effect of fitness was observed for PETCO2 (p = 0.013), with Hi-Fit individuals displaying greater values throughout exercise. There was no observed fitness by time interaction on PETCO2 (p = 0.757) 40 Figure 3.1 Ratio of V̇O2 During the Constant Load Exercise Test to V̇O2max Abbreviations: V̇O2, oxygen uptake; V̇O2max, maximum oxygen uptake. No significant fitness difference (p > 0.05) 41 Table 3.3 Representative Participants Avg-Fit (Participant 15) Hi-Fit (Participant 13) Height, cm 160 165 Weight, Kg 51.4 54.0 TLC, L 4.69 5.47 VA, L 4.21 4.88 V̇O2max, mL/kg/min 40.4 61.5 V̇O2, % predicted 94.3 143.3 V̇Emax, L/min 83.0 140.3 TTE, seconds 540 592 V̇O2 Average TTE, mL/kg/min 36.2 56.5 V̇O2 Average/ V̇O2Max, % 90 92 Cumulative WB, Joules 1007.2 2979.7 Cumulative PTPeso, cmH2O/s 4164.4 5656.1 Cumulative PTPdi, cmH2O/s 3455.8 4409.5 5 min post TTE Pdi,tw, % of baseline 89.8 83.6 Comparison of two participants of similar size. Abbreviations: TLC, total lung capacity; VA, alveolar volume; V̇Emax, maximum minute ventilation; V̇O2, oxygen uptake; TTE, time to exhaustion test (constant load exercise test); WB, work of breathing; PTPeso, esophageal pressure time product; PTPdi, diaphragmatic pressure time product; Pdi,tw, transdiaphragmatic twitch pressure. 42 Table 3.4 Cardiorespiratory Data During the Constant Load Exercise Test to Exhaustion Relative Time to Exhaustion p - value Group 20% 40% 60% 80% 100% Time Fitness Fitness*Time HR, Avg-Fit 169±8 176±9 181±8 183±9 182±10 <0.001* 0.391 0.382 beats/min Hi-Fit 164±14 171±12 176±11 179±10 180±9 V̇O2, Avg-Fit 1.9±0.4 2.1±0.4 2.1±0.4 2.1±0.4 2.0±0.4 <0.001* <0.001* 0.045* L/min Hi-Fit 3.1±0.4 3.3±0.4 3.4±0.4 3.5±0.4 3.3±0.4 V̇CO2, Avg-Fit 2.1±0.4 2.3±0.5 2.3±0.4 2.3±0.5 2.1±0.4 <0.001* <0.001* 0.222 L/min Hi-Fit 3.2±0.4 3.6±0.5 3.7±0.5 3.7±0.5 3.5±0.5 RER Avg-Fit 1.08± 0.07 1.13± 0.06 1.12± 0.08 1.08± 0.06 1.04± 0.04 0.004* 0.314 0.457 Hi-Fit 1.06± 0.06 1.09± 0.07 1.07± 0.07 1.05± 0.06 1.04± 0.06 V̇E, Avg-Fit 61±10 75±12 81±13 87±18 84±15 <0.001* <0.001* 0.185 L/min Hi-Fit 83±9 97±11 109±11 116±8 116±10 fb, Avg-Fit 37±10 44±12 48±13 55±15 57±13 <0.001* 0.686 0.810 breaths /min Hi-Fit 35±8 41±11 46±11 52±9 57±7 VT, L Avg-Fit 1.7±0.4 1.7±0.4 1.6±0.3 1.6±0.3 1.5±0.3 <0.001* 0.006* 0.200 Hi-Fit 2.3±0.5 2.4±0.6 2.3±0.5 2.2±0.5 2.0±0.4 PETCO2, mmHg Avg-Fit 35.1± 4.0 31.5± 3.7 28.7± 3.5 26.0± 3.6 25.0± 3.2 <0.001* 0.013* 0.757 Hi-Fit 39.4± 3.5 36.2± 4.0 32.8± 3.9 29.9± 3.0 28.4± 3.1 V̇E/ V̇O2 Avg-Fit 32±4 37±5 39±5 42±6 43±7 <0.001* 0.002* 0.462 Hi-Fit 27±2 30±3 32±4 34±3 35±4 V̇E/ V̇CO2 Avg-Fit 30±3 32±4 35±4 38±4 41±5 <0.001* 0.001* 0.213 Hi-Fit 26±2 27±3 30±3 32±3 34±4 Values are means ± SD. Abbreviations: HR, heart rate; V̇O2, oxygen uptake; V̇CO2, carbon dioxide production; RER, respiratory exchange ratio; V̇E, minute ventilation; fb, breathing frequency; VT, tidal volume; PETCO2, end-tidal pressure of carbon dioxide. * = significant effect (p < 0.05). 43 3.4 Diaphragmatic Function 3.4.1 Supramaximal Stimulation CMS recruitment curves are shown in Figure 3.3. In the Hi-Fit group, no significant difference was observed between the 95% and 100% stimulation intensities (p = 0.151). In the Avg-Fit group no significant difference was observed between the 90% and 100% (p = 0.460), and the 95% and 100% stimulation intensities (p = 0.935). 3.4.2 Diaphragmatic Fatigue In the present study the threshold for EIDF was set at >15% reduction in Pdi,tw, relative to baseline, approximately three times greater than the observed coefficient of variation of Pdi,tw during the potentiated twitch protocol (6.15%). According to this criterion five Hi-Fit participants and three Avg-Fit participants experienced EIDF. Twitch control characteristics are shown in Table 3.6. Relative and absolute Pdi,tw are shown in Table 3.5 and Figure 3.4. The main effect of fitness on absolute Pdi,tw was significant (p = 0.005) and the fitness by time interaction was not (p = 0.186). There was no significant main effect of fitness on Pdi,tw relative to baseline (p = 0.186), and no time by fitness interaction (p = 0.930). There was no clear relationship (r = -0.215; p = 0.391) between aerobic fitness and relative Pdi,tw 5-min post exercise (Figure 3.5). 44 Figure 3.2 Illustration of Potentiated Twitches Unaltered trace of a single potentiated twitch at each measurement time of a participant who did not experience a >15% post-exercise reduction in Pdi,tw. All traces are from participant 23. Participant 23 was a Hi-Fit participant (V̇O2max = 51.9 mL/kg/min). Participant 23 had a cycling time to exhaustion of 449 seconds. The legend includes relative percentage of Pdi,tw when compared to baseline. Abbreviations: Pdi,tw, transdiaphragmatic twitch pressure. 45 Figure 3.3 CMS Recruitment Curves Graphs show Pdi,tw ± SD for each level of CMS intensity. Mean data of each group is depicted in Panel A. Group mean data and individual data for the Avg-Fit and Hi-Fit participants are shown in Panel B and C, respectively. Abbreviations: Pdi,tw, transdiaphragmatic twitch pressure. * = significantly different than 100% intensity (p < 0.05) 46 Table 3.5 Absolute and Relative Pdi,tw Measurement Time p - value Group Baseline 5-Min Post 30-Min Post 60-Min Post Time Fitness F*T Absolute Pdi,tw, cmH2O Avg-Fit 30.8  3.7 26.9  2.7 30.1  3.3 31.3  3.6 <0.001* 0.005* 0.186 Hi-Fit 40.9  7.3† 33.8  8.2† 38.2  7.7† 39.4  5.9† Relative Pdi,tw, % Baseline Avg-Fit 100 87.8  8.5 97.7  6.9 101.8  5.0 <0.001* 0.186 0.930 Hi-Fit 100 82.5  12.0 93.9  9.8 97.5  5.6 Values are means ± SD. Abbreviations: Pdi,tw, transdiaphragmatic twitch pressure; Hi-Fit, highly-aerobically fit; Avg-Fit, averagely-aerobically fit. * = significant effect (p < 0.05), † = significant fitness difference (p < 0.05). 47 Figure 3.4 Relative and Absolute Pdi,tw Panel A, Pdi,tw relative to baseline. Panel B, absolute Pdi,tw . Abbreviations: Pdi,tw , transdiaphragmatic twitch pressure. * = significant fitness difference (p < 0.05) 48 Figure 3.5 Relationship Between EIDF and Aerobic Fitness Abbreviations: Pdi,tw , transdiaphragmatic twitch pressure; V̇O2max, maximum oxygen uptake. * = significant relationship (p < 0.05). 49 Figure 3.6 Relationship between EIDF and Exercise Time Abbreviations: Pdi,tw, transdiaphragmatic twitch pressure. * = significant relationship (p < 0.05). 50 Figure 3.7 Relationship Between EIDF and Respiratory Mechanics Abbreviations: WB, work of breathing; PTPdi, diaphragmatic pressure time product; Pdi,tw, transdiaphragmatic twitch pressure. * = a significant relationship (p < 0.05). 51 3.5 Breathing Mechanics The WB and its components during exercise are displayed in Figure 3.9. There was a significant fitness by time interaction on WB (p < 0.001); a greater time dependent increase in WB was observed in the Hi-Fit group. A significant fitness by time interaction was observed in inspiratory resistive WB (p < 0.001), inspiratory elastic WB (p < 0.001), and expiratory WB (p = 0.006), all of which were due to a greater time dependent increase in the Hi-Fit group. At end exercise cumulative WB was significantly greater in the Hi-Fit group compared to the Avg-Fit group (p = 0.002) (Figure 3.11). Respiratory pressure generation variables during exercise are displayed in Figure 3.10. There was a significant fitness by time interaction on PTPeso (p = 0.002), due to a greater time dependent increase in the Hi-Fit group. Furthermore, a significant main effect of fitness on PTPeso (p < 0.001) was observed, as values were greater in the Hi-Fit group throughout exercise. A significant fitness by time interaction was observed on PTPdi (p < 0.001) because PTPdi increased at a greater rate in the Hi-Fit group. But no main effect of fitness observed on PTPdi (p = 0.354). There was no fitness by time interaction (p = 0.197) on PTPeso/PTPdi. But a significant main effect of fitness was observed on PTPeso/PTPdi (p = 0.001); the Avg-Fit group displayed a greater ratio throughout exercise. At end exercise cumulative PTPeso was slightly greater in the Hi-Fit group, however this difference was not significant (p = 0.075). Furthermore, cumulative PTPdi at end exercise was similar between groups (p = 0.751). Correlations comparing the post exercise reduction in Pdi,tw to breathing mechanics are detailed in Figure 3.7. No significant relationship was observed between the cumulative WB or cumulative PTPdi with Pdi,tw post exercise (r = 0.027; p = 0.914 and r = 0.020; p = 0.938, respectively). Nor with the relationship between average WB and Pdi,tw post exercise (r = -0.367; 52 p = 0.135). A significant relationship was observed between average PTPdi and Pdi,tw post exercise (r = -0.750; p < 0.001). Figure 3.8 WB versus V̇E for All Participants During the Constant Load Exercise Test Each data point corresponds to a 10% TTE increment during the constant load exercise test. There are ten data points for each participant. Abbreviations: WB, work of breathing; V̇E, minute ventilation; TTE, time to exhaustion.53 Figure 3.9 WB and Components of WB During the Constant Load Exercise Test Abbreviations: WB, work of breathing. * = significant fitness difference (p < 0.05) 54 Figure 3.10 Rate of Pressure Produced During the Constant Load Exercise Test Abbreviations: PTPeso, esophageal pressure time product; PTPdi, diaphragmatic pressure time product. * = significant fitness difference (p < 0.05) 55 Figure 3.11 Cumulative Mechanical Load at Exercise Cessation Abbreviations: WB, work of breathing; PTPeso, esophageal pressure time product; PTPdi, diaphragmatic pressure time product. * = significant fitness difference (p < 0.05) 56 Figure 3.12 Relationship between Cumulative Diaphragmatic Work, the Rate of Diaphragmatic Pressure Production, and Time Abbreviations: PTPdi, diaphragmatic pressure time product. * = a significant relationship (p < 0.05). 57 Table 3.6 Potentiated Twitch Control Measures Electrical (top panel) and mechanical (bottom panel) characteristics of potentiated twitches. * = significant difference from baseline (p < 0.05). M-Wave Characteristic Avg-Fit (n = 7) Hi-Fit (n = 7) Baseline 5 Min 30 Min 60 Min Baseline 5 Min 30 Min 60 Min Right Hemi-Diaphragm Amplitude (V) 1.0±0.2 1.1±0.2* 1.1±0.2* 1.0±0.2 1.2±0.3 1.2±0.3 1.1±0.4 1.1±0.4 Area (V·ms) 5.7±0.9 6.0±1.2 5.8±0.9 5.4±0.8 6.9±1.9 7.3±1.7* 6.9±1.8 6.8±1.8 Latency (ms) 6.6±0.6 6.4±0.8 6.6±0.8 6.6±0.8 6.4±0.6 6.5±0.7 6.4±0.7 6.5±0.7 Duration (ms) 25.9±3.2 25.4±3.3 25.7±3.6 25.1±3.0* 27.3±2.7 27.4±1.5 27.1±1.2 26.6±1.3 Left Hemi-Diaphragm Amplitude (V) 1.0±0.2 1.1±0.3 1.1±0.2 1.1±0.2 1.0±0.3 1.1±0.2 1.1±0.3 1.0±0.3 Area (V·ms) 5.8±1.2 6.1±1.0 5.9±1.0 5.8±1.0 6.2±1.2 6.9±1.1* 6.4±1.3 6.5±1.6 Latency (ms) 6.4±0.6 6.2±0.4 6.3±0.5 6.3±0.5 6.9±0.9 6.7±0.9 6.8±0.9 6.8±0.8 Duration (ms) 26.8±3.3 26.5±3.5 26.0±3.0* 25.9±3.2* 25.1±2.2 27.0±1.8* 25.6±2.0 25.7±1.9 Twitch Characteristic Avg-Fit (n = 9) Hi-Fit (n = 9) Baseline 5 Min 30 Min 60 Min Baseline 5 Min 30 Min 60 Min Contraction Time (ms) 132 ± 6 120±10* 129±11 133±10 138±9 120±8* 131±10* 135±8 ½ Relaxation Time (ms) 91±12 70±5* 82±9 87±10 83±15 71±11* 82±14 83±14 58 Chapter 4: Discussion 4.1 Main Findings The purpose of this thesis was to determine the effect of aerobic fitness on the development of EIDF in females. EIDF was assessed following intense cycle exercise to exhaustion in two groups of healthy females, one highly-aerobically fit (Hi-Fit) and one averagely-aerobically fit (Avg-Fit). It was hypothesized that the Hi-Fit females would experience a reduced incidence and magnitude of EIDF when compared to Avg-Fit females. Contrary to our hypothesis, aerobic fitness does not appear to affect the incidence and magnitude of EIDF in females. The greater ventilatory demands in the Hi-Fit group during exercise were predominately met through an increase in accessory respiratory muscle contribution to inspiratory pressure generation as indicated by the ratio of PTPdi to PTPeso. Reliance on accessory muscles likely serves to minimize the pressure output of the diaphragm, which is a determinate of EIDF. Indeed, a strong relationship between average diaphragmatic pressure output during exercise and the magnitude of EIDF was observed. Greater absolute diaphragmatic strength in the Hi-Fit group did not result in EIDF resistance. It is possible that intramuscular adaptations within the diaphragm occur but do not result in a lower incidence of EIDF during exhaustive exercise. Together our findings suggest that irrespective of aerobic fitness in females, high-intensity whole-body dynamic exercise to exhaustion results in a similar mismatch between diaphragmatic demands and aerobic energy supply. Chronic whole-body endurance training and the exercise hyperpnea experienced during training do not appear to result in adaptations that mitigate EIDF. 59 4.2 EIDF and Aerobic Fitness No effect of aerobic fitness on the relative magnitude of EIDF in females was observed following high-intensity exercise or during recovery. These results suggest that previously reported sex-differences in EIDF are likely independent of the fitness of female subjects. Our findings agree with Babcock et al. (1996) who also reported no significant effect of aerobic fitness on EIDF, albeit in an almost exclusively male cohort. While these results were unexpected, the magnitude of EIDF observed in the Hi-Fit group, was similar to previously reported values in aerobically fit females. Guenette et al. (2010) and Welch et al. (2018) investigated EIDF in a population of females with an average V̇O2max of 57.1 mL/kg/min and 52.3 mL/kg/min, respectively. Following exercise to exhaustion, Guenette et al. (2010) and Welch et al. (2018) reported a 13.0% and 13.5% reduction in Pdi,tw, respectively. While slightly less than the 17.5% reduction observed in the Hi-Fit group in the present study these values are similar, especially when post-exercise measurement times are considered. The initial post-exercise measurements in Guenette et al. (2010) occurred 10-minutes after exercise cessation, whereas in the present study and in Welch et al. (2018) initial post-exercise measurements occurred 5-minutes after exercise cessation. Given the rapid recovery of diaphragmatic contractile function in females (<60 minutes), a 5-minute difference in the initial post-exercise contractile function measurement could result in substantial EIDF recovery. Previous investigations of EIDF that included a cohort of averagely-aerobically fit females have reported conflicting EIDF values (Ramsook et al., 2022; Reinhard et al., 2023). For example, the magnitude of EIDF observed in the Avg-Fit group in the present study aligns with Ramsook et al. (2022). They reported a 14.8% reduction in relative Pdi,tw following exercise to 60 exhaustion in a cohort of averagely-aerobically fit females (V̇O2max = 40.1 mL/kg/min) which is similar to the 12.2% observed in the Avg-Fit group (Table 3.5). We observed a substantially lower amount of EIDF than Reinhard et al. (2023). They observed a 21.5% reduction in relative Pdi,tw, following exercise to exhaustion in a group of females with a slightly above average aerobic fitness (V̇O2max = 45.4 mL/kg/min). Reinhard et al. (2023), reported that EIDF measurements were taken immediately after exercise cessation. Again, the immediate post-exercise time window may be critical for EIDF recovery. Indeed, at 10 minutes post-exercise Reinhard et al. (2023) reported a reduction in Pdi,tw of 12.7% in females, akin to the 12.2% value observed in our Avg-Fit group at 5-minutes after exercise cessation. Considerable within group variability in the occurrence of EIDF was present in this study and in previous investigations of EIDF in females. As defined by a greater than 15% post exercise reduction in Pdi,tw, Guenette et al. (2010) observed EIDF in 8 out of 19 females (42%), Welch et al. (2018) observed EIDF in 3 out of 9 females (33%), and in the present study EIDF was observed in 5 out of 9 Hi-Fit females (56%) and 3 out of 9 Avg-Fit females (33%). The large variability of EIDF incidence in females is not reflected in the mean post-exercise reduction in Pdi,tw. Therefore, when comparing relatively small sample sizes of individuals, this variability could have a substantial effect on the mean reduction in Pdi,tw.. It is worth noting that the total incidence of EIDF reported in the present study differs from incidence of EIDF reported by Babcock et al. (1996) in their investigation on the effect of aerobic fitness on EIDF. In the present study, 8 of the 18 participants (44%) experienced EIDF, whereas 21 of the 24 participants (88%) reported EIDF in Babcock et al. (1996). The discrepancy in the incidence of EIDF between these two studies may be due to sex differences in EIDF: females were exclusively used in the present study versus a majority male cohort (20 males, 4 females) used in 61 the Babcock et al. (1996) study. It is also possible that the use of electrical phrenic nerve stimulation in Babcock et al. (1996) allowed for a more accurate assessment of the magnitude of EIDF because CMS, which was used in the present study, may underestimate EIDF (Angus et al., 2023). In the present study, M-wave amplitude and total rectified area tended to increase relative to baseline during the 5-minute post-exercise measurements in both hemidiaphragms and in both groups (Table 3.6). Ramsook et al. (2022) reported a significant post-exercise increase in M-wave amplitude, but other investigations have not (Guenette et al., 2010; Welch et al., 2018; Reinhard et al., 2023). According to Rodrigues and Falces (2017), increases in M-wave amplitude may be evidence of impaired sarcolemma excitability. Since an M-wave is a composite of all action potentials within a muscle the broadening of individual potentials, caused by performance fatigability, can result in a greater summative peak and therefore a greater amplitude (Rodriguez and Falces, 2017). However, increases, non-significant changes, and decreases in M-wave amplitude have all been reported when performance fatigability has occurred (Dimitrova & Dimitrov, 2003) which makes it difficult to assess if impaired sarcolemma excitability contributed to the post-exercise reduction in Pdi,tw in the present study. 4.3 Ventilatory Response and Mechanical Cost Endurance training has been demonstrated to reduce the ventilatory response to exercise and is likely due to a decrease in multiple stimulating factors including circulating metabolites, neural feedforward, and neural feedback mechanisms (Sheel & Romer, 2012). In the present study the ventilatory response relative to metabolic demand was different between groups; throughout exercise we observed a lower V̇E/V̇CO2 and V̇E/V̇O2 ratio in the Hi-Fit subjects (Table 3.4). A greater PETCO2 was also observed in the Hi-Fit individuals throughout the entirety 62 of the constant load exercise test to exhaustion, providing further evidence of a reduced hyperventilatory response. Babcock et al. (1996) also reported a lower V̇E/V̇CO2 ratio in their aerobically fit group when compared to their less aerobically fit group. Fitness differences in respiratory efficiency provide another explanation for the difference in V̇E/V̇CO2. Since fB was not different between groups at any point during the constant load exercise test, the observed group difference in V̇E was due to the Hi-Fit individuals having a greater VT. An elevation in VT would reduce dead space fraction, increasing the proportion of VA thereby improving ventilatory efficiency. Indeed, when VT is expressed as a proportion of VA it is greater in the Hi-Fit group when compared to the Avg-Fit group. Irrespective of the cause, a reduced ventilatory response to a given metabolic demand would reduce the total force required by the respiratory muscles, which may protect against EIDF. While the Hi-Fit group had a reduced ventilatory response relative to their metabolic demand they also had a significantly greater metabolic demand during exercise which resulted in a significantly higher V̇E. The relationship between V̇E and mechanical work is well documented; as V̇E increases WB increases in an exponential fashion (Otis, 1950). Due to sex differences in average airway size in females the increase in WB for a given increase in V̇E is even greater than that seen in males. (Peters et al., 2021; Dominelli et al., 2015). As expected, WB was significantly greater in the Hi-Fit group when compared to the Avg-Fit group across all timepoints during exercise (Figure 3.9). The fitness difference in WB was so pronounced that at exercise cessation cumulative WB in the Avg-Fit group (1378 J) was ~55% of the value observed in the Hi-Fit group (2525 J) (Figure 3.11). Despite exercising for similar amounts of time the Hi-Fit group performed almost double the mechanical work. Why can the Hi-Fit group sustain a greater WB, a determinate of EIDF (Babcock et al., 2002) without 63 a concomitant increase in EIDF? Babcock et al. (1996) proposed two explanations as to why Hi-Fit individuals do not experience more EIDF despite greater mechanical work: 1) a lower diaphragmatic contribution to total inspiratory pressure generation, and 2) a training induced increase in the aerobic capacity of their diaphragm. 4.4 Aerobic Fitness and Respiratory Mechanics Despite greater ventilatory requirements Hi-Fit individuals appear to be placing a similar load on the diaphragm as the Avg-Fit individuals. Throughout exercise diaphragmatic pressure output, as measured by PTPdi, was similar between groups, the Hi-Fit group was only significantly greater at 100% TTE (Figure 3.10). As a result, cumulative PTPdi was not significantly different between groups at exercise cessation (Figure 3.11). A likely explanation for the similar PTPdi between groups is a greater use of accessory inspiratory muscles in the Hi-Fit participants. In the present study, inspiratory muscle activity was not measured using EMG. But the ratio of PTPdi to PTPeso was used to determine the relative amount of pressure that the diaphragm and accessory muscles generate during inspiration. Hi-Fit females consistently had a lower PTPdi/PTPeso throughout exercise when compared to Avg-Fit females (Figure 3.10). The lower PTPdi/PTPeso provides evidence of a greater relative accessory muscle contribution to inspiratory pressure generation in the Hi-Fit females. Furthermore, since PTPdi was similar between groups for most of exercise (20%-80% TTE), the greater ventilatory demands of the Hi-Fit group were almost entirely met through greater accessory muscle contribution. The breathing strategy employed by the Hi-Fit individuals minimizes diaphragmatic pressure output, a determinate of EIDF (Babcock et al., 1995), which should protect this group against the fatiguing consequences of high respiratory muscle work. Furthermore, minimizing the development of EIDF should also improve exercise tolerance 64 (Welch et al., 2018). EIDF is associated with activation of type III and IV afferents arising from the diaphragm (Hill, 2000). The activation of these afferents is believed to cause vasoconstriction in active locomotor muscles due to a sympathetically mediated metaboreflex, hastening the development of locomotor fatigue by limiting oxygen delivery (Romer & Polkey, 2008). EIDF is also associated with exercise intolerance due to the sensation of dyspnea (Boyle et al., 2020). It is possible that the post-exercise reduction in Pdi,tw in the Avg-Fit group could have been further minimized if they had a PTPdi/PTPeso ratio that was lower, similar to that of the Hi-Fit group. While speculative, by reducing diaphragmatic work, the Avg-Fit group may have been able to increase exercise tolerance by delaying the onset of EIDF. However, according to the principle of minimum effort, respiratory mechanics at a given V̇E are well suited to minimize the mechanical cost of breathing (Otis, 1950). Since the Avg-Fit group has a much lower V̇E throughout exercise, fitness differences in respiratory muscle contribution could be due to a ventilatory strategy which minimizes the mechanical work at a given V̇E. Thus, reducing total mechanical work may take precedence over reducing diaphragmatic force requirements. During exercise in the Hi-Fit group, PTPdi and PTPeso steadily increased reaching a maximum at 100% TTE (Figure 3.10). The rise in PTPdi and PTPeso occurred in proportion with each other keeping the ratio of PTPdi/PTPeso stable throughout the duration of exercise past the 20% TTE mark. The stability of the PTPdi/PTPeso ratio throughout exercise in females is similar to previously reported results (Guenette et al., 2010; Welch et al., 2018; Reinhard et al., 2023). In the Avg-Fit group a gradual decrease in PTPdi/PTPeso was observed from 20% to 80% TTE, but from 80% to 100% TTE the ratio remained stable (Figure 3.10). The initial gradual decrease in PTPdi/PTPeso was due to a gradual increase in PTPeso, while PTPdi remained constant. However, 65 no fitness by time interaction was observed for PTPdi/PTPeso, so the observed gradual decrease in the Avg-Fit group may not actually differ from the stability observed in the Hi-Fit group. 4.5 Diaphragmatic Fatigue and Absolute Diaphragmatic Load Notably, we observed a strong relationship between average PTPdi during exercise and EIDF, but a weak relationship between cumulative PTPdi at the end of exercise and EIDF (Figure 3.7). A similar finding was reported by Johnson et al. (1993), who observed a significant relationship between the magnitude of EIDF and the percentage change of PTPdi from rest to PTPdi in the final 20% of exercise, but no relationship between cumulative PTPdi and EIDF. The rate of diaphragmatic pressure production appears to be more important to the development of EIDF than the total amount of pressure produced over the duration of exercise. The reason PTPdi is correlated with EIDF but cumulative PTPdi is not, is difficult to determine due to the multiple factors which cause EIDF during intense whole-body exercise. A possible explanation could be related to the concept of critical power. Critical power is the greatest metabolic rate which can be achieved through oxidative phosphorylation with substrate-level phosphorylation remaining in a steady state (Poole et al., 2016). A metabolic rate greater than the threshold of critical power results in the rapid development of a physiological environment associated with performance fatigability (Grassi et al., 2015). While commonly used to discuss whole-body exercise, the concept of critical power also applies to intramuscular metabolism (Jones et al., 2008). The diaphragm should have a critical power, when surpassed diaphragmatic fatigue occurs progressively. Whole-body exercise appears to reduce the critical power of the diaphragm. In the absence of whole-body exercise, when high-intensity exercise hyperpnea is replicated at rest EIDF does not occur (Babcock et al., 1995). For EIDF to reach similar levels to that seen during 66 high-intensity exercise to exhaustion, PTPdi must be 1.5-2 times greater than that observed during high-intensity exercise (Babcock et al., 1995). It is worth noting that during inspiratory loading tasks, in the absence of whole-body exercise, task failure is highly dependent on the tension-time index of the diaphragm: if the inspiratory portion of the duty cycle is increased, a lower Pdi can cause task failure (Bellemare & Grassino, 1982). During inspiration blood flow to the diaphragm is mechanically impeded, so a greater inspiratory time relative to the duty cycle results in more blood flow limitation making the diaphragm more vulnerable to performance fatigability (Bellemare et al., 1983). A tension-time index of greater than 0.15 is likely to result in ventilation being unsustainable and task failure will occur (Bellemare & Grassino, 1982). However, the tension-time index of the diaphragm during intense whole-body exercise to exhaustion is a poor predictor of the magnitude of EIDF experienced (Babcock et al., 1995; Johnson et al., 1993). The onset of EIDF during intense whole-body exercise is also likely related to diaphragmatic blood flow. At rest ample blood can be diverted to the diaphragm to support the replication of exercise hyperpnea. But during intense exercise the respiratory and locomotor muscles appear to compete for a limited supply of blood (Sheel et al., 2018). For example, high respiratory muscle work decreases blood flow to the legs during maximal exercise (Harms et al., 1997), whereas high locomotor muscle work appears to limit blood flow to the respiratory muscles during intense whole-body exercise (Vogiatzis et al., 2009). The reduction in blood flow in response to muscular work is believed to be mediated by global sympathetic vasoconstriction caused by activation of type III and IV afferents located in the exercising musculature (Sheel et al., 2018). Thus, during intense exercise cardiac output must be rationed between competing musculature, with neither respiratory nor locomotor muscles receiving a satisfactory share; the 67 lack of adequate oxygen supply promotes performance fatigability in both the respiratory and locomotor muscles (Sheel et al., 2018). Further supporting the assertation that inadequate oxygen supply promotes diaphragmatic fatigue, when blood flow in the phrenic artery is experimentally increased the magnitude of diaphragmatic fatigue is reduced (Supinski et al., 1987). It is possible that intense whole-body exercise limits blood flow to the diaphragm, blood flow limitation would reduce the power output that the diaphragm can maintain without the onset of performance fatigability. The critical power of the diaphragm is likely reduced during exercise. During high intensity exercise the power output of the diaphragm may exceed the critical power of the diaphragm, causing the onset of EIDF. Average PTPdi is likely closely related to the magnitude of EIDF because individuals with a greater average PTPdi are more likely to have a diaphragmatic power output that approaches or exceeds the critical power of their diaphragm. Cumulative PTPdi likely shows little relationship with EIDF (Figure 3.7) because it closely relates to absolute exercise time (Figure 3.12) but does not relate to average PTPdi (Figure 3.12). Therefore, individuals who exercised for longer can have a large cumulative PTPdi, but a relatively low average PTPdi. When participants exercise to exhaustion average PTPdi is a greater predictor of EIDF than cumulative PTPdi because average PTPdi more accurately reflects the intensity of diaphragmatic contractions. 4.6 Diaphragmatic Intramuscular Adaptions It is well recognized that regular endurance training of locomotor muscles results in numerous intramuscular adaptations that improve performance fatigability resistance. The diaphragm, like locomotor muscles, is a skeletal muscle, therefore it is reasonable to predict that the diaphragm of the Hi-Fit individuals would be resistant to performance fatigability due to chronic exposure to exercise hyperpnea during whole-body endurance training sessions. 68 However, in humans, intramuscular adaptations within the diaphragm in response to endurance training are unknown. Currently, direct evidence for intramuscular adaptations within the mammalian diaphragm in response to endurance training comes from rodent studies. In rodents it is well established that endurance training can induce intramuscular adaptations that improve the oxidative capacity of the diaphragm (Powers et al., 1997). Evidence from rodent models currently provides mechanistic insight into how the mammalian diaphragm responds to regular endurance training. In rodents, endurance adaptations of the costal diaphragm appear to be independent of exercise intensity (Powers et al., 1994). Furthermore, in rodents, the pre-training oxidative capacity of the diaphragm is greater than that of locomotor muscles with a similar fibre type composition (Powers et al., 1990) and increases in oxidative capacity within the diaphragm are less than those observed in locomotor muscles (Powers et al., 1997). We did observe a possible training related diaphragmatic adaptation in the Hi-Fit participants. Absolute Pdi,tw was greater at all time points in the Hi-Fit group when compared to the Avg-Fit group (Figure 3.4), suggesting that regular endurance training produces diaphragmatic strength gains. Interestingly, fitness differences in absolute Pdi,tw were not observed by Babcock et al. (1996) in their investigation of the effect of aerobic fitness on EIDF. This discrepancy may be related to the fitness of the included cohorts. In Babcock et al. (1996) the lower fitness group had a V̇O2max = 50.4 ± 1.7 mL/kg/min. For context, the lower fitness group in the Babcock study was mostly males with an average age of 34, the average V̇O2max for males 30-39 is 42.4 ± 4.7 mL/kg/min (Myers et al., 2017). Whereas, in the current study the lower fitness group was slightly below average fitness (Table 3.2). The lower fitness group in Babcock et al. (1996) may have been exposed to enough regular exercise hyperpnea to stimulate strength gains, whereas the Avg-Fit group in the present study has not. It is also possible that 69 electrical stimulation of the phrenic nerves used in Babcock et al. (1996) produced a maximal diaphragmatic contraction in all participants, whereas CMS used in the present study was incapable of maximally stimulating the diaphragm of all participants (Angus et al., 2023). In the present study, greater diaphragmatic strength occurred without parallel improvements in the ability of the diaphragm to resist EIDF (Figure 3.4). If the diaphragm in the Hi-Fit participants had endurance related intramuscular adaptations, it would be expected that EIDF would be reduced under conditions where the PTPdi and/or cumulative PTPdi was similar between groups. However, both the Hi-Fit and Avg-Fit group had a similar PTPdi and cumulative PTPdi and EIDF was not lower in the Hi-Fit group. While it may appear counterintuitive, there are explanations as to why the diaphragm of Hi-Fit individuals does not appear to be more resistant to EIDF. Adaptations within a muscle are in part mediated by the metabolic and mechanical stress it experiences during training. Perhaps the training intensities commonly used by endurance athletes are insufficient to generate a V̇E that results in substantial metabolic and/or mechanical demand on the diaphragm. For example, many athletes do not regularly exercise under conditions known to cause EIDF (>85% V̇O2max to exhaustion) and therefore would not experience the mechanical and metabolic demands on their diaphragm which cause performance fatigability. However, as previously mentioned adaptations within the diaphragm of a rodent are independent of exercise intensity (Powers et al., 1994). If the human diaphragm is similar, insufficient training intensity could not explain the apparent lack of adaptation. Alternatively, it is possible that the diaphragm of a non-endurance trained individual already has a substantial endurance capacity, and any additional endurance adaptations from training are insignificant. In female humans this seems particularly relevant, 70 given the relatively low magnitude of EIDF that has been observed in non-aerobically trained individuals, in the present study, and in others (Ramsook et al., 2022). It is also possible that the diaphragm does experience endurance related intramuscular adaptations to regular endurance training, but other fatiguing influences present during high-intensity whole-body exercise cause EIDF to be similar between fitness groups at exhaustion. First, even though diaphragmatic blood flow was not measured during this study brief comment it merited to contextualize the results. High-intensity exercise to exhaustion would have likely caused blood flow competition between the respiratory and locomotor muscles, limiting oxygen supply to the diaphragm (Sheel et al., 2018). Reduced oxygen supply would limit the critical power of the exercising muscle (Poole et al., 2016). Even if Hi-Fit individuals had substantial endurance related diaphragmatic adaptations, limitations in diaphragmatic blood flow may cause a similar degree of EIDF by the time whole-body exhaustion is reached. Second, the uptake of circulating metabolites by the diaphragm has been previously identified as a potential causative factor in EIDF (Fregosi & Dempsey., 1986). During high-intensity exercise to exhaustion, elevated levels of anaerobic energy production would raise circulating metabolites levels in each group. High levels of circulating metabolites, and its subsequent uptake by the diaphragm could impair diaphragmatic function similarly in each group. To summarize, perhaps intramuscular adaptations within the diaphragm occur but are inconsequential to protect against EIDF when faced against the homeostatic challenges induced by whole-body exercise to exhaustion. 4.7 Methodological Considerations In this thesis we did not exclude participants if they were on any form of contraceptive, and we did not attempt to assess participants in a specific phase of their menstrual cycle. We also did not attempt to track menstrual cycle phase through subjective or objective measures. It is 71 possible that hormonal differences related to the reproductive status the of participants may have confounded the results and obscured the possible effect of fitness on EIDF. Theoretically sex hormones may affect EIDF through multiple mechanisms including affecting ventilatory control and the performance fatigability of skeletal muscles, but current evidence suggests that this is unlikely. First, while progesterone has been previously demonstrated to increase V̇E at rest (MacNutt et al., 2012), during submaximal exercise progesterone has no appreciable effect on V̇E (MacNutt et al., 2012). It is possible that the feedforward and feedback mechanisms that control exercise hyperpnea override the influence of progesterone on V̇E. It is currently not known if hormonal differences in females affect ventilatory mechanics at rest or during exercise. Second, hormonal differences related to oral contraceptive use or menstrual cycle phase have no clear effect on fatigue. While some studies have shown an effect of the menstrual cycle on TTE performance, most studies have not (Pereira et al., 2020). Oral contraceptive use may have minor effects on anaerobic capacity, aerobic capacity, and muscle strength, but these effects are subjects to subject to high-interindividual variability (Burrows & Peters, 2007). We cannot rule out that hormonal differences in our participants may have confounded the results, but we believe that it is unlikely. In the current study we did not attempt to make EMG measurements of accessory respiratory muscles, such as the scalene or sternocleidomastoid, during exercise. We inferred a greater relative contribution of accessory muscles to inspiratory pressure generation in the Hi-Fit group because of their lower PTPdi to PTPeso ratio. While PTPdi/PTPeso does correlate with EMG activity of the scalene and sternocleidomastoid muscles (Molgat-Seon et al., 2018), we had no direct measure of accessory muscle activity. The inclusion of a direct measure of accessory muscle activity would have enhanced the respiratory mechanics data. 72 The ERS Statement on respiratory muscle testing lists supramaximal stimulation as a requirement to validly assess EIDF using CMS (Laveneziana et al., 2019). The ability of CMS to achieve supramaximal stimulation has recently been called into question (Angus et al., 2023; Dominelli et al., 2018). When using two-stimulators attached to a single coil absolute Pdi,tw was significantly greater than using a single stimulator (Angus et al., 2023). The maximum magnetic field of a single stimulator may be inadequate to supramaximally stimulate the phrenic nerves, which could overestimate diaphragmatic fatigue (Angus et al., 2023). In the present study careful attention was taken to only include participants that demonstrated a plateau in Pdi,tw with increasing stimulator intensity. Only including participants that reached a plateau in Pdi,tw should minimize the potential error associated with using a single stimulator. 4.8 Unresolved Questions and Future Directions The results of this thesis leave many unresolved questions regarding the response of the diaphragm to chronic endurance training and the implications of enhanced endurance of the diaphragm on whole-body exercise tolerance. It is unclear if the diaphragm gains substantial intramuscular adaptations to regular endurance training. Histologic analysis and respirometry performed on human diaphragmatic tissue would be the obvious answer to resolve this knowledge gap. However, unlike muscles of the limb, muscle biopsies of the diaphragm cannot be performed in healthy human subjects in a research setting. Until this is possible, proxies of diaphragmatic adaptations must be used. Non-invasive imaging techniques of diaphragm could provide a measure of thickness that may be indicative of diaphragmatic hypertrophy in response to training. However, hypertrophy of the diaphragm may not necessarily translate to increased fatigability resistance. The development of 73 non-invasive imaging techniques that can measure muscle fibre type composition would be useful in investigations examining diaphragmatic adaptations to training. Currently, the most actionable investigation to determine if aerobic fitness results in endurance improvements in the diaphragm, is to study the endurance of the diaphragm in an environment where blood flow limitation to the diaphragm is not occurring, such as in the absence of whole-body exercise. For example, using pressure-threshold loading to expose the diaphragm to a given absolute load and various loads relative to the maximal pressure producing capacity of the diaphragm would reveal if individuals with high aerobic fitness can sustain diaphragmatic work for longer than those with average aerobic fitness. If there is evidence of endurance-related diaphragmatic adaptations to regular endurance training, two questions need to be asked. First, if the human diaphragm does respond to regular endurance training with improvements in its endurance capacity, why do these adaptations not manifest as a reduced incidence and magnitude of EIDF? Second, if the human diaphragm does respond to regular endurance training with improvements in its endurance capacity, then what are the consequences of these adaptations on whole-body exercise tolerance? Experiments that can precisely quantify diaphragmatic blood flow and diaphragmatic V̇O2 during exercise would provide insight on these questions. 74 Chapter 5: Conclusion This thesis sought to determine if regular exposure to endurance training and high-aerobic fitness would provide females additional protection from EIDF. We hypothesized that greater aerobic fitness would reduce the incidence and magnitude of EIDF in females. However, no aerobic fitness-based difference in EIDF was observed. Hi-Fit females employ multiple strategies that reduce the mechanical demands of exercise hyperpnea. Hi-Fit females tend to have a lower V̇E relative to their metabolic demand. However, V̇E and WB are still significantly greater in Hi-Fit females throughout exercise. Even though the mechanical demands on the entire respiratory musculature are greater, Hi-Fit females spare their diaphragm from additional work. 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Thesis/Dissertation
eng
Kinesiology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Sheel, A. William
The effect of aerobic fitness on exercise-induced diaphragmatic fatigue in females
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