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Respiratory mechanics and diaphragmatic fatigue during exercise in men and women Guenette, Jordan Ali 2010-12-31

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    RESPIRATORY MECHANICS AND DIAPHRAGMATIC FATIGUE DURING EXERCISE IN MEN AND WOMEN   by  JORDAN ALI GUENETTE  B.H.K., The University of British Columbia, 2004 M.Sc., The University of British Columbia, 2006     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF   DOCTOR OF PHILOSOPHY   in   THE FACULTY OF GRADUATE STUDIES  (Human Kinetics)       THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)     February 2010   © Jordan Ali Guenette, 2010     ii ABSTRACT  Purpose:  The purpose of this thesis was to determine the underlying mechanisms associated with a higher total WOB in women (Study 1) and to determine if women experience greater levels of diaphragmatic fatigue relative to men (Study 2).  Methods:    Study 1:  Sixteen endurance-trained subjects (8M:8F) underwent a progressive cycling test to exhaustion while esophageal pressure and lung volumes were measured.  Modified Campbell diagrams were used to calculate the inspiratory and expiratory resistive and elastic components at 50, 75, 100 l·min-1 and maximal ventilations and also at standardized mass-corrected work-rates.  Study 2:  Thirty-eight endurance-trained subjects (19M:19F) underwent a constant-load cycling test at 90% of peak work-rate until exhaustion.  Pressure-time product of the diaphragm (PTPdi) was calculated during exercise.  Trans-diaphragmatic pressure twitches (Pdi,tw) were assessed using cervical magnetic stimulation before and 10, 30 and 60 minutes after exercise.  Diaphragm fatigue was defined as a ≥ 15% reduction in Pdi,tw post-exercise.  Results:   Study 1:  The inspiratory resistive WOB was higher in women at all absolute ventilations (P<0.05).  The expiratory resistive WOB was higher in women at 75 l·min-1 (P<0.05).  There were no sex-differences in the elastic WOB.  However, the total WOB was significantly higher in men at relative percentages of maximal ventilation (P<0.05) but this sex-difference was reversed when the WOB was standardized for a given work-rate to body mass ratio.  Study 2:  Diaphragm fatigue was present in 11 males and 8 females.  The reduction in Pdi,tw at 10 and 30 min following exercise was significantly greater in men relative to women (P<0.05).  Men consistently had higher absolute values for PTPdi during exercise but this sex-difference was reversed when body mass was taken into account.  Over time, men continued to have a reduced contribution of the diaphragm to total inspiratory force output whereas diaphragmatic contribution in women remained relatively constant over time.    Conclusions:  The higher total WOB in women is due to an increased resistive WOB which is likely attributable to their smaller airways.  Despite a respiratory system that may have a higher mechanical cost of breathing, women appear to be more resistant to exercise-induced diaphragmatic fatigue.            iii TABLE OF COglyph1197TEglyph1197TS  ABSTRACT.......................................................................................................................ii  TABLE OF COglyph1197TEglyph1197TS................................................................................................ iii  LIST OF TABLES............................................................................................................vi  LIST OF FIGURES.........................................................................................................vii  ACKglyph1197OWLEDGEMEglyph1197TS.............................................................................................ix  CO-AUTHORSHIP STATEMEglyph1197T.................................................................................x  CHAPTER I – Introduction ...............................................................................................1   Sex Differences in Respiratory Anatomy....................................................2 Sex Differences in Breathing Mechanics ....................................................8 Sex Differences in Skeletal Muscle Fatigue..............................................15 Sex Differences in Muscle Mass and Morphology....................................16 Sex Differences in Substrate Utilization ...................................................17 Sex Differences in Neuromuscular Activation..........................................17 Summary....................................................................................................18       PURPOSE..............................................................................................................19  RESEARCH QUESTIONS ...................................................................................20  HYPOTHESES......................................................................................................21   REFERENCES ......................................................................................................22  CHAPTER II - Sex differences in the resistive and elastic work of breathing during exercise in endurance-trained athletes .................................................................................................26   INTRODUCTION .................................................................................................27  METHODS............................................................................................................29  Subjects......................................................................................................29 Experimental Overview.............................................................................29 Pulmonary Function...................................................................................30 Maximal Cycle Exercise............................................................................30 Flow, Volume and Pressure.......................................................................30 End-Expiratory Lung Volume...................................................................30 Work of Breathing .....................................................................................31 Data Processing .........................................................................................33 Statistical Analysis.....................................................................................33   iv RESULTS..............................................................................................................34  Subject Characteristics...............................................................................34 Work of Breathing vs. Absolute Minute Ventilation ................................35 Work of Breathing vs. Relative Submaximal Minute Ventilation ............37 Work of Breathing vs. External Muscular Work.......................................39 Breathing Pattern .......................................................................................41 Lung Size vs. Work of Breathing ..............................................................44 Flow vs. Work of Breathing ......................................................................46   DISCUSSION........................................................................................................48  Resistive Work of Breathing vs. Minute Ventilation ................................48 Elastic Work of Breathing vs. Minute Ventilation ....................................49 Total Work of Breathing vs. Minute Ventilation.......................................50 Work of Breathing vs. External Muscular Work.......................................52 Sex vs. Size Differences ............................................................................52 Methodological Considerations.................................................................54 Conclusions ...............................................................................................57  REFERENCES ......................................................................................................58  CHAPTER III - Sex differences in exercise-induced diaphragmatic fatigue in endurance-trained athletes ...............................................................................................................................60  INTRODUCTION .................................................................................................61  METHODS............................................................................................................63  Subjects......................................................................................................63 Experimental Overview.............................................................................63 Pulmonary Function...................................................................................64 Incremental Exercise Test..........................................................................64 Pressure Measurements .............................................................................64 Diaphragm Fatigue ....................................................................................65 Exercise Breathing Mechanics ..................................................................69 Symptom Evaluation .................................................................................69 Statistical Analyses....................................................................................69  RESULTS..............................................................................................................71  Subject Characteristics...............................................................................71 Supramaximal Stimulation ........................................................................73 Diaphragm Fatigue ....................................................................................73 Time to Exhaustion Test............................................................................76 Ratings of Perceived Exertion ...................................................................76 Ventilation and Breathing Mechanics During Exercise ............................77    v DISCUSSION........................................................................................................81  Diaphragm Fatigue ....................................................................................81 Fatigue Resistance Mechanisms................................................................82 Diaphragmatic Force Production and Respiratory Muscle Recruitment...83 Muscle Morphology and Substrate Utilization..........................................86 Consequences of Diaphragm Fatigue ........................................................87 Methodological Considerations.................................................................88 Conclusions ...............................................................................................90  REFERENCES ......................................................................................................91  CHAPTER IV – Conclusions ..........................................................................................95  Overall Summary.......................................................................................96 Significance ...............................................................................................97 Pulmonary Limitations in Women ............................................................98 Strengths and Limitations..........................................................................98 Future Research .........................................................................................99 Overall Conclusions.................................................................................101  REFERENCES ....................................................................................................102   APPEglyph1197DIX I – Informed consent forms........................................................................104  APPEglyph1197DIX II – Physical activity readiness and health questionnaires ........................114  APPEglyph1197DIX III – Reprints of selected publications.......................................................117  APPEglyph1197DIX IV– Individual diaphragm fatigue response in men and women...............149  APPEglyph1197DIX V – Certificates of ethical approval ...........................................................151  APPEglyph1197DIX VI – Candidate’s list of research publications...........................................154                vi LIST OF TABLES   Table 2.1:  Pulmonary function data.................................................................................34  Table 2.2:  Maximal exercise data on day 1 .....................................................................35  Table 3.1:  Descriptive characteristics of the subjects......................................................71  Table 3.2:  Maximal incremental exercise data on day 1 .................................................72                                         vii LIST OF FIGURES   Figure 1.1:  Three dimensional reconstruction of an airway tree using computed          tomography..........................................................................................................................4  Figure 1.2:  Three dimensional reconstruction of an airway tree using computed tomography with a highlighted section showing measured branches......................................................5  Figure 1.3:  Percent differences in various airway areas between men and women matched for lung size...............................................................................................................................6  Figure 1.4:  Airway tree with assigned labels showing significant size differences between men and women...........................................................................................................................7    Figure 1.5:  Theoretical ventilatory response to progressive exercise in an age and height matched man and woman ....................................................................................................9  Figure 1.6:  Regulation of lung volumes in men and women during progressive exercise to exhaustion..........................................................................................................................12  Figure 1.7:  Work of breathing in men and women..........................................................13    Figure 2.1:  Example of a modified Campbell diagram obtained from a male subject during exercise at ~75% of maximum ventilation (115 l·min-1)...................................................32  Figure 2.2:  Total work of breathing (A), inspiratory resistive work (B), expiratory resistive work (C), inspiratory elastic work (D) and expiratory elastic work (E) vs. minute ventilation in men (○) and women (●).....................................................................................................36  Figure 2.3:  Modified Campbell Diagrams from an individual male and female subject matched approximately for absolute minute ventilation (100 vs. 101 l·min-1(STPD)), tidal volume (2.1 vs. 2.2 liters), breathing frequency (52 vs. 49 breaths·min-1), age (24 vs. 25 years) and mass (64.6 vs. 64.2 kg), respectively.........................................................................................................37  Figure 2.4:  Total work of breathing vs. relative percentages of maximal minute ventilation in men (○) and women (●).....................................................................................................38  Figure 2.5:  Total work (A), inspiratory resistive work (B), and expiratory resistive work of breathing (C) vs. work rate in men (○) and women (●) ....................................................40  Figure 2.6:  Breathing frequency (A) and tidal volume (B) vs. minute ventilation in men (○) and women (●) .........................................................................................................................42  Figure 2.7:  Inspiratory elastic work of breathing vs. tidal volume (A) and breathing frequency (B) at the four ventilatory points (i.e., 50, 75, 100 l·min-1and maximal ventilation) in men (○) and women (●)...................................................................................................................43   viii Figure 2.8:  Regression analysis of the total work (A), inspiratory resistive work (B), and expiratory resistive work (C) vs. forced vital capacity (FVC) in men (○) and  women (●) .........................................................................................................................45  Figure 2.9:  Inspiratory resistive work of breathing vs. average inspiratory flow in individual male (thin lines) and female (thick lines) subjects ............................................................47  Figure 3.1:  Twitch potentiation protocol.........................................................................68  Figure 3.2:  Trans-diaphragmatic pressure response to increasing cervical magnetic stimulation intensities in men and women............................................................................................73  Figure 3.3:  Response of twitch trans-diaphragmatic pressure during recovery in men and women................................................................................................................................75  Figure 3.4:  Example M-wave in an individual female subject at baseline and 10 min after exercise ..............................................................................................................................76  Figure 3.5:  Perception of leg discomfort and breathlessness in men and women...........77  Figure 3.6:  Ventilatory and work of breathing response to exercise in men and  women................................................................................................................................79  Figure 3.7:  Diaphragmatic and esophageal pressure-time product response to exercise in men and women.........................................................................................................................80  Figure A.IV.1:  Individual diaphragm fatigue response in men and women .................150                        ix ACKglyph1197OWLEDGEMEglyph1197TS   This thesis would not have been possible without the exceptional mentorship of Dr. Bill Sheel.  I would like to offer my gratitude to Dr. Sheel for taking the time to inspire me as a third year undergraduate student.  You gave me direction in life, allowed me to realize my passion for science and mentored me during my master’s and PhD where I now have the intense desire to run my own research program.  It is my sincere hope that I will one day be able to offer my students the same level of training and the incredible mentorship that you have given me.  I would also like to thank the contribution of Drs. Don McKenzie, Neil Eves and Jeremy Road for serving on my thesis committee and Dr. Lee Romer for being such an outstanding collaborator.  I am also indebted to Dr. Romeo Chua for his extreme generosity and willingness to develop several data analysis programs for me without expecting anything in return.  Your brilliance is only shadowed by your kind heart and generosity.  It is Professors like you that keep me motivated to stay involved in research and teaching.    I would like to acknowledge all members of the Health and Integrative Physiology (HIP) Laboratory for creating a working environment that fosters collaboration and friendship.  Lastly, I would like to thank my amazing parents and my incredibly patient and supportive wife for giving me the strength to get through so many years of post-secondary education.         x CO-AUTHORSHIP STATEMEglyph1197T   A version of Chapter 2 has previously been published as: Guenette JA, Querido JS, Eves ND, Chua R and Sheel AW.  Sex differences in the resistive and elastic work of breathing during exercise in endurance trained athletes.  American Journal of Physiology; Regulatory, Integrative and Comparative Physiology.  297(1):R166-175, 2009.  Guenette JA was the primary author and played the principle role in identification and design of the research program, performance of research, data analysis and manuscript preparation.  Querido JS assisted with data analysis.  Eves ND assisted in identification and design of the research program.   Chua R assisted with data analysis.  Sheel AW assisted in identification and design of the research program and manuscript preparation.   A version of Chapter 3 will be submitted for publication as:  Guenette JA, Romer LM, Querido JS, Chua R, Eves ND, Road JD, McKenzie DC and Sheel AW.  Sex-differences in exercise-induced diaphragmatic fatigue in endurance-trained athletes.    Guenette JA was the primary author and played the principle role in identification and design of the research program, performance of research, data analysis and manuscript preparation.  Romer LM assisted in identification and design of the research program.  Querido JS assisted with data analysis.  Chua R assisted with data analysis.  Eves ND assisted in identification and design of the research program.  Road JD assisted in identification and design of the research program.  McKenzie DC assisted in identification and design of the research program.  Sheel AW assisted in identification and design of the research program and manuscript preparation.     1           CHAPTER I: Introduction                          2 A recent symposium (Sept. 2006) dealing with sex differences in exercise biology was held at the American College of Sports Medicine Integrative Physiology of Exercise Conference.  Day (2008) introduced the symposium papers by stating that “the number of publications addressing sex differences and exercise physiology has increased from 12, before 1970, to as many as 1344 through October 2007.”  Clearly, sex-based physiological studies have become increasingly recognized as an important area of research among exercise physiologists.  Our research group has been particularly interested in understanding sex differences in respiratory exercise physiology and how anatomical differences may have functional consequences during exercise in healthy women (Guenette et al., 2004; Richards et al., 2004; Sheel et al., 2004; Guenette et al., 2006; Guenette & Sheel, 2007a; Guenette et al., 2007a; Guenette et al., 2007b; Sheel & Guenette, 2008; Guenette et al., 2009; Sheel et al., 2009).  The purpose of this introduction is four-fold.  First, to discuss fundamental sex differences in respiratory anatomy with a focus on the airways.  Second, to present a theoretical framework on how these anatomical sex differences influence resting pulmonary function and ventilatory mechanics during exercise.  Third, to summarize the existing physiological literature that supports our theoretical framework and finally, to discuss sex differences in skeletal muscle fatigue.    Sex Differences in Respiratory Anatomy:  There are a number of important sex differences in respiratory structure and resting pulmonary function that may influence the ventilatory response to exercise.  Perhaps the most important sex difference in respiratory anatomy comes from studies suggesting that women have smaller lungs and airways relative to size-matched males.  Direct anatomical evidence suggests that the pattern of airway-parenchymal growth is different between boys and girls.  Thurlbeck (1982) obtained post-mortem lungs from boys and girls (6 weeks to 14 years) and showed that boys have larger lungs than girls starting at approximately 2 years of age.  Even when lung volume was corrected for differences in body length, it appears  3 that with increasing age, boys continue to have larger lungs.  If there is perfectly proportional growth of the airways and lung parenchyma, then the ratio of airway area to lung volume should be constant and independent of lung volume.  However, the large variability in maximal expiratory flow rates in individuals with comparable lung volumes (Green et al., 1974) suggests that their may be a dissociation between airway and lung size.  This dissociation was termed “dysanapsis” (Mead, 1980).  Mead (1980) indirectly assessed the relationship between airway size (estimated from maximal expiratory flow ÷ static recoil pressure at 50% vital capacity) and lung size (vital capacity) in men (n=21), boys (n=5) and women (n=7).  He concluded that “the airways of men were approximately 17% larger in diameter than the airways of women,” and speculated that “the adult sex difference in airway size develops relatively late in the growth phase.”  Additional evidence for dysanpsis comes from cross-sectional studies that have made estimates of tracheal area using acoustic reflectance in healthy men (n=26) and women (n=28) (Martin et al., 1987).  Martin et al. (1987) found that the tracheal cross-sectional area was 29% smaller in women even when matched for total lung capacity.  These studies have given important insight into basic sex differences in airway anatomy but are limited in their indirect approaches to measuring airway size.  Moreover, these studies and others (Griscom & Wohl, 1986; Hoffstein, 1986) have limited their assessment to areas above the tracheal carina.  Accordingly, we recently used high resolution computed tomography (CT) to measure airway luminal areas in men and women (Sheel et al., 2009) [see APPENDIX III for journal reprint].  Figure 1.1 shows a three-dimensional reconstruction of an airway tree in an individual subject and Figure 1.2 shows a similar airway tree with a highlighted section to demonstrate the various branches in which airway luminal area can be reliably determined based on resolution quality.   4  Figure 1.1:  Three dimensional reconstruction of an airway tree using computed tomography.       5   Figure 1.2:  Three dimensional reconstruction of an airway tree using computed tomography with a highlighted section showing the branches that can be successfully measured.  We obtained images from 57 older ex-smokers (28 men and 27 women) that were being screened for lung nodules using CT scans.  It was not possible to obtain such scans in healthy individuals due to risk of radiation exposure without diagnostic justification.  Nevertheless, our relatively large sample size allowed us to compare airway luminal areas in all subjects, but more importantly, in a subset of subjects matched for lung capacity.  We found that women had significantly smaller tracheal areas but also smaller diameter airways beyond the tracheal carina.  Figure 1.3 shows the percentage difference in airway luminal area between men and women for specific airways.  6   Figure 1.3:  Percent differences in various airway areas between men and women matched for lung size. Trachea  19.5%  18.1%  23.8%  25.4%  14.8%  31.3%   7  Figure 1.4:  Airway tree with assigned labels.  Labels refer to segments but are assigned to terminating branchpoint of respective segment.  Drawing based on Boyden (1955).  Post, posterior; lat, lateral; ant, anterior. * Significant differences between men and women of varying body size (P < 0.05).  † Significant differences between subjects matched for lung size (P < 0.05).  Reproduced with permission from Sheel et al. (2009).   This study confirmed previous reports that have shown women, matched for lung volume, have smaller tracheal areas than men.  However, this study demonstrates that these sex differences persist beyond the tracheal carina which may have important implications for the flow resistive work of breathing (WOB), which will be discussed later.  Collectively, these findings coupled with those of others (Mead, 1980; Thurlbeck, 1982; Martin et al., 1987) suggest that women have smaller lungs and airways, even when matched for body size or lung capacity.   How these anatomical differences influence ventilatory mechanics during exercise is discussed below.  8 Sex Differences in Breathing Mechanics: Given the aforementioned disparity in pulmonary structure, differences in resting pulmonary function between men and women are predictable.  As previously described, women typically have smaller lung volumes and smaller diameter airways.  Smaller airways mean that women will have lower maximal expiratory flow rates relative to men.  Smaller lungs and lower flow rates result in a smaller maximum flow volume loop (MFVL) and thus, a reduced capacity to generate flow and volume during exercise.   A smaller MFVL might make women susceptible to developing expiratory flow limitation (EFL) despite the fact that they achieve lower levels of minute ventilation during exercise relative to men.  EFL is defined as the inability to increase expiratory flow despite increases in trans-pulmonary pressure and is quantified as the percent of the tidal volume that meets or exceeds the expiratory boundary of the MFVL (Johnson et al., 1991a; Johnson et al., 1991b).  The presence of EFL may cause reflex inhibition of the hyperventilatory response and/or a significant alteration in breathing pattern.    Figure 1.5 provides a theoretical framework that illustrates the functional consequences of having smaller lungs and airways and how these factors may influence breathing during exercise.     9 7 6 5 4 3 2 17 6 5 4 3 2 136912-3-6-9Volume (L) Volume (L)Flow (L/sec)FEMALE MALEAge: 25 yrsEFLEELV EELVHeight: 175 cmFVCpred: ~ 4.4 LPEFpred: ~ 8.9 L/secAge: 25 yrsHeight: 175 cmFVCpred: ~ 5.4 LPEFpred: ~ 11.6 L/secFlow (L/sec) Figure 1.5:  Theoretical response to progressive exercise in age and height matched men and women.  Based on predictive equations, women have a smaller forced vital capacity (FVC) and peak expiratory flows (PEF).  Shown are increasing tidal volumes and the presence of flow limitation in women when the expiratory tidal flow-volume loop intersects the volitional maximal flow-volume loop.  At maximal exercise there is a greater increase in end-expiratory lung volume (EELV) in women relative to men.  This leftward shift in EELV back towards resting values is indicative of dynamic hyperinflation.  Reproduced with permission from Sheel and Guenette (2008).   Figure 1.5 shows a male and female matched for age and standing height.  Tidal flow-volume loops are plotted within the MFVL.  The forced vital capacity (FVC) and peak expiratory flow values are based on established prediction equations.  It can be seen that the female has a smaller MFVL, lower maximal flow rates and thus, a smaller capacity to generate minute ventilation during exercise.  With increasing exercising intensity, the female tidal flow-volume loop increases to the point of intersecting the boundary of the MFVL suggesting the presence of EFL.  This response does not occur in an age and height matched male.  It should be stressed however that Figure 1.5 is a theoretical schematic only and does not necessarily suggest that men do not  10 develop EFL.  The concepts in this schematic are supported by the work of McClaran et al. (1998) who were the first to demonstrate that healthy women develop significant EFL during heavy exercise because of their smaller lungs and lower maximal expiratory flow rates.    The few studies that have assessed EFL in women have used a technique whereby tidal flow volume loops are positioned within the MFVL based on a measurement of EELV.  EFL is considered present if the tidal breath meets or exceeds the expiratory boundary of the MFVL.  This technique provides an excellent visual representation of EFL and the regulation of lung volumes during exercise.  However, this technique can overestimate the magnitude of EFL or even falsely detect its occurrence due to exercise-induced bronchdodilation and thoracic gas compression artefacts.  An alternative method is to apply a negative expiratory pressure at the mouth, and compare the flow volume curve during the ensuing expiration with that of the preceding control breath.  If the negative pressure does not increase expiratory flow then the subject is considered flow limited.  This particular technique alleviates some of the limitations associated with the method of superimposing tidal breaths within a MFVL.  We recently used the negative expiratory pressure technique to measure EFL in a group of highly trained male and female endurance athletes (Guenette et al., 2007b).  We found that 9 out of 10 women experienced significant EFL during maximal cycle exercise which is consistent with the work of McClaran et al. (1998), who observed EFL in 86% of their fit women during treadmill running to exhaustion.  In contrast, we found evidence of EFL in only 43% of our male subjects.  Although we had a relatively small sample size, the preliminary results of this study suggest that EFL may be more common in women.    With the onset of EFL, end-expiratory lung volume (EELV) may increase back to and sometimes beyond resting values resulting in dynamic hyperinflation of the lungs (Figure 1.5).   11 This dynamic hyperinflation permits increases in flow rate (Pellegrino et al., 1993) at the expense of an increased elastic work because lung compliance is reduced as lung volume increases.  Hyperinflation may then lead to earlier fatigue of the respiratory muscles by requiring them to contract from a shorter length, which means that the muscular force required to ventilate the lungs is closer to the muscle’s maximal capacity to generate force (Roussos et al., 1979).  This will further reduce inspiratory muscle length and may substantially increase the work and O2 cost of breathing, thus decreasing inspiratory muscle endurance time (Tzelepis et al., 1988).  Secondary to the hyperinflation-induced fatigue, a relative ischemia to the diaphragm may further exacerbate diaphragm fatigue (Bellemare & Grassino, 1982).  A potential consequence of diaphragm fatigue and a high work of breathing (WOB) is the sympathetically mediated vasoconstriction and reduction in locomotor muscle blood flow (Dempsey et al., 2003).  These effects were demonstrated by mechanically loading or unloading the respiratory muscles at maximum exercise (Harms et al., 1997).  Changes in leg blood flow were observed which indicate a competitive relationship between locomotor and respiratory muscles for a limited cardiac output and this may be associated with reductions in exercise performance.    Figure 1.6 shows sex differences in the regulation of lung volumes in trained men and women.  EELV and end inspiratory lung volume (EILV) follow the same pattern during submaximal exercise.  However, as the subjects approached maximal exercise, women increased EELV back towards resting values whereas men did not.  EILV in these women approached 90% of their FVC indicating that there would be an increased elastic load on the inspiratory muscles relative to their male counterparts whose EILV was only 82% of FVC.  When expressed as a percentage of FVC, women had significantly higher EELV and EILV compared with men at maximal exercise.  Based on the higher relative values for EILV and EELV in women, it would be predicted that the WOB would also be higher in women compared with men.  McClaran et al.  12 (1998) examined the relationship between EFL and operational lung volumes by having women breathe a low density gas mixture (i.e., helium) to increase the size of the MFVL.  Increasing the size of the MFVL can eliminate EFL which resulted in subjects being able to maintain a lower EELV.  This suggests an important association between EFL and the regulation of EELV and EILV during exercise.                Figure 1.6:  Regulation of lung volumes in men and women during progressive exercise to exhaustion (Guenette et al., 2007b).  Shown are end-inspiratory lung volume (EILV) and end-expiratory lung volume (EELV) expressed as % forced vital capacity (FVC) at rest and during progressive exercise to maximal workload (Wmax) in men and women.  Values are means ± SE.  * Significantly different from men (P < 0.05).  Reproduced with permission from Sheel and Guenette (2008).   Given the smaller lungs and airways in women, coupled with the finding of greater EFL and higher operational lung volumes during high intensity exercise, it may be predicted that the mechanical WOB would be higher in women.  There have been few attempts to compare the WOB between sexes (Holmgren et al., 1973; Guenette et al., 2007b).  Figure 1.7 shows the total Wmax (%)0 20 40 60 80 100FVC (%)020406080100MENWOMENRESTEILVEELV** 13 WOB in endurance-trained men and women across a range of ventilations measured during an incremental cycle test to exhaustion.    04008001200160020000 50 100 150 200Minute Ventilation (l min-1)Work of Breathing (J min-1 ) WomenMen Figure 1.7:  Work of breathing in men and women.  Each curve represents a mean curve relating the work of breathing vs. minute ventilation.  Each curve has been extrapolated to 200 l·min-1 for theoretical purposes.  The work of breathing is essentially the same at rest and during very low levels of ventilation.  As ventilation increases with increasing intensity, the work of breathing in women significantly increases out of proportion relative to men.  Reproduced with permission from Guenette et al. (2007b).     The total WOB was not different at low intensity exercise up to 50 l·min-1.  However, the WOB increased disproportionally in women as ventilation increased beyond 50 l·min-1.  When minute ventilation exceeded 90 l·min-1 the WOB in women was approximately twice that of men.  Therefore, the work and presumably the O2 cost of moving a given volume of air through the lungs is substantially higher in women.  It should also be noted that both curves in Figure 1.7 have been extrapolated to 200 l·min-1 for theoretical purposes only.  In addition to the data  14 presented in Figure 1.7, Guenette et al. (2007b) also assessed the relationship between minute ventilation and the WOB according to the following equation described by Otis et al. (1950):    Work of breathing = aV˙  E 3+ bV˙  E2  The term bV˙  E2 represents the mechanical work done in overcoming the viscous resistance offered by the lung tissues to deformation and by the respiratory tract to the laminar flow of air whereas term aV˙  E 3 represents the work done in overcoming the resistance to turbulent flow.  A value for constant ‘a’ and ‘b’ was then determined for each individual subject.  Constant ‘a’ was significantly higher in women, suggesting that perhaps the higher WOB in women is associated with the additional work needed to overcome the resistance to turbulent airflow.  This may explain why the magnitude of the difference between men and women increased out of proportion with increasing levels of minute ventilation, and thus airflow.  It would be expected that subjects with larger lung volumes would have lower pulmonary resistance and thus a lower WOB for a given level of ventilation.  The women in this study had significantly smaller lungs (and presumably airways) compared to the men which may be one of many major reasons for their higher WOB.  Consistent with the concept of differences in lung volumes was the observation that when men and women were pooled together, there was a significant, albeit modest, correlation between FVC and constant ‘a’ (r = - 0.54, P < 0.05).  Perhaps more importantly was the finding that constant ‘a’ was significantly correlated with peak expiratory flow rates in women (r = - 0.76, P < 0.05) and when all subjects were pooled together (r = - 0.68, P < 0.05).  Peak expiratory flow rates may serve as a crude surrogate for airway size, which may explain, in part, the increased work needed to overcome the resistance to turbulent airflow in women relative to men (Guenette et al., 2007b).    15 Based on the aforementioned differences in respiratory anatomy and breathing mechanics, one may conclude that the respiratory muscles of females may be placed under greater mechanical stress relative to males.  This is particularly the case if women are breathing at higher lung volumes.  This would place the diaphragm in a sub-optimal contractile position along its length tension relationship resulting in a higher WOB for a given level of minute ventilation.  We have hypothesized in previous articles that these sex differences might make the female diaphragm more prone to fatigue (Guenette & Sheel, 2007b; Guenette et al., 2007b; Sheel & Guenette, 2008) but this continues to remain an untested hypothesis and is based solely on speculation.  This hypothesis is also difficult to reconcile with previous studies showing that women are actually less susceptible to non-respiratory muscle fatigue.  The following section will briefly summarize the literature as it pertains to sex differences in skeletal muscle fatigue.  Sex Differences in Skeletal Muscle Fatigue:  The deleterious effects of skeletal muscle fatigue on exercise performance has been a topic of great interest to exercise and muscle physiologists over the last century.  Muscle fatigue can be defined as a loss in the capacity for developing force and/or velocity resulting from muscle activity under load and which is reversible by rest (NHLBI, 1990) and can be further characterized as either peripheral or central fatigue.  Peripheral fatigue occurs at or distal to the neuromuscular junction while central fatigue refers to a reduction in motor output from the central nervous system.  The precise contribution of central or peripheral fatigue to the reduced force-generating capacity of the muscle remains controversial.  Studies examining sex differences in skeletal muscle fatigue have typically shown that women have greater relative fatigue resistance compared to their male counterparts (Maughan et al., 1986; Miller et al., 1993; West et al., 1995; Fulco et al., 1999).  The question germane to the present study is why would women be less susceptible to muscle fatigue compared with men and is this fatigue resistance a finding that can be extended to the muscles of  16 respiration?  Hicks et al. (2001) recently reviewed potential mechanisms of this apparent sex difference in muscle fatigue although none of the studies mentioned dealt specifically with the muscles of respiration.  The most common mechanisms associated with greater fatigue resistance in women relative to men include differences in muscle mass, muscle morphology, substrate utilization and neuromuscular activation.    Sex Differences in Muscle Mass and Morphology:  It is well known that women have smaller muscle mass compared with men and this fact has been proposed as one of the key contributors to explain the greater fatigue resistance found in women.  Lower muscle mass translates directly into lower absolute force generation in females when performing at the same relative intensity as males.  This lower absolute force production means there will be a decreased O2 demand, a decrease in mechanical compression of the local vasculature and less intramuscular occlusion of blood flow.  However, most studies have ignored the greater absolute force of submaximal contraction in men (Maughan et al., 1986; Miller et al., 1993) and therefore the proposed sex differences may simply be due to contraction conditions eliciting a greater degree of imbalance between muscle O2 supply and demand in men (Fulco et al., 1999).  Fulco et al. (1999) addressed this issue by comparing muscle performance in men and women at the same absolute force development and matched subjects for maximal voluntary contraction of the adductor pollicis muscle.  Despite matching for maximal muscle strength, these authors still found that females exhibit less fatigue than males.     In terms of muscle morphological differences, there is some evidence to suggest that there are sex differences in muscle fibre type composition such that women have more slow oxidative type I fibres (Nygaard, 1981; Simoneau et al., 1985; Simoneau & Bouchard, 1989; Mannion et al., 1997; Jaworowski et al., 2002).  Type I muscle fibres have a slower contraction  17 speed and thus a slower rate of energy utilization (Stienen et al., 1996; Hamada et al., 2003).  This in turn means that type I fibres fatigue at slower rates compared to fast glycolytic type II fibres (Hamada et al., 2003).  These potential sex differences in muscle fibre type composition may explain, in part, why female muscles are more fatigue resistant than male muscles.    Sex Differences in Substrate Utilization:  Sex differences in substrate utilitzation during exercise may also contribute to potential sex differences in muscle fatigue.  It has been established that males have a higher glycolytic capacity and a greater reliance on glycolytic pathways than females (Hicks et al., 2001).  Muscle biopsy studies have also revealed that women have lower activities of common glycolytic enzymes, which in turn would translate into a decreased potential for anaerobic glycolysis (Tarnopolsky, 1999).  As pointed out by Hicks et al. (2001), these differences may mean that women have a greater reliance on β-oxidation of fatty acids, thus prolonging endurance during certain types of exercise and perhaps improving their ability to resist fatigue.  Sex Differences in glyph817euromuscular Activation:  One of the few studies to systematically examine sex differences in neuromuscular activation and its association with muscle fatigue was conducted by Häkkinen (1993).  The purpose of this study was to examine acute neuromuscular fatigue and short-term recovery from fatigue in men and women following an intense resistance exercise protocol.  The relative loading intensity and volume of exercise was kept the same for both sexes.  Häkkinen (1993) found significant decreases in maximal voluntary EMG in males but not in the females, suggesting a greater impairment in neuromuscular activation in males relative to females after fatiguing resistance exercise.  Despite this finding, the potential role of sex differences in neuromuscular activation as a mechanism for the increased fatigue resistance in women still requires additional research.  18 Summary:  There are clear differences in respiratory anatomy, such that women have smaller lungs and airways, even when matched for body size.  Smaller lungs and airways in women translate directly into a reduced capacity to generate flow and volume, and thus ventilation during exercise.  Some have suggested that these anatomical factors may be associated with the notion that women may be more susceptible to pulmonary system limitations during exercise.  For example, women may be more susceptible to EFL and have greater increases in operational lung volumes during high intensity exercise.  Moreover, women have been shown to have a higher WOB during exercise for a given level of absolute minute ventilation.  However, there are no studies that have examined possible mechanisms associated with a higher WOB in women.  The aforementioned observations point to a female respiratory system that may be at a mechanical disadvantage relative to their male counterparts during exercise.  This mechanical disadvantage might make the primary inspiratory muscle (i.e., the diaphragm) more susceptible to exercise-induced fatigue which has important physiological consequences in both health and diseased populations.  However, there are no studies that have systematically compared the diaphragmatic fatigue response in women with that of men.            19 PURPOSE  1.  To provide a comprehensive assessment of the mechanical WOB in men and women and to determine the mechanisms associated with a higher WOB in women.  2.  To characterize diaphragmatic pressure production and recruitment during exercise and to determine if there are sex differences in the severity of exercise-induced diaphragmatic fatigue.                    20 RESEARCH QUESTIOglyph1197S  1.  Which WOB component(s) is responsible for the higher total WOB in women for a given absolute ventilation?  2.  Are there sex differences in the total WOB and its constituent components when comparisons are made at relative percentages of maximal ventilation?  3.  Are there sex differences in the total WOB and its constituent components when comparisons are made at absolute mass-corrected workloads?  4.  Is the severity of exercise-induced diaphragmatic fatigue different between men and women?  5.  Are the absolute and mass-corrected esophageal and trans-diaphragmatic pressure-time products different between sexes during exercise?            21 HYPOTHESES  1.  The higher total WOB during exercise for a given absolute ventilation will be due to a combination of inspiratory and expiratory resistive and elastic components.  2.  There will be no sex differences in the WOB when comparisons are made at relative percentages of maximal ventilation.    3.  The total WOB and its constituent components will be higher in women for a given mass-corrected workload.  4.  The severity of exercise-induced diaphragmatic fatigue will be greater in women.  5.  Absolute and mass-corrected esophageal and diaphragmatic pressure-time products will be higher in women during exercise                    22 REFEREglyph1197CES  Bellemare F & Grassino A. (1982). 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Sex differences in human skeletal muscle fatigue. Exerc Sport Sci Rev 29, 109-112.  Hoffstein V. (1986). Relationship between lung volume, maximal expiratory flow, forced expiratory volume in one second, and tracheal area in normal men and women. Am Rev Respir Dis 134, 956-961.  Holmgren A, Herzog P & Astrom H. (1973). Work of breathing during exercise in healthy young men and women. Scand J Clin Lab Invest 31, 165-174.  Jaworowski A, Porter MM, Holmback AM, Downham D & Lexell J. (2002). Enzyme activities in the tibialis anterior muscle of young moderately active men and women: relationship with body composition, muscle cross-sectional area and fibre type composition. Acta Physiol Scand 176, 215-225.  Johnson BD, Reddan WG, Pegelow DF, Seow KC & Dempsey JA. (1991a). Flow limitation and regulation of functional residual capacity during exercise in a physically active aging population. Am Rev Respir Dis 143, 960-967.  Johnson BD, Reddan WG, Seow KC & Dempsey JA. (1991b). Mechanical constraints on exercise hyperpnea in a fit aging population. Am Rev Respir Dis 143, 968-977.  Mannion AF, Dumas GA, Cooper RG, Espinosa FJ, Faris MW & Stevenson JM. (1997). Muscle fibre size and type distribution in thoracic and lumbar regions of erector spinae in healthy subjects without low back pain: normal values and sex differences. J Anat 190 ( Pt 4), 505-513.  Martin TR, Castile RG, Fredberg JJ, Wohl ME & Mead J. (1987). Airway size is related to sex but not lung size in normal adults. J Appl Physiol 63, 2042-2047.  Maughan RJ, Harmon M, Leiper JB, Sale D & Delman A. (1986). Endurance capacity of untrained males and females in isometric and dynamic muscular contractions. Eur J Appl Physiol Occup Physiol 55, 395-400.  McClaran SR, Harms CA, Pegelow DF & Dempsey JA. (1998). Smaller lungs in women affect exercise hyperpnea. J Appl Physiol 84, 1872-1881.   24 Mead J. (1980). Dysanapsis in normal lungs assessed by the relationship between maximal flow, static recoil, and vital capacity. Am Rev Respir Dis 121, 339-342.  Miller AE, MacDougall JD, Tarnopolsky MA & Sale DG. (1993). Gender differences in strength and muscle fiber characteristics. Eur J Appl Physiol Occup Physiol 66, 254-262.  NHLBI. (1990). NHLBI Workshop summary. Respiratory muscle fatigue. Report of the Respiratory Muscle Fatigue Workshop Group. Am Rev Respir Dis 142, 474-480.  Nygaard E. (1981). Skeletal muscle fibre characteristics in young women. Acta Physiol Scand 112, 299-304.  Otis AB, Fenn WO & Rahn H. (1950). Mechanics of breathing in man. J Appl Physiol 2, 592-607.  Pellegrino R, Violante B, Nava S, Rampulla C, Brusasco V & Rodarte JR. (1993). Expiratory airflow limitation and hyperinflation during methacholine-induced bronchoconstriction. J Appl Physiol 75, 1720-1727.  Richards JC, McKenzie DC, Warburton DE, Road JD & Sheel AW. (2004). 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Skeletal muscle histochemical and biochemical characteristics in sedentary male and female subjects. Can J Physiol Pharmacol 63, 30-35.  Stienen GJ, Kiers JL, Bottinelli R & Reggiani C. (1996). Myofibrillar ATPase activity in skinned human skeletal muscle fibres: fibre type and temperature dependence. J Physiol 493 ( Pt 2), 299-307.  Tarnopolsky MA. (1999). Gender differences in metabolism: practical and nutritional considerations. Boca Raton, CRC Press.  25  Thurlbeck WM. (1982). Postnatal human lung growth. Thorax 37, 564-571.  Tzelepis G, McCool FD, Leith DE & Hoppin FG, Jr. (1988). Increased lung volume limits endurance of inspiratory muscles. J Appl Physiol 64, 1796-1802.  West W, Hicks A, Clements L & Dowling J. (1995). The relationship between voluntary electromyogram, endurance time and intensity of effort in isometric handgrip exercise. Eur J Appl Physiol Occup Physiol 71, 301-305.                                          26       CHAPTER II: Sex differences in the resistive and elastic work of breathing during exercise in                                                            endurance-trained athletes *                    ________________________ * A version of this chapter has been published as: Guenette JA, Querido JS, Eves ND, Chua R and Sheel AW.  Sex differences in the resistive and elastic work of breathing during exercise in endurance trained athletes.  American Journal of Physiology; Regulatory, Integrative and Comparative Physiology.  297(1):R166-175, 2009. [see APPENDIX III for journal reprint]  27 Iglyph1197TRODUCTIOglyph1197  A growing number of investigations aimed at characterizing the healthy female respiratory response to exercise have reported sex-based differences in pulmonary gas exchange (Harms et al., 1998; Richards et al., 2004) and respiratory mechanics (McClaran et al., 1998; Guenette et al., 2007).  These studies show that young adult women free from respiratory disease may be more susceptible to pulmonary limitations during exercise which is likely associated with women having smaller lungs and airways relative to size-matched males (Mead, 1980; Thurlbeck, 1982).     There have been few attempts to systematically compare the work of breathing (WOB) between sexes (Holmgren et al., 1973; Guenette et al., 2007).  We recently demonstrated that the WOB was higher in endurance trained women at moderate to high levels of minute ventilation compared with trained males with no differences in the total WOB when comparisons were made at different percentages of maximal aerobic capacity (V˙  O2max) (Guenette et al., 2007).  On average, women had a WOB that was approximately twice as high as men at ventilatory rates above 50 l·min-1.  However, the analysis used in this previous study was limited in that it did not provide specific information regarding the individual components that make up the total WOB.  The WOB can be sub-divided into the work of the respiratory muscles to overcome the elasticity of the lungs during inspiration, the work required to overcome airflow resistance during inspiration, the work of the expiratory muscles to overcome the elastic outward recoil of the chest wall and the work required to overcome airflow resistance during expiration.  It is currently unknown which of these factors contribute to the higher total WOB in women compared with men.    28 Based on mechanical grounds, we hypothesized that the resistive WOB would be higher in women because of their inherently smaller diameter airways (Mead, 1980; Martin et al., 1987).  However, this hypothesis may be an oversimplification because it is unknown if women adopt a unique breathing pattern to minimize one WOB component at the expense of another.  To this end, we re-analyzed data from our previous investigation (Guenette et al., 2007) by partitioning the respiratory pressure-volume data into 4 distinct WOB components across a range of ventilations and also at 3 different body mass corrected work rates achieved by all subjects.  The WOB was compared at different mass corrected work rates to determine if the WOB is higher for given level of external muscular work.  Furthermore, we examined sex differences in breathing pattern to determine the effect of tidal volume and breathing frequency on the WOB.                 29 METHODS  Subjects:  Sixteen endurance trained athletes (8 men and 8 women) volunteered to participate in this study.  Endurance trained athletes were used instead of untrained individuals because they are capable of generating higher levels of minute ventilation compared to their untrained counterparts.  This permits physiological comparisons across a wider range of values.  Moreover, the mechanical work of breathing appears to be independent of fitness level (Milic-Emili et al., 1962).  The subjects gave informed written consent [APPENDIX I] and all experimental procedures received institutional ethical approval and conformed to the Declaration of Helsinki.  All subjects were healthy non-smokers and did not have any previous history of cardiopulmonary disease.  Subjects with a forced expired volume in one second (FEV1.0) to forced vital capacity (FVC) ratio of < 80% of predicted were excluded from the investigation.    Experimental Overview:  Subjects participated in two testing sessions separated by a minimum of 48 hours.  All women were tested during the early follicular phase (days 3 to 8) of the menstrual cycle as determined via a self-reported menstrual history/health questionnaire [APPENDIX II].  On the first day, subjects performed general spirometry to assess lung function and an incremental cycle test to exhaustion to determine V˙  O2max.  They also received extensive practice on how to perform inspiratory capacity maneuvers at rest and during exercise.  The second day served as the primary testing day which included 10 min of seated quiet breathing followed by an incremental cycle test to exhaustion using the identical exercise protocol as used on Day 1.   30 Pulmonary Function:  FVC, FEV1.0, FEV1.0/FVC and peak expiratory flow were obtained using routine spirometry according to standardized procedures and expressed using prediction equations (ATS, 1995).    Maximal Cycle Exercise:  Subjects performed an incremental test to exhaustion on a cycle ergometer using a step protocol.  Men and women began cycling at 200 W and 100 W respectively, with the work rate increasing by 30 W every 3 min.  Ventilatory and mixed expired metabolic parameters were assessed using a customized metabolic cart consisting of a calibrated pneumotachograph (model 3813, Hans Rudolph, Kansas City, MO) and calibrated CO2 and O2 analyzers (Models CD-3A and Model S-3-A/I respectively, Applied Electrochemistry, Pittsburgh, PA).    Flow, Volume and Pressure:  Inspiratory and expiratory flow was measured using a heated and calibrated pneumotachograph (model 3813, Hans Rudolph, Kansas City, MO) attached to a mouthpiece.  Inspiratory and expiratory volume was obtained through numerical integration of the flow signal.  Esophageal pressure (Pes) was obtained by placing a 10-cm long latex balloon (no. 47-9005, Ackrad Laboratory, Cranford, NJ) ~45 cm down from the nostril (Milic-Emili et al., 1964) after application of a local anesthetic.  All air was removed from the balloon by having subjects perform a valsalva maneuver.  The balloon was then inflated with 1-ml of air as per manufacturer specifications.  Pes was measured using a calibrated piezoelectric pressure transducer (± 100 cmH2O; Raytech Instruments, Vancouver, BC, Canada).    End Expiratory Lung Volume:  End expiratory lung volume (EELV) was determined by having subjects perform inspiratory capacity (IC) maneuvers at rest and during exercise as previously described (Guenette et al., 2007).   Two to three IC maneuvers were obtained near the middle  31 and end of each 3 minute exercise bout and additional IC maneuvers were performed immediately prior to exhaustion.  End-expiratory lung volume (EELV) was calculated as the difference between FVC and the IC volume.  FVC was used to calculate EELV rather than total lung capacity because it was not possible to measure residual lung volume in our subjects.  FVC maneuvers were performed before and immediately after exercise with the largest FVC value being used for the analysis.    Work of Breathing:  The muscular WOB was determined using modified Campbell diagrams as described by Roussos and Campbell (1986) and using the technique of Yan et al. (1997).  Flow, volume and pressures from several breaths (~5-20) corresponding to approximately 50, 75 and 100 l·min-1 and 25, 50, 75 and 100% of maximal ventilation were selected for each subject and ensemble averaged using a customized software program (Bibo, LabVIEW software V6.1, National Instruments).  The same procedure was performed to compare the WOB at 3.0, 3.5 and 4.0 W·kg-1.  These ventilatory rates and workloads were selected because nearly all male and female subjects were successfully able to reach these values.  Additional Campbell diagrams were also generated for each stage of exercise in order to determine the relationship between average inspiratory flow and the inspiratory resistive WOB.  An example of the modified Campbell diagram in a representative male subject at approximately 75% of maximal minute ventilation (i.e., 115 l·min-1) is shown in Figure 2.1.     32 EILVEELVEsophageal Pressure (cmH2O)Volume (l)Cl CcwFRC-25 -15 -5 5 1522.53.5344.50Volume (l) Figure 2.1:  Example of a modified Campbell Diagram obtained from a male subject during exercise at approximately 75% of maximum ventilation (115 l·min-1).  Oblique hatching represents the inspiratory resistive work of breathing.  Horizontal hatching represents the inspiratory elastic work of breathing.  Stippling represents the expiratory resistive work of breathing.  Vertical hatching represents the expiratory elastic work of breathing.  FRC, functional residual capacity; EELV, end-expiratory lung volume; EILV, end-inspiratory lung volume; Cl, dynamic lung compliance; Ccw, chest wall compliance.  Upward arrow represents inspiration and downward arrow represents expiration.  Small open circles represent zero flow points.   A line was drawn connecting the points of zero flow (i.e., EELV and EILV) representing the dynamic compliance of the lung.  The compliance of the chest wall was derived using previously published data (Estenne et al., 1985) based on both age and sex as done by others (Yan et al., 1997; Sliwinski et al., 1998; Butcher et al., 2006; Eves et al., 2006).  The chest wall compliance line was positioned through functional residual capacity (EELV at rest) and extended to EELV and EILV.  The muscular WOB was then partitioned into 4 separate components.  The area  33 inside of the Pes-loop to the left of the lung compliance line (oblique hatching) represents the work needed to overcome airflow resistance during inspiration (i.e., inspiratory resistive work).  The area enclosed by the lung compliance and chest wall compliance lines (horizontal hatching) represents the work needed to overcome lung elasticity (i.e., inspiratory elastic work).  The area to the right of the chest wall compliance line (stippling) represents the active muscular work needed to overcome airflow resistance during expiration (i.e., expiratory resistive work).  Lastly, the area between the lung and chest wall compliance lines below functional residual capacity (vertical hatching) represents the work needed to overcome the outward elastic recoil of the chest wall to maintain EELV below functional residual capacity (i.e., expiratory elastic work).  The sum of all four areas shown in Figure 2.1 represents the total WOB.  All WOB values were multiplied by breathing frequency representing a unit of power (i.e., J·min-1).  However, as conventionally used, we will refer to this throughout the manuscript as the WOB rather than the power of breathing.    Data Processing:  All raw data was recorded continuously at 200 Hz using a 16-channel data acquisition system (PowerLab/16SP model ML 795, ADI, Colorado Springs, CO) and stored on a computer for subsequent analysis (Chart v5.3, ADInstruments, Colorado Springs, CO).   Statistical Analysis:   Descriptive characteristics were compared between sexes using unpaired t-tests.  Pre-planned comparisons were used to compare men and women for the various WOB components and the ventilatory parameters at the target ventilations and work rates using unpaired t-tests.  Linear regression analysis using Pearson correlations was performed to test for associations between specific WOB components and pulmonary function parameters.  The α level was set a priori at 0.05 for all statistical comparisons.  Values are presented throughout the manuscript as means ± SD unless otherwise stated.  34 RESULTS  Subject Characteristics:  The subjects used in the present investigation participated in a study that has been previously published (Guenette et al., 2007) [see APPENDIX III for journal reprint].  The present study has 8 males and 8 females while our previous manuscript reported data on 8 males and 10 females.  Two females were excluded from the present analysis because they did not have Pes data (n=1) or were unable to correctly perform IC maneuvers (n=1).  Males and females were not different for age (25.9 ± 4.9 vs. 24.9 ± 3.1 years, respectively) but men were taller (183.9 ± 6.6 vs. 168.8 ± 4.0 cm, P < 0.0001) and heavier (76.6 ± 9.8 vs. 64.3 ± 3.6 kg, P < 0.01).  Table 2.1 summarizes the pulmonary function data for the present study and table 2.2 summarizes the maximal exercise data obtained on day 1.  As expected, women had smaller FVC, FEV1.0 and peak expiratory flows compared with men.  All subjects were within normal values for all pulmonary function measures except peak expiratory flows which were typically >120% of predicted.  There were no significant sex differences in percent predicted values for any pulmonary function parameters.  Table 2.1:  Pulmonary function data                           Men                                      (n=8) Women                             (n=8)  FVC (l) 6.0 ± 1.0 (4.5-7.2) 4.5 ± 0.5 (3.8-5.4) *  FVC (%predicted) 105 ± 14 (84-125) 108 ± 14 (92-134)  FEV1.0 (l) 5.1 ± 1.0 (3.5-6.4) 3.8 ± 0.4 (3.0-4.5) *  FEV1.0 (%predicted) 106 ± 18 (80-133) 106 ± 14 (80-131)  PEF (l·s-1)  12.6 ± 1.4 (10.4-14.8) 8.1 ± 1.2 (6.5-10.0) *  PEF (%predicted) 124 ± 11 (109-138) 118 ± 19 (91-148)  FEV1.0/FVC (%) 85.3 ± 4.2 (78.3-88.6) 84.6 ± 5.9 (74.3-92.4)   FEV1.0/FVC (%predicted) 101 ± 5 (92-106) 98 ± 7 (86-109)            FVC, forced vital capacity; FEV1.0, forced expired volume in 1 second; PEF, peak expiratory flow.  * Significantly different from men.  Ranges are presented in parentheses.   35 Table 2.2:  Maximal exercise data on day 1                            Men                           (n=8)   Women                          (n=8)    V˙  O2 (ml·kg-1·min-1) 69.5 ± 7.8  59.2 ± 4.7 *  V˙  O2 (l·min-1) 5.3 ± 0.7  3.8 ± 0.3 *  V˙  CO2 (l·min-1) 5.7 ± 0.6  4.1 ± 0.4 *  RER 1.08 ± 0.07  1.07 ± 0.05   Fb (breaths·min-1) 59 ± 9  59 ± 6   VT (l) 3.1 ± 0.4  2.3 ± 0.3 *  V˙  E (l·min-1) 161.2 ± 25.1  122.3 ± 17.3 *  V˙  E/V˙  O2  30.6 ± 4.9  32.1 ± 3.1   V˙  E/V˙  CO2  28.2 ± 3.0  30.2 ± 3.1   HR (bpm) 189 ± 8  191 ± 13   Exercise duration (s) 1273 ± 189  1213 ± 143   Work-rate (W) 380 ± 29  271 ± 22 *                              V˙  O2, oxygen consumption; V˙  CO2, carbon dioxide production; RER, respiratory exchange ratio; Fb, breathing frequency; VT, tidal volume;  V˙  E, minute ventilation; V˙  E/V˙  O2, ventilatory equivalent for oxygen; V˙  E/V˙  CO2, ventilatory equivalent for carbon dioxide; HR, heart rate.          * Significantly different from men.    Work of Breathing vs. Absolute Minute Ventilation:  Figure 2.2 shows the total WOB (panel A) and the constituent components of the WOB (panels B-E) at comparable absolute ventilations and at maximal ventilation in men and women.  The total WOB was significantly higher in women at 75 and 100 l·min-1 but not at 50 l·min-1.  The total WOB at 50, 75 and 100 l·min-1 was 23, 33 and 48% higher in women, respectively, but was 45% higher in men at maximal ventilations.  The inspiratory resistive WOB was 67, 89 and 109% higher in women at 50, 75 and 100 l·min-1, respectively (P < 0.05), while the expiratory resistive WOB was only significantly higher in women at 75 l·min-1.  The expiratory resistive WOB at 75 l·min-1 was 131% higher in women.  There was no significant difference in the elastic WOB during inspiration or expiration at any absolute ventilation.  However, the inspiratory elastic WOB was 42% higher in men at maximal ventilations (P = 0.05).    36 Minute Ventilation (l min-1)0 50 75 100 125 150 175 200Expiratory Resistive Work(J—min-1 )050100150200250300350Minute Ventilation (l—min-1)0 50 75 100 125 150 175 200Expiratory Elastic Work(J—min-1)05101520Minute Ventilation (l—min-1)0 50 75 100 125 150 175 200Inspiratory Elastic Work(J—min-1 )050100150200250300350Minute Ventilation (l—min-1)0 50 75 100 125 150 175 200Inspiratory Resistive Work(J—min-1)050100150200250300350Minute Ventilation (l—min-1)0 50 75 100 125 150 175 200Total Work(J—min-1 )02004006008001000*CE *A *  ***BD † * Figure 2.2:  Total work of breathing (A), inspiratory resistive work (B), expiratory resistive work (C), inspiratory elastic work (D) and expiratory elastic work (E) versus minute ventilation in men (○) and women (●).  The last data point represents maximal minute ventilation.  Values are mean ± SE.  * Significantly different between groups (P < 0.05); † P = 0.05.       37 Figure 2.3 shows Campbell diagrams for an individual male and female subject matched for minute ventilation, tidal volume and breathing frequency.  This figure demonstrates the significantly higher pressures needed to maintain the same ventilatory loads resulting in a much higher inspiratory and expiratory resistive work with little difference in elastic work.  Esophageal Pressure (cmH2O)-30 -20 -10 0 10 20Volume (l)012345Esophageal Pressure (cmH2O)-30 -20 -10 0 10 20Volume (l)012345 BACl CcwCl CcwIe: 70 J—min-1 Ir: 56 J—min-1Ee: 10 J—min-1 Er: 28 J—min-1Total WOB: 164 J—min-1Ie: 78 J—min-1 Ir: 143 J—min-1Ee: 14 J—min-1 Er: 82 J—min-1Total WOB: 317 J—min-1MALE FEMALEVolume (l)Volume (l) Figure 2.3:  Modified Campbell Diagrams from an individual male and female subject matched approximately for absolute minute ventilation (100 vs. 101 l·min-1(STPD)), tidal volume (2.1 vs. 2.2 liters), breathing frequency (52 vs. 49 breaths·min-1), age (24 vs. 25 years) and mass (64.6 vs. 64.2 kg), respectively.  The male was slightly taller than the female subject (181 vs. 167 cm, respectively).  Oblique hatching represents the inspiratory resistive work of breathing (Ir).  Horizontal hatching represents the inspiratory elastic work of breathing (Ie).  Stippling represents the expiratory resistive work of breathing (Er).  Vertical hatching represents the expiratory elastic work of breathing (Ee).  Cl, dynamic lung compliance; Ccw, chest wall compliance.  Upward arrow represents inspiration and downward arrow represents expiration.  Small open circles represent zero flow points.   Work of Breathing vs. Relative Submaximal Minute Ventilation:  The total WOB plotted against percentages of maximal minute ventilation is shown in figure 2.4.  Men had a significantly higher total WOB for any given percentage of maximal ventilation compared with  38 women.  The total WOB was 120, 60 and 50% higher in men at 25, 50 and 75% of maximal minute ventilations, respectively.  While each component of the WOB was higher in men for a given percentage of minute ventilation, the largest (and statistically significant) differences were seen with the inspiratory elastic WOB (data not shown).  The absolute ventilations in men versus women corresponding to 25, 50 and 75 % of maximal ventilations were: 48.4 ± 6.5 vs. 32.8 ± 7.0 l·min-1, 90.3 ± 14.7 vs. 62.9 ± 12.2 l·min-1, and 134.9 ± 22.1 vs. 94.9 ± 18.4 l·min-1, respectively.  Thus, the absolute ventilations were, on average, 31% higher in men when comparing sexes at the aforementioned relative minute ventilations.  The tidal volumes were 75, 37 and 36% higher in men at 25, 50 and 75% of maximal minute ventilations (P < 0.05), respectively with little to no difference in breathing frequency.    Minute Ventilation (% maximum)0 25 50 75 100Total Work (J—min-1 )0100200300400500*** Figure 2.4:  Total work of breathing versus relative percentages of maximal minute ventilation in men (○) and women (●).  Values are mean ± SE.  * Significantly different between groups    (P < 0.05).   39 Work of Breathing vs. External Muscular Work:  Figure 2.5 shows the total WOB (panel A), inspiratory resistive WOB (panel B) and expiratory resistive WOB (panel C) versus work rate (corrected for mass) in men and women.  Panel A demonstrates that the total WOB was significantly higher in women at approximately 3.5 W·kg-1 (239 ± 31 vs. 173±12 J·min-1, P  < 0.05) and 4.0 W·kg-1 (387 ± 53 vs. 243 ± 36 J·min-1, P < 0.05).  The inspiratory resistive WOB was higher in women at approximately 3.0 W·kg-1 (56 ± 8 vs. 41 ± 4 J·min-1, P = 0.05), 3.5 W·kg-1 (93 ± 15 vs. 56 ± 5 J·min-1, P < 0.05) and 4.0 W·kg-1 (162 ± 24 vs. 81 ± 2 J·min-1, P < 0.05).  The expiratory resistive WOB was higher in women at approximately 3.5 W·kg-1 (44 ± 11 vs. 20 ± 3 J·min-1, P < 0.05) and 4.0 W·kg-1 (67 ± 16 vs. 35 ± 8 J·min-1, P = 0.05).  There were no significant differences in the inspiratory or expiratory elastic components at any work rate.                40 Workload (W—kg-1)0.0 3.0 3.2 3.4 3.6 3.8 4.0Inspiratory Resistive Work (J—min-1)0255075100125150175200*†*BWorkload (W—kg-1)0.0 3.0 3.2 3.4 3.6 3.8 4.0Expiratory Resistive Work (J—min-1 )020406080100*†CWorkload (W—kg-1)0.0 3.0 3.2 3.4 3.6 3.8 4.0Total Work (J—min-1 )0150200250300350400450**A Figure 2.5:  Total work (A), inspiratory resistive work (B) and expiratory resistive work of breathing (C) versus work rate in men (○) and women (●).  Values are mean ± SE.                      * Significantly different between groups (P < 0.05); † P = 0.05.   41 Breathing Pattern:  Figure 2.6 shows the breathing frequency (panel A) and tidal volume (panel B) response to exercise.  It can be seen that men achieved a maximal minute ventilation that was considerably higher than women (180.2 ± 28.7 vs. 126.2 ± 24.2 l·min-1, respectively).  Generally, women breathed with a significantly higher breathing frequency and lower tidal volume to achieve the same absolute minute ventilation as men.  Breathing frequency was significantly different between sexes at 75 and 100 l·min-1 while tidal volume was attenuated in women at all ventilations above 50 l·min-1.  Figure 2.7 shows the effect of tidal volume (panel A) and breathing frequency (panel B) on the inspiratory elastic WOB at the 4 venitlatory points (i.e., 50, 75, 100 l·min-1 and maximal ventilation).  For any given tidal volume, the inspiratory elastic WOB is considerably higher in women.  However, the inspiratory elastic WOB is lower in women for any given breathing frequency.  While these are physiologically significant observations, specific statistical procedures could not be performed on the data presented in Figure 2.7.    42 Minute Ventilation (l—min-1)0 50 75 100 125 150 175 200Breathing Frequency (breaths—min-1 )020304050607080Minute Ventilation (l—min-1)0 50 75 100 125 150 175 200Tidal Volume (l)0.01.52.02.53.03.5AB * * *  * * Figure 2.6:  Breathing frequency (A) and tidal volume (B) versus minute ventilation in men (○) and women (●).  The last data point represents maximal minute ventilation.  Values are mean ± SE.  * Significantly different between groups (P < 0.05).    43 Tidal Volume (l)0.0 1.6 2.0 2.4 2.8 3.2 3.6Inspiratory Elastic Work (J—min-1)050100150200250300350 ABreathing Frequency (breaths—min-1)0 20 30 40 50 60 70Inspiratory Elastic Work (J—min-1)050100150200250300350 B Figure 2.7:  Inspiratory elastic work of breathing versus tidal volume (A) and breathing frequency (B) at the four ventilatory points (i.e., 50, 75, 100 l·min-1 and maximal ventilation) in men (○) and women (●).  Values are mean ± SE.    44 Lung Size vs. Work of Breathing:  Figure 2.8 summarizes the relationships between different components of the WOB and FVC with all subjects pooled together.  The WOB values shown in this figure are from a minute ventilation corresponding to 100 l·min-1.  We chose to report data at 100 l·min-1 for the regression analysis for 3 reasons.  Firstly, we wanted to report data at the highest range of absolute ventilations when the work and metabolic cost of breathing are highest.  Secondly, this ventilation tended to show the largest sex-based difference in the total WOB and inspiratory resistive WOB.  Finally, all subjects achieved 100 l·min-1 of ventilation during exercise.  FVC was used as a surrogate for total lung capacity (less residual volume) in order to determine the relationship between lung size and the WOB.  FVC was significantly and linearly related to the total WOB, the inspiratory resistive WOB and the expiratory resistive WOB.  When partitioned into individual groups, the correlation coefficients relating FVC to the total WOB, the inspiratory resistive WOB and expiratory resistive WOB in women was 0.92 (P = 0.001), 0.78 (P = 0.02) and 0.73 (P = 0.04)  whereas the men were 0.46 (P = 0.25), 0.27 (P = 0.52)  and 0.75 (P = 0.03), respectively.               45 FVC (l)0 3 4 5 6 7Total Work(J—min-1)0100200300400500 A r  = -0.74P = 0.001FVC (l)0 3 4 5 6 7Inspiratory Resistive Work(J—min-1 )050100150200 B r  = -0.71P = 0.002FVC (l)0 3 4 5 6 7Expiratory Resistive Work(J—min-1)020406080100 C r  = -0.72P = 0.002 Figure 2.8:  Regression analysis of the total work (A), inspiratory resistive work (B) and expiratory resistive work (C) versus forced vital capacity (FVC) in men (○) and women (●).  Work of breathing values are obtained at a minute ventilation of 100 l·min-1.   46 Flow vs. Work of Breathing:  The inspiratory resistive WOB was plotted against the corresponding average inspiratory flow throughout all exercise intensities in each individual subject as shown in Figure 2.9 (panel A).  All of the raw data points from panel A were fitted with a 2nd order polynomial (mean r2 for all subjects = 0.99 ± 0.01) in order to produce a mean curve for all men and women as shown in panel B.  Panels A and B show that the inspiratory resistive WOB was higher in women for any given flow rate above ~2 l·s-1 and the magnitude of this difference increased disproportionally in women with increasing flow.  Figure 2.9B also shows the response of an individual female subject with FVC and peak expiratory flows that were 134 and 148% above predicted values, respectively.                  47 Average Inspiratory Flow (l—s-1)0 1 2 3 4 5 6 7 8Inspiratory Resistive Work (J—min-1)0100200300400500Average Inspiratory Flow (l—s-1)0 1 2 3 4 5 6 7 8Inspiratory Resistive Work (J—min-1)0100200300400500AB Figure 2.9:  Inspiratory resistive work of breathing versus average inspiratory flow in individual male (thin lines) and female (thick lines) subjects (A).  Panel B shows the mean inspiratory resistive work of breathing versus average inspiratory flow in women (thick line) and men (thin line) and an individual female subject (dashed line) with larger than average FVC and peak expiratory flow values.  48 DISCUSSIOglyph1197   Our present understanding of the WOB during exercise is primarily based on studies conducted in males but a recent study by our group demonstrated significant differences in the total WOB between men and women for a given absolute minute ventilation (Guenette et al., 2007).  The present study adds to the previous literature by systematically measuring the elastic and resistive WOB in exercising women.  The novel findings from this study are four-fold.  First, the inspiratory resistive WOB was higher in women for any given absolute minute ventilation while the expiratory resistive WOB was higher in women at only 75 l·min-1.  There were no sex differences in the inspiratory or expiratory elastic WOB across any absolute minute ventilation.  However, the total WOB was actually higher in men when compared across relative percentages of maximal ventilations, due to their higher absolute tidal volumes and thus higher minute ventilations.  Second, the total WOB and the inspiratory and expiratory resistive WOB were higher in women when performing the same relative external muscular work.  Third, the WOB was inversely related to lung size and presumably airway size.  Lastly, the inspiratory resistive WOB was considerably higher for a given level of inspiratory flow compared with men, demonstrating the importance of airway size in determining the mechanical cost of breathing.  We interpret our findings to mean that the higher total WOB observed in exercising women at absolute ventilations is due to a higher resistive WOB which can be attributed to relatively smaller lungs and airways.  Resistive Work of Breathing vs. Minute Ventilation:  In our previous work (Guenette et al., 2007) we plotted the total WOB  against a range of ventilatory rates and fit the data points to the following equation as originally described by others (Otis et al., 1950; Margaria et al., 1960):   49 WOB = aV˙  E3 + bV˙  E2      The term bV˙  E2  describes the mechanical work done in overcoming the viscous resistance offered by the lung tissues to deformation and by the respiratory tract to the laminar flow of air.  The term aV˙  E3 represents the work done in overcoming the resistance to turbulent flow.  We found that the constant a was significantly higher in women meaning that the higher total WOB in women is associated with the additional resistive work due to turbulent airflow.  While this is an instructive analysis it does not permit a quantitative measure of the individual factors that make up the total WOB at specific time or physiological points.  By using a more extensive approach we have now partitioned the total WOB into its individual components at specific values of minute ventilation and at standardized work rates.  During progressive exercise we found that both inspiratory and expiratory resistive work were significantly higher in women over a range of ventilatory rates (Figure 2.2B and C).  Interestingly, we observed significant differences in inspiratory resistive work at low levels of minute ventilation (50 l·min-1) and the magnitude of difference increased as ventilation increased up to 100 l·min-1.  Figure 2.3 provides a compelling example of the high pressures that are needed in a female subject in order to achieve the same minute ventilation as a male subject.  It is important to note that the male and female subject shown in Figure 2.3 have been matched for breathing frequency, tidal volume, age and body mass.  Despite the fact that both subjects are breathing at the same volume and rate, the inspiratory and expiratory resistive work components are considerably higher in the female subject.    Elastic Work of Breathing vs. Minute Ventilation:  The elastic work required to increase and decrease the volume of the lung is related to the elastic forces that develop in the tissues of the lung and chest wall.  Unique to this study, we found that there were no sex differences in the  50 elastic WOB at any absolute ventilation.  However, with further examination, it can be seen in Figure 2.7 (panel A) that the inspiratory elastic WOB is substantially higher in women for a given tidal volume.  This can be attributed to the fact that women are breathing at a higher percentage of their total lung capacity for a given level of ventilation which reduces the compliance of the lungs.  Perhaps more important is the observation that women adopt a higher breathing frequency for a given minute ventilation which acts to reduce the inspiratory elastic WOB (Figure 2.7B).  This type of breathing pattern comes at the expense of an increased resistive WOB.  This is an important observation if one is to consider how breathing patterns are regulated in humans in an effort to minimize the total WOB.  The “principle of minimum effort” was first used to describe that for a given alveolar ventilation there is a breathing frequency that is optimal (Otis et al., 1950).  This pattern is adopted because if the breathing frequency is too low then large amounts of elastic work are required whereas if the breathing frequency is too high then respiratory muscle work is expended to ventilate dead space (i.e., wasted ventilation).  During exercise the diaphragm generates most of the inspiratory driving force and appears to remain within the favourable part of its length-tension curve (Grimby et al., 1976).  This suggests that under spontaneously breathing conditions, the diaphragm tension or O2 cost is what is being minimized during exercise.  Based on the present study, it appears that women use a higher breathing frequency to minimize the elastic WOB which comes at the expense of a higher resistive WOB.  Total Work of Breathing vs. Minute Ventilation:  The total WOB was higher in women at any absolute ventilation comparison above 50 l·min-1 which is consistent with our previous findings using a different analysis technique (Guenette et al., 2007).  In our previous work we also compared the total WOB between men and women at different percentages of V˙  O2max and found that the total WOB was modestly higher in men at V ˙ O2max but there was no statistically  51 significant difference (Guenette et al., 2007).  However, in the present study we have shown that the total WOB is significantly higher in men when compared at maximal ventilations (Figure 2.2) and also at submaximal relative percentages of maximal ventilation (Figure 2.4).  We attribute this discrepant finding to the fact that the Modified Campbell Diagram technique takes into account the compliance of the chest wall which allows us to calculate the additional part of the inspiratory elastic WOB which extends beyond the area directly within the pressure-volume loop (see Figure 2.1).  Indeed, it can be seen in Figure 2.2 that the only component to approach statistical significance at maximal ventilations was the inspiratory elastic WOB (P = 0.05).  The primary driving force for the higher total WOB at submaximal relative ventilations was also the inspiratory elastic WOB (data not shown).  This is due to the fact that for a given relative percentage of maximal ventilation, the tidal volume is considerably higher in men with little to no difference in breathing frequency.  This will substantially increase the inspiratory elastic WOB and thus the total WOB when comparisons are made at relative intensities.  We have purposely limited the majority of our analysis and interpretation in this study and in our previous work (Guenette et al., 2007) to absolute ventilations in order to determine if the mechanical cost of moving a given amount of air in and out of the lungs is different between sexes.  Examining the mechanics of breathing between sexes at relative ventilations is a difficult comparison because men are utilizing a much higher tidal volume and thus have higher minute ventilations than women.  For example, the men in this study were breathing 54 l·min-1 higher than women at maximal exercise.  Despite the fact that maximal ventilations were 43% larger in our male subjects, it is interesting to note that there were no significant differences in the resistive WOB components.  This lends further support to the finding that women have a substantially higher resistive WOB than men.     52 Work of Breathing vs. External Muscular Work:  Figure 2.5 shows the WOB required to perform the same relative external work on the cycle ergometer.  Rather than using absolute work rate (i.e., power) in Watts, we have normalized the work rate by expressing it in Watts per kg of body mass which provides a physiologically relevant comparison because it minimizes the potential confounding effect of body size differences.  Moreover, it allows us to compare the physiological cost of breathing betweens sexes for a given standardized external work load.  Even when normalized for mass, the total WOB is higher at 3.5 and 4.0 W·kg-1 with the inspiratory and expiratory resistive WOB components accounting for the vast majority of this difference (Figure 2.5).  At 3.5 and 4.0 W·kg-1, the inspiratory resistive WOB was 67 and 100% higher in women while the expiratory resistive WOB was 123 and 89% higher in women, respectively.  It is important to note that these comparisons do not take into account lean body mass since body composition was not measured in these subjects.  It has been suggested that sex differences are minimized or completely abolished in laboratory-based experiments when comparing sexes for a given power to lean body mass ratio (Stefani, 2006).  Although all subjects were lean endurance athletes of similar training status, it would be expected that the women would still have a higher percentage of body fat and therefore less muscle mass.  Therefore, it is important to acknowledge that this interpretation has its limitations because the female participants are still working at a slightly higher percentage of their maximum output relative to their male counterparts.  This will certainly account for some of the differences observed in our WOB values.  However, we cannot directly assess the impact of this limitation in our study without a measurement of lean body mass and therefore do not attempt to overstate these findings.  Sex vs. Size Differences:  We observed statistically significant associations between the resistive WOB at 100 l·min-1 and FVC (see Figure 2.8).  As would be expected, women had lower FVC  53 values than men, which were inversely related to a higher resistive WOB.  Therefore, those with the smallest lungs and presumably the smallest airways had the highest resistive WOB.  We do not have a direct measurement of airway size in our subjects but similar correlation coefficients were also observed when relating the resistive WOB components against peak expiratory flows which may serve as a crude surrogate for airway size.  We are cognizant of the limits of correlative evidence and therefore do not attempt to overstate these findings.  However, in an effort to provide a more mechanistic understanding of the higher resistive WOB in women, particularly on inspiration, we performed additional analyses as shown in Figure 2.9.  In this analysis, panel A shows the inspiratory resistive WOB for a given level of inspiratory flow in individual subjects while panel B represents the group average.  This data shows that for a given level of flow, the resistive WOB is higher in women and the magnitude of this difference increases disproportionally with increasing flow.  Panel B includes one female subject superimposed with the mean curves.  This subject had unusually large lungs and peak expiratory flows (> 130% predicted).  In fact, her FVC and peak expiratory flows were relatively close to the group mean values for men.  Interestingly, her inspiratory resistive WOB response for a given level of flow was nearly identical to the average curve for the male subjects.  These observations in conjunction with correlative evidence points to an anatomical basis (i.e., smaller lungs and airways) for the WOB differences we observed during exercise.  Additional physiological and performance based consequences of these anatomical differences in lung and airway size have been reviewed elsewhere (Sheel & Guenette, 2008).    Our sex-based comparisons were made between men and women of significantly different statures.  It could be argued that our findings simply reflect size differences rather than a true male-female difference in lung and airway size.  However, there is reason to suggest that our findings would be similar between men and women of comparable sizes (i.e., men and  54 women matched for total lung capacity).  We make this claim based on two lines of anatomical evidence.  First, in healthy young men and women matched for total lung capacity, women have significantly smaller tracheal areas (2.79 vs 1.99 cm2) as assessed by acoustic reflectance (Martin et al., 1987).  As such, the reduced female tracheal area would result in a higher WOB for a given level of minute ventilation.  Airflow is determined, in part, by Poiseuille's Law and the factors governing it are internal diameter, length, gas viscosity and airflow pressure where radius is raised to the fourth power.  As such, even a small difference in airway radius is magnified and would have an effect on airflow resistance and the accompanying WOB.  Second, the relationship between airway size (estimated from maximal expiratory flow ÷ static recoil pressure at 50% vital capacity) and lung size (vital capacity) shows that adult men have airways that are 17% larger than those of women (Mead, 1980).  This has been termed “dysanapsis” to reflect the dissociation between airway size and lung parenchymal size (Green et al., 1974).  Given the brief summary presented above, coupled with the findings of the present study, it appears that the higher resistive WOB seen in women is due to inherently smaller airways.    Methodological Considerations:  Our measures of the work done by the respiratory muscles do not take into account the distorting forces of the chest wall observed at high levels of minute ventilation.  Volume displacement of the rib cage and abdomen can be independent of one another (Roussos & Campbell, 1986).  Phrased differently this means that all of the respiratory muscles do not necessarily shorten during inspiration nor do all of the muscles of expiration shorten during expiration.  We recognize this as a critique of the modified Campbell approach and that our measure of the WOB may be underestimates.  However, it is unlikely that this systematic underestimation applied to all subjects equally would have had any substantive effect on our overall conclusion that women have a higher WOB during exercise owing to a greater resistive WOB.  This is supported by optoelectronic plethysmography measures which suggest  55 that men and women utilize muscles of the ribcage compartment and those of the abdomen to the same extent (Vogiatzis et al., 2005).  The compliance of the chest wall is required to determine the various WOB components using the modified Campbell Diagram method.  It is typically very difficult to reliably measure the compliance of the chest wall because naive subjects have a difficult time completely relaxing their respiratory muscles.  Therefore, we based our chest wall compliance values on previously published data taking into account both age and sex (Estenne et al., 1985) as done by others (Yan et al., 1997; Sliwinski et al., 1998; Butcher et al., 2006; Eves et al., 2006).  There are limitations with this approach that warrant discussion.  For example, the chest wall compliance values we used were obtained in healthy volunteers with normal static lung volumes and FEV1.0 values and it is assumed that the values were measured in untrained individuals.  Therefore, we are making the assumption that the compliance of the chest wall is similar between trained and untrained subjects.  To our knowledge, there are no studies that have studied the effect of fitness on the compliance of the chest wall.  While there are inherent limitations in using “normative” data and applying it to elite athletes, we do not think that this had an effect on our main conclusions regarding sex differences in the WOB.  We base this assumption on several factors.  Firstly, according to Estenne et al. (1985), there are no sex differences in the compliance of the chest wall (at least in untrained individuals).  Our men and women were of similar relative fitness levels so it is reasonable to assume that this lack of a sex difference should persist along the fitness continuum and any potential errors in our chest wall compliance values would likely be a systematic error across all subjects.  Secondly, the vital capacity values in our men (6.0 ± 1.0 l) and women (4.5 ± 0.5 l) were nearly identical to the age-matched men (6.0 ± 0.9 l) and women (4.3 ± 0.4 l) from whom we derived are chest wall compliance values.  Finally, we have calculated that even a ± 10% change in the compliance of the chest wall would only affect the  56 elastic components by approximately ± 3-5%, the inspiratory resistive WOB by less than ± 0.5% and the expiratory resistive WOB by approximately ± 1.5-3%.  The elastic WOB measurements that we and others (Yan et al., 1997; Sliwinski et al., 1998) have made may be underestimations because the calculations are based on tidal volume measured at the mouth which ignores gas compression (Otis, 1964).  Gas compression is typically negligible in healthy individuals at rest or during exercise.  However, under conditions where expiratory flow limitation is present, this could increase the magnitude of underestimation.  We did observe expiratory flow limitation in many of our female and male subjects (Guenette et al., 2007) during high levels of exercise but we do not believe that this had a major influence on our findings or overall interpretation of the present study, particularly at submaximal workloads.  Specifically, we observed no demonstrable difference between men and women in the elastic WOB at low levels of ventilation (50 l·min-1) when expiratory flow limitation is not present.  This absence held true at higher levels of ventilation suggesting that any underestimation was most likely consistent across all ventilatory rates.  Thus any underestimation due to gas compression likely had a negligible influence on our findings and conclusion that the elastic WOB is similar between men and women for a given level of ventilation.  The current study design does not allow us to determine the direct role of expiratory flow limitation and the corresponding changes in operational lung volumes between sexes because flow limitation was typically observed at maximal to near-maximal intensities.  As such, it would be difficult to isolate the effects of EFL on our WOB values in men and women because any potential differences would be masked by the fact that men have much higher tidal volumes and thus ventilations at maximal exercise.  A novel study design would be required to assess sex-based differences in expiratory flow limitation and the direct corresponding effect on the WOB.     57 Conclusions:  This study is the first to systematically assess the mechanisms of a higher WOB in women during dynamic exercise.  Describing sex differences in breathing mechanics poses a significant challenge because of the inherent difficulties in comparing men and women due to known differences in body size and an incomplete understanding of the most appropriate allometric scaling factor to use.  In the present study, we have made a number of unique physiological comparisons.  First, comparing the WOB for a given level of ventilation allowed us to quantify the additional cost of breathing necessary to move a fixed amount of air through smaller lungs and airways in women.  Second, comparing men and women at different percentages of maximal ventilations allowed us to determine the cost of breathing for a given relative intensity.  Third, comparing sexes at a standardized size-corrected work rate enabled us to determine if there are differences in the WOB for a given level of external muscular work.  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Am J Respir Crit Care Med 156, 807-813.             60          CHAPTER III: Sex differences in exercise-induced diaphragmatic fatigue in endurance-trained athletes*                     ________________________ * A version of this chapter will be submitted for publication as: Guenette JA, Romer LM, Querido JS, Chua R, Eves ND, Road JD, McKenzie DC and Sheel AW.  Sex differences in exercise-induced diaphragmatic fatigue in endurance-trained athletes.    61 Iglyph1197TRODUCTIOglyph1197  The effects of skeletal muscle fatigue on the ability to perform muscular work has been a topic of interest to exercise and muscle physiologists for over a century.  Muscle fatigue can be defined as a loss in the capacity for developing force and/or velocity resulting from muscle activity under load and which is reversible by rest (NHLBI, 1990).  Studies examining sex differences in peripheral skeletal muscle fatigue have shown that women have greater relative fatigue resistance compared to their male counterparts (Maughan et al., 1986; Miller et al., 1993; West et al., 1995; Fulco et al., 1999).  Commonly cited mechanisms associated with greater fatigue resistance in women include differences in muscle mass/morphology, substrate utilization and neuromuscular activation (Hicks et al., 2001).  The majority of studies examining sex differences in skeletal muscle fatigue have focussed on the muscles involved in moving the elbow, finger, knee, thumb, ankle, back and neck.  To our knowledge, no study has systematically assessed sex differences in fatigue of the human diaphragm.  The diaphragm is embryologically, morphologically and functionally a striated skeletal muscle.  However, it remains distinct from other skeletal muscles because it is under both voluntary and involuntary control and like the heart, contracts rhythmically across the entire life span.  As such, the diaphragm has a primary role in breathing and in sustaining human life.  Contraction of the diaphragm is important if we consider physiological conditions that require high levels of ventilation such as during strenuous exercise.  However, the diaphragm is similar to other muscles in that it is susceptible to exercise-induced muscle fatigue (Johnson et al., 1993; Vogiatzis et al., 2008).  Moreover, there are important hemodynamic and exercise performance consequences associated with fatigue of the human diaphragm as recently reviewed by Romer & Polkey (2008).  While it appears that many non-respiratory skeletal muscles are more fatigue  62 resistant in women, the diaphragm may be an exception due to known differences in respiratory structure and function that may predispose women to greater pulmonary limitations relative to men.  We base this suggestion on four lines of evidence.  First, there are a number of sex differences in respiratory structure and resting pulmonary function that may influence the ventilatory response to exercise in women.  Compared to men, women have smaller lung volumes, lower maximal expiratory flow rates and a decreased capacity for lung diffusion even when corrected for age and standing height (Mead, 1980; Schwartz et al., 1988; McClaran et al., 1998).  Second, women have smaller diameter airways even when matched for lung volume (Martin et al., 1987; Sheel et al., 2009).  Third, these anatomical differences may explain, in part, why women have a higher work of breathing relative to men during exercise (Guenette et al., 2007; Guenette et al., 2009).  Lastly, smaller lungs and airways may also predispose women to expiratory flow limitation during exercise (McClaran et al., 1998; Guenette et al., 2007).  Based on the aforementioned differences, the respiratory muscles of exercising females may be placed under greater mechanical stress relative to males which may result in greater fatigue.    Previous work from our laboratory (Guenette et al., 2007; Sheel & Guenette, 2008)  and others (Harms, 2006) have postulated that these anatomical and functional sex differences in pulmonary physiology might make the female diaphragm more prone to fatigue.  This hypothesis has not been directly tested, but is complicated by the findings of previous studies showing that women may be less susceptible to fatigue of the limb muscles (Hicks et al., 2001; Hunter, 2009) and perhaps even cardiac muscle (Scott et al., 2007).   Therefore, the purpose of this study was to determine if there are sex differences in the magnitude of exercise-induced diaphragmatic fatigue.  We hypothesized, that the magnitude of diaphragm fatigue following high-intensity cycling exercise would be greater in trained women relative to trained men.     63 METHODS  Subjects:  Thirty eight (19 males and 19 females) healthy subjects gave informed written consent [APPENDIX I].  Thirty one subjects were competitive endurance-trained cyclists while the remaining 7 subjects were non-competitive endurance-trained individuals.  “Competitive” was defined as regular participation in cycling and/or triathlon races.  All of the women were tested randomly throughout the menstrual cycle.  Subjects were excluded from participating if they were smokers or had a history of cardiopulmonary disease.  Subjects with nasal septum deviation, esophageal ulcers, or allergies to local anaesthetics or latex were also excluded from participating.  All procedures received institutional ethical approval and conformed to the Declaration of Helsinki.    Experimental Overview:  The experiment was conducted on two separate days with a minimum of 48 h rest between each testing session.  On day 1, subjects underwent basic anthropometric measures followed by pulmonary function testing, general experimental familiarization and an incremental cycle test to exhaustion to determine peak work-rate and associated variables (e.g., maximum oxygen uptake, V˙  O2max).  Day 2 served as the primary testing day, which involved the assessment of diaphragmatic fatigue in response to constant load cycling.  On day 2, subjects were instrumented with diaphragmatic surface electromyography (EMG) electrodes, and balloon-tipped catheters were placed in the esophagus and stomach.  They then underwent cervical magnetic stimulation (CMS) of the phrenic nerves following a 10 min period of quiet breathing.  Upon completion of all baseline measures, subjects performed a self-selected warm-up on a cycle ergometer followed by steady-state cycling at 90% of peak work-rate as determined on day 1.  CMS was then performed 10, 30 and 60 min after exercise.  64  Pulmonary Function:  Forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1.0), FEV1.0/FVC, peak expiratory flow (PEF) and forced expiratory flow between 25 and 75% of FVC (FEF25-75%) were measured using a portable spirometer (Spirolab II, Medical International Research, Vancouver, BC) according to ATS/ERS guidelines (2002).  Actual values were compared to predicted normal values.  Subjects with an FEV1.0/FVC < 80% of predicted were excluded from participating in the investigation.    Incremental Exercise Test:  Subjects performed a 10 min warm-up at a self-selected work-rate on an electromagnetically-braked cycle ergometer (Excalibur Sport, Lode, Groningen, The Netherlands).  Males and females started the test at 200 W and 100 W, respectively, with the work-rate increasing in a stepwise fashion by 30 W every 3 min until the subjects could no longer maintain cadence ≥ 60 rpm despite verbal encouragement.  Peak work-rate was calculated as the sum of the final completed exercise stage and an extrapolated work-rate depending on the time spent in the final non-completed stage.  To determine V˙  O2max, subjects wore a nose clip and breathed through a mouthpiece connected to a non-rebreathing valve (model 2700B, Hans-Rudolph, Kansas City, MO).  Mixed expired gases were measured using calibrated CO2 and O2 analyzers (Model CD-3A and Model S-3-A/I respectively, Applied Electrochemistry, Pittsburgh, PA) measured at a port located in a mixing chamber, while inspiratory flow was measured using a calibrated pneumotachograph (model 3813, Hans Rudolph, Kansas City, MO).  Heart rate was recorded every 30 s using a commercially available heart rate monitor (Polar Electro, Kempele, Finland).    Pressure Measurements:  Mouth pressure (Pm) was monitored at a port located in the mouthpiece and connected to a piezoelectric pressure transducer (Raytech Instruments,  65 Vancouver, BC).  Esophageal pressure (Pes) and gastric pressure (Pga) were measured using balloon-tipped catheters (no. 47-9005, Ackrad Laboratory, Cranford, NJ) attached to piezoelectric pressure transducers (Raytech Instruments Inc).  The transducers were calibrated across the physiological range using a digital manometer (2021P, Digitron, Torquay England).  Viscous lidocaine (2%) was applied to the nasal and pharyngeal passages to minimize discomfort during catheter placement.  Both catheters were first inserted into the stomach.  The esophageal catheter was withdrawn until a negative pressure deflection was observed during inspiration and then withdrawn an additional 10 cm to ensure that it was completely within the esophagus.  After the balloons were inserted, all air was evacuated by subjects performing a Valsalva maneuver.  One and two milliliters of air were injected into the esophageal and gastric balloons, respectively.  Validity of the esophageal balloon position was verified using the occlusion technique (Baydur et al., 1982).  Trans-diaphragmatic pressure (Pdi) was obtained online as the difference between Pes and Pga.    Diaphragm Fatigue:  The CMS technique was used to assess diaphragmatic fatigue following exercise (Similowski et al., 1989).  Both phrenic nerves were stimulated using a 90 mm circular coil attached to a magnetic stimulator (Magstim 200 Mono Pulse, MagStim, Whitland, Wales).  With the subject seated in a chair, their neck was flexed and the coil was placed over the cervical spine.  The site of optimal stimulation was determined by positioning the coil between C5 and C7 until the largest Pdi was achieved.  The position of the coil was then marked with indelible ink on the subject’s neck to ensure the coil was in an identical position for all subsequent stimulations.  Considerable care was also taken to ensure that subjects were seated in the same position throughout the experiment with the same level of neck flexion.  Since lung volume can influence twitch amplitude, all twitches were performed at end-expiratory lung volume (EELV) with the glottis closed.  EELV was verified by examining end-expiratory Pes prior to stimulation.   66 Supramaximal stimulation was assessed by charging the stimulator to progressively increasing levels of its maximal power output (i.e., 50, 60, 70, 80, 85, 90, 95, and 100%).  Three twitches were performed at each power output with each twitch separated by 30 s of quiet breathing to avoid twitch potentiation.  A plateau in mean twitch Pdi (Pdi,tw) with increasing power output was an indication that the phrenic nerves were maximally stimulated.  Surface EMG of the right and left costal diaphragm was recorded using surface electrodes (Soft-E H59P: Kendall-LTP, Chicopee, MA, USA).  The electrodes were placed on the anterior axillary line on the sixth to eighth intercostal spaces and re-positioned if necessary to optimize M-wave characteristics (Glerant et al., 2006).  Peak-to-peak amplitudes of the M-waves were measured for every twitch.  EMG signals were amplified, band-pass filtered and the analog signals were A/D converted (PowerLab/16SP model ML 795, ADI, Colorado Springs, CO) and recorded simultaneously using PowerLab data acquisition software (Chart v6.1.3, ADInstruments, Colorado Springs, CO).  Pressure and EMG signals were sampled at 1kHz.      The protocol for this experiment included a series of 5 potentiated twitches that were performed at 100% of the stimulator output before (baseline) and 10, 30 and 60 min after the exercise bout.  Potentiated twitches involved a maximal inspiration for ~5 sec initiated at functional residual capacity against a device which incorporated a 2mm orfice to prevent glottic clousre.  The phrenic nerves were then stimulated at the end of the second tidal expiration following the maximal inspiratory effort.  The subject then immediately repeated this procedure a total of 5 times (Figure 3.1).  We discarded the first two twitches from the analysis because twitch amplitude was still rising.  Individual twitches were rejected if there was evidence from the raw Pes trace of deviation from relaxed FRC or esophageal contraction.  The primary outcome variable for this study was the change in Pdi,tw.  Additional fatigue and contractility characteristics of the diaphragm included contraction time and half relaxation time.  Contraction  67 time was determined as the time interval between the initiation of twitch tension and peak tension and half relaxation time was determined as the time for Pdi to decrease to one-half of the peak tension (Figure 3.1).  Fatigue of the diaphragm was considered present if there was a ≥ 15% reduction in Pdi,tw relative to the pre-exercise baseline values as used previously by others (Kufel et al., 2002).  This conservative definition of fatigue is based upon an approximate 2-3 fold increase in the coefficient of variation of Pdi,tw as observed in the present study.                        68                      Figure 3.1: Twitch potentiation protocol.  Example of the twitch potentiation protocol in an individual subject.  Each of the 5 maximal inspiratory efforts is followed by a twitch performed at end-expiratory lung volume.  The top 4 graphs include mouth pressure (Pm), esophageal pressure (Pes), gastric pressure (Pga) and trans-diaphragmatic pressure (Pdi).  Bottom graph is an example of an individual trans-diaphragmatic pressure twitch (Pdi,tw) using cervical magnetic stimulation to stimulate the phrenic nerves.  ct, contraction time; ½rt, half-relaxation time; thick vertical arrow represents the onset cervical magnetic stimulation.  PesPes 69 Exercise Breathing Mechanics:  Subjects breathed through a mouthpiece connected to a bi-directional heated pneumotachograph (model 3813, Hans Rudolph, Kansas City, MO).  Inspiratory and expiratory air flow was continuously monitored and integrated to obtain volume.  Approximately 10 breaths during exercise were selected at various percentages of time to exhaustion (TTE) and ensemble averaged using customized software.  The work of breathing against the lung was estimated from the area within the tidal Pes-volume loop with the addition of that portion of a triangle describing the work that fell outside the tidal Pes-volume loop representing part of the elastic work of breathing (McGregor & Becklake, 1961).  The work of breathing was then multiplied by breathing frequency and converted into joules per minute.  Diaphragmatic pressure-time product (PTPdi) and esophageal pressure-time product (PTPes) were determined by integrating Pdi and Pes, respectively during inspiration with respect to time and then multiplying these values by breathing frequency.    Symptom Evaluation:  Ratings of perceived exertion for breathlessness and leg discomfort were measured each minute during exercise using Borg’s 0-10 category ratio scale (Borg, 1982).  The scale’s endpoints were anchored such that ‘0’ represented “no respiratory (or leg) discomfort” and ‘10’ represented “the most severe respiratory (or leg) discomfort you have ever experienced or could ever imagine experiencing.”    Statistical Analyses:  Descriptive characteristics, pulmonary function and maximal exercise data were compared between groups using unpaired t-tests.  Pre-planned comparisons were used to determine sex differences at each time point during the constant-load cycle test using unpaired t-tests with Bonferroni corrections for multiple comparisons.  When 5 comparisons were made, a P value of < 0.01 was considered statistically significant and a P value of < 0.017 was considered statistically significant when 3 comparisons were made.  P values ≤ 0.05 are also  70 reported throughout the manuscript.  One way repeated-measures ANOVA with a Dunnet post hoc test was used to determine if there were significant differences in Pdi,tw at all submaximal stimulation intensities compared to maximal stimulation intensity (100%).  Linear regression analysis was performed to determine the relationship between diaphragm fatigue and selected exercise parameters.  Values are presented as mean ± S.E.M.                         71 RESULTS  Subject Characteristics:  Table 3.1 summarizes basic descriptive characteristics and pulmonary function data.  Men and women were not different for age, but men were taller and heavier.  As expected, men had larger absolute values for FVC, FEV1.0, PEF and FEF25-75% but there were no sex differences in percent predicted values for any of these parameters.  Table 2 summarizes maximal exercise data for men and women on day 1.  Men had a higher absolute and relative       V˙  O2max and higher peak work-rates compared with women.  The larger minute ventilation in men was due exclusively to a higher tidal volume.  At peak exercise, women reported lower levels of perceived exertion for both breathlessness and leg discomfort.   Table 3.1:  Descriptive characteristics of the subjects                      Men Women          Age (years) 27 ± 1 28 ± 1  Height (cm) 182 ± 2 167 ± 2*  Mass (kg)  77 ± 2 62 ± 2*  BMI (kg·m2) 23.3 ± 0.6 22.3 ± 0.4  BSA (m2) 1.98 ± 0.03 1.69 ± 0.03*  FVC (l) 6.0 ± 0.2 4.2 ± 0.1*  FVC (% predicted) 110 ± 3 113 ± 2  FEV1.0 (l) 4.9 ± 0.1 3.7 ± 0.1*  FEV1.0 (% predicted) 107 ± 2 111 ± 2  FEV1.0/FVC (%) 81.8 ± 1.6 86.2 ± 1*  FEV1.0/FVC (% predicted) 99 ± 2 103 ± 1  PEF (l·sec-1) 11.3 ± 0.3 8.1 ± 0.3*  PEF (% predicted) 111 ± 2 112 ± 4  FEF25-75% (l·sec-1) 5.0 ± 0.3 4.4 ± 0.3*  FEF25-75% (% predicted) 98 ± 5 108 ± 7                  Values are mean ± S.E.M.  BMI, body mass index; BSA, body surface area; FVC, forced vital capacity; FEV1.0, forced expired volume in 1 second; PEF, peak expiratory flow; FEF25-75%, forced expiratory flow between 25 and 75% of FVC. * Significant difference between men and women.    72 Table 3.2:  Maximal incremental exercise data on day 1                     Men Women         V˙  O2 (ml·kg-1·min-1) 64.0 ± 1.9 57.1 ± 1.5* V˙  O2 (l·min-1) 4.9 ± 0.1 3.5 ± 0.1* V˙  CO2 (l·min-1) 5.4 ± 0.1 3.8 ± 0.1* RER  1.12 ± 0.01 1.10 ± 0.01 V˙  E (l·min-1) 154.7 ± 3.9 109.4 ± 3* Fb (breaths·min-1) 62.0 ± 2.4 62.7 ± 2.3 VT (l)  3.2 ± 0.1 2.2 ± 0.1* HR (bpm) 190 ± 2 185 ± 2 Work-rate (W)  364 ± 10 269 ± 9* Breathlessness (Borg scale) 9.1 ± 0.2 8.2 ± 0.4* Leg discomfort (Borg scale) 9.3 ± 0.2 8.5 ± 0.3*                         Values are mean ± S.E.M.  V˙  O2, oxygen consumption; V˙  CO2, carbon dioxide production; RER, respiratory exchange ratio; V˙  E, minute ventilation; Fb, breathing frequency; VT, tidal volume;  HR, heart rate.  * Significant difference between men and women.                73 Supramaximal Stimulation:  There was a proportional increase in Pdi,tw as the power output of the stimulator increased to 85-90% and then began reaching a plateau thereafter in men and women (Figure 3.2).  Clear evidence of a plateau was observed in all of the female subjects and in 11 of the male subjects. Stimulation Intensity (%max)65 70 75 80 85 90 95 100P di,tw (%max)020406080100120MenWomen* ***** **50 6 0 Figure 3.2: Trans-diaphragmatic pressure response to increasing cervical magnetic stimulation intensities in men and women.  Values are mean ± S.E.M.  * Significantly different from 100% stimulation intensity.  Diaphragm Fatigue:  Diaphragm fatigue was present in 11 out of 19 males (58%) and 8 out of 19 females (42%).  Figure 3.3A shows the reduction in potentiated Pdi,tw in all men and women.  The percent drop in Pdi,tw at 10, 30 and 60 min following exercise in men (n = 19) was 20.4 ± 3.3, 12.0 ± 3.1 and 8.2 ± 3.2%, respectively.  The drop in Pdi,tw in women (n = 19) at the same time points was 10.0 ± 2.3, 6.7 ± 2.2 and 4.5 ± 2.6% respectively.  Figure 3.3B shows the reduction in  74 potentiated Pdi,tw in those subjects who developed diaphragm fatigue as defined by a ≥15% reduction in Pdi,tw  [for individual responses see APPENDIX IV].  The percent drop in Pdi,tw at 10, 30 and 60 min following exercise in these men were 30.6 ± 2.3, 20.7 ± 3.2 and 13.3 ± 4.5%, respectively.  The drop in Pdi,tw in the women that developed fatigue at the same time points was 21.0 ± 2.1, 11.6 ± 2.9 and 9.7 ± 4.2% respectively.  Men consistently had greater reductions in Pdi,tw relative to women with the largest differences seen at 10 min post-exercise.  The data presented in Figure 3.3 points to a female diaphragm that has greater resistance to exercise-induced diaphragmatic fatigue.  Peak-to-peak M-wave amplitude for the right and left side of the diaphragm at 10, 30 and 60 min post-exercise were not different relative to baseline values in men or women (see also Figure 3.4).  Mean coefficient of variation in Pdi,tw  at baseline and 10, 30 and 60 min post-exercise was 5.5, 5.9, 5.5 and 5.5% in men (P > 0.05) and 6.7, 5.8, 5.9 and 6.5% in women, respectively (P > 0.05) with no differences between sexes.  There were no significant sex differences for diaphragmatic contraction time or half-relaxation time at any time point following exercise.             75  Recovery Time (min)0 10 20 30 40 50 60 70Pdi,tw (% Baseline)65707580859095100105Men (n = 11) Women (n = 8) Recovery Time (min)0 10 20 30 40 50 60 70Pdi,tw (% Baseline)65707580859095100105Men (n = 19)Women (n = 19)AB††*BB Figure 3.3:  Response of twitch trans-diaphragmatic pressure (Pdi,tw) during recovery in men and women.  Values are mean ± S.E.M.  Top graph shows all subjects.  Bottom graph shows only those subjects that developed diaphragm fatigue (defined as a drop in Pdi,tw of  ≥ 15%.  B, baseline.  * Significant difference between men and women (P < 0.01). † (P < 0.05)    76             Figure 3.4:  Example M-wave in an individual female subject at baseline and 10 min after exercise.   Time to Exhaustion Test:  The work-rate for the time to exhaustion (TTE) test in men and women was 327 ± 9 W and 242 ± 8 W, respectively which corresponded to 90% of their peak work-rate as determined on day 1.  Men cycled for 13.7 ± 0.9 min (range: 9.3-22.9 min) whereas women cycled for 11.4 ± 0.7 min (range: 7.8-18.0 min) (P = 0.051).  For any given time (expressed as a percentage of TTE), women consistently cycled at a higher percentage of their maximum HR.  During the latter half of the test, men averaged 95% and women averaged 97% of their maximum HR (P < 0.01).    Ratings of Perceived Exertion:  Figure 3.5 shows the ratings of perceived leg discomfort and breathlessness for men and women during the TTE test.  There were no significant differences in Time (ms)0 20 40 60 80mV-1.5-1.0-0.50.00.51.01.5Pre-exercise10 min Post-exerciseStimulus ArtifactM-wave 77 perceived exertion, despite women reporting lower levels of breathlessness and leg discomfort at maximal exercise during the incremental test on day 1 (Table 3.2).  A BTTE (%max)0 20 40 60 80 100Leg Discomfort (Borg)012345678910MenWomenTTE (%max)0 20 40 60 80 100Breathlessness (Borg)012345678910"Severe""Very Severe""Very Very Severe""Moderate" Figure 3.5:  Perception of leg discomfort and breathlessness in men and women.  Values are mean ± S.E.M.  TTE, time to exhaustion.  No differences in leg discomfort or breathlessness were detected at any time point between men and women.   Ventilation and Breathing Mechanics During Exercise:  Breathing frequency, tidal volume, minute ventilation, minute ventilation expressed as a percentage of maximum minute ventilation (as determined on day 1) and the work of breathing are shown in Figure 3.6.  Women relied on a higher breathing frequency during the first 60% of the exercise test with no sex differences over the last 40% of the test (Figure 3.6A).  Men consistently had higher tidal volumes (Figure 3.6B) and ventilations (Figure 3.6C).  Women utilized a larger fraction of their maximum ventilation   (V˙  E/V˙  Emax) during the first 40% of the exercise test but utilized a lower fraction at the end of the exercise test relative to men (Figure 3.6D).  The V˙  E/V˙  Emax ratio at the end of the exercise test (i.e., 100%TTE) was significantly correlated with the magnitude of diaphragm fatigue in all subjects (r = 0.52, P < 0.001).  Thus, those utilizing the largest fraction of their exercise ventilatory capacity at the end of the exercise test tended to demonstrate the greatest  78 diaphragmatic fatigue.  The mechanical work of breathing was consistently higher in men throughout the entire exercise test and rose disproportionally compared to women with increasing time (Figure 3.6E).  The absolute- and mass-corrected pressure-time-products of the diaphragm and esophagus are shown in Figure 3.7.  Men consistently had higher absolute values for PTPdi (Figure 3.7A).  Women have a relative plateau in PTPdi whereas men continue to increase PTPdi towards exhaustion.  Taking mass into account normalizes the PTPdi response such that there is no significant sex difference across time (Figure 3.7B).  There were no differences in absolute PTPes at any time point until exhaustion where PTPes rose disproportionally in men relative to women (Figure 3.7C).  However, this lack of difference in PTPes throughout the majority of the test was reversed when body mass was accounted for (Figure 3.7D).  Mass corrected PTPes was, on average, approximately 15% higher in women across the entire TTE test.  The relative contribution of the diaphragm to total inspiratory muscle force output (PTPdi/PTPes) was higher in men at the start of the cycle test with the data converging upon reaching exhaustion (Figure 3.7E).  However, over time, men continued to have a reduced contribution of the diaphragm to total inspiratory force output whereas diaphragmatic contribution in women changed very little over time.  There was a 12% reduction in PTPdi/PTPes from 20%TTE to 100%TTE in men and only a 3% reduction in women.  To further characterize this response, mean slopes were calculated across the entire duration of exercise for all subjects (Figure 3.7F).  The mean slope for men was significantly higher than for women (P < 0.05), suggesting a progressive decrease in diaphragmatic pressure contribution during exercise and an increase in accessory muscle recruitment in order to generate the increasing levels of ventilation shown in Figure 3.6C.  Regression analysis on the mean values relating PTPdi/PTPes to TTE (%max) showed a significant association for men (r2 = 0.89; P <  0.001) but not for women (r2 = 0.37;  P > 0.05).      79 TTE (% max)0 20 40 60 80 100VE / VEmax (%)0708090100110TTE (% max)0 20 40 60 80 100Fb (breaths—min-1 )03040506070MenWomen—TTE (% max)0 20 40 60 80 100VE (l—min-1)075100125150175TTE (% max)0 20 40 60 80 100VT (l)0.01.52.02.53.03.5**********†**CBAD——**†TTE (% max)0 20 40 60 80 100Work of Breathing (J—min-1 )0150300450600750*****E Figure 3.6:  Ventilatory and work of breathing response to exercise in men and women.  Values are mean ± S.E.M.  TTE, time to exhaustion; Fb, breathing frequency; VT , tidal volume; V˙  E, minute ventilation, V˙  Emax, maximal minute ventilation from the incremental cycle test to exhaustion (day 1). * Significant difference between men and women (P < 0.01).  † (P < 0.05).     80 A BC DTTE (% max)0 20 40 60 80 100PTPdi (cmH 2O—s—min-1 )0300400500600MenWomenTTE (% max)0 20 40 60 80 100PTPes (cmH 2O—s—min-1 )   0400500600700800TTE (% max)0 20 40 60 80 100PTPdi (cmH 2O—s—min-1 —kg-1 )06810TTE (% max)0 20 40 60 80 100PTPes (cmH 2O—s—min-1 —kg-1 )0681012†††††††**ETTE (% max)0 20 40 60 80 100PTPdi /  PTPes0.000.750.800.850.900.95 †‡TTE (% max)0 20 40 60 80 100PTPdi /  PTPes0.000.750.800.850.900.95F Figure 3.7:  Diaphragmatic and esophageal pressure-time product response to exercise in men and women.  Values are mean ± S.E.M.  TTE, time to exhaustion; PTPdi, trans-diaphragmatic pressure-time product; PTPes, esophageal pressure-time product.  * Significant difference between men and women (P < 0.01).  † (P < 0.05), ‡ (P = 0.05).     81 DISCUSSIOglyph1197  The purpose of this study was to determine if there are sex differences in the magnitude of exercise-induced diaphragm fatigue in healthy trained men and women.  Our findings do not support our original hypothesis that women would develop more diaphragm fatigue than men.  We have shown that fewer women developed diaphragmatic fatigue and that the magnitude of fatigue was significantly greater in men.  To our knowledge, this is the first study to measure Pdi during exercise in a large group of women and the first to assess diaphragm fatigue in women using phrenic nerve stimulation.      Diaphragm Fatigue:  The reduction in Pdi,tw 10 min following exercise and the pattern of recovery at 30 and 60 min in our male subjects is consistent with previous studies (Babcock et al., 1995; Babcock et al., 1996; Vogiatzis et al., 2007; Vogiatzis et al., 2008).  The time-course of Pdi,tw in response to phrenic nerve stimulation was nearly identical between men and women (Figure 3.3).  That is, the greatest reductions in Pdi,tw were seen 10 min following exercise with Pdi,tw approaching baseline levels 60 min following exercise.  However, the percent drop in Pdi,tw was lower in women, particularly at 10 and 30 min following exercise suggesting greater fatigue resistance in women.  The literature concerning the prevalence of exercise induced diaphragmatic fatigue is controversial due to the wide range of methodologies used, the large variation in subject characteristics and the relatively small sample sizes used.  Thus, it is difficult to determine the prevalence of exercise induced diaphragm fatigue.  In the present study, we show that the fatigue response of the diaphragm is variable with only about half of the subjects showing evidence of diaphragm fatigue (as defined by a ≥ 15% reduction in Pdi,tw).  It is difficult to determine why some subjects in a relatively homogenous population develop diaphragm fatigue while others do not.  Nevertheless, the present study suggests that the magnitude of  82 fatigue appears to be greater in men.  Potential mechanisms for this apparent fatigue resistance in the female diaphragm are discussed below.   Fatigue Resistance Mechanisms:  The men and women in this study were exercising at the same relative intensity (90% of peak work-rate) but the absolute work-rate was higher in men.  Therefore it would be expected that the absolute load on the male respiratory system would be higher to accommodate their higher levels of absolute ventilation.  Indeed, the total work of breathing was higher in men across the entire duration of the constant load cycling test (Figure 3.6D) which is an observation that is consistent with our recent data (Guenette et al., 2009) showing a higher total work of breathing in men compared to women at the same relative levels of minute ventilation.  Women generally have less muscle mass compared to men and this has been proposed as one of the key contributors to explain the greater fatigue resistance found in women (Hicks et al., 2001).  Lower muscle mass translates directly into lower absolute force generation in females when exercising at the same relative intensity as males.  This lower absolute force production in women means there will be a decreased O2 demand, a decrease in mechanical compression of the local vasculature and less intramuscular occlusion of blood flow.  The question relevant to the present study is whether or not we observed greater levels of diaphragmatic fatigue in men because they had higher absolute diaphragmatic force production during exercise.  Thus our proposed sex difference may simply be due to contractile conditions eliciting a greater degree of imbalance between muscle O2 supply and demand in men.  While we cannot completely rule out this possibility, we do not believe that this mechanism alone can fully explain our findings.  First, the absolute PTPdi in men was only modestly higher than women (~14%) during exercise.  Second, when we corrected the PTPdi for body mass in an attempt to normalize for size differences, we found that men and women were actually well matched for PTPdi (Figure 3.7B).  Third, we were able to match men and women for maximal  83 voluntary contraction of the diaphragm by removing some males with the highest maximal Pdi values (data not shown) and we still show that men have greater levels of fatigue 10 min following exercise.  Finally, Fulco et al. (1999) compared muscle performance in men and women at the same absolute force development and matched subjects for maximal voluntary contraction of the adductor pollicis muscle.  Despite matching for maximal muscle strength, these authors still found that females exhibit less fatigue than males.  Other potential mechanism to explain our findings include differences in muscular recruitment, muscle morphology and substrate utilization (see below).  Diaphragmatic Force Production and Respiratory Muscle Recruitment:  A close examination of Figures 3.6 and 3.7 point to some important sex differences in the ventilatory response to exercise that may explain why men develop greater levels of diaphragmatic fatigue than women.  These potential differences stem, in large part from the response of minute ventilation as shown in Figure 3.6C.  Men have a progressive and linear increase in minute ventilation with increasing time spent at the same absolute work-rate.  The male ventilatory response to this constant load cycling test is nearly identical to what has been previously reported by Johnson et al. (1993) in fit males exercising at 95% of their V˙  O2max to exhaustion.  The percent increase in minute ventilation from the early stages of exercise (20%TTE) to exhaustion (100%TTE) in our male subjects was 46%.  This contrasts sharply to the modest increase in minute ventilation of 21% in women from 20%TTE to 100%TTE.  In fact, the minute ventilation rises only 4% from 60%TTE to 100%TTE and is not different between 80 and 100%TTE.  Thus women have a plateau in minute ventilation despite performing the same muscular task and therefore use a smaller fraction of their maximal exercise ventilatory capacity at 100%TTE (Figure 3.6D) compared with men.  In fact, the V˙  E/V˙  Emax ratio at 100%TTE was significantly, albeit modestly related to the magnitude of diaphragmatic fatigue.  Thus the higher ventilatory requirements in men during  84 exercise might make the diaphragm more susceptible to fatigue and compromise its role as the primary inspiratory muscle.  Why do men increase ventilation linearly over time whereas women do not?  There are several possible explanations for this.  Firstly, the women may have been mechanically constrained due to the potential presence of expiratory flow limitation.  While expiratory flow limitation was not assessed in the present study, we (Guenette et al., 2007) and others (McClaran et al., 1998) have shown that women may be particularly susceptible to mechanical ventilatory constraints which may be related to their smaller lungs and airways (Sheel & Guenette, 2008).  However, this is unlikely to fully explain our findings since women appeared to have a slightly larger ventilatory reserve at 100%TTE compared with men.  Alternatively, it is possible that the blunted ventilatory response in women during exercise was due to a reduced chemical drive to breathe.  We have previously shown that chemosensitivity is not different between healthy men and women (Guenette et al., 2004) under resting conditions but it is currently unknown if there are sex differences in chemosensitivity during exercise.  Finally, it is entirely possible that the female ventilatory response was perfectly adequate for gas exchange and therefore did not need to increase over time.  Unfortunately, we do not have the required data to address this hypothesis.    What effect does this difference in the ventilatory response have on respiratory mechanics and muscle recruitment?  Figure 3.7A demonstrates a continual rise in PTPdi across time for men whereas women plateau between 80 and 100%TTE.  Figure 3.7E demonstrates the relative contribution of the diaphragm to total inspiratory muscle force output and Figure 3.7F shows the slopes of these relationships for men and women.  In relative terms, men rely less on the diaphragm over time suggesting an increased recruitment of accessory inspiratory muscles in order to enable them to continue increasing minute ventilation.  Women on the other hand show very little change in diaphragmatic contribution to total inspiratory force output shown by the  85 slope in Figure 3.7F.  This is likely a function of their reduced ventilatory requirement.  This change in recruitment pattern in men may be a physiological response to the onset of diaphragm fatigue which persists well into recovery.    The potential role that PTPdi and respiratory muscle recruitment has on diaphragmatic fatigue remains difficult to determine.  While these potential sex differences in breathing mechanics are an attractive hypothesis to explain our findings, there are some important factors that must be considered.  For example, diaphragmatic work is partially related to exercise-induced diaphragmatic fatigue because fatigue was prevented when diaphragmatic work was reduced during exercise using a mechanical ventilator (Babcock et al., 2002).  However, diaphragmatic work alone cannot fully explain diaphragm fatigue during exercise because Babcock et al. (1995) found that fatigue did not occur when resting subjects mimicked the magnitude and duration of diaphragmatic work incurred during exercise.  Fatigue only occurred when pressures developed by the diaphragm were voluntarily increased to levels that were twice as high as those required during whole-body exercise.  The lower fatigue threshold for diaphragmatic force production during exercise compared with rest suggests an important contribution from mechanisms directly related to the whole-body exercise itself.  For example, recent work by Vogiatzis et al. (2009) suggests that intercostal muscle blood flow increases linearly with the work of breathing during voluntary hyperpnea but decreases at the same work of breathing during whole body exercise at intensities above 80% of maximal work-rate.  Thus the circulatory system is unable to meet the demands of both locomotor and respiratory (intercostal) muscles during heavy exercise, which likely contributes to respiratory muscle fatigue.  While we have shown differences in diaphragmatic force production between sexes and differences in respiratory muscle recruitment, we recognize that there are other crucial factors such as blood flow competition that may explain our findings.  Recent work has utilized near  86 infrared spectroscopy and a light absorbing tracer (indocyanine green) to measure respiratory muscle blood flow during voluntary hyperpnea and exercise (Guenette et al., 2008; Vogiatzis et al., 2008; Vogiatzis et al., 2009) but no such measurements have been made in women.  Future work in this area is required to help explain the mechanisms underlying the greater fatigue resistance in women.     Muscle Morphology and Substrate Utilization:  Some evidence suggests that there are sex differences in muscle fibre type composition such that women have more slow twitch oxidative fibres (Miller et al., 1993).  Slow twitch oxidative fibres fatigue at slower rates compared to fast twitch glycolytic fibres (Hamada et al., 2003).  These potential sex differences in muscle fibre type composition may explain, in part, why female muscles are more fatigue resistant than male muscles (Hicks et al., 2001).  In the present investigation, we are interested in the primary muscle of inspiration.  The human diaphragm is composed of 76% high-oxidative fibres (55% slow twitch and 21% fast twitch) and 24% low-oxidative fast twitch fibres (Lieberman et al., 1973).  However, we are unaware of any studies that have looked specifically at sex differences in diaphragmatic fibre type composition in healthy humans, particularly in endurance-trained individuals.  There are also sex differences in substrate utilitzation during exercise which may also contribute to potential sex differences in muscle fatigue.  It has been established that males have a higher glycolytic capacity and a greater reliance on glycolytic pathways than females with muscle biopsy studies revealing that women have lower activities of common glycolytic enzymes, which in turn would translate into a decreased potential for anaerobic glycolysis (Tarnopolsky, 1999).  As pointed out by Hicks et al. (2001), these differences may mean that women have a greater reliance on β-oxidation of fatty acids, thus prolonging endurance during certain types of exercise and perhaps improving their ability to resist fatigue.     87 Consequences of Diaphragm Fatigue:  There are numerous studies pointing to a female respiratory system that may be more susceptible to specific pulmonary limitations such as expiratory flow limitation (McClaran et al., 1998; Guenette et al., 2007) and exercise induced arterial hypoxaemia (Harms et al., 1998; Richards et al., 2004; Guenette & Sheel, 2007).  However, the data from the present study points to a female respiratory system that has a distinct advantage over their male counterparts.  That is, women are more resistant to exercise-induced diaphragmatic fatigue.  There are several important physiological and performance based consequences of diaphragmatic fatigue which have recently been reviewed by Romer and Polkey (2008).  We will briefly discuss some of these consequences in the context of the present data.    The greater reliance on accessory inspiratory muscles in men with progressive exercise may result in chest wall distortion (Goldman et al., 1976; Grimby et al., 1976) and reduce the mechanical efficiency of breathing (Hart et al., 2002).  The reliance and recruitment of accessory inspiratory muscles may lead to an increase in sensory input to the central nervous system resulting in an increased sensation of breathlessness (Romer & Polkey, 2008).  However, despite the greater levels of fatigue in men, there appeared to be no sex differences in ratings of perceived exertion for either breathing or leg discomfort.  While respiratory muscle fatigue may increase the sensation of breathlessness (Gandevia et al., 1981; Supinski et al., 1987; Ward et al., 1988), it is likely that this effect is specific to the accessory muscles because diaphragm fatigue does not increase neural respiratory drive as assessed by esophageal diaphragm EMG (Luo et al., 2001).   Another potential consequence related to diaphragm fatigue is a sympathetically mediated metaboreflex that originates from fatiguing inspiratory muscles.  Fatigue inducing inspiratory contractions result in a time-dependent increase in muscle sympathetic nerve activity  88 (St Croix et al., 2000) and a reduction in arterial blood flow to the resting limb (Sheel et al., 2001).  This metaboreflex response may also become active during whole body endurance exercise.  Harms et al. (1997) found that reducing the inspiratory work of breathing using a mechanical ventilator causes vascular conductance and blood flow in the exercising limb to increase.  Thus the sympathetically mediated vasoconstriction of locomotor limb muscle vasculature may lead to an exacerbation of peripheral fatigue, increase effort perceptions and ultimately limit exercise performance (Dempsey et al., 2002).  It is possible that men have a more pronounced inspiratory muscle metaboreflex response relative to women given that they tend to exhibit greater levels of diaphragm fatigue.  A sex-based comparison of the inspiratory metaboreflex is required to fully address this hypothesis.  Methodological Considerations:  The CMS technique is considered to be as effective as electrical stimulation for detecting diaphragmatic fatigue (Laghi et al., 1996).  Moreover, it has been shown to be reproducible, safe and painless (Similowski et al., 1989).  We chose the CMS technique over electrical stimulation for two main reasons.  First, magnetic stimulation does not activate cutaneous pain receptors and is therefore less painful for subjects than electrical stimulation.  Second, CMS is easier than electrical stimulation in terms of finding the optimal stimulation site, which is important for consistency and reproducibility between pre- and post-stimulations.  However, the technique does have certain limitations that require attention.  It is more difficult to demonstrate supramaximal stimulation with CMS compared with electrical stimulation.  Supramaximal stimulation can be confirmed by showing a plateau in Pdi,tw with increasing power output of the stimulator.  Figure 3.2 shows, that on average, men and women demonstrated a levelling off in Pdi,tw at 90-95% of stimulator output.  However, fewer men reached a plateau in Pdi,tw compared with women, likely because the men were generally taller with thicker necks.  However, we do not believe that submaximal stimulation in some of our  89 men would influence our primary finding of greater fatigue resistance in women for several reasons.  First, Verges et al. (2006) found that only 4 out of 11 subjects showed evidence of a plateau when comparing Pdi,tw at 94% of the stimulator output to 100%;  however, a plateau was seen in 8 out of 11 subjects when comparing Pdi,tw at 98% of the stimulator output to 100%.  We made our measurements at larger increments on the stimulator output than Verges et al. (2006).  Our more conservative approach probably led to an underestimation of the number of subjects showing a plateau in Pdi,tw, and thus suggesting that stimulations may have been submaximal.  Had we performed our stimulations at 98% of the stimulator power output, we believe we would have demonstrated a plateau in most of our male subjects.  Second, M-wave amplitude was unchanged following exercise in both men and women, suggesting that the reduction in Pdi,tw was not due to decrecruitment of muscle fibres or to transmission failure.  Third, the stimulator output was set at 100% of maximal power output for all stimulations and we paid particular attention to ensure that the subjects were in the same position before and after exercise.  In addition to visual inspection of the subject while seated, we also marked the coil position on the back of the neck enabling us to reposition the coil in the exact location for all stimulations.  The consistency of our stimulations before and after exercise can be seen by the lack of change in the coefficient of variation in Pdi,tw.  Fourth, any twitches that were not initiated from resting EELV were discarded from analysis.  Therefore, while supramaximal stimulations were not obtained in all men, we are confident that our stimulations were constant and that this limitation would not influence our primary finding regarding sex differences in diaphragmatic fatigue.  Finally, when the male subjects that did not reach supramaximal stimulation were removed from the analysis, we found that the magnitude of fatigue was still greater in males compared with females (data not shown).     90 Conclusions:  This study is the first to measure diaphragm function during exercise in healthy trained women and the first to use objective measures to investigate diaphragm fatigue in a large group of women.  The data from the present study points to a female diaphragm that is more resistant to exercise-induced fatigue compared with men.  We have also shown sex differences in the ventilatory response to high-intensity constant load cycling exercise that may be related, in part, to the greater fatigue resistance observed in women.                       91 REFEREglyph1197CES  ATS/ERS (2002). Statement on respiratory muscle testing. Am J Respir Crit Care Med 166, 518-624.  Babcock MA, Pegelow DF, Harms CA & Dempsey JA. (2002). 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This is particularly the case when dealing with the respiratory response to exercise.  Studying sex differences in respiratory exercise physiology is a discipline that is still in its infancy and before we can understand sex differences in clinical populations, it is important to first characterize the “normal” responses to exercise in healthy women.  The work in this thesis, coupled with previous work from our laboratory (Guenette et al., 2004; Richards et al., 2004; Guenette et al., 2007a; Guenette et al., 2007b) and others (Harms et al., 1998; McClaran et al., 1998; Harms et al., 2000; Olfert et al., 2004) provides an important first step in characterizing the normal respiratory response to exercise in healthy women.  This thesis provides unique insight into the mechanisms associated with a higher mechanical work of breathing (WOB) in women.  Specifically, we have shown that women have a higher WOB for a given absolute ventilation due to a higher inspiratory and expiratory resistive WOB rather than differences in the elastic WOB.  This is presumably due to the inherently smaller diameter airways in women (Sheel et al., 2009).  This study and others (McClaran et al., 1998; Guenette et al., 2007b) point to a female diaphragm that may be at a mechanical disadvantage relative to their male counterparts.    The findings of greater expiratory flow limitation, higher operational lung volumes and a higher WOB in women during exercise led to the subsequent hypothesis that women would be more susceptible to diaphragmatic fatigue.  This thesis presents the first study to systematically measure diaphragmatic function during exercise and its ensuing fatigue in exercising women.  Contrary to our original hypothesis, the female diaphragm was actually more resistant to fatigue than the male diaphragm.  We have also demonstrated clear sex differences in the ventilatory  97 response to constant load cycling which directly influenced diaphragmatic recruitment during exercise.  These sex differences may explain, in part, why women were able to preserve the diaphragm’s force generating ability, despite having higher body mass-corrected esophageal pressure-time products during exercise.  Not only was the relative load on the respiratory muscles greater in women, but they were also exercising at a higher percentage of their maximum heart rate, even though they were all exercising at the same relative external work-rate.  This makes the finding of greater diaphragmatic fatigue resistance in women even more impressive.  Is this an evolutionary or physiological adaptation to a respiratory system that may be at a mechanical disadvantage (i.e., smaller lungs, smaller airways, etc.) during strenuous exercise?  Perhaps the diaphragm is simply behaving like any other skeletal muscle that shows greater fatigue resistance in women.  The answers to these questions remain unknown.  Significance:  Recent review’s by Hicks et al. (2001) and Hunter (2009) provide a comprehensive list of studies that support the idea that women have greater skeletal muscle fatigue resistance relative to males.  The majority of these studies have focussed on muscles involved in moving the elbow, finger, knee, thumb, ankle, back and neck.  These studies have provided important insight into sex differences in skeletal muscle fatigue.  The data presented in this thesis adds to the existing literature by studying the single most important and complex skeletal muscle in the human body; the diaphragm.  The diaphragm is the primary muscle involved in breathing and is required to contract for the entire duration of one’s life.  As such, its ability to resist fatigue may have important clinical implications, particularly in the intensive care unit where individuals are undergoing mechanical ventilation and where weaning success depends on the ability of the diaphragm to maintain a given force production.   98 This research is also significant because it is the first to comprehensively assess respiratory mechanics and diaphragmatic function during an actual bout of high-intensity exercise.  Despite performing the same external muscular work (i.e., 90% of peak work-rate), women had a very different ventilatory response to exercise with different respiratory muscle recruitment patterns.  This may give some initial insight into why women have greater diaphragmatic fatigue resistance relative to men.  Pulmonary Limitations in Women:  Three of the primary pulmonary limitations that occur in healthy athletes during exercise include: exercise induced arterial hypoxaemia, expiratory flow limitation and diaphragmatic fatigue (Dempsey et al., 2008).  Studies have shown that women may be more susceptible to exercise-induced arterial hypoxaemia (Harms et al., 1998; Richards et al., 2004) and that it occurs in relatively untrained women, a phenomenon that is not typically observed in men (Harms et al., 1998; Richards et al., 2004).  Furthermore, there is evidence to suggest that expiratory flow limitation may also be more common in women (McClaran et al., 1998; Guenette et al., 2007b).  These studies have led some to conclude that women are more susceptible to pulmonary system limitations during exercise relative to men, which may be related, in part, to their smaller lungs and airways and their reduced surface areas for diffusion.  However, the present study suggests that diaphragmatic fatigue is not a pulmonary system limitation that is more common in women.  The force preserving ability of the female diaphragm may be a physiological adaptation to counteract the potential deleterious effects of hypoxaemia and expiratory flow limitation on diaphragm fatigue and or exercise performance.    Strengths and Limitations:  The primary strengths of the studies presented in this thesis relate to study design and the methodological approaches to answering our research questions.  Accurately quantifying respiratory mechanics and diaphragmatic fatigue often requires highly  99 invasive and uncomfortable procedures for the research participants.  All of our primary outcomes were measured using the most objective and validated methods.  An additional strength to this research, particularly the diaphragm fatigue study (Chapter 3) is the large sample size used.  To our knowledge, 38 subjects is one of the largest sample sizes used for any respiratory mechanics or diaphragm fatigue study in the current literature.  This gave us excellent statistical power and allowed us to partition our data to look at responses to various sub-groups (i.e., those that did and those that did not develop diaphragm fatigue).    The studies comprising this thesis have certain limitations beyond those discussed in detail within chapters 2 and 3.  Specifically, we made our measurements on very well-trained endurance athletes with a high aerobic capacity.  We used athletes because they are able to stress their cardio-respiratory system well-beyond their untrained or diseased counterparts.  Thus, athletes provide an excellent model to study the limitations of the human respiratory system during exercise.  However, endurance athletes represent a small fraction of the general population and thus the generalizability of our findings are limited to the population from which we derived our data.  How our findings fit to other populations such as healthy ageing, obesity and those with chronic diseases remains unknown.  Another limitation to this research is that we did not assess the functional consequences or the non-mechanical mechanisms associated with respiratory muscle fatigue (e.g., perfusion).  These ideas are discussed below in “future research.”   Future Research:  There are several unanswered questions related to this thesis that will require unique experimental designs and methodologies.  For example, what are the functional consequences of expiratory flow limitation, diaphragm fatigue and a high WOB?  Unloading the respiratory muscles using proportional assist ventilation or breathing low density gas mixtures  100 such as heliox may provide unique insight into the exercise performance-based implications of these pulmonary limitations during exercise in men and women.    Perfusion to a muscle is also an important factor involved in the fatigue process.  Are the respiratory muscles in women receiving adequate perfusion to avoid fatigue?  Perhaps there are sex differences in respiratory muscle blood flow regulation which may provide some mechanistic data to support the finding of greater fatigue resistance in women.  Are there sex differences in blood flow competition between respiratory and locomotor muscles for a limited cardiac output during exercise?  All of these unique questions have not been tested because a method for measuring respiratory muscle blood flow in conscious humans was not available.  However, we recently developed a method to measure blood flow to superficial respiratory muscles using near-infrared spectroscopy and a light absorbing tracer (Guenette et al., 2008).  This innovative method may provide insight into some of the underlying mechanism associated with pulmonary system limitations during exercise in humans.  As highlighted above, the data from the present thesis can only be generalized to healthy humans with a relatively high aerobic capacity.  Therefore, future research is required to expand on these findings to different populations such as chronic obstructive pulmonary disease, obesity, chronic heart failure and healthy ageing, among others.  For example, population studies in patients with cardiopulmonary diseases have shown that women experience greater levels of respiratory discomfort, greater exercise intolerance and poor perceived health status relative to men when matched for disease severity (Watson et al., 2004; de Torres et al., 2005; Han et al., 2007).  Recent work also suggests that women experience greater levels of dyspnea with advancing age (Ofir et al., 2008).  These findings point to the importance of studying sex-based physiological differences since biological sex can play a direct role in disease.  Finally, future  101 research is required to determine how greater fatigue resistance in women influences athletic performance.  Perhaps the findings of the present study may explain, in part, why women tend to outperform men in ultra-endurance events (Speechly et al., 1996).  Overall Conclusion:  The purpose of this thesis was to provide a comprehensive assessment of respiratory mechanics and diaphragm fatigue in men and women during exercise.  We found that women have a higher WOB for a given level of ventilation due to their smaller diameter airways which act to increase the inspiratory and expiratory resistive WOB.  We also observed that that the female diaphragm is more resistant to fatigue following high intensity exercise, which may be related, in part, to sex differences in respiratory muscle recruitment and differences in the ventilatory response to exercise.  Future work is required to expand on these observations to different patient populations.               102 REFEREglyph1197CES de Torres JP, Casanova C, Hernandez C, Abreu J, Aguirre-Jaime A & Celli BR. (2005). Gender and COPD in patients attending a pulmonary clinic. Chest 128, 2012-2016.  Dempsey JA, McKenzie DC, Haverkamp HC & Eldridge MW. (2008). Update in the understanding of respiratory limitations to exercise performance in fit, active adults. Chest 134, 613-622.  Guenette JA, Diep TT, Koehle MS, Foster GE, Richards JC & Sheel AW. (2004). Acute hypoxic ventilatory response and exercise-induced arterial hypoxemia in men and women. Respir Physiol glyph817eurobiol 143, 37-48.  Guenette JA, Sporer BC, Macnutt MJ, Coxson HO, Sheel AW, Mayo JR & McKenzie DC. (2007a). Lung Density Is Not Altered Following Intense Normobaric Hypoxic Interval Training In Competitive Female Cyclists. J Appl Physiol.  Guenette JA, Vogiatzis I, Zakynthinos S, Athanasopoulos D, Koskolou M, Golemati S, Vasilopoulou M, Wagner HE, Roussos C, Wagner PD & Boushel R. (2008). Human respiratory muscle blood flow measured by near-infrared spectroscopy and indocyanine green. J Appl Physiol 104, 1202-1210.  Guenette JA, Witt JD, McKenzie DC, Road JD & Sheel AW. (2007b). Respiratory mechanics during exercise in endurance-trained men and women. J Physiol 581, 1309-1322.  Han MK, Postma D, Mannino DM, Giardino ND, Buist S, Curtis JL & Martinez FJ. (2007). Gender and chronic obstructive pulmonary disease: why it matters. Am J Respir Crit Care Med 176, 1179-1184.  Harms CA, McClaran SR, Nickele GA, Pegelow DF, Nelson WB & Dempsey JA. (1998). Exercise-induced arterial hypoxaemia in healthy young women. J Physiol 507 ( Pt 2), 619-628.  Harms CA, McClaran SR, Nickele GA, Pegelow DF, Nelson WB & Dempsey JA. (2000). Effect of exercise-induced arterial O2 desaturation on VO2max in women. Med Sci Sports Exerc 32, 1101-1108.  Hicks AL, Kent-Braun J & Ditor DS. (2001). Sex differences in human skeletal muscle fatigue. Exerc Sport Sci Rev 29, 109-112.  Hunter SK. (2009). Sex differences and mechanisms of task-specific muscle fatigue. Exerc Sport Sci Rev 37, 113-122.  McClaran SR, Harms CA, Pegelow DF & Dempsey JA. (1998). Smaller lungs in women affect exercise hyperpnea. J Appl Physiol 84, 1872-1881.  Ofir D, Laveneziana P, Webb KA, Lam YM & O'Donnell DE. (2008). Sex differences in the perceived intensity of breathlessness during exercise with advancing age. J Appl Physiol 104, 1583-1593.  103  Olfert IM, Balouch J, Kleinsasser A, Knapp A, Wagner H, Wagner PD & Hopkins SR. (2004). Does gender affect human pulmonary gas exchange during exercise? J Physiol 557, 529-541.  Richards JC, McKenzie DC, Warburton DE, Road JD & Sheel AW. (2004). Prevalence of exercise-induced arterial hypoxemia in healthy women. Med Sci Sports Exerc 36, 1514-1521.  Sheel AW, Guenette JA, Yuan R, Holy L, Mayo JR, McWilliams AM, Lam S & Coxson HO. (2009). Evidence for Dysanapsis Using Computed Tomographic Imaging of the Airways in Older Ex-Smokers. J Appl Physiol, In Press.  Speechly DP, Taylor SR & Rogers GG. (1996). Differences in ultra-endurance exercise in performance-matched male and female runners. Med Sci Sports Exerc 28, 359-365.  Watson L, Vestbo J, Postma DS, Decramer M, Rennard S, Kiri VA, Vermeire PA & Soriano JB. (2004). Gender differences in the management and experience of Chronic Obstructive Pulmonary Disease. Respir Med 98, 1207-1213.                              104                APPEglyph1197DIX I:  Informed consent forms                 105  T H E  U N I V E R S I T Y  O F  B R I T I S H  C O L U M B I A         Iglyph1197FORMED COglyph1197SEglyph1197T FORM  Title of Project: Ventilatory responsiveness and the work of breathing in men and     women with exercise induced arterial hypoxaemia.  Principal Investigator: William Sheel, Ph.D.  Co-Investigators:  Donald McKenzie, Ph.D., M.D.   Mike Koehle, M.D.   Jeremy Road, M.D.   Meaghan McNutt, Ph.D. candidate    Sara Jane Lusina, M.Sc. candidate   Jonathan Witt, M.Sc. candidate   Jordan Guenette, M.Sc. candidate   Institution:  School of Human Kinetics    The University of British Columbia   Contact Person: Jordan Guenette (office phone)  (604) 822-4384    24 hour emergency contact:   (604) 617-4644       Background: Some athletes develop low oxygen content in their blood during exercise (hypoxaemia) and investigators have suggested that this is due to insufficient breathing during exercise.  Some researchers have suggested that there is a gender difference, however, there is limited scientific information available to make accurate conclusions.  Purpose: The purpose of this study is to investigate how breathing changes in response to low oxygen content in the blood during rest and exercise and how breathing mechanics influences these responses.  Specifically, we are concerned with gender differences between elite male and female cyclists and triathletes.       School of Human Kinetics 210, War Memorial Gym 6081 University Boulevard Vancouver, B.C., Canada V6T 1Z1  Tel: (604) 822-3838  Fax: (604) 822-6842    106 Page 2 of 4                         version 4 (July 14, 2005)  Procedures:   You are being invited to participate in one data collection test day and your participation in the study is entirely voluntary.  The session will take place at the   Health and Integrative Physiology Laboratory at the Osborne Center (Unit 2, Room 202) on the University of British Columbia campus.  The study will require approximately two (2) hours of your time.  Before any measurements are taken, a physical activity readiness questionnaire (PAR-Q) will be administered.  The experiment is divided into three parts.  First, your height and weight will be measured.  You will then undergo a simple, non-invasive breathing test to ensure that you do not have any obstructive lung disease (i.e., asthma).  This requires you to breathe deeply and exhale quickly through a mouthpiece.  Another simple, non-invasive test of your wrist will be performed by a physician to ensure adequate circulation.  If your circulation is deemed inadequate, you will not be able to participate in the study.  If you are currently using anti-inflammatory medication or have a history of bruising easily or having blood clotting problems, you will be excluded from the study.         You will then be required to lie comfortably on a bed while listening to music and wearing a nose clip in which you will breathe through a two-way valve so that expired gases and flow can be monitored.  A small plastic clip will be attached to your ear.  This will permit us to measure the amount of oxygen in your blood.  After 10 minutes of breathing normal air, experimenters will slowly and progressively add nitrogen gas to the air you are breathing.  We will measure the amount that your breathing (rate and depth) increases in response to this.  This experiment will simulate high altitude exposure and will take approximately 15-20 minutes.  You will then perform a warm-up exercise session on a bicycle (5-10 minutes).  After you have warmed up and stretched you will then perform a maximal cycling test.  The test will progressively become more difficult and will last approximately 10-15 minutes until you are tired and must stop.  It will be necessary that you breathe through a mouthpiece while wearing a nose clip so that expired gases and flow can be monitored.    A thin and flexible tube (catheter) will be placed in the brachial or radial artery of your arm to allow the experimenters to take multiple blood samples throughout the test.  Approximately 10 samples will be taken throughout the test with each sample containing 3mL of blood for a total of 30mL.  To minimize discomfort, a local anesthetic will be applied.  In order to measure temperature in the body during exercise, a temperature measuring probe will be placed through your nose into the lower one-third of the esophagus.  The esophagus is the tube that connects your mouth to your stomach.  To continually measure the amount of oxygen in your blood, a small clip will be attached to your ear and finger.  Also, two additional thin flexible tubes will be inserted through nose into the esophagus and stomach so that breathing pressures can be obtained.  A local anesthetic will be applied in your throat to minimize discomfort.  If you have any diseases such as an ulcer in your esophagus or a tumor you will be excluded from the investigation.  You will also be excluded if you have had recent nasopharyngeal surgery.       107 Page 3 of 4                         version 4 (July 14, 2005) Risks:  Due to the unpredictable response of some individuals to exercise, unforeseen difficulties may arise which would necessitate treatment.  You are asked to report any unusual symptoms during the test.  You may stop the test when you wish to because of personal feelings of discomfort and tiredness.  Every effort will be made to conduct the tests in such a way as to minimize discomfort and risk.  However, there exists the possibility of   Potential risks from maximal exercise such as vomiting (5%), abnormal blood pressure (less than 1%), fainting (less than 1%), disorders of heartbeat (less than 0.1%), and very rare instances of heart attack (less than 0.001%).  If you experience any of these, you will receive immediate care from a physician (co-investigator Dr. McKenzie or Dr. Koehle) at no cost.  If you feel that you are experiencing any side effects as a result of any procedures you should immediately report this to the principal investigator.  You may also experience mild discomfort due to the catheter in your arm and the catheters in your oesophagus.    Potential risks associated with the catheter in your arm include bleeding (less than 1%), bruising (14%), and infection (less than 1%).  Other potential risks associated with an arterial line include artery aneurysms, blood clotting, brief tightening of a blood vessel, death of skin tissue over the catheter site, and line disconnection.   When collecting blood and measuring temperature, the utmost care will be taken to ensure your comfort.  Catheter placement in your arm will be performed by a trained physician.    There are no risks associated with decreased oxygen supply simulating mild high altitude exposure (approximately 10,000 feet).  Benefits:  As a result of your participation in this study, you will receive detailed fitness assessments that can be used to help you with your personal training program.  Confidentiality:  Your rights to privacy are protected by the Freedom of Information and Protection of Privacy Act of British Columbia.  This Act lays down rules for the collection, protection, and retention of your personal information by public bodies, such as the University of  British Columbia and its affiliated teaching hospitals.  Further details about this Act are available upon request.  Your confidentiality will be respected.  No information that discloses your identity will be released or published without your specific consent to the disclosure.  However, research records and medical records identifying you may be inspected in the presence of the Investigator or his or her designate by representatives of the UBC Research Ethics Board or Health Canada for the purpose of monitoring the research.  However, no records which identify you by name or initials will be allowed to leave the Investigators’ offices.  You are encouraged to ask for an explanation or         108 Page 4 of 4                          version 4 (July 14, 2005)  clarification of any of the procedures or other aspects of this study before signing this consent form or at any time during your participation in the study.    YOU MAY DECLINE TO ENTER THIS STUDY OR WITHDRAW FROM THE EXPERIMENT AT ANY TIME.  If you have any concerns or questions about your rights or experience as a research subject, you may contact the Research Subject Information Line in the UBC Office of Research Services at (604) 822-8598.  Consent:  In signing this form you are consenting to participate in this research project and acknowledge receipt of a copy of this form.  Signing this consent form in no way limits your legal rights against the sponsor, investigators, or anyone else.  ________________________________ ___________________________ Signature of Subject   Date  ________________________________  Printed Name of Subject  ________________________________ ___________________________ Signature of Witness   Date  ________________________________  Printed Name of Witness  ________________________________ ___________________________ Signature of Investigator  Date   ________________________________  Printed Name of Investigator                109 T H E  U N I V E R S I T Y  O F  B R I T I S H  C O L U M B I A         INFORMED CONSENT FORM  Title of Project:  Respiratory muscle fatigue in men and women  Principal Investigator: A. William Sheel, Ph.D.  Co-Investigators:  Jordan A. Guenette, B.H.K., M.Sc. PhD candidate   Donald McKenzie, Ph.D., M.D.   Jeremy Road, M.D.   Meaghan McNutt, Ph.D. candidate    Jordan Querido, Ph.D. candidate   Simone Tomczak, M.Sc. candidate      Institution:  School of Human Kinetics    The University of British Columbia   Contact Person: Jordan Guenette (office phone)  (604) 822-4384    24 hour emergency contact:   (604) 617-4644       Background: It is well known that women, on average, have smaller lungs and airways compared to men of similar stature.  There is recent evidence to suggest that these anatomical differences may predispose women to certain breathing limitations and that the energy cost of breathing may be higher in women compared to men.  The higher energy cost of breathing may cause the breathing muscles to become fatigued during exercise to a greater degree in women relative to men.  However, there are no research studies that have measured fatigue of the breathing muscles in women and compared their responses to men after exercise.  Purpose: The purpose of this study is to determine if the breathing muscles (diaphragm and abdominal muscles) are more prone to fatigue in women compared to men following high intensity cycling exercise.      School of Human Kinetics 210, War Memorial Gym 6081 University Boulevard Vancouver, B.C., Canada V6T 1Z1 Tel: (604) 822-3838  Fax: (604) 822-6842    110 Page 2 of 5                         version 3 (May 6, 2008)  Procedures: You are being invited to participate in two data collection testing days and your participation in the study is entirely voluntary.  Both testing days will take place at the Health and Integrative Physiology Laboratory at the Osborne Center (Unit 2, Room 202) on the University of British Columbia campus.  The study will require approximately six to seven (6-7) hours of your time.  Before any measurements are taken, a physical activity readiness questionnaire (PAR-Q) and medical history questionnaire will be administered.  Female participants will also be required to fill out an additional menstrual cycle history questionnaire.  You are not required to answer any questions that make you feel uncomfortable.    Day 1:  This testing day is divided into two parts.  First, your height and weight will be measured.  You will then undergo a simple, non-invasive breathing test to ensure that you do not have any obstructive lung disease (i.e., asthma).  This requires you to breathe deeply and exhale quickly through a mouthpiece.  You will also be required to perform some additional breathing exercises which consist of taking maximal inspirations (breathing in) and additional exercises which require maximal expirations (breathing out).   The second part of this day requires you to perform exercise on a stationary bicycle.  You will perform a warm-up exercise session on the bicycle (15 minutes).  After you have warmed up you will then perform a maximal cycling test.  The test will progressively become more difficult and will last approximately 10-20 minutes until you are tired and must stop.  It will be necessary that you breathe through a mouthpiece while wearing a nose clip so that expired gases and air flow can be monitored.  We will also place a clip on your ear and finger to measure the amount of oxygen in your blood.    The entire testing session will take approximately 2 hours.  Day 2:  A trained respiratory physiologist (Jordan A. Guenette) will insert two thin flexible tubes through your nose into your oesophagus and stomach.  The oesophagus is the tube that connects your mouth to your stomach.  This will allow us to measure breathing pressures.  A local anesthetic will be applied to your nose and throat to minimize discomfort.  If you have any diseases such as an ulcer in your oesophagus or a tumor you will be excluded from the investigation.  You will also be excluded if you have had recent nasopharyngeal surgery or if you have a nasal septum deviation.  We will then place sticky electrodes on your stomach and chest in order to monitor muscle activity of your breathing muscles.  Once the catheters and electrodes are secured and positioned correctly, you will then rest for 20 minutes.  After this rest period, we will place a circular coil at the back of your neck while you are seated comfortably in a chair.  This coil will be activated which uses a magnet to non-invasively stimulate the nerve that attaches to your diaphragm.  This will cause you to take a quick inspiration and it will feel like a hiccup.  We will also perform the same test but this time the coil will be placed in the          111 Page 3 of 5                         version 3 (May 6, 2008)  middle of your back while you lay face down on an incline bench.  You will be excluded if you have any metal inside of your body or if you have a cardiac pacemaker.  Upon completion of several stimulations, you will then be required to perform a cycling test at a constant workload until you can no longer continue.  The resistance on the bicycle will correspond to approximately 90% of the resistance you achieved on the first  day of testing.  You will then undergo the same stimulations 10, 30, 60 and 90 minutes after the exercise test.    The entire testing session will take approximately 4-5 hours.  Risks:  Due to the unpredictable response of some individuals to exercise, unforeseen difficulties may arise which would necessitate treatment.  You are asked to report immediately any unusual symptoms during the test.  You may stop the test when you wish to because of personal feelings of discomfort and tiredness.  Every effort will be made to conduct the tests in such a way as to minimize discomfort and risk.  Potential risks from maximal exercise include vomiting (5%), abnormal blood pressure (less than 1%), fainting (less than 1%), disorders of heartbeat (less than 0.1%), and very rare instances of heart attack (less than 0.001%).  If you experience any of these, you will receive immediate care from a physician (co-investigator Dr. McKenzie) at no cost.  If you feel that you are experiencing any side effects as a result of any procedures you should immediately report this to the principal investigator.    You may feel mild discomfort or soreness in the nostrils and upper airway during the placement of the tubes in your oesophagus and stomach. You may also experience slight discomfort as a result of “gagging” while swallowing the tubes and during the removal of the tubes. This will resolve once the tubes are in position. A numbing gel called Lidocaine will be used to minimize the discomfort. Adverse reactions to Lidocaine are extremely rare but include light-headedness, blurred/double vision, euphoria, confusion, dizziness, convulsions, sensations of heat, cold or numbness.  You will not be allowed to participate in the study if you are known to be sensitive to local anaesthetics or if you have allergies to latex. We are unaware of any laboratory that has experienced any of the aforementioned adverse reactions to such a small amount of lidocaine.  There is also a small risk that the catheters in your oesophagus may be placed in the wrong position.  In some extremely rare cases, the catheter can enter your trachea (wind pipe).  You may experience mild discomfort in the back of your throat and you may gag.  When this occurs the catheter will be pulled out and re-positioned.    Although extremely rare, you may feel nausea, headache, mild discomfort and annoyance with the magnetic stimulation procedure.  You may also experience muscular contractions, involuntary movements (such as the arms) and a mild tingling sensation in your arms.        112 Page 4 of 5                          version 3 (May 6, 2008)  Benefits:  As a result of your participation in this study, you will receive detailed fitness assessments that can be used to help you with your personal training program.  You will receive an explanation of your test results and recommendations will be given on how this information can be used to assist you in your training.    Remuneration:  You will receive a $100 honorarium for your participation in this study.  The honorarium will be based on your participation on the second day of testing.  Confidentiality:  Your rights to privacy are protected by the Freedom of Information and Protection of Privacy Act of British Columbia.  This Act lays down rules for the collection, protection, and retention of your personal information by public bodies, such as the University of  British Columbia and its affiliated teaching hospitals.  Further details about this Act are available upon request.  Your confidentiality will be respected.  No information that discloses your identity will be released or published without your specific consent to the disclosure.  However, research records and medical records identifying you may be inspected in the presence of the Investigator or his or her designate by representatives of the UBC Research Ethics Board or Health Canada for the purpose of monitoring the research.  However, no records which identify you by name or initials will be allowed to leave the Investigators’ offices.  You are encouraged to ask for an explanation or clarification of any of the procedures or other aspects of this study before signing this consent form or at any time during your participation in the study.    YOU MAY DECLINE TO ENTER THIS STUDY OR WITHDRAW FROM THE EXPERIMENT AT ANY TIME WITHOUT PROVIDING ANY REASONS FOR YOUR DECISION AND WITHOUT AFFECTING YOUR MEDICAL CARE IN ANY WAY.  Should you have any questions about the procedures or your involvement in this study, please contact Dr. A.W. Sheel at 604-822-9451.  If you have any concerns or questions about your rights or experience as a research subject, you may contact the Research Subject Information Line in the UBC Office of Research Services at (604) 822-8598.              113 Page 5 of 5                         version 3 (May 6, 2008)  Consent:  In signing this form you are consenting to participate in this research project and acknowledge receipt of a signed and dated copy of this form.  Signing this consent form in no way limits your legal rights against the sponsor, investigators, or anyone else.   ________________________________ ___________________________ Signature of Subject   Date  ________________________________  Printed Name of Subject  ________________________________ ___________________________ Signature of Witness   Date  ________________________________  Printed Name of Witness  ________________________________ ___________________________ Signature of Investigator  Date   ________________________________  Printed Name of Investigator                114              APPEglyph1197DIX II:  Physical activity readiness and health questionnaires                115     116  Menstrual History Questionnaire  1.  Are you having regular periods?  YES/NO  2.  How long is your cycle length?  _________ (days)  3.  How many days long is your flow? _________ (days)  4. Can you usually tell, by the way you feel, that your period is coming?     YES/NO  5.  Do you usually experience the following symptoms?   Breast tenderness YES/NO  Appetite changes YES/NO  Mood changes  YES/NO  Fluid retention  YES/NO  6.  How many times did you menstruate in the past year? __________  7.  How many periods have you missed in the last five years?  ___________  8.  Are you currently taking oral contraceptives?  YES/NO  • If yes, for how long?   ____________  • What is the name of the oral contraceptive pill which you are taking?     __________________  9.  When was the last start of your period (DAY 1)?   ______________________  Medical History  1.  Are you currently taking any medications (excluding oral contraceptives)?   Please List:  ____________________________________________________  2.  Do you currently smoke?  YES/NO  3.  Are you a past smoker?  YES/NO  4.  When was the last time you had a cold?  ___________________  5.  Do you have asthma, other lung problems or significant illness?  Please List: _____________________________________________________________________  6.  Do you have a cardiac pacemaker or any metal inside of your body? YES/NO  7. Have you had recent nasopharyngeal surgery?  YES/NO  8.  Do you have an ulcer or tumour in your oesophagus?  YES/NO  Physical Activity History  Type of Physical Activity:  _______________________________________________ Average Duration:  _____________________________________________________ Average Frequency:  ___________________________________________________  117               APPEglyph1197DIX III:  Reprints of selected publications                118       119       120       121       122       123      124       125     126     127     128     129     130    131      132     133     134      135      136      137       138     139      140       141       142       143       144      145       146       147       148         149               APPEglyph1197DIX IV:  Individual diaphragm fatigue response in men and women                            150                                          Figure A.IV.1:  Response of twitch trans-diaphragmatic pressure (Pdi,tw) during recovery in the individual male (A) and female (B) subjects that developed diaphragm fatigue (defined as a drop in Pdi,tw of  ≥ 15%).  B, baseline.        0204060801001200 10 20 30 40 50 60 70Time (min)Pdi,tw (% Baseline)0204060801001200 10 20 30 40 50 60 70Time (min)Pdi,tw (% Baseline)∆∆BBABPdi,tw (% Baseline)Pdi,tw (% Baseline)∆∆Pdi,tw (% Baseline)Pdi,tw (% Baseline)∆∆ 151                       APPEglyph1197DIX V:  Certificates of ethical approval                 152                              153      154              APPEglyph1197DIX VI:  Candidate’s list of research publications                 155 REFEREED JOURglyph1197AL PUBLICATIOglyph1197S  1.  Guenette JA, Dominelli PB, Reeve SS, Durkin CM,Eves ND and Sheel AW.  Effect of thoracic gas compression and bronchodilation on the evaluation of expiratory flow limitation during exercise in healthy humans.  Respiratory Physiology and glyph817eurobiology.  In Press.  2.  Koehle MS, Guenette JA and Warburton DER.  Oximetry, heart rate variability, and the diagnosis of mild to moderate acute mountain sickness.  European Journal of Emergency Medicine.  In Press.  3.  Sheel AW, Guenette JA, Yuan R, Mayo JR, McWilliams A, Lam S and Coxson HO.  Evidence for dysanapsis using computed tomographic imaging of the airways in older ex-smokers.  Journal of Applied Physiology. 107: 1622:1628, 2009.  4.  Guenette JA, Querido JS, Eves ND, Chua R and Sheel AW.  Sex differences in the resistive and elastic work of breathing during exercise in endurance trained athletes.  American Journal of Physiology; Regulatory, Integrative and Comparative Physiology.  297(1): R166-175, 2009.  5.  Vogiatzis I, Athanasopoulos D, Boushel R, Guenette JA, Koskolou M, Vasilopoulou M, Wagner H, Roussos C, Wagner PD and Zakynthinos S.  Contribution of respiratory muscle blood flow to exercise-induced diaphragmatic fatigue in trained cyclists.  Journal of Physiology (London). 586: 5575-5587, 2008.  6.  Sheel AW and Guenette JA.  Mechanics of breathing during exercise in men and women: sex or body size differences?  Invited Review: Exercise and Sports Sciences Reviews.  36 (3):128-134, 2008.  7.  Vogiatzis I, Zakynthinos S, Boushel R, Athanasopoulos D, Guenette JA, Wagner H, Roussos C and Wagner PD.  The contribution of intrapulmonary shunts to the alveolar to arterial oxygen difference during exercise is very small.  Journal of Physiology (London).  586 (9): 2381-2391, 2008.  8.  Guenette JA, Vogiatzis I, Zakynthinos S, Athanasopoulos D, Koskolou M, Vassilopolou M, Wagner H, Roussos C, Wagner PD and Boushel R.   Human respiratory muscle blood flow measured by near-infrared spectroscopy and indocyanine green.  Journal of Applied Physiology.  104: 1202-1210, 2008.    • Invited Editorial:  Kuebler WM. How NIR is the future in blood flow monitoring?  J Appl Physiol. 104: 905-906, 2008.  9.  Monrieux G, Guenette JA, Sheel AW and Sanderson DJ.  Influence of cadence, power output and hypoxia on the joint moment distribution during cycling.  European Journal of Applied Physiology. 102 (1): 11-19, 2007. 10.  Guenette JA and Sheel AW.  Exercise induced arterial hypoxaemia in healthy active women. Applied Physiology glyph817utrition and Metabolism. 32: 1263-1273, 2007.  11.  Guenette JA and Sheel AW.  Physiological consequences of a high work of breathing during heavy exercise in humans. Journal of Science and Medicine in Sport. 10: 341-350, 2007.  156  12.  Witt JD, Guenette JA, Rupert JL, McKenzie DC and Sheel AW.  Respiratory muscle training attenuates the respiratory muscle metaboreflex in healthy humans.  Journal of Physiology (London). 584 (3): 1019-1028, 2007.    • Invited Editorial:  Harms CA. Insights into the role of the respiratory muscle metaboreflex. J Physiol (London). 584 (3): 711, 2007.   13.  Guenette JA, Sporer BC, Macnutt MJ, Sheel AW, Coxson HO, Mayo JR and Mckenzie DC.  Lung density is not altered following intense normobaric hypoxic interval training in competitive female cyclists.  Journal of Applied Physiology. 103: 875-882, 2007.  14.  Guenette JA, Witt JD, Road JD, McKenzie DC and Sheel AW.  Respiratory mechanics during exercise in endurance trained men and women.  Journal of Physiology (London). 581 (3): 1309-1322, 2007.  15.  Macnutt MJ, Guenette JA, Witt JD, Yuan R, Mayo JR and McKenzie DC. Intense hypoxic cycle exercise does not alter lung density in competitive male cyclists.  European Journal of Applied Physiology. 99 (6): 623-31, 2007.  16.  Koehle MS, Wang P, Guenette JA and Rupert JL.  No association between variants in the ACE and angiotensin II receptor 1 genes and acute mountain sickness in Nepalese pilgrims to the Janai Purnima Festival at 4380 metres.  High Altitude Medicine and Biology. 7 (4): 281-289, 2006.   17.  Witt JD, Fisher JRKO, Guenette JA, Cheong KA, Wilson BJ and Sheel AW. Measurement of exercise ventilation by a portable respiratory inductive plethysmograph.  Respiratory Physiology and glyph817eurobiology. 154: 389-395, 2006.  18.  Guenette JA, Martens AM, Lee AL, Tyler GD, Richards JC, Foster GE, Warburton DER and Sheel AW. Variable effects of respiratory muscle training on cycle exercise performance in men and women.  Applied Physiology, glyph817utrition and Metabolism. 31 (2): 159-166, 2006.  19.  Sheel AW, Koehle MS, Guenette JA, Foster GE, Sporer BC, Diep TT and McKenzie DC. Human ventilatory responsiveness to hypoxia is unrelated to maximal aerobic capacity.  Journal of Applied Physiology. 100: 1204-1209, 2006.  20.  Guenette JA, Diep TT, Koehle MS, Foster GE, Richards JC and Sheel AW. Acute hypoxic ventilatory response and exercise-induced arterial hypoxemia in men and women.  Respiratory Physiology and glyph817eurobiology. 143 (1): 37-48, 2004.   21.  Sheel AW, Richards JC, Foster GE and Guenette JA. Sex differences in respiratory exercise physiology.  Sports Medicine. 34 (9): 567-79, 2004.        157 ABSTRACTS Aglyph1197D COglyph1197FEREglyph1197CE PROCEEDIglyph1197GS  1.  Guenette JA, Vogiatzis I, Zakynthinos S, Boushel R, Wagner PD and Roussos C.  Influence of respiratory muscle blood flow and hypoxemia on exercise induced diaphragmatic fatigue in humans. Medicine and Science in Sports and Exercise.  41: S55, 2009.  Invited oral presentation (Featured Science Symposium), Seattle, Washington, USA.  2.  Vogiatzis I, Athanasopoulos D, Boushel R, Guenette J, Koskolou M, Wagner H, Roussos C, Wagner P and Zakynthinos S.  Contribution of respiratory muscle blood flow to exercise-induced diaphragmatic fatigue in trained cyclists.  European Respiratory Journal.  44: 263, 2008.  Oral Presentation, Berlin, Germany.  3.  Guenette JA, Querido JS, Eves ND, Chua R and Sheel AW.  Why is the total muscular work of breathing higher in women during exercise compared to men?  Applied Physiology glyph817utrition and Metabolism. 33: S38, 2008.  Oral Presentation, Banff, Alberta, Canada.  4.  Rossi A, Guenette JA, Augustensen H, Dela F, Belhage B, Pott FC and Boushel R.  Time course of oxy- and total-hemoglobin concentration during arterial infusion of adenosine.  Applied Physiology glyph817utrition and Metabolism. 33: S85, 2008.  Oral Presentation, Banff, Alberta, Canada.   5.  Sheel AW, Guenette JA, Yuan R, Holy L, Mayo JR and Coxson HO.  Computed tomographic (CT) imaging of the airways: evidence for dysanapsis in women.  Applied Physiology glyph817utrition and Metabolism. 33: S91, 2008.  Oral Presentation, Banff, Alberta, Canada.  6.  Reid WD, Shadgan B, Guenette JA and Sheel AW.  Tissue oxygenation of limb and respiratory muscles during progressive inspiratory loading.  The Physiologist.  51 (6): 49, 2008.  Poster Presentation, Hilton Head, South Carolina, USA.    7.  Sanderson DJ, Monrieux G, Guenette JA and Sheel AW. Influence of cadence, power output and hypoxia on the joint powers and muscle excitation during cycling. Proceedings of the glyph817orth American Congress on Biomechanics, 2008.  Poster Presentation, Ann Arbor, Michigan, USA.  8.  Guenette JA, Vogiatzis I, Athanasopoulos D, Koskolou M, Vasilopoulou M, Wagner H, Zakynthinos S, Roussos C, Wagner PD and Boushel R.  Human respiratory muscle blood flow measured by near-infrared spectroscopy and indocyanine green. Medicine and Science in Sports and Exercise.  40: S1801, 2008.  Poster presentation, Indianapolis, Indiana, USA.  9. Wang P, Guenette JA, Koehle MS and Rupert JL.  Genetic association studies of acute mountain sickness susceptibility (2008).  The Western Canadian Conference on Environmental Ergonomics and Physiology.  Oral Presentation, Vancouver, British Columbia, Canada.  10.  Guenette JA, Durkin CM, Eves ND and Sheel AW.  What is the best method of generating a maximum flow volume loop for evaluating expiratory flow limitation during exercise?  Applied Physiology glyph817utrition and Metabolism. 32: S39, 2007.  Oral Presentation, London, Ontario, Canada.    158 11.  Guenette JA, Comtois AS, Sheel AW, Larsen B, Heyer L, Kjaer M and Boushel R.  Human diaphragmatic blood flow measured by near-infrared spectroscopy and indocyanine green.  Applied Physiology glyph817utrition and Metabolism. 32: S39, 2007. Poster Presentation, London, Ontario, Canada.  12.  Rossi A, Guenette JA, Augustensen H, Della F, Belhage B, Pott FC and Boushel R.  Arterial adenosine infusion overrides local muscle vasodilatory autoregulation during exercise.  Applied Physiology glyph817utrition and Metabolism. 32: S77, 2007. Oral Presentation, London, Ontario, Canada.  13.  Rossi A, Guenette JA, Augustensen H, Della F, Belhage B, Pott FC and Boushel R.  Muscle blood flow heterogeneity increased at rest and during exercise by arterial adenosine infusion, mediated in part by nitric oxide. Applied Physiology glyph817utrition and Metabolism. 32: S77, 2007. Oral Presentation, London, Ontario, Canada.  14.  Witt JD, Guenette JA, Rupert JL, McKenzie DC and Sheel AW.  Inspiratory muscle training attenuates the human respiratory muscle metaboreflex.  Applied Physiology glyph817utrition and Metabolism. 32: S93, 2007. Oral Presentation, London, Ontario, Canada.  15.  Guenette JA, Witt JD, McKenzie DC, Road JD and Sheel AW.  Expiratory flow limitation and the regulation of lung volumes in aerobically trained men and women.  Applied Physiology glyph817utrition and Metabolism. 31: S35, 2006.  Oral Presentation, Graduate Student Competition Symposium (Winner), Halifax, Nova Scotia, Canada.  16.  Guenette JA, Macnutt MJ, Witt JD, Giles LV, Yuan R, Zbogar D, Hodges AN, von der Porten F, Houghton KM, Schwab N, Mayo JR and McKenzie DC. Does strenuous hypoxic exercise induce pulmonary oedema?  Canadian Journal of Applied Physiology. 30: S32, 2005.  Oral Presentation, Gatineau, Quebec, Canada.   17.  Witt JD, Fisher JRKO, Guenette JA, Cheong KA, Wilson BJ and Sheel AW. Measurement of exercise ventilation by a respiratory inductive plethysmograph. Canadian Journal of Applied Physiology. 30: S86, 2005.  Oral Presentation, Gatineau, Quebec, Canada.   18.  Guenette JA, Martens A M, Lee AL, Tyler GD, Richards JC, Foster GE, Warburton DER and Sheel AW.  Inspiratory muscle training: variable effects on exercise performance and no effect of gender. Canadian Journal of Applied Physiology. 29: S53, 2004.  Oral Presentation, Saskatoon, Saskatchewan, Canada.   19.  Guenette JA, Koehle MS, Diep TT, Richards JC, Foster GE and Sheel AW.  Hypoxic ventilatory response in trained male and female cyclists. Medicine and Science in Sports and Exercise.  36(5): S265, 2004.  Poster Presentation, Indianapolis, Indiana, USA.    

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