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Sex differences in left ventricular mechanics in response to acute physiological stress Williams, Alexandra Mackenzie 2016

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  SEX DIFFERENCES IN LEFT VENTRICULAR MECHANICS IN RESPONSE TO ACUTE PHYSIOLOGICAL STRESS   by  Alexandra Mackenzie Williams  B.MSc., The University of Western Ontario, 2009 M.Sc., The University of Western Ontario, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE COLLEGE OF GRADUATE STUDIES  (Interdisciplinary Studies)   THE UNIVERSITY OF BRITISH COLUMBIA  (Okanagan)  October 2016   © Alexandra Mackenzie Williams, 2016    ii Thesis Committee The undersigned certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled:   Sex differences in left ventricular mechanics in response to acute physiological stress  submitted by              Alexandra Mackenzie Williams in partial fulfilment of the requirements of the degree of   Doctor of Philosophy .    Dr. Neil Eves, Faculty of Healthy and Social Development, UBC’s Okanagan Campus  Supervisor, Professor (please print name and faculty/school above the line)  Prof. Philip Ainslie, Faculty of Healthy and Social Development, UBC’s Okanagan Campus  Supervisory Committee Member, Professor (please print name and faculty/school in the line above)  Prof. Robert Shave, School of Sport, Cardiff Metropolitan University  Supervisory Committee Member, Professor (please print name and faculty/school in the line above)  Dr. Christopher West, School of Kinesiology, UBC  University Examiner, Professor (please print name and faculty/school in the line above)  Dr. David Oxborough, Liverpool John Moores University  External Examiner, Professor (please print name and university in the line above)   October 19, 2016  (Date Submitted to Grad Studies)   iii Abstract  Left ventricular (LV) mechanics, including rotation, twist and strain, specifically describe the patterns of myocardial deformation across the cardiac cycle. The twisting motion of the ventricle results from the contraction of helically oriented LV myofibres, and contributes to efficient systolic and diastolic function. Left ventricular mechanics are highly sensitive to alterations in preload, afterload and contractility, and therefore are profoundly altered when the heart is acutely stressed. While sex-related differences in LV structure and hemodynamics have been established, there is a paucity of data comparing LV mechanics between males and females. This thesis therefore investigated sex differences in LV mechanics in response to acute physiological stress. In Chapter 3, LV preload was reduced during progressive lower body negative pressure (LBNP). LV twist increased in both sexes during LBNP, but females had greater LV apical rotation and twist during -60 mmHg, and these sex differences may have resulted from differences in adrenergic stimulation, vagal withdrawal or LV geometry. In Chapter 4, LV mechanics were compared between the sexes, during increases and reductions to adrenergic stimulation with post exercise ischemia (PEI) and ß1-adrenergic receptor (ß1-AR) blockade, respectively. LV twist was not altered in either of the sexes during PEI, likely due to concomitant increases in LV afterload. However, during ß1-AR blockade, LV apical rotation and twist were reduced in males, but not females, suggesting that LV twist mechanics in males are more sensitive to altered adrenergic stimulation. Chapter 5 then assessed the potential contribution of vagal autonomic control to sex differences in LV mechanics during LBNP and submaximal exercise. During -60 mmHg LBNP, females had greater LV apical rotation and twist than males, and these differences persisted during vagal blockade with glycopyrrolate. LV rotation and twist increased in both sexes during exercise up to 50% peak power, but were not different between the sexes in the control or blocked conditions. While sex differences in LV mechanics may not result from differences in cardiac autonomic control, it is possible that variations in other key regulators of LV mechanics, including LV myofibre configuration and chamber structure, determine sex differences in LV twist mechanics.        iv Preface  All experimental chapters of this thesis (Chapters 3, 4 and 5) were approved by the University of British Columbia clinical research ethics board (H12-03531, H13-03472, H15-02490). Data collection for experimental Chapter 5 was completed at Cardiff Metropolitan University’s Cycoed Campus (United Kingdom), and this study was approved by the Cardiff Metropolitan University research ethics committee.   A version of the study presented in Chapter 3 has been published: Williams AM, Shave RE, Stembridge M & Eves ND (2016). Females have greater left ventricular twist mechanics than males during acute reductions to preload. Am J Physiol Heart Circ Physiol 311:H76-H84. Dr. Neil Eves and myself were responsible for the conception and design of the study. I was primarily responsible for data collection, with the help of Dr. Eves and Dr. Mike Stembridge who performed the echocardiographic imaging. I completed data analysis with the help of Dr. Stembridge and Prof. Rob Shave. I was primarily responsible for the interpretation of data and drafting of the manuscript with help from Dr. Eves. Prof. Shave and Dr. Stembridge also contributed to the analysis and interpretation of data, and editing of the manuscript. Data from this chapter were presented at The Physiological Society’s 2015 Annual Meeting (Cardiff, UK).   Chapter 4 will be submitted for publication to The Journal of Physiology: Williams AM, Shave RE & Eves ND (2016). The influence of adrenergic stimulation on sex differences in left ventricular twist mechanics. The study design was conceived by Dr. Eves and myself, with input from Prof. Shave and Prof. Phil Ainslie. I performed all data collection and analysis, and was primarily responsible for the interpretation of data and preparation of the manuscript. Dr. Eves contributed to the interpretation of data and drafting of the manuscript, and Prof. Shave contributed to the editing of the manuscript.  A draft of the manuscript has been prepared for Chapter 5. I was primarily responsible for the conception and design of this study, with the help of Dr. Eves and Prof. Shave. I additionally performed all data collection with the help of Dr. James Coulson who administered the infusions of glycopyrrolate. Dr. Stembridge also assisted with the data collection. I was primarily responsible for the interpretation of data and drafting of the manuscript, with the assistance of Dr. Eves.   v Table of Contents  Thesis Committee ......................................................................................................................... ii	  Abstract ........................................................................................................................................ iii	  Preface ........................................................................................................................................... iv	  Table of Contents ........................................................................................................................... v	  List of Tables ................................................................................................................................. ix	  List of Figures ............................................................................................................................... ix	  List of Symbols and Abbreviations .............................................................................................. x	  Acknowledgements ...................................................................................................................... xv	  Dedication .................................................................................................................................... xvi	  Chapter 1. Review of Literature .................................................................................................. 1	  1.1	   Introduction .................................................................................................................................... 1	  1.2	   Left ventricular anatomy and mechanics .................................................................................... 2	  1.2.1	   Historical perspective of left ventricular anatomy and fibre architecture ................................ 2	  1.2.2	   Current understanding of LV myocardial architecture and mechanical deformation .............. 6	  1.2.3	   Definitions of mechanical deformation .................................................................................... 9	  1.2.4	   Sequence of mechanical deformation ..................................................................................... 10	  1.3	   Early investigations of LV mechanics ........................................................................................ 12	  1.4	   Influences of preload, afterload and contractility on LV mechanics: animal studies ........... 14	  1.5	   Influences of preload, afterload and contractility on LV mechanics: human studies ........... 15	  1.5.1	   LV mechanics during alterations to preload ........................................................................... 16	  1.5.2	   LV mechanics during alterations to afterload ........................................................................ 17	  1.5.3	   LV mechanics during alterations to adrenergic stimulation ................................................... 17	  1.5.4	   LV mechanics during dynamic exercise ................................................................................. 18	  1.6	   Sex differences in LV mechanics ................................................................................................ 22	  1.7	   Sex differences in LV structure and function at rest ................................................................ 22	  1.7.1	   Sex differences in LV mass and structure .............................................................................. 22	  1.7.2	   Sex differences in global LV systolic and diastolic function and heart rate. ......................... 24	  1.8	   Sex differences in LV responses to acute physiological stress ................................................. 26	  1.8.1	   LV responses to acute preload challenges .............................................................................. 26	  1.8.1.1	   General hemodynamic responses to central hypovolemia .................................................. 26	  1.8.1.2	   Sex differences in the LV responses to central hypovolemia ............................................... 26	  1.8.1.3	   Implications for sex differences in LV mechanics during acute preload challenges .......... 29	  1.8.2	   Alterations to adrenergic stimulation ..................................................................................... 29	  1.8.2.1	   General autonomic and adrenergic control of the heart ..................................................... 29	  1.8.2.2	   Sex differences in the autonomic control of the heart ......................................................... 30	  1.8.2.3	   Sex differences the LV responses to adrenergic and muscarinic stimulation ..................... 31	  1.8.2.4	   Implications for sex differences in LV mechanics during altered adrenergic stimulation .. 32	  1.8.3	   LV responses during isometric handgrip exercise and post-exercise ischemia ..................... 33	  1.8.3.1	   General sympathetic and hemodynamic responses to post-exercise ischemia ................... 33	  1.8.3.2	   Sex differences in the autonomic and metabolic responses to post-exercise ischemia ....... 34	  1.8.3.3	   Implications for sex differences in LV mechanics during post-exercise ischemia .............. 34	  1.8.4	   LV responses during dynamic exercise .................................................................................. 35	  1.8.4.1	   General alterations to LV hemodynamics during dynamic exercise ................................... 35	    vi 1.8.4.2	   Sex differences in the LV responses during dynamic exercise ............................................ 35	  1.8.4.3	   Implications for sex differences in LV mechanics during dynamic exercise ....................... 36	  1.9	   Specific aims and hypotheses ...................................................................................................... 37	  Chapter 2. Techniques for Measurement of Cardiac Structure, Global Ventricular Function And Left Ventricular Mechanics ............................................................................... 39	  2.1	   Ultrasound Technology ............................................................................................................... 39	  2.1.1	   Basic principles ...................................................................................................................... 39	  2.2	   Basic forms of echocardiography ............................................................................................... 41	  2.2.1	   Two-dimensional B-mode echocardiography ........................................................................ 41	  2.2.2	   Doppler echocardiography ..................................................................................................... 42	  2.2.3	   Speckle tracking echocardiography ........................................................................................ 42	  2.3	   Image acquisition and optimization for cardiac assessment .................................................... 44	  2.3.1	   General procedures, standardization and optimization for image acquisition ....................... 44	  2.3.2	   Measurement of LV structure and geometry .......................................................................... 45	  2.3.3	   Measurement of global LV systolic and diastolic function .................................................... 46	  2.3.4	   Measurement of LV mechanics using speckle tracking analysis ........................................... 47	  2.4	   Assessment of LV hemodynamics .............................................................................................. 50	  Chapter 3. Sex differences in LV mechanics during acute alterations to preload ................ 51	  3.1	   Background .................................................................................................................................. 51	  3.2	   Methods ......................................................................................................................................... 52	  3.2.1	   Ethical approval and study participants .................................................................................. 52	  3.2.2	   Study design ........................................................................................................................... 53	  3.2.3	   Specific methodology ............................................................................................................. 53	  3.2.3.1	   Screening and familiarization ............................................................................................. 53	  3.2.3.2	   Total blood volume .............................................................................................................. 54	  3.2.3.3	   Transthoracic echocardiography and analysis of LV mechanics and hemodynamics ........ 54	  3.2.4	   Statistical analysis and power calculation .............................................................................. 54	  3.3	   Results ........................................................................................................................................... 55	  3.3.1	   Participant characteristics ....................................................................................................... 55	  3.3.2	   LV mechanics and structure during altered preload ............................................................... 56	  3.3.3	   LV volumes and hemodynamics during altered preload ........................................................ 62	  3.4	   Discussion ..................................................................................................................................... 66	  3.4.1	   Sex differences in LV responses to altered preload ............................................................... 66	  3.4.2	   Sex differences in LV adrenergic stimulation ........................................................................ 67	  3.4.3	   Sex differences in LV geometry ............................................................................................. 68	  3.4.4	   Sex differences in the Frank-Starling relationship ................................................................. 68	  3.4.5	   Limitations .............................................................................................................................. 69	  3.5	   Summary and significance .......................................................................................................... 70	  Chapter 4. The influence of adrenergic stimulation on sex differences in left ventricular twist mechanics ............................................................................................................................ 72	  4.1	   Background .................................................................................................................................. 72	  4.2	   Methods ......................................................................................................................................... 73	  4.2.1	   Study participants ................................................................................................................... 73	  4.2.2	   Study design ........................................................................................................................... 73	  4.2.3	   Specific methodology ............................................................................................................. 74	  4.2.3.1	   Isometric handgrip and post-exercise ischemia .................................................................. 74	  4.2.3.2	   β1-AR blockade .................................................................................................................... 74	  4.2.3.3	   Transthoracic echocardiography, analysis of LV mechanics, and measurements    vii  of blood pressure and heart rate ......................................................................................... 74	  4.2.3.4	   Analysis of LV hemodynamics ............................................................................................. 75	  4.2.4	   Statistical analysis and sample size calculation ...................................................................... 75	  4.3	   Results ........................................................................................................................................... 76	  4.3.1	   Baseline characteristics, LV structure and hemodynamics .................................................... 76	  4.3.2	   LV mechanics in response to altered adrenergic stimulation ................................................. 77	  4.3.2.1	   LV mechanics during post exercise ischemia ...................................................................... 78	  4.3.2.2	   LV mechanics during β1-AR blockade ................................................................................. 78	  4.3.3	   Hemodynamic responses to altered adrenergic stimulation ................................................... 80	  4.3.3.1	   Hemodynamics responses during post exercise ischemia ................................................... 80	  4.3.3.2	   Hemodynamic responses during β1-AR blockade ................................................................ 81	  4.3.4	   LV structure and geometry during altered adrenergic stimulation ......................................... 83	  4.3.4.1	   Relationships of LV mechanics with structure and geometry ............................................. 84	  4.4	   Discussion ..................................................................................................................................... 86	  4.4.1	   Effects of post exercise ischemia on sex differences in LV mechanics ................................. 86	  4.4.2	   Effects of ß1-AR blockade on sex differences in LV mechanics ........................................... 88	  4.4.3	   Relationships between LV twist mechanics and chamber geometry ..................................... 89	  4.4.4	   Limitations .............................................................................................................................. 90	  4.5	   Summary and significance .......................................................................................................... 90	  Chapter 5. The influence of vagal control on sex differences in left ventricular mechanics during alterations to stroke volume ........................................................................................... 91	  5.1	   Background .................................................................................................................................. 91	  5.2	   Methods ......................................................................................................................................... 92	  5.2.1	   Study participants ................................................................................................................... 92	  5.2.2	   Study design ........................................................................................................................... 93	  5.2.3	   Specific methodology ............................................................................................................. 94	  5.2.3.1	   Screening and familiarization ............................................................................................. 94	  5.2.3.2	   Incremental exercise testing ................................................................................................ 95	  5.2.3.3	   Vagal blockade .................................................................................................................... 95	  5.2.3.4	   Transthoracic echocardiography, analysis of LV mechanics and hemodynamics, and measurement of blood pressure and heart rate ................................................................... 95	  5.2.4	   Statistical analysis and power calculation .............................................................................. 95	  5.3	   Results ........................................................................................................................................... 97	  5.3.1	   Baseline characteristics, LV structure and hemodynamics .................................................... 97	  5.3.2	   Lower body negative pressure ................................................................................................ 99	  5.3.2.1	   LV mechanics during lower body negative pressure ........................................................... 99	  5.3.2.2	   LV hemodynamics during lower body negative pressure .................................................. 105	  5.3.2.3	   LV structure and geometry during lower body negative pressure .................................... 109	  5.3.3	   Submaximal exercise ............................................................................................................ 111	  5.3.3.1	   LV mechanics during exercise ........................................................................................... 111	  5.3.3.2	   LV hemodynamics during exercise .................................................................................... 116	  5.3.3.3	   LV structure and geometry during exercise ...................................................................... 120	  5.3.4	   Relationship of LV torsion to SV⋅BSA-1.5 during LBNP and exercise. ................................ 120	  5.3.5	   Relationships of LV mechanics with structure and geometry during LBNP and exercise. . 122	  5.4	   Discussion ................................................................................................................................... 126	  5.4.1	   Vagal control of LV mechanics during reductions to preload ............................................. 126	  5.4.1.1	   Influence of LV volumes on LV mechanics during reductions to preload ......................... 127	  5.4.1.2	   Influence of LV structure, geometry and myofibre orientation on LV mechanics during reductions to prelaod ......................................................................................................... 128	    viii 5.4.2	   Vagal control of LV mechanics during submaximal exercise .............................................. 129	  5.4.3	   Effect of vagal blockade on LV mechanics at rest ............................................................... 131	  5.5	   Summary and significance ........................................................................................................ 131	  Chapter 6. General Discussion and Conclusions .................................................................... 133	  6.1	   Sex differences in the LV twist-to-volume relationships ........................................................ 133	  6.2	   Sex differences in the alterations to LV preload and afterload ............................................. 135	  6.2.1	   Sex differences in reductions to preload .............................................................................. 135	  6.2.2	   Differences in LV afterload .................................................................................................. 136	  6.3	   Sex differences related to sympathovagal balance and adrenergic stimulation .................. 137	  6.3.1	   Sex differences in sympathovagal balance and the chronotropic responses to acute stress . 137	  6.3.2	   Sex differences in adrenergic stimulation ............................................................................ 140	  6.4	   Sex differences related to LV structure and geometry ........................................................... 140	  6.5	   Determinants of LV twist at rest in males and females .......................................................... 143	  6.6	   Considerations ............................................................................................................................ 147	  6.7	   Relevance and implications and directions for future study ................................................. 147	  6.7.1	   Influences of LV structure and geometry ............................................................................. 147	  6.7.2	   Influences of adrenergic stimulation .................................................................................... 148	  6.7.3	   Influences of arterial and cardiopulmonary baroreflexes ..................................................... 148	  6.7.4	   Relevance for clinical and sport applications ....................................................................... 149	  6.8 	   Overall conclusions ................................................................................................................... 151	     ix List of Tables  Table 1.1. Effects of altered preload, afterload and adrenergic stimulation on LVaaaa mechanics in healthy human study populations. ....................................................... 20	  Table 1.2. Comparison of cardiac structure and volumes at rest in males and females ............... 23	  Table 1.3. Comparison of global LV function at rest in males and females ................................ 25	  Table 2.1. Technical error of measurement of the sonographer (A. Williams) foraaaaa measurements of LV structure and function. ............................................................. 49	  Table 3.1. Baseline participant characteristics and echocardiographic measurements ................ 56	  Table 3.2. LV mechanics, structure and geometry during altered preload. .................................. 59	  Table 3.3. Cardiovascular responses to altered preload. ............................................................... 64	  Table 4.1. Baseline characteristics, LV hemodynamics, structure and geometry ........................ 77	  Table 4.2. LV mechanics during altered adrenergic stimulation. ................................................. 80	  Table 4.3. LV hemodynamics during altered adrenergic stimulation. .......................................... 82	  Table 4.4. LV structure and geometry during altered adrenergic stimulation. ............................. 84	  Table 5.1. Participant demographics and baseline characteristics ................................................ 98	  Table 5.2. LV mechanics during LBNP with and without vagal blockade ................................ 102	  Table 5.3. LV hemodynamics during LBNP with and without vagal blockade ......................... 107	  Table 5.4. LV structure and geometry during LBNP with and without vagal blockade ............ 110	  Table 5.5. LV mechanics during submaximal exercise with and without vagal blockade ......... 113	  Table 5.6. LV hemodynamics during submaximal exercise with and without vagalaaaaaaa blockade ................................................................................................................... 118	  Table 5.7. LV structure and geometry during submaximal exercise with and without vagal blockade ................................................................................................................... 121	  Table 6.1. Baseline characteristics, LV hemodynamics, structure and geometry ...................... 146	    x List of Figures  Figure 1.1. Left panel: the “Golden Section”, characterized by the Golden Ratio of 1.618 (172). Right panel: the formation of a logarithmic spiral as a harmony of proportional elements (27) (images reproduced with © permission). ......................... 2	  Figure 1.2. Anatomical structure and characterization of myocardial bands proposed by Mall and MacCallum (132)(images reproduced with © permission from (173)). .......................................................................................................................... 3	  Figure 1.3. Torrent-Guasp’s schematic of the ventricular myocardial band, showing the intact ventricle (A), separation of the pulmonary artery (B) and right free wall (C), and finally the unraveled band (D and E)(figure reproduced with © permission from (209)). ............................................................................................... 4	  Figure 1.4. Gradient of fibre angles across the canine LV wall, transitioning from +90º to -90º from the endocardium to epicardium, respectively (figure reproduced with © permission from (199)). ........................................................................................... 5	  Figure 1.5. Visualization of LV architecture and myofibre orientations using diffusion tensor magnetic resonance imaging (figure reproduced with © permission from (174)). .................................................................................................................. 6	  Figure 1.6. Myocardial fibre orientations and structural organization across the LV wall. Long oval structures depict myofibres, joined by endomysial collagen and organized into laminar sheets in the perimysium (figure reproduced with © permission from (174)). ............................................................................................... 8	  Figure 1.7. Left ventricular myofibre and sheet reorientation during systole. Alterations to helix angle (αhelix) (left) and sheet angle (ß’) (right; A and C depict diastole, B and D depict systole) contribute to myocardial deformation in the circumferential, radial and longitudinal planes (images reproduced with © permission from (38)). ................................................................................................. 8	  Figure 1.8. Sequence of regional myocardial deformation and rotation across the cardiac cycle. Markers to the right and left denote clockwise and counterclockwise rotations, respectively (as viewed from the apex) (figure reproduced with © permission from (26)). ............................................................................................... 12	  Figure 1.9. Alterations to LV pressure and apical rotation during volume infusion (left) and vena caval occlusion (right). “ED” indicates end-diastole, and “ES” indicated end-systole (figures reproduced with © permission from (78)). ................ 14	  Figure 1.10. (A) Frank-Starling curves for males and females, relating pulmonary capillary wedge pressure (estimate of LV end-diastolic pressure) to absolute (upper panel) and indexed (lower panel) stroke volume. (B) Reductions to wedge   xi pressure in males and females during progressive LBNP (figures reproduced with © permission from (73)). ................................................................................... 27	  Figure 1.11. Regional distributions of autonomic nerve efferents (left), and adrenergic and muscarinic receptors (right). TH-positive: sympathetic nerve efferent; AChE-positive: vagal nerve efferent; M2: muscarinic receptor. Based on data from (23, 58, 85, 110, 111, 147, 222). ................................................................................ 30	  Figure 2.1. Reflective properties of diffuse (e.g. myocardium) and specular (e.g. pericardium) interfaces (left panel reproduced with © permission from (190)). ...... 40	  Figure 2.2. Two-dimensional B-mode (brightness mode) images generated from echoes of multiple successive scan lines (figure reproduced with © permission from (228)). ........................................................................................................................ 41	  Figure 2.3. Assessment of LV rotation using speckle tracking echocardiography. Systolic rotation across consecutive frames is counterclockwise at the apex (left) and clockwise at the base (right) (figure reproduced with © permission from (154)). ........................................................................................................................ 43	  Figure 2.4. Visualization of echocardiographic imaging planes in apical (panels 1, 2), long axis (panel 3) and short axis (panels 4, 5, 6) orientations, according to the American Society of Echocardiography and European Society of Echocardiography (figure reproduced with © permission from (118)). .................... 46	  Figure 2.5. Progression of analysis using speckle tracking software for the assessment of LV mechanics (example at LV apex). ....................................................................... 48	  Figure 3.1. Graphical representation of mean LV twist mechanics during LBNP. Blue and red lines represent data for males and females, respectively. Top panel: dotted and dashed lines represent rotations of the LV apex and base, respectively. Middle panel: solid lines represent LV twist. Lower panel: solid lines represent twist and untwisting velocities. Standard deviations are provided in Table 3.2. *p<0.05 males vs. females. ....................................................................... 58	  Figure 3.2. LV torsion in males (blue circles) and females (red circles) during LBNP. Points represent means ± SD. *p<0.05 vs. males for given stage of LBNP. ............. 61	  Figure 3.3. Upper panel: the relationship of LV twist to LVSV (A) and LV torsion to SV⋅BSA-1.5 (B) Lower panel: the relationship of LV twist to LVEDV (C) and LV torsion to EDV⋅BSA-1.5 (D). Points represent group means at baseline and LBNP (closed, n=20 males, n=20 females) and following saline infusion (open, n=8 males, n=6 females). A trend toward a difference between the sexes was observed in the relationships of twist to LVSV (A) (b1, p=0.077; b2, p=0.073) and LV torsion to SV⋅BSA-1.5 (B) (b1, p=0.073; b2, p=0.074). ................. 61	    xii Figure 4.1. Graphical representation of mean left ventricular (LV) twist mechanics at baseline, during post exercise ischemia and ß1-AR blockade (bisoprolol). Blue and red lines represent mean data for males and females, respectively. Top: dotted and dashed lines represent rotations of the LV apex and base, respectively. Middle: solid lines represent LV twist. Bottom: solid lines represent twist and untwisting velocities. SDs are provided in Table 4.2. *p<0.05 males vs. females. ........................................................................................ 79	  Figure 4.2. Relationships for LV twist mechanics with chamber structure and geometry. Data include measures during baseline, post exercise ischemia and ß1-AR blockade. Blue and red represent data for males and females, respectively. Top: closed circles represent LV twist. Bottom: open triangles and circles represent LV rotation at the apex and base, respectively. *Significant relationship (p<0.05). ................................................................................................ 85	  Figure 5.1. Schematic of methodology for control (CON) and vagal blockade (GLY) trials. Infusion denotes administration of glycopyrrolate during GLY trials. Echo denotes collection of echocardiographic images. LBNP: lower body negative pressure; EX: submaximal exercise. .......................................................................... 94	  Figure 5.2. Graphical representation of mean left ventricular (LV) rotation and twist at rest (left), -40 mmHg (middle) and -60 mmHg (right) LBNP. Upper and lower panels depict data during CON and GLY (post-infusion). Blue and red lines represent mean data for males and females, respectively. Dotted lines represent rotations of the LV base (negative) and apex (positive). CON: control; GLY: glycopyrrolate. SD for peak data are provided in Table 5.2. *p<0.05 males vs. females. .................................................................................................................... 104	  Figure 5.3. Graphical representation of mean LV rotation and twist at rest (left), 25% (middle) and 50% (right) of peak power in supine cycling exercise (EX). See Figure 5.2 for symbol legends. SD for peak data are provided in Table 5.5. *p<0.05 males vs. females. ...................................................................................... 115	  Figure 5.4. The relationships of LV torsion to SV⋅BSA-1.5 during (A) LBNP and (B) exercise. Red and blue points represent group means (SD) for females and males, respectively. Closed and open circles represent data during glycopyrrolate infusion (GLY) and control (CON) conditions. *Significantly different relationships for males vs. females (p<0.05). ........................................... 122	  Figure 5.5. Relationships for LV twist mechanics with LV structure and geometry during LBNP. Data include measures during CON-LBNP and GLY-LBNP. Blue and red represent data for males and females, respectively. Top: closed circles represent LV twist. Bottom: open triangles and circles represent LV rotation at the apex and base, respectively. *Significant relationship (p<0.05). ...................... 124	    xiii Figure 5.6. Relationships for LV twist mechanics with LV structure and geometry during exercise. Data include measures during CON-LBNP and GLY-LBNP. See Figure 5.5 for symbol legends. *Significant relationship (p<0.05). ........................ 125	  Figure 6.1. Relationships between ∆LV twist with ∆SV (upper panel) and ∆torsion and ∆SV·BSA-1.5 (lower panel). Red and blue points represent individual data for females and males, respectively. *Significantly different relationships for males vs. females (p<0.05). ..................................................................................... 134	  Figure 6.2. Relationships between ∆LV twist mechanics and ∆HR. Closed circles represent individual twist data (left), and open symbols represent individual peak rotation data (right) at the base (circles) and apex (triangles). ....................... 139	  Figure 6.3. Relationships for ∆LV twist mechanics with ∆Lengthd (upper panel) and ∆LVIDd (lower panel). See Figure 6.2 for figure legend. ....................................... 142	  Figure 6.4. Relationships for ∆LV twist mechanics with ∆sphericity index (upper panel) and ∆relative wall thickness (lower panel). See Figure 6.2 for figure legend. ........ 143	    xiv List of Symbols and Abbreviations 2D – two-dimensional αhelix – myofibre helix angle A – atrial diastolic filling velocity A’ – atrial diastolic tissue velocity AR – adrenergic receptor ß’ – myolaminar sheet angle B-mode – brightness mode BMI – body mass index BSA – body surface area CS – circumferential strain DBP – diastolic blood pressure E – early diastolic filling velocity E’ – early diastolic tissue velocity EDV – end-diastolic volume EF – ejection fraction ESV – end-systolic volume HF – high frequency HR – heart rate HRV – heart rate variability IVST – intraventricular septal wall thickness LBNP – lower body negative pressure Lengthd – length at end-diastole LF – low frequency LS – longitudinal strain  LV – left ventricle LVIDd – left ventricular internal diameter at end-diastole LVIDs – left ventricular internal diameter at end-systole M2 – muscarinic receptors MAP – mean arterial pressure MSNA – muscle sympathetic nerve activity  MVC – maximal voluntary contraction PCWP – pulmonary capillary wedge pressure PEI – post exercise ischemia PWT – posterior wall thickness Q – cardiac output S’ – systolic tissue velocity SBP – systolic blood pressure STE – speckle tracking echocardiography SV – stroke volume TPR – total peripheral resistance   xv Acknowledgements   The ‘journey’ of this PhD has been a pretty remarkable four years, and I am very fortunate to have met and worked with so many outstanding individuals during this time.   To Neil. I’ve looked back at where I started and compared it to where I am now, and I undoubtedly owe my development as a researcher to your influence. But more importantly, I have developed a true passion for this research, and I am so thankful to you for that. I know that I can always run into your office blathering about new data at 100 mph, and that you’ll share that excitement.   To Phil Ainslie. Thanks for your great support, and especially for being my initial link to this awesome group. My first visit to the lab in 2011 solidified my decision to pursue this degree and move to British Columbia.   To Rob Shave and Mike Stembridge. I owe so many of the great experiences from this research to you both. Without you, I probably would not have learned echo and this story would have been very different. You have been wonderful mentors, and become great friends.   To my labmates. Thanks for all the smiles and laughs we’ve shared every day in the office. Ayla Graham, Nia Lewis, Megan Harper, Spencer Cheyne, and Daniela Flueck, I’ve had such a great time working with you, and you have become wonderful friends. Lindsey Boulet, Josh Tremblay and Heather Hackett, thank you especially for the great music, coffee, and dance moves.  To my incredible friends and family. Flaviu Dobrescu, Joey Lanthier, Mimi Fong and Rob McKeough, thank you for your visits, our travels, and for the countless Skype catch-ups. To Ian and Alicia, the best siblings ever, I love you so much and I never laugh as much as when I’m with you both. To my dad, the real Dr. Williams, I’m so thankful for our great adventures across the world throughout this process.   Finally, to my mom. You are a glowing spirit with unbelievable amounts of positivity. You have taught me to see the bright side of every aspect in life, and to have patience when things don’t go to plan. You know I could not have done this without you.     xvi Dedication  To my parents. Thank you for your love, support and encouragement in this and every other aspect of my life. I am so lucky and grateful for everything I’ve learned from you, and for every moment we’ve had together.   To an individual very dear to me, who I lost to illness. They know how thankful I am for their love, the great adventures we shared and the countless smiles they brought to my life.   For my grandfather, Mack. He is the reason I came to love science. Grandpa had incredible passion for forestry. Growing up, he taught me about life, nature, and wisdom. During my studies at The University of Western Ontario, we would chat back and forth in emails. In a certain message, he wrote,    You mention wisdom. Did I tell you one difference between knowledge and wisdom? Knowledge is to know that a tomato is a fruit. Wisdom is to know you don't use it in a fruit salad.   Later, upon completing my undergraduate degree, he wrote,   So: how does it feel to be a graduate of one of Canada's leading universities? Wear it well! Build on it! Give back to the world of your wisdom and knowledge! And enjoy the moment! Imagine: Alexandra Williams, B.MSc. Just a thought: you have received a precious gift, made up of your degree, but more importantly what you have learned while earning that degree, in the classroom, in the lab, and in the world; the supportiveness of each of your parents, and most of all the intellect and health that have allowed you to achieve.  Your challenge will be to use that gift, cherish and build on it, so that the world will be a better place for your having done so.  Love, Grandpa. XX  Grandpa, thank you for always challenging me to become a better version of myself.    1 Chapter 1. Review of Literature  1.1 Introduction  In the mid-17th century, physiologist Robert Lower outlined the current controversies of cardiac ejection: “famous men . . . doubt if the Heart caused its own movement, or if it is not rather put into motion by the blood” ((128), pp 60-61). During this period, it was commonly believed that ejection resulted from “ebullition”, or the chemically-induced boiling and swelling of blood in the heart. However, in a series of seminal experiments, Lower disproved this theory by demonstrating that the extracted heart, devoid of its blood supply, continued to beat outside of the body. He therefore concluded that ejection resulted from the forceful contraction of the cardiac musculature (61).  Lower was also one of the first to describe a structure-function relationship in the heart. Comparing the myocardium in systole to the wringing of a cloth, he acknowledged the incredible complexity of the heart’s functional anatomy. In the following centuries, physiologists and anatomists continued to dissect the structural intricacies of the heart (27). To the present day, our understanding of cardiac anatomy has evolved and it has been well established that the left ventricular (LV) myocardium is composed of obliquely-oriented helical myofibres, which produce a twisting or “wringing” motion to ultimately support efficient systolic and diastolic function (184).   Although the male and female hearts were previously considered to be similar, work in the past several decades has determined key differences in LV function and morphology (29, 41, 91). More recently, sex differences in LV mechanics (rotation, twist and strain) have additionally been reported (9, 103, 119), however there is a paucity of data comparing LV mechanics between the sexes in response to acute physiological stress. Therefore, the experiments performed within this thesis aimed to investigate sex differences in LV mechanics when the male and female hearts are challenged during acute reductions to preload, increases and decreases to adrenergic stimulation, and alterations to sympathovagal balance.   2 1.2 Left ventricular anatomy and mechanics  1.2.1 Historical perspective of left ventricular anatomy and fibre architecture The helical structure and arrangement of myocardial fibres was first characterized in the 1600’s. In 1669, Lower described a “vortex” of muscle fibres at the apex, arranged in a clockwise orientation from the outside-in, and a counterclockwise orientation from the inside-out (27). While Lower was the first to specifically identify this geometry in the heart, the formation of logarithmic spirals had been identified much earlier in history. From the times of Pythagoras, Plato and Euclid, the “Golden Section Principle” of harmony mathematics introduced the concept of self-organizing structures (193). The Golden Ratio is nature’s “perfect number” (Figure 1.1), and characterizes a harmony that occurs within the logarithmic spirals of many natural structures: the structure of DNA, opposing spirals of our fingertips, and the oblique orientations of myocardial bands (27).    Figure 1.1. Left panel: the “Golden Section”, characterized by the Golden Ratio of 1.618 (172). Right panel: the formation of a logarithmic spiral as a harmony of proportional elements (27) (images reproduced with © permission).   After Lower, a growing interest developed amongst anatomists in cardiac anatomy, and specifically the discrete muscle bands that comprise the ventricles. The arrangement most commonly agreed upon up to the twentieth century closely resembles that defined by Mall and MacCallum (132). This model is characterized by superficial layers, including the bulbospiral band of the “bulbar” left heart (Figure 1.2 A) and the sinospiral band of the right heart (Figure 1.2 B). The deep layers include the sinospiral muscle encircling both ventricles with transverse fibres (Figure 1.2 C), and the bulbospiral muscle specific to the left ventricle (Figure 1.2 D)(173).   3   Figure 1.2. Anatomical structure and characterization of myocardial bands proposed by Mall and MacCallum (132)(images reproduced with © permission from (173)).  These structural models continued to evolve throughout the twentieth century. Rushmer et al. (176) described a model with three distinct and interconnected fibre layers which twisted into a vortex at the apex. This model proposed that the transverse constrictor muscle provides circumferential compression during ejection, and that the simultaneous contraction of inner and outer oblique muscles results in storage of potential energy during ejection. In 1957, anatomist Torrent-Guasp (210) unraveled the myocardium into a “simple” flattened rope-like structure extending from the pulmonary artery to the aorta. Similar to Rushmer’s model, Torrent-Guasp described a vortex at the apex, in which subepicardial fibres transitioned into subendocardial fibres (209). The unraveled myocardial band was characterized by a basal loop with left and right segments, and an apical loop with a descending endocardial segment and ascending epicardial segment (Figure 1.3). The apical loop comprised of conical or oblique helical fibres in a “figure of eight” configuration (81), whereas the basal loop comprised of circumferential fibres. Wrapping around both ventricles, this basal loop was proposed to act as an external “buttress” or supportive shell for the oblique apical segments (209).   4  Figure 1.3. Torrent-Guasp’s schematic of the ventricular myocardial band, showing the intact ventricle (A), separation of the pulmonary artery (B) and right free wall (C), and finally the unraveled band (D and E)(figure reproduced with © permission from (209)).  In 1969, Streeter et al. (199) fixed canine hearts in situ during systole and diastole, and determined fibre orientation at multiple sites of the LV free wall. This work established several key concepts of myocardial fibre orientation in the LV. First, at a constant fraction of wall thickness, fibre orientation was fairly constant along the longitudinal plane. Second, fibre angle was variable across the LV wall. Specifically, the myofibre helix angle (αhelix) continually transitioned from +90º at the endocardium, through 0º at the midwall, and to -90º at the epicardium (Figure 1.4). Third, during the transition from diastole to systole, there was a constant increase in αhelix angles across the LV wall.      5  Figure 1.4. Gradient of fibre angles across the canine LV wall, transitioning from +90º to -90º from the endocardium to epicardium, respectively (figure reproduced with © permission from (199)).  Similar experiments were later performed by Greenbaum et al. (81) in postmortem human hearts. In agreement with the models of Rushmer and Torrent-Guasp, Greenbaum et al. described an endo- to epicardial continuity of myofibres at apex, as well as a clear transition of fibre orientation across the surface of the subepicardium. They additionally observed that circumferential fibres were most prominent in the LV midwall, and thickest at the basal level.   More recently, the application of diffusion tensor imaging has allowed for the high-quality reconstruction of the LV myofibre architecture, and studies utilizing this technique have confirmed key elements of the earlier anatomical and histological models in the human heart. Data from Rohmer et al. (174)(Figure 1.5) provide a clear visualization of the twisted fibre   6 bundles described by Torrent-Guasp, and the smooth transition of fibre angles from +60º at the endocardium to -60º at the epicardium, similar to those described by Streeter and Greenbaum.    Figure 1.5. Visualization of LV architecture and myofibre orientations using diffusion tensor magnetic resonance imaging (figure reproduced with © permission from (174)).   1.2.2 Current understanding of LV myocardial architecture and mechanical deformation It is presently accepted that the LV’s myocardial architecture is characterized by a transmural continuum of fibres that transition from a right-handed (counterclockwise) helical orientation in the endocardium to a left-handed (clockwise) helical orientation in the epicardium (38, 148, 215). Transverse circumferential fibres additionally form a “buttress” or transverse shell around the ventricular helices (25). When myofibre groups with different orientations simultaneously contract, the resulting movement will occur in the direction of the more dominant helix (i.e. with a greater moment arm), or can be overpowered by the contraction of circumferential fibres (25, 205).   The structural arrangement of cardiac myocytes is supported by a complex extracellular scaffold with endomysial, perimysial and epimysial components (4). Myocytes themselves are long, thin mononucleated cells, and are joined together by intercalated disks. An endomysial layer or “weave” provides support to the individual myocytes, and additionally bundles groups of these cells together to form myofibres in the perimysium (4). In addition to providing structural integrity, the endomysial and perimysial weave attachments play an integral role in   7 accommodating shearing forces between myofibres, and transmitting contractile forces from the myocytes to the matrix and cardiac chambers (4, 139).  In the perimysium, adjacent myofibres are bound by collagen into laminar layers, or “sheets” (Figure 1.6). These myolaminar sheets are generally 4 cells thick, and slide alongside one another throughout the cardiac cycle (120, 185). The specific orientations of myofibres and laminar sheets may be quantified relative to different planes. The myofibre’s helix angle, αhelix, describes the angle between the local circumferential direction and the projection of the fibre path onto the circumferential-longitudinal plane. As outlined earlier in this section, αhelix in the human LV transitions continuously from +60º to -60º across the ventricular wall (174, 192). The myolaminar sheets are characterized by cleavage planes or “sheet angles” in the longitudinal-radial plane (ß’), which have been shown to vary across the LV wall and differ between the apical and basal regions (Figure 1.7 C) (38, 174).   The arrangement and orientation of LV myofibres and sheets provide the structural basis and support for LV deformation or mechanics throughout the cardiac cycle. During the transition from diastole to systole, αhelix increases as myofibres become more longitudinally oriented, whereas sheet angle ß’ become lesser in magnitude (Figure 1.7) (38, 63). These patterns of myofibre reorientation are directly responsible for wall thickening, circumferential and longitudinal shortening, and the generation of ventricular twist (38, 120). Therefore, any alterations to myofibre helix and sheet angles will impact myocardial deformation. This has been demonstrated as reductions to LV αhelix result in reductions LV twist and wall thickening for a given change to sheet angle (120).    8  Figure 1.6. Myocardial fibre orientations and structural organization across the LV wall. Long oval structures depict myofibres, joined by endomysial collagen and organized into laminar sheets in the perimysium (figure reproduced with © permission from (174)).   Figure 1.7. Left ventricular myofibre and sheet reorientation during systole. Alterations to helix angle (αhelix) (left) and sheet angle (ß’) (right; A and C depict diastole, B and D depict systole) contribute to myocardial deformation in the circumferential, radial and longitudinal planes (images reproduced with © permission from (38)).    9 The twisting motion of the LV allows for a uniform distribution of LV fibre stress and strain (fibre shortening) across the ventricular wall during systole, and therefore lowers myocardial oxygen demand (16, 186). The LV’s myofibre architecture specifically underpins systolic efficiency, and it has been demonstrated that a 13% decrease in myofibre length can produce a 50% increase in ventricular wall thickness (192). Diastolic efficiency is also enhanced by ventricular twist, as potential energy stored in the LV myocardial matrix during systole is released during diastole and generates the intraventricular pressure gradients in the early filling phase (168, 186).   1.2.3 Definitions of mechanical deformation  Rotation describes myocardial angular motion around a vertical axis (25). In the LV, rotation is conventionally expressed as being viewed from the apex, where clockwise and counterclockwise rotations occur in negative and positive degrees, respectively (185). During systole, rotation at the base is primarily clockwise, and rotation at the apex is primarily counterclockwise, and rotation at the base is substantially lower in magnitude than that of the apex (185).  Left ventricular twist describes the base-to-apex gradient in rotation along its longitudinal axis (25). Earlier literature has used the term “torsion” to describe the difference between apical and basal rotations; for that reason, “twist” and “torsion” have been used interchangeably in studies of LV mechanics (185). More recent clarification and standardization of these parameters have defined “LV twist” as the absolute base-to-apex difference in rotation angle in degrees, and “LV torsion” as the twist for a given LV length, expressed as degrees per centimeter (145).   While twist is a parameter that characterizes LV systolic function, measures related to ventricular untwisting have been used to assess diastolic function. Numerous parameters have been assessed to characterize diastolic untwisting in canine models, including peak untwisting velocity, intraventricular pressure gradient, the rate of pressure decay and the maximal change in pressure over time (153). Of these, peak untwisting velocity is most commonly used to characterize diastolic untwisting mechanics in humans as this may be measured non-invasively, and directly corresponds to the rapid fall in pressure during the isovolumic phase in both canines (153) and humans (152).    10 Myocardial strain or deformation is defined by the Lagrangian formula: 𝜀 𝑡 = 𝐿 𝑡 − 𝐿 𝑡!𝐿 𝑡!  where 𝜀 𝑡   is strain at instantaneous time t, L(t) is the length at instantaneous time t, and L(to) is the original length of the segment (25). Strain is positive or negative when the segment length exceeds or is less than its original length, respectively. Strain can be measured in the longitudinal, circumferential and radial axes. Based on the law of conservation of mass, shortening through the longitudinal and circumferential axes results in a relative thickening of the radial axis. However, it is important to note that LV strain (as commonly assessed using speckle tracking echocardiography) does not result from the simple shortening of myocytes in the longitudinal and circumferential planes, but is rather an effect of the shearing that occurs between laminar sheets of myocytes (185).   Because the assessment of global LV strain involves the measurement of shortening, narrowing or thickening within a single plane, ‘strain’ represents the net effect of shearing between myolaminar sheets of myocyctes, and the shortening of oblique, longitudinal and circumferential fibers (38). For example, LV twist during systole primarily results from the contraction of the oblique helices, and produces a shortening of the LV through its long axis and thus contributes to augmenting longitudinal strain. However, the responses or alterations to LV twist and strain can become uncoupled, where LV twist increases but strain is reduced, e.g. during large reductions to preload and LV filling (see section 1.5 for further detail). In those cases, reduced LV strain is not necessarily indicative of ‘dysfunction’ but is likely more reflective of altered loading.   1.2.4 Sequence of mechanical deformation Due to the regional heterogeneities of electrical activation and myocardial architecture, the mechanical sequence of activation in the LV is complex and specific to each phase of the cardiac cycle (185). It is important to recall that when the three myocardial layers (the oblique helices and transverse circumferential layer) contract simultaneously, the resulting deformation will depend on which movement dominates, and the circumferential wrap can overpower the movement of either helix (25, 205). The mechanical events of each phase are outlined in the following paragraphs, and illustrated in Figure 1.8.    11 During the isovolumic contraction phase, fibres of the circumferential muscle and endocardial right-handed helix shorten at the same time (25). The circumferential fibres dominate the movement and act as an outer shell, compressing the oblique helices. This results in lengthening and narrowing of the ventricle, and a rotation of the entire ventricle in a counterclockwise direction (25). The shortening of the right-handed endocardial helix produces a stretching of the left-handed epicardial helix, which is proposed to “load” the myocardium through the protein titin (a molecular “spring”) and produce a Starling effect for ejection (34, 185).  During ejection, circumferential fibres continue to shorten and co-contract with oblique fibres of the endocardial and epicardial helices (25). Although greater shortening occurs in the right-handed endocardial helix, a greater moment arm of the left-handed epicardial helix results in a counterclockwise rotation at the apex and clockwise rotation at the base (207). The shortening of both helices therefore causes shortening of the LV chamber, and constriction of the circumferential fibres leads to further narrowing of the ventricle. The contraction of circumferential fibres additionally acts as a “buttress” to counteract against forces from the twisting helical segments and essentially prevent an outward “explosion” at the base (25).   During the isovolumic relaxation phase, the LV begins to widen as circumferential fibres have stopped contracting and begin to recoil. The right-handed endocardial helix begins to relax, while the left-handed epicardial helix continues to contract (25, 184). Therefore while the base continues to rotate in a clockwise direction, the apex rapidly untwists or recoils in a clockwise direction, re-lengthens the ventricle, and generates an intraventricular pressure gradient for diastolic filling (149). It is important to note that the earlier studies describing “suction” or a vacuum effect from untwisting specifically refer to the negative pressures measured in canine models (101, 149, 200); however, this negative pressure isn’t always observed even in the canine heart (159), and may not occur in human hearts (7, 88, 201). Nonetheless, the term “suction” has been used to describe the reductions to LV filling pressures in the early filling phase, rather than a true negative pressure (153, 168). For clarity, this review will not associate the term “suction” with LV untwisting and early filling in humans.     12 Approximately 40-50% of untwisting occurs prior to mitral valve opening and the rapid filling phase (60, 168). After this point, further untwisting results from the relaxation or recoil of the left-handed subepicardial helix (25).    Figure 1.8. Sequence of regional myocardial deformation and rotation across the cardiac cycle. Markers to the right and left denote clockwise and counterclockwise rotations, respectively (as viewed from the apex) (figure reproduced with © permission from (26)).  1.3 Early investigations of LV mechanics  In addition to their work in LV anatomical modeling, Rushmer et al. (176) assessed LV myocardial deformation by tracking the movement of implanted markers in the canine ventricle using cinefluorographic imaging. The current thinking at that time was that the myofibre arrangement would produce a rotation of the LV chamber during systole. However, their data did not provide evidence for rotation about the longitudinal axis. The authors proposed that the simultaneous shortening of the inner oblique layers might have counteracted each other to result only in shortening through the long axis. Interestingly, although the authors reported that there were no indications of ventricular rotation, they actually noted a “slight rotation around the longitudinal axis” but state that “it could not have exceeded 10° of angular rotation” ((176), pp   13 166). This was, in fact, a realistic amplitude of rotation given that apical rotation routinely does not exceed 10° in the canine model (152).  It wasn’t until the 1970’s that LV twist mechanics were assessed in the human heart. At this time, cineangiography and cineradiography were used to track the movement of surgically implanted markers in patient populations (105, 140). McDonald (140) described a slight counterclockwise rotation and “descent” of the base toward the apex during the pre-ejection phase, followed by a clockwise rotation at the apex as the ventricle narrowed and shortened during systole. He postulated that rotation might result from a “sequential activation of the LV myocardium from the endocardial to epicardial surface.” Shortly after, Ingels et al. (105) noted that apical segments rotated opposite to basal segments, with little rotation in midventricular segments, producing a “wringing motion” of the LV during ejection.    Ingels’ group would become leaders of LV twist mechanics research in humans over the next few decades. They studied LV deformation in cardiac allograft recipients, by implanting helical tantalum wire coils at 12 specific sites in the donor hearts, 5 mm below the LV epicardial surface. In an initial study (87), they established twist angles at each of the 12 sites, and differentiated deformation among the basal, midventricular and apical levels of the anterior, posterior and inferior walls. They described non-uniform deformation angles across different regions of the myocardium, with the apical region having substantially greater rotation angles than the mid-level and base. These investigators also sought to determine the influences of inotropic stimulation, pressure loading and volume loading on LV twist (87, 88, 144). Increases to inotropic stimulation using dobutamine increased peak twist angles (87, 88) and early diastolic untwisting velocities (144). In contrast, both pressure and volume loading did not alter peak twist angles from baseline (87, 88). The authors concluded that peak twist was highly dependent on myocardial contractility, and independent of preload and afterload (88); however, it was acknowledged that these findings were limited to volume loading that raised filling pressures by 10-20 mmHg. Also, given that there were no alterations to LV ejection fraction (EF), cardiac output (Q) or stroke volume (SV) during pressure and volume loading, they could not entirely preclude the influence of altered loading on LV twist.     14 1.4 Influences of preload, afterload and contractility on LV mechanics: animal studies In their seminal study, Gibbons-Kroeker et al. (78) performed specific manipulations of loading parameters in an integrative in-vivo model using open-chest canines. Sonocrystals were used to measure apical rotation and midwall shortening, although the authors made a major assumption that apical rotation alone determined LV twist, with the argument that rotation at the base was minimal. Accordingly, “twist” data from this study represent measurements of apical rotation alone, and are not necessarily the true base-to-apex gradient in rotation. Alterations to preload were accomplished by occluding the vena cava and saline loading through the jugular vein. These interventions shifted end-diastolic and end-systolic volumes and pressures, but there was no change to “twist amplitude” which was assessed as the difference in end-diastolic to end-systolic apical rotation (Figure 1.9). However, when visualizing the changes to the pressure-apical rotation loops, peak systolic apical rotation appears to be increased during both increases and reductions to preload.   Figure 1.9. Alterations to LV pressure and apical rotation during volume infusion (left) and vena caval occlusion (right). “ED” indicates end-diastole, and “ES” indicated end-systole (figures reproduced with © permission from (78)).  In the same study, alterations to afterload were accomplished using single-beat occlusions of the aorta, with the advantage that they do not simultaneously alter preload (78). The occlusions led to reductions in twist amplitude due to reductions in end-systolic apical rotation. Epinephrine infusion and paired-pacing were additionally used to increase contractility with and without   15 altering heart rate (HR), respectively, and both interventions resulted in increases to LV twist amplitude. Finally, to investigate the effects of altered HR, atrial pacing was used to alter HR from 90-180 bpm. Increasing HR resulted in leftward shifting of the pressure-apical rotation relationships, with increases to peak apical rotation but reductions to twist amplitude.   In a similar study, Dong et al. (59) alternatively utilized a servopump to independently manipulate preload, afterload and contractility in a blood-perfused ejecting canine heart. These authors assessed LV twist with magnetic resonance imaging and tissue tagging. In their model, increases to preload increased LV end-diastolic volume (EDV) and end-systolic volume (ESV) but did not alter SV, EF or twist. Similarly, reductions to preload reduced EDV and ESV, but did not alter twist. The authors explained that the mixed effects without “pure” alterations to preload likely accounted for the unchanged twist. To address this point, they reported that with a constant LVESV, a greater EDV resulted in greater twist, demonstrating an effect of “pure” alterations to preload on LV twist. During alterations to afterload with a constant LVEDV, increases and decreases to LVESV resulted in reductions and increases to twist, respectively. Using multiple regression analysis, the authors demonstrated that the effects of preload on LV twist were approximately two-thirds as great as those of afterload. Finally, twist was augmented during inotropic stimulation with fixed LV volumes. These findings collectively highlight the load-dependency of LV twist, and demonstrate that inotropic state affects LV twist in a force-dependent but also volume-independent manner.  1.5 Influences of preload, afterload and contractility on LV mechanics: human studies In the early 2000’s, advancements in ultrasound technology allowed for the development of speckle tracking echocardiography (STE) as a newer, noninvasive method for quantifying LV rotation, twist and strain. This approach has been validated against magnetic resonance image tagging in healthy humans (93, 154) and sonicometry in canines during the resting state, dobutamine infusion and acute ischemia (93). Using STE, a number of studies over the past decade have examined the influence of alterations in preload, afterload and contractility on LV mechanics. Table 1.1 summarizes the population characteristics, interventions and key findings amongst investigations with young, healthy individuals.    16 1.5.1 LV mechanics during alterations to preload A variety of interventions have been utilized to determine the influence of preload on LV mechanics in humans. Weiner et al. (220) used saline infusion (1200mL·body surface area (BSA), administered over 2 hours) to augment LV preload in a mixed-sex cohort. Following infusion, the augmentations to LV volumes (EDV, ESV and SV) were accompanied by increases to LV apical rotation, twist and untwisting velocity, as well as increases in longitudinal and circumferential strain. These increases to twist and strain mechanics are likely reflective of a Frank-Starling effect, where a greater initial passive stretch of the myocardium produces a greater active shortening of the oblique, longitudinal and circumferential myofibers (220).   To date, the study by Weiner et al. (220) is the only study to investigate how LV mechanics change in response to increased preload. Other investigations have used dehydration (197) or lower body negative pressure (LBNP) (98, 99) to reduce preload. Both interventions were effective in reducing LV volumes, and resulted in increases to LV twist as well as reductions to longitudinal and circumferential strain. However, the increases to LV twist were attributed to increases in apical rotation during LBNP (98, 99), but reflected increases in basal rotation during dehydration (197). The increases to rotation exclusively at the base during dehydration are somewhat surprising, given that alterations to LV twist mechanics during acute stress are commonly more pronounced at the apex (2, 99, 152, 184). It is important to note that the alterations to LV twist in both interventions likely resulted in part from alterations to adrenergic stimulation (more detail in section 1.5.3). In the study by Stohr et al. (197), HR was elevated with increasing levels of dehydration, and it is likely that the preceding cycling exercise augmented sympathetic activity in their participants. Moreover, LBNP unloads the arterial and cardiopulmonary baroreceptors, and therefore results in augmented sympathetic activation. However, the reductions to longitudinal and circumferential strain observed in these studies (98, 99, 197) are not consistent with increases to adrenergic stimulation (see section 1.5.3 for further detail) and likely result from the reductions to venous return, rather than any impairment to intrinsic myocardial function (197). Although dehydration and LBNP clearly do not produce alterations to preload alone, the findings from these investigations demonstrate that both increases and reductions to preload result in increased LV twist and reduced strain amplitudes in young, healthy human participants.    17 1.5.2 LV mechanics during alterations to afterload Two studies have investigated the effects of altered afterload in healthy participants. To elicit large changes in blood pressure, Weiner et al. (221) had participants perform three minutes of isometric handgrip exercise at 40% of their maximum voluntary contraction (MVC). During the handgrip exercise, LVESV and SV were reduced, as were longitudinal strain, apical rotation and LV twist. The reductions to both twist and strain mechanics are likely reflective of reduced active fiber shortening in the oblique, longitudinal and circumferential myofibres. Although this intervention produced significant increases to afterload, it also produced increases in HR. Thus, Balmain et al. (11) sought to determine the effects of increased systemic afterload independent of increases in HR, by additionally examining the responses of LV mechanics during the activation of the muscle metaboreflex with post-exercise ischemia (PEI). During handgrip exercise identical to that used by Weiner et al. (221), LVSV, apical rotation and twist were reduced. Circumferential strain was reduced, however longitudinal strain was unaltered. During the subsequent bout of PEI, blood pressure was further augmented whereas HR declined to baseline levels. LV volumes (EDV, ESV and SV) were not different from baseline, but LV apical rotation, twist and untwisting velocity were further reduced from the handgrip exercise phase. Although Balmain et al. intended to remove the influence of increased HR, they did not account for the fact that both IHG and PEI result in increases in adrenergic stimulation (17, 50), and PEI has actually been shown to augment LVSV (49, 50). Moreover, increased adrenergic stimulation has been shown to increase LV apical rotation, twist and untwisting velocities (more detail in section 1.5.3), which entirely contradict the reductions to LV twist mechanics observed in Balmain’s study. Of course, it is possible that in that specific cohort of participants, the potential increases to SV and LV twist during PEI were countered by large elevations to LV afterload.  1.5.3 LV mechanics during alterations to adrenergic stimulation In the preceding sections it has become clear that interventions primarily aimed at altering preload or afterload often inherently result in alterations in adrenergic stimulation and LV contractility. For example, increasing levels of LBNP produce increases to HR, muscle sympathetic nerve activity (MSNA) and circulating catecholamines (43, 75, 113). During high-intensity isometric handgrip exercise or PEI, elevations to blood pressure, HR and systemic vascular resistance (11, 221) result from increased sympathetic activity to the heart and   18 vasculature (49, 50, 189). Consequently, it can be difficult to delineate the independent effects of preload and afterload from those related to an altered inotropic state.    Most investigations primarily aimed at altering adrenergic stimulation have been performed in canines (59, 153, 168) or in patient populations (12, 88, 144). One study by Akagawa et al. (2) specifically examined the effects of altered adrenergic stimulation on LV mechanics in healthy humans. Infusions of dobutamine at 5 µg·kg–1·min–1 and 10 µg·kg–1·min–1 produced increases to LVSV and LV twist, as well as increases to radial strain at the base and apex. While apical rotation and basal rotation were both augmented, apical rotation was increased to a greater extent than at the base (+6.3º versus +3.1º). Also, apical rotation became increased with 5 µg·kg–1·min–1 dobutamine, whereas basal rotation was only increased with 10 µg·kg–1·min–1 dobutamine. The greater responsiveness of twist mechanics at the apex compared to the base may be reflective of regional heterogeneities of adrenergic receptor densities, which are further discussed in section 1.8.2.  1.5.4 LV mechanics during dynamic exercise Dynamic exercise is a unique integrative model of acute stress that involves simultaneous alterations to preload, afterload and contractility, which ultimately contribute to changes in SV, and accordingly LV twist (59, 175). Both Doucende et al. (64) and Stohr et al. (198) have examined LV mechanics during incremental supine cycling exercise up to 40% and 90% of maximal exercising workload, respectively. Similar to the common plateau in SV ~50% of maximal exercise (8, 214), LV systolic and diastolic twist mechanics reach a plateau at moderate exercise intensities (64) without further increases at higher exercise intensities (198). The increase in LV twist reflects the augmentation of both basal and apical rotations (64, 198), although the increases to rotation at the apex exceed those at the base. This is similar to the observations of Akagawa et al. (2) where apical rotation increased to a greater degree than basal rotation during infusions of  5 µg·kg–1·min–1 and 10 µg·kg–1·min–1 dobutamine.   In parallel to the alterations to LV twist, diastolic untwisting velocity also progressively increases during exercise until a plateau is reached at ~50% of maximal workloads. The increases in peak untwisting velocity result from faster apical untwisting rather than basal   19 untwisting (198). In agreement with the concept that LV twist and untwisting are tightly coupled, there is a strong correlation between the increases in twist and untwist velocity that occur during dynamic exercise (64, 198). Given that untwisting produces the intraventricular pressure gradients for early diastolic filling, the increases in untwisting velocities likely help to increase or maintain EDV in the face of reduced filling time during exercise (64, 152). Moreover, the increases to LV twist and untwisting are proposed to reflect a “torsional reserve” during exercise (64), and the concomitant plateaus of LV twist and SV may be indicative of a mechanical limitation to increasing SV at higher exercise intensities (198).     20 Table 1.1. Effects of altered preload, afterload and adrenergic stimulation on LV mechanics in healthy human study populations. Reference  Primary challenge and intervention Population Key findings LV mechanics  LV hemodynamics and dimensions Notomi et al. 2006 (152) Submaximal exercise: at HR ~100 bpm Mixed sex n=20, 8 females; 34±7 yrs ↑ApRot (+10º) ↑BRot (+4º) ↑Twist (+13º) ↑Untwisting velocity (+200º/s)  ↑EDV  ↓ ESV ↑SV  ↑EF Akagawa et al. 2007 (2) Increased adrenergic stimulation:  dobutamine infusion  (5 and 10 µg·kg–1·min–1) “Normal volunteers” (no info on sex) n=13; 30±2.6 yrs During 10 µg·kg–1·min–1: ↑ApRot (+6.3º) ↑BRot (+3.1º) ↑Twist (+9.8º) During 10 µg·kg–1·min–1: ↑HR   ↔ EDV and SV   ↔ LVIDd, ↓LVIDs ↑Fractional shortening Doucende et al. 2010 (64) Incremental exercise: 20%, 30% and 40% peak power Sedentary males n=20; 25±9 yrs Up to 40% peak power: ↑ApRot (+4º) ↑BRot (+6º) ↑Twist (+5.3º)  ↑Untwisting velocity (+94º/sec) ↑CS (+5%) ↑LS (+5%) ↑EDV at 20% only, then plateau  ↓ ESV at 20%, 30% and 40% ↑SV at 20% only, then plateau  Weiner et al. 2010 (220) Increased preload: saline infusion  (2.1±0.3 L) over 2 hours Mixed sex  n=8, 6 females;  25±3 yrs ↑ApRot (+3.0º)  ↔ BRot ↑Twist (+3.7º) ↑Untwisting velocity (+65º/sec) ↑LS (+2.4%) and CS (+3.5%)  ↔ HR, SBP and DBP ↑EDV, ESV and SV ↑LVIDd, ↔ LV length ↑Q  ↔ EF Hodt et al. 2011 & 2015 (98, 99) Decreased preload: LBNP  (-20 and -40 mmHg) Males n=12; 22±3 yrs ↑ApRot (+3.5º)  ↔ BRot ↑ Twist (+4.2º) ↑Untwisting velocity (+45º/sec)  ↓ LS (-2.5% at mid LV)  ↔ CS (-2.7% at base, -2.2% at apex) ↑HR  ↓ EDV, ESV and SV   ↓ Q and EF  Stohr et al. 2011 (197) Decreased preload: dehydration  (2% and 3.5%)  Active males  n=8; 20±2 yrs During 3.5% dehydration: ↑BRot (+4.2º)  ↔ ApRot ↑Twist (+5.9º)  ↔ LS and CS  ↔ Untwisting velocity  During 3.5% dehydration:  ↑HR  ↓ EDV, ESV and SV  ↔ Q and EF     21 Table 1.1 (cont). Effects of altered preload, afterload and adrenergic stimulation on LV mechanics in healthy human study populations. Reference  Primary challenge and intervention Population Key findings LV mechanics  LV hemodynamics and dimensions Stohr et al. 2011 (198) Incremental exercise: up to 90% peak power Recreationally active males  n=9; 26±4 yrs Up to 50% peak power: ↑BRot (+2.1º) ↑ApRot (+8.3º) ↑Twist (+9.9º) ↑Untwisting velocity (+170º/sec) ↑EDV to 50%, then plateau  ↓ ESV to 50%, then plateau ↑SV to ~50%, then plateau Progressive↑HR, Q and MAP Weiner et al. 2012 (221) Increased afterload: isometric handgrip exercise  (3 minutes, 40% MVC) Mixed sex n=18, 3 females; 29.7±2.7 yrs  ↓ ApRot (-3.3º)  ↔ BRot  ↓ Twist (-4.1º)  ↓ LS (-2.4%) ↔ Untwisting velocity ↑SBP and DBP  ↑HR  ↔ EDV ↑ESV and SV   ↓ EF Balmain et al. 2016 (11) Increased afterload: isometric handgrip exercise  (3 minutes, 40% MVC), then post-exercise ischemia (3 minutes) Males n=19; 23±2 yrs Isometric handgrip exercise (vs. rest):  ↓ ApRot (-1.5º)  ↔ BRot  ↓ Twist (-2º)  ↔ Untwisting velocity  ↓ CS (-3%)  ↔ LS Isometric handgrip exercise (vs. rest): ↑SBP, DBP and MAP  ↑HR  ↔ EDV and ESV  ↓ SV ↑Q Post-exercise ischemia (vs. rest):  ↓ ApRot (-4º)   ↔ BRot  ↓ Twist (-2.5º)  ↓ Untwisting velocity (-20º/s) ↔ CS and LS Post-exercise ischemia (vs. rest): ↑DBP, SBP, MAP and SVR  ↔ HR  ↔ EDV and ESV  ↔ SV and Q LV mechanics data represent peaks across the cardiac cycle. Alterations to LV mechanics are estimated from mean data in respective publications, and represent absolute magnitude of change.↑ increase; ↓ decrease; ↔ no change; LV: left ventricle; MVC: maximal voluntary contraction; SBP: systolic blood pressure; DBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; EDV; end-diastolic volume; ESV: end-systolic volume; SV: stroke volume; Q: cardiac output; EF: ejection fraction; LVIDd: LV internal diastolic diameter; LVIDs: LV internal systolic diameter; ApRot: apical rotation; BRot: basal rotation; LS: longitudinal strain; CS: circumferential strain.    22 1.6 Sex differences in LV mechanics Although LV mechanics have been relatively well characterized in healthy mixed-sex cohorts across various age ranges (162, 203, 204, 206, 231, 233), little data exist comparing LV mechanics between males and females. A few studies have reported greater longitudinal and circumferential strain in females compared to males at rest (9, 103, 119). Foll et al. (72) have also reported a reduced “systolic twist” in females compared to males. However, twist was measured in cm/s and therefore represented an assessment of twist velocity, not twist amplitude.  While studies have investigated sex differences in LV hemodynamics during challenges to preload (45, 73, 188, 224), alterations to adrenergic stimulation (36, 68, 106, 211), and dynamic exercise (86, 94, 157, 202, 223), sex-related differences in LV twist mechanics during these stressors have not been determined. Given the important role of LV mechanics in supporting efficient LV systolic and diastolic function, sex differences in LV rotation, twist and strain likely coincide with the known differences in LV hemodynamics during periods of acute physiological stress.   1.7 Sex differences in LV structure and function at rest 1.7.1 Sex differences in LV mass and structure  The female heart is most commonly smaller than that of males, and this difference is primarily attributed to large differences in body size between the sexes (108, 179). Even so, LV internal dimensions, mass and volumes often remain larger in males when controlling for differences in body size, indicating that factors other than body size likely contribute to sex differences in cardiac morphology. Table 1.2 outlines the key differences in LV mass, volumes and structure between the sexes.     23 Table 1.2. Comparison of cardiac structure and volumes at rest in males and females  Reference Pop. LV Mass LVIDd Wall Thickness LVEDV LVESV Structure and Geometry Abs Idx Abs Idx Abs Idx Abs Idx Abs Idx Chung et al. 2006 (41) n=2618 44±9 yrs M>F M>F     M>F M>F M>F M>F Concentricity (LVMass/LVEDV) M>F Sandstede et al. 2000 (179) n=18 30±7 yrs M>F M>F     M>F M>F M>F M>F  Cain et al. 2009  (33) n=76 45±16 yrs       M>F M>F M>F M>F  Marcus et al. 1999 (134) n=61 22±2 yrs M>F M>F     M>F M>F    Daimon et al. 2011 (51) n=700 44±14 yrs M>F M>F     M>F M>F M>F M>F  Kaku et al. 2011 (108) n=61 20-39 yrs M>F M>F     M>F M=F M>F M>F Ellipsoid (LVEDV/ spherical volume) M<F Claessens et al. 2006 (42) n=291 38-48 yrs M>F M>F   M>F  M>F    Relative wall thickness M>F Celeteno et al. 2003 (37) n=243 35±12 yrs M>F M>F M>F  M>F  M>F M>F M>F M>F Relative wall thickness M>F Hutchinson et al. 1991  (104) n=31 29±3 yrs M>F  M>F  M>F  M>F  M=F   M=F: no difference between males and females; M>F: greater in males than females; M<F: greater in females than males; Pop: population; LV: left ventricle; LVIDd: LV end-diastolic internal diameter; LVEDV: LV end-diastolic volume; LVESV: LV end-systolic volume; RWT: relative wall thickness; LVM: LV mass; Abs: absolute; Idx: index.   24 Compared to females, males have greater LV mass, internal dimensions, wall thickness and volumes, both as absolute measures and when indexed to BSA. Differences in LV geometry, specifically the shape of the LV chamber, have also been reported. Sphericity index is commonly determined as the ratio of LV length (long-axis) to the LV internal diameter (short-axis) at end-diastole (92, 212), or vice versa (83). Kaku et al. (108) assessed LV geometry as the ratio of LVEDV to the spherical volume (4/3 ∙ π ∙ ((long  axis  diameter)/2)!) of the ventricle, and reported an augmented ratio in males, suggestive of a more spherical LV geometry in males compared to females.   1.7.2 Sex differences in global LV systolic and diastolic function and heart rate. Table 1.3 outlines the comparisons of global LV systolic function and hemodynamics between the sexes. Although LV volumes are greater in males, females have a greater EF at rest than males (33, 37, 41, 42, 108, 134, 141, 179), and thus a larger SV for a given EDV. This provides evidence for sex differences in intrinsic LV function that are independent of differences in LV size (41). Additionally, some studies have reported higher resting HR in females (13, 37, 41, 44, 170), although these differences are not always observed (179).  Most investigations in Table 1.3 have demonstrated a greater LVSV in males, however these sex differences are less common when LVSV is indexed to body size (33, 37, 108). In a study by Celentano et al. (37), SV indexed to BSA was not different between the sexes, yet females had a greater LVEF, fractional shortening (ratio of LV end-diastolic to end-systolic diameter), and midwall shortening. The differences in fractional shortening and midwall shortening remained when corrected for circumferential end-systolic stress, an index of afterload. Thus, women appear to have “higher” systolic function in the rested state, which could result in a lowered functional reserve for females during acute changes in homeostasis (227).    25 Table 1.3. Comparison of global LV function at rest in males and females  Reference Pop. LVSV LVEF HR  Cardiac Output Abs Idx Abs Idx Chung et al. 2006  (41) n=2618 44±9 yrs   M<F M<F M>F M=F Sandstede et al. 2000  (179) n=18 30±7 yrs   M<F M=F M=F M=F Cain et al. 2009 (33) n=76 45±16 yrs M>F M=F M<F    Marcus et al. 1999 (134) n=76 45±16 yrs M>F M>F M<F  M>F M=F Daimon et al. 2011(51) n=61 22±2 yrs M>F M>F     Kaku et al. 2011  (108) n=700 44±14 yrs M=F M=F M<F    Claessens et al. 2006 (42) n=61 20-39 yrs M>F  M<F    Celentano et al. 2003 (37) n=243 35±12 yrs M>F M=F M<F M<F   M=F: no difference between males and females; M>F: greater in males than females; M<F: greater in females than males; Pop: population; LV: left ventricle; LVSV: LV stroke volume; LVEF: LV ejection fraction: Abs: absolute; Idx: index.  Doppler ultrasound assessment of LV inflow and tissue velocities are commonly used to assess diastolic and systolic function. Studies comparing these measures have observed greater indices of diastolic filling and relaxation in females, including higher early diastolic filling velocities (E) (51, 100), early diastolic tissue velocities (E’) (51), and greater ratios of early diastolic to atrial diastolic filling (A) and tissue (A’) velocities (E/A and E’/A’) compared to males (51). Conversely, a report by Holland et al. (100) observed greater systolic tissue velocities in males than females, but these sex differences disappeared when velocities were corrected for LV length. The larger early diastolic tissue and filling velocities are indicative of greater diastolic function and enhanced myocardial relaxation kinetics in females compared to males. More recently, work using magnetic resonance tissue phase mapping has demonstrated greater peak diastolic long-axis velocities in females (+11%) compared to males (72). Peak long-axis velocities during the isovolumic relaxation period precede LV filling, and are highly indicative of LV relaxation and early diastolic pressure decay (153) providing further evidence for superior diastolic function and myocardial relaxation compared to males.     26 1.8 Sex differences in LV responses to acute physiological stress 1.8.1 LV responses to acute preload challenges 1.8.1.1 General hemodynamic responses to central hypovolemia Reductions to central venous pressure and cardiac filling occur during orthostatic challenges such as head up tilt or simulated hemorrhage using LBNP. These challenges sequester blood toward the lower limbs and thorax, effectively reducing central venous pressure, venous return and consequently LV preload (46). The alterations to hemodynamics during central hypovolemia commonly involve reductions to LVSV and increases to HR, as well as increases to total peripheral resistance (TPR) that support mean arterial pressure (MAP) even in the face of a lowered Q (45, 113, 196). Orthostatic intolerance can be characterized by signs of presyncope (large reductions to systolic blood pressure, symptoms of lightheadedness, nausea, diaphoresis), and usually occurs with severe reductions to preload during high levels or sustained periods or LBNP (46). This intolerance is thought to reflect the individual or combined contributions of increased lower limb compliance (130), reduced blood volume (44), impaired baroreflex function (47), and decreased EDV and filling pressures, which ultimately result in large reductions to SV and Q (123).   1.8.1.2 Sex differences in the LV responses to central hypovolemia Females often have lower tolerance to central hypovolemia than males, potentially reflecting differences in any or all of the mechanisms outlined above (section 1.8.1.1) (45, 73, 224). Fu et al. (73) determined sex differences in the hemodynamic regulation of cardiac filling during acute preload challenges with LBNP and demonstrated that females had greater increases to HR, but lower absolute and indexed SV at -60 mmHg. The authors additionally matched a subgroup of males and females for baseline LVSV, and found that females still had greater reductions to SV and proportionally larger increases to HR. Using pulmonary capillary wedge pressure (PCWP) to estimate LV end-diastolic pressure, the authors were able to compare the Frank-Starling relationships between the sexes (Figure 1.10 A). Cardiac filling was reduced with LBNP and increased with a rapid infusion of saline (15 and 30 ml/kg, infused ~100ml/min), and maximal slopes of the curves were steeper in females than males (∆SV/∆PCWP was 12.5±2.0 and 7.1±1.5   27 ml/mmHg in females and males, respectively; ∆stroke index/∆PCWP was 6.3±0.8 and 4.3±0.6 ml/m2/mmHg in females and males, respectively). Although females had more rapid reductions to wedge pressure at lower levels of LBNP, sex differences in wedge pressure did not occur during higher levels of LBNP (Figure 1.10 B). These findings support that females have greater reductions to SV compared to males, despite similar reductions to wedge pressure (and presumably LV preload) from baseline to -60 mmHg. Additional support for a steeper Frank-Starling relationship in females is provided by later investigations demonstrating an upward- and left-shifted relationship of the pressure-volume relationship (wedge pressure to indexed LVEDV) in females compared to males (102, 165).   Figure 1.10. (A) Frank-Starling curves for males and females, relating pulmonary capillary wedge pressure (estimate of LV end-diastolic pressure) to absolute (upper panel) and indexed (lower panel) stroke volume. (B) Reductions to wedge pressure in males and females during progressive LBNP (figures reproduced with © permission from (73)).      28 Fu et al. (73) suggested that the relative tachycardia in females during LBNP might be indicative of a reflex response to a smaller SV rather than a strategy to maintain MAP. In the subset of males and females matched for baseline SV, females had a lower EDV and therefore a smaller LV. With respect to the steeper slope of the Frank-Starling relationship observed in females and previous reports of lower diastolic compliance in females (91), Fu et al. proposed that the smaller “less distensible” female LV could be more sensitive to fluid shifts, and potentially unable to further reduce ESV and maintain SV during acute preload challenges. In other words, females may have a lower functional reserve for SV compared to males. This postulate is supported by studies reporting greater reductions to SV (230) and lower SV ‘reserve’ in females during central hypovolemia (35).   The augmented chronotropic response in females during acute reductions to preload has been proposed to reflect greater vagal withdrawal, whereas males may have greater sympathetically-mediated peripheral vascular responses to maintain blood pressure (45). A greater sympathetic response to central hypovolemia is supported by observations of greater increases to circulating catecholamines (45, 73, 75) and MSNA burst amplitude in males (188). However, the alterations to MSNA burst frequency and total activity have been reported not to differ between the sexes (35, 188), and in some cases may be elevated in females compared to males (75, 188). Similarly, the increases to peripheral vascular resistance have been found to be similar between the sexes (73, 230) or augmented in females (45, 84), thus the evidence for sex differences in the sympathetic and peripheral responses to central hypovolemia is not definitive. Nonetheless, the reductions to Q during central hypovolemia do not appear to differ between the sexes (75, 224, 230), indicating that females may utilize greater increases to HR to counterbalance a smaller SV, and ultimately maintain Q similar to males. Sex differences in the vagal autonomic control of the heart may therefore contribute to the reported differences in ventricular hemodynamics during acute preload challenges (73, 188).    29 1.8.1.3 Implications for sex differences in LV mechanics during acute preload challenges  The literature discussed in sections 1.4 and 1.5 highlights the load-dependent characteristics of LV mechanics. In humans, both increases and reductions to preload result in augmented LV twist mechanics (98, 99, 197, 220). In the current section, it has been established that females commonly have greater reductions to SV and increases to HR than males during acute reductions to preload. With a steeper slope of the Frank-Starling relationship, females may have a lower functional reserve to cope during large reductions to LV preload (73, 230). Thus, potentially greater increases to contractility and chronotropy may be required in females to maintain Q and MAP during preload challenges, and contribute to greater LV mechanics compared to males.   1.8.2 Alterations to adrenergic stimulation  1.8.2.1 General autonomic and adrenergic control of the heart The autonomic control of the heart is shared between the parasympathetic (vagal) and sympathetic branches of the autonomic nervous system. Fibres from the vagus nerve release acetylcholine, which binds to postjunctional muscarinic (M2) receptors results in reductions to sinoatrial node firing (chronotropy) (124), atrioventricular nodal conduction (dromotropy) (167) and myocardial contractility (inotropy) (114). Fibres from the sympathetic branch release norepinephrine, which primarily binds to ß1 adrenergic receptors (ß1-ARs) in the nodal tissues, conducting tissues and myocardium (114). The activation of these receptors results in increases to chronotropy, dromotropy and inotropy. ß2-ARs also exist in the heart and may be bound by both norepinephrine and epinephrine (107).   There is considerable regional heterogeneity of cardiac innervation and receptor densities. First, histochemical work in autopsied human hearts has identified greater vagal (~7:1) and sympathetic (~1.5:1) innervation in the atrium versus ventricle, and greater vagal (~2:1) and sympathetic innervation (~1.5:1) in the LV base versus apex (Figure 1.11) (110, 222). Second, while the numbers of ß-ARs are fairly comparable amongst the human atrial and ventricular tissues (22), greater ß-AR densities have been identified at   30 the apex compared to the base in an animal model (146). Potentially similar distributions in the human LV might contribute to the augmented mechanical responses at the apex during increases to adrenergic stimulation (outlined in section 1.5.3) (2, 152, 198). Finally, in human hearts, the numbers of M2 receptors are ~2.5 times greater in the atria compared to the ventricles (58). Base-to-apex variations in vagal innervation and M2 receptors have not been established in the human LV, but Hanckock et al. (85) have demonstrated in an animal model that cholinergic innervation was generally limited to the basal area, and low densities of M2 receptors occurred throughout the ventricular myocardium.    Figure 1.11. Regional distributions of autonomic nerve efferents (left), and adrenergic and muscarinic receptors (right). TH-positive: sympathetic nerve efferent; AChE-positive: vagal nerve efferent; M2: muscarinic receptor. Based on data from (23, 58, 85, 110, 111, 147, 222).  1.8.2.2 Sex differences in the autonomic control of the heart While differences in receptor densities and distributions have not been determined in the males and female hearts, there is evidence for sex differences in cardiac autonomic control. For example, measures of heart rate variability (HRV) have commonly been used to compare indices of sympathovagal balance between the sexes (143). High frequency (HF) variability is reflective of respiratory sinus arrhythmia and vagal tone (71), whereas low frequency (LF) variability is jointly influenced by vagal and sympathetic control   31 (15). The ratio of LF/HF is believed to estimate the relative contribution of sympathetic modulation, or the overall balance of sympathetic-to-parasympathetic control of the heart (54, 133, 143). The vast majority of sex difference studies measuring HRV have reported greater LF indices in males (13, 82, 116, 177) and greater HF indices in females (13, 82, 170), resulting in an overall greater LF/HF ratio in males compared to females in the resting state. These differences persist even when data are adjusted for differences in basal HR, which is often higher in females (170). Estrogen is suggested to contribute to the vagal dominance observed in females (65), as sex differences in HRV and higher HF indices in females do not occur following menopause  (116). To determine the influence of estrogen status on HRV in women, Liu et al. (127) compared HRV amongst premenopausal women, postmenopausal women with and without hormone replacement therapy, and males. In agreement with other reports, premenopausal women had greater HF indices, lower LF indices and reduced LF/HF ratio compared to men, whereas HRV in postmenopausal women without hormone therapy was not different from males. When comparing postmenopausal women with and without hormone therapy, those receiving therapy had lower LF and greater HF indices than those without, and were not significantly different from the premenopausal women supporting that estrogen likely facilitates greater vagal and reduced sympathetic modulation of HR in females.  1.8.2.3 Sex differences the LV responses to adrenergic and muscarinic stimulation Pharmacological receptor agonists and antagonists are commonly used to investigate the influence of specific adrenergic and muscarinic receptors and their related pathways on cardiac function. The simultaneous administration of propranolol (ß-AR blockade) and atropine (muscarinic receptor blockade) has been used to determine potential sex differences in the autonomic control of the sinus node. Double blockade attenuates the transduction of both sympathetic and cholinergic signaling, and results in increases to HR and LF/HF from baseline (30). While the changes in HR do not differ between the sexes, the differences in LF/HF that occur at baseline are abolished (30). These data support the notion of predominant parasympathetic control in females, and indicate that sex differences in HR aren’t specifically related to intrinsic differences in properties of the sinus node (30).    32  Investigations utilizing pharmacological receptor antagonists have identified some sex differences in the influences of sympathetic and vagal control on cardiac function, however the findings from these studies are not always in agreement. During ß-AR blockade, some reports have demonstrated larger reductions to HR in females (69), while others have reported no differences between the sexes (36, 79). On the other hand, during graded increases in ß1-AR stimulation (i.e. administration of dobutamine or isoproterenol), females have greater slopes of the increase in HR (45, 183), whereas males have larger increases to indexes of contractility (183, 211). Thus, while sex differences during ß-AR blockade aren’t always observed, the majority of these data provide an indication that males have greater positive inotropic responses, and females have greater positive chronotropic responses to β1-AR stimulation.   During the administration of atropine (vagal blockade), the alterations to LV dimensions, fractional shortening and contractility index appear to be comparable between the sexes, and SV is reduced to a similar extent in males and females (69). However, females have been reported to have larger increases to HR during vagal blockade (211). Along with the higher HF indices of HRV (outlined in section 1.8.2.2), this observation further supports the contention that females have a greater vagal control of the heart compared to males.   1.8.2.4 Implications for sex differences in LV mechanics during altered adrenergic stimulation The findings from studies measuring HRV and utilizing pharmacological agonists or antagonists provide strong evidence for sex differences in the autonomic control of the heart and LV responses to adrenergic stimulation. Males appear to have greater sympathetic control of the heart, and enhanced inotropic responses to ß1-AR stimulation. In contrast, females appear to be more vagally-mediated, and commonly have more pronounced chronotropic responses to altered adrenergic or muscarinic stimulation. As outlined in sections 1.4 and 1.5.3, increases and reductions to adrenergic stimulation augment and attenuate LV twist, and to a greater extent at the apex than at the base (2, 59, 153). Therefore, if males have more pronounced alterations to LV contractility during   33 alterations to adrenergic stimulation, it is plausible that males will also have greater alterations to LV mechanics compared to females.    1.8.3 LV responses during isometric handgrip exercise and post-exercise ischemia 1.8.3.1 General sympathetic and hemodynamic responses to post-exercise ischemia The muscle metaboreflex involves the isolated activation of metabolically sensitive skeletal muscle afferents, using PEI following handgrip exercise (135, 217). Group IV muscle afferents are highly sensitive to chemical stimulation which occurs with the accumulation of muscle metabolites, altered muscle pH or chemical milieu (109). Increases in MSNA during rhythmic handgrip exercise and PEI have a strong inverse relationship with forearm muscle pH, providing strong evidence that reflex sympathetic activation is tightly coupled to the accumulation of hydrogen ions in the working muscle (216). The metaboreflex is a unique physiological response in which exercise-induced elevations in MAP and MSNA are maintained but HR returns to resting values (135, 217). The return of HR to baseline occurs due to an enhancement of vagal tone, which balances the increased sympathetic activity during metaboreflex activation (151, 156). However, HR may be augmented compared to baseline if the preceding handgrip exercise produces robust muscle activation and sympathetic activation (i.e. when performing handgrip at >40% MVC) (70).  Although vagal control dominates chronotropy during PEI (151), the elevated sympathetic activation is capable of modulating LV contractility (49). This is evidenced by Crisafulli et al. (49, 50) who observed increases in SV to ~130% of baseline values during PEI in males, even though HR had returned to baseline levels. In females, Shoemaker et al. (189) examined the metaboreflex response, using 2 minutes of isometric handgrip exercise at 40% of MVC followed by 2 minutes of PEI. During the exercise, Q, SV, HR, MAP and MSNA were elevated, although only MAP and MSNA remained elevated during PEI, and SV tended to be elevated in comparison to baseline. These findings demonstrate that metaboreflex activation is capable of augmenting adrenergic stimulation and LV contractility in both sexes, without significant alterations to HR.      34 1.8.3.2 Sex differences in the autonomic and metabolic responses to post-exercise ischemia For the same relative intensity and duration of handgrip exercise, males have greater increases to blood pressure than females, and greater increases to MSNA during PEI following higher handgrip exercise intensities (40% MVC) (68, 106). This has been attributed to greater accumulation of metabolites and lower pH in the occluded muscle, resulting in greater metaboreceptor stimulation. Differences in muscle mass between the sexes do not seem to contribute to the sex-differences in these neurohormonal responses, as males have greater increases to MSNA and MAP during fatiguing handgrip exercise with the adductor pollicis muscle, which has a similar MVC and muscle mass in males and females (68).   Jarvis et al. (106) used a unique approach to match the “metabolic end-point” between the sexes by having participants perform static handgrip exercise at 40% of MVC and occluding at the point of muscular fatigue (force <80% of target for >2s). Despite the larger MVC in males, time to fatigue was similar between the sexes. During PEI, HR returned to baseline levels in both groups, but males had greater blood pressure and MSNA responses than females. There were no differences in MSNA responses between the sexes during the cold pressor test, suggesting that the sex differences during PEI are not related to differences in the central integration and efferent signaling of vasomotor processes.   1.8.3.3 Implications for sex differences in LV mechanics during post-exercise ischemia The literature presented in section 1.8.2 provides evidence for sex differences in the autonomic and adrenergic control of the heart. Specifically, LV function appears to be influenced to a greater extent by sympathetic autonomic control in males, and may also be more sensitive to altered ß1-AR stimulation in comparison to females (183, 211). As it is well-established that LV mechanics are strongly influenced by adrenergic stimulation (2, 153), males might be expected to have larger increases to LV twist and strain than females during increases to adrenergic stimulation with PEI.     35 1.8.4 LV responses during dynamic exercise  1.8.4.1 General alterations to LV hemodynamics during dynamic exercise During dynamic exercise, increases in Q are achieved by increases to SV and HR (175). Stroke volume is augmented by increases to EDV (increased preload) and reductions to ESV (increased contractility). The increases to venous return and LV preload are primarily determined by adjustments to the peripheral circulation, including the mechanical effects of the muscle and respiratory pumps which displace peripheral vascular volume back to the heart, as well as the increases to sympathetic neural activity which produce constriction to peripheral vascular beds and augment central venous pressure (175). Heart rate is primarily elevated by dynamic alterations to sympathovagal balance, wherein the reductions to vagal drive are reciprocated by increasing sympathetic drive (225). A continuum of sympathovagal balance occurs with increasing HR, and it has been demonstrated that both branches make equal contributions (i.e. 1:1 ratio) at HRs ~140 beats per minute (225). While HR continually increases until maximum workloads are achieved, LVSV commonly reaches a plateau at ~40-50% of VO2max (8, 214).   1.8.4.2 Sex differences in the LV responses during dynamic exercise Early radionucleotide studies using both supine (1, 86) and upright (94, 141) cycling exercise have reported greater increases to EF in males, both when resting EF was similar between the sexes (1, 95) or elevated in females (86, 141). These observations provided preliminary evidence for a greater systolic functional reserve in males compared to females during dynamic exercise. Additionally, while LVSV, HR and Q were reported to increase similarly in males and females (1), SV appeared to be augmented by different means between the sexes. From rest to peak exercise, Hanley et al. (86) and Higginbotham et al. (94) reported that males had either small increases or no change to LVEDV, whereas females had 20-30% increases in EDV at lower exercise intensities that were maintained to peak exercise.   Other investigations have focused to a greater extent on the LV responses to stepwise and constant-load upright exercise. In males and females with similar relative maximal rates of O2 consumption (VO2max), Sullivan et al. (202) observed greater HRs in females but   36 similar Q between the sexes for a given absolute work rate. The increases to SV index were similar in both sexes and reached a plateau at 50% of VO2peak. However, the increases to SV were entirely due to increases in EDV in both sexes, as ESV was slightly increased compared to baseline. Additionally, while EF at rest tended to be higher in females, there were no sex differences in the increases to EF during exercise. Ogawa et al. (157) also reported a plateau in SV at 50% of VO2max in both sexes, but there were no differences in HR, SV index or Q index at 25% or 50% VO2max. Recently, Wheatley et al. (223) reported a trend of greater HR in females during exercise at 40% of peak workload, and a greater SV index and Q index in males. Comparisons amongst the findings of these studies are difficult given differences in the exercise modality (supine vs. upright cycling), intensities (peak exercise vs. moderate intensities), study design (incremental vs. constant-load vs. stepwise exercise) and measurement techniques (radionucleotide vs. acetylene rebreathe). Despite these methodological considerations, and a lack of consensus regarding sex differences in hemodynamics responses to exercise, the findings from these studies highlight the potential for sex differences in filling and ejection patterns. It therefore seems plausible that the adjustments to Q and SV during dynamic exercise are achieved through different mechanisms in males and females.  1.8.4.3 Implications for sex differences in LV mechanics during dynamic exercise It has been demonstrated that LV apical and basal rotation, twist and untwisting velocity are increased with increasing exercise intensities up to ~50% peak power (64, 197), at which point LVEDV and SV also reach a plateau. The increases to LV twist therefore appear to mechanically support the increases to SV during exercise. In the current section, the hemodynamic responses to dynamic exercise were compared between the sexes, and the patterns of alterations to LV hemodynamics may differ between males and females(86, 94, 202). Additionally, females may have greater increases to HR for a given submaximal workload (202, 223). Provided the well-established link between alterations to LV volumes and twist (59), and a proposed functional link between HR and LV twist (198), it is feasible that sex differences in LV twist mechanics occur during submaximal exercise.     37 1.9 Specific aims and hypotheses The literature discussed in this review has provided a significant body of evidence to suggest that sex differences in LV mechanics exist when the heart is challenged during periods of acute physiological stress. Therefore, the three experimental chapters of this thesis will aim to assess the potential sex differences in LV mechanics during alterations to preload, altered adrenergic stimulation and during dynamic exercise.   Chapter 3: Sex differences in LV mechanics during acute alterations to preload Aim:  To investigate the sex differences in LV mechanics in response to acute challenges to preload.  Hypothesis: During decreases and increases to preload, females will have greater LV apical rotation and twist than males, and females will have greater longitudinal strain and circumferential strain than males.  Chapter 4: The influence of adrenergic stimulation on sex differences in LV mechanics Aim: To investigate sex differences in LV mechanics with altered adrenergic stimulation, using activation of the muscle metaboreflex with post-exercise ischemia and β1-AR blockade (bisoprolol) to augment and attenuate adrenergic stimulation, respectively.  Hypotheses:  1. During increases to adrenergic stimulation, LV twist and untwisting velocity will be lower in females than males. 2. During reductions to adrenergic stimulation, females will have greater twist and faster untwisting than males.  Chapter 5: The influence of vagal control on sex differences in left ventricular mechanics during alterations to stroke volume Aim: To determine the sex differences in LV twist mechanics during decreases and increases to SV, with and without vagal muscarinic blockade.      38 Hypotheses:  1. During -60 mmHg LBNP and exercise at 50% of maximal workload, females will have larger LV twist than males. 2. Sex differences in LV twist will be minimized during -60 mmHg LBNP and 50% exercise during vagal blockade with glycopyrrolate.     39 Chapter 2. Techniques for Measurement of Cardiac Structure, Global Ventricular Function And Left Ventricular Mechanics  2.1 Ultrasound Technology 2.1.1 Basic principles Echocardiography is a form of ultrasound imaging used for the non-invasive assessment of cardiac structure and function. Ultrasound is a form of sound wave which is compressed and decompressed as it travels through a transmitting medium (166). Because ultrasound is propagated at frequencies higher than audible sound, these waves move in more of a straight line and therefore can be reflected and focused similar to beams of light (166). When the emitted ultrasound is perpendicular to a specular interface (smooth, e.g. pericardium), most of the ultrasound signal will be reflected back along its initial path to produce strong “echoes” (Figure 2.1). However, when the ultrasound meets a non-specular interface (diffuse, e.g. myocardium), it becomes scattered and less of the ultrasound returns along the original wave path (28). As a result, myocardial echoes generate granular patterns called “speckles”, which serve as tissue markers for the assessment of myocardial deformation (more detail in section 2.2.3). Ultrasound waves may additionally be refracted and change direction as they meet a nonperpendicular interface, or attenuated as they are absorbed and scattered further from their point of origin (28). As bone tissue and air almost entirely absorb or attenuate ultrasound waves (228), the transducers utilized for transthoracic echocardiography have a small scanning footprint to access the narrow intercostal “windows”, and avoid the ribcage and lungs (166).    In echocardiography, ultrasound waves are generated by a transducer with piezoelectric crystal elements. These transducers convert electrical energy into mechanical energy by electrically stimulating the piezoelectric crystals, whose mechanical vibrations generate an acoustic or ultrasound signal (164). The returning ultrasound waves then mechanically deform the crystal elements to produce an electric signal which is interpreted by the system’s software (164). Presently, transducers are most commonly phased array, meaning that they comprise of a string of crystal elements arranged in a row, all of which   40 are fired for a single scan line, and the specific timing of excitation of the crystals determines the beam’s direction (164).   Figure 2.1. Reflective properties of diffuse (e.g. myocardium) and specular (e.g. pericardium) interfaces (left panel reproduced with © permission from (190)).   Ultrasound waves are characterized by their amplitude, wavelength and cycle duration (or frequency), and this relationship is described by the formula: λ = V/𝑓 where λ is the wavelength, V is the velocity through the transmitting medium, and f is the transducer frequency (166). As the velocity of ultrasound waves is relatively constant in soft tissues, the manipulation of wavelength and frequency determines the image resolution or ability to distinguish between two points (166). For example, higher frequency probes have shorter wavelengths and augmented spatial resolution, however they also have increased attenuation and poorer penetration, therefore the balance of these parameters is required to optimize focal depth and resolution (164).   Two-dimensional (2D) images are constructed from a series of multiple adjacent scan lines (Figure 2.2)(164). In B-mode (brightness mode), returning ultrasound echoes are displayed as dots along a scan line representing the depth into the insonated medium (28).   41 Each scan line is generated from a single return trip of the ultrasound beam, or pulse; therefore, increasing pulse repetition frequency allows for a greater amount of scan lines to be generated per unit time, and primarily determines frame rate or temporal resolution of the imagining (164). Frame rate is also a function of image sector depth, width and scan line density, thus manipulations to any of these factors will impact temporal resolution (166). In addition to temporal resolution, 2D imaging quality is also determined by spatial resolution, both axial and lateral. Axial resolution describes the ability to distinguish between structures along the length of the ultrasound beam, and can be as precise ~0.5-1.0 mm using high frequency transducers (allowing for smaller wavelengths) (166). Lateral resolution, the ability to distinguish between structures perpendicular to the ultrasound beam, has relatively lower precision and becomes reduced with increasing distance from the transducer (160).   Figure 2.2. Two-dimensional B-mode (brightness mode) images generated from echoes of multiple successive scan lines (figure reproduced with © permission from (228)).   2.2 Basic forms of echocardiography 2.2.1 Two-dimensional B-mode echocardiography In B-mode echocardiography, the brightness of a given point along a scan line is determined by the strength or amplitude of the returning ultrasound signal (28). The properties of the returning echo cumulatively represent the reflection, refraction and   42 attenuation of the outgoing ultrasound signal and the acoustic impedances of structural interfaces (164).   2.2.2 Doppler echocardiography Doppler ultrasound utilizes the Doppler shift principle to assess the velocity and direction of motion relative to the ultrasound probe (28). When ultrasound waves interact with moving targets, such as blood or myocardial tissue, the reflected ultrasound waves will have a different frequency from the originally transmitted waveform. This difference between the outgoing and received ultrasound frequencies, the Doppler shift, is proportional to the velocity of the moving target (164, 166). Motion of the target towards and away from the transducer produces positive and negative frequency shifts, respectively (164). The best Doppler velocities are measured when the direction of motion is parallel to the ultrasound beam (i.e. 0º or 180º), but when the motion angle becomes less parallel to the ultrasound beam, the ability to detect that motion is reduced, and when motion angle is perpendicular to the transducer (i.e. 90º), no motion can be detected (166).  Doppler echocardiography is commonly used to measure the motion of blood and myocardial tissue (161). Pulsed-Doppler mode is used to assesses the velocity of blood flow by measuring higher-frequency, lower-amplitude signals from the rapid movement of blood cells, whereas tissue Doppler imaging assesses the lower-frequency, higher-amplitude signals from myocardial tissue motion (97). The apex is relatively stationary during the cardiac cycle, therefore tissue Doppler imaging of the mitral annulus has been utilized as a surrogate of LV contraction and relaxation in the longitudinal plane (97).  2.2.3 Speckle tracking echocardiography Speckle tracking echocardiography is a more recently developed method used for the analysis of myocardial deformation and LV mechanics. In B-mode images, this software uses spatial and temporal image processing algorithms to detect natural acoustic markers, or “speckles”, and track them frame-by-frame across the cardiac cycle (93, 121). These speckles are equally distributed within the myocardium, and comprise of 20-40 pixels   43 with stable acoustic patterns that aren’t significantly changed between adjacent frames (121). Superior to tissue-Doppler imaging, STE allows for the assessment of regional shortening (strain) or rotation in multiple regions of interest for a given imaging plane (Figure 2.3)(161). The assessment of LV strain, rotation and twist using STE has been validated against sonicometry in canines (r=0.94 for strain and twist), and magnetic resonance imaging in humans (r=0.90 for strain, r=0.87 for twist)(3, 93).   Figure 2.3. Assessment of LV rotation using speckle tracking echocardiography. Systolic rotation across consecutive frames is counterclockwise at the apex (left) and clockwise at the base (right) (figure reproduced with © permission from (154)).  The optimization of speckle tracking analysis is highly dependent on the spatial and temporal resolution of the ultrasound image (121). Lower frame rates reduce tracking quality, as speckles may move out of the imaging plane or beyond the region of interest. Moreover, although higher frame rates improve temporal resolution, this may come at the cost of reducing spatial resolution and image quality. For this reason, frame rates >70 frames per second are recommended, but should be adjusted based on participant’s heart rate for the best spatial resolution possible to ensure optimal speckle tracking quality (93, 145). Speckle tracking echocardiography is also highly sensitive to acoustic reverberations and image shadowing, which can lead to the underestimation of true myocardial deformation (145).      44 2.3 Image acquisition and optimization for cardiac assessment 2.3.1 General procedures, standardization and optimization for image acquisition In each experiment, echocardiographic imaging was performed by a single experienced sonographer for the assessment of LV structure, global function and mechanics in accordance with current imaging guidelines (117, 118). Images were recorded on a commercially available ultrasound system (Vivid q in Chapter 3, Vivid E9 in Chapters 4 and 5; GE Healthcare) using M5S 1.5-4.6 MHz and 4V 1.5-4.0 MHz transducers. Participants were positioned in the left lateral decubitus position, and images were recorded at end-expiration to minimize the lateral displacement of LV structures related to respiration. Five consecutive cardiac cycles were recorded for each image and saved for offline analysis (EchoPAC, GE Healthcare).   The echocardiographic imaging planes utilized in these experiments are illustrated in Figure 2.4 (118). In order to standardize the echocardiographic assessment acquisition, the sonographer performed a similar sequence of imaging across all experiments. The exam began with imaging of the LV parasternal long-axis images, by placing the transducer in the second or third intercostal space, near to the sternal border and with the transducer marker angled at the right shoulder. Images were collected with the mitral valve near the center of the image and the septal and posterior LV walls clearly defined. From this position, the transducer was rotated 90º toward the left shoulder. Imaging of parasternal short-axis images were performed at the base, with leaflets of the mitral valve and the full myocardial wall visible. For imaging at the apex, the probe was shifted distally to the point at which the apical beat could be visually identified. In cases where that was not apparent, the transducer was placed in the sixth or seventh intercostal space, and further shifted to center the LV in the image. Images at the apex were acquired at the level of the apex, just proximal to end-systolic luminal obliteration (213). From this position, the sonographer angled the footprint of the transducer upwards (toward the participant’s head) to visualize the apical 4-chamber view, with a focus on the LV that included the full LV myocardial area and mitral annulus. Pulsed-Doppler and tissue Doppler recordings were performed in the apical 4-chamber view (see section 2.3.3 for further detail). In Chapter 3, images were collected in the apical 2-chamber view by   45 slightly rotating the transducer in a counter-clockwise direction, for the assessment of LV volumes using Simpson’s biplane method. In Chapters 4 and 5, a triplane images were acquired in the apical 2-, 3- and 4-chamber views using 4V 1.5-4.0 MHz transducer for the assessment of LV volumes using a modified Simpson’s biplane method (see section 2.3.3 for further detail)(187).  During the echocardiographic assessment, ultrasound settings were optimized based on the measurements specific to the image. For all images, the sector width and depth were adjusted to include the full LV myocardial area of interest, and to exclude additional structures that were not being assessed. The focal point was adjusted to the depth of the structures of interest, e.g. in the short-axis views, the focal point was aligned with the center of the myocardial circumference. During triplane imaging in the apical window, small adjustments and rotations were made to the transducer position to clearly visualize the LV endocardial borders. In images used for speckle-tracking analysis (further detail in section 2.3.4), the frame rate was adjusted to 70-100 frames per second. Higher frame rates were selected for images collected during elevated HR’s.   2.3.2 Measurement of LV structure and geometry  From parasternal long-axis images, intraventricular septal wall thickness (IVST), posterior wall thickness (PWT) and LV internal diameter at end-diastole (LVIDd) were measured in a line perpendicular to the long axis, at tips of the mitral valve leaflets. For the estimation of end-systolic wall stress (Chapter 4), LV parasternal long-axis images were analyzed at end-systole for IVST, PWT and LVIDs. LV length at end-diastole (lengthd) was determined as the mean length from the mitral plane to the apical subendocardium in the apical two- and four-chamber views. Relative wall thickness was calculated as 2⋅PWT/LVIDd, and sphericity index was calculated as LV lengthd/LVIDd (212). To account for sex-related differences in LV size, LV dimensions were allometrically scaled to BSA0.5. A coefficient of 0.5 was specifically utilized to scale one-dimensional (linear) LV dimensions to a two-dimensional body size scaling variable (14).     46  Figure 2.4. Visualization of echocardiographic imaging planes in apical (panels 1, 2), long axis (panel 3) and short axis (panels 4, 5, 6) orientations, according to the American Society of Echocardiography and European Society of Echocardiography (figure reproduced with © permission from (118)).  2.3.3 Measurement of global LV systolic and diastolic function Left ventricular EDV, ESV, SV and EF were determined using Simpson’s biplane method in the apical 2- and 4-chamber views (Chapter 3), or a modified Simpson’s technique in triplane recordings of the apical 2-, 3- and 4-chamber views (Chapters 4 and 5)(187). To account for sex differences in LV size, LV volumetric data were allometrically scaled to body surface area BSA1.5. A coefficient of 1.5 was specifically used to scale three-dimensional LV volumetric data to a two-dimensional body size scaling variable (14).  Pulsed Doppler recordings were performed in the apical 4-chamber view, with the sample volume placed at the tips of the mitral valve for the assessment of LV diastolic inflow velocities (E and A). The image angle was adjusted to ensure the sampling cursor was as perpendicular as possible to the direction blood flow. Tissue Doppler imaging was additionally performed in the apical 4-chamber view. The septum was centered in the image, and the sector width was reduced as much as possible to focus only on the mitral septal annulus. The sample volume placed at the level of the mitral septal annulus for the measurement of myocardial velocities during systole (S’), and diastole (E’ and A’). Data   47 from for diastolic inflow velocities and tissues velocities were determined as the peak trace velocities for the respective phase, and were averaged across three cardiac cycles.  2.3.4 Measurement of LV mechanics using speckle tracking analysis Images for speckle tracking analysis were acquired at rate of 70-90 frames·s-1. Parasternal short-axis images were acquired at the base for the assessment of basal rotation and circumferential strain, and at the apex for the assessment of apical rotation and circumferential strain. Longitudinal strain was assessed in the apical 4-chamber view. For all studies, a single experienced sonographer who was blinded to group allocation and experimental condition performed all analyses. Measurements of LV strain (negative for myocardial shortening) and rotation (positive at the apex, negative at the base) parameters were performed using speckle-tracking software (EchoPAC v.113, GE, Fairfield, CT). The observer manually traced the endocardial border (Figure 2.5 A), and the width of the region of interest was adjusted to include the full myocardial area (Figure 2.5 B), with trabeculations and papillary muscles excluded. The software provided feedback on tracking adequacy for six myocardial segments, which was visually confirmed by the observer (Figure 2.5 C). When the automated tracking appeared nonphysiologic or disagreed with the visual impression of wall motion, the region of interest was manually adjusted to optimize tracking quality. Regions of interest were excluded for segments with insufficient speckle tracking (i.e. did not appropriately track myocardial motion) due to image dropout, reverberation or drift (93), and images with inadequate tracking in ≥2 segments were excluded from analysis.    48   Figure 2.5. Progression of analysis using speckle tracking software for the assessment of LV mechanics (example at LV apex).   Frame-by-frame LV strain and rotation curves were generated by the speckle tracking software for three cardiac cycles (Figure 2.5 D). To account for intra- and inter-individual variability in HR and imaging frame rate, raw data were time-aligned and transformed to 1200 points (600 each for systole and diastole) using cubic spline interpolation (2D Strain Analysis Tool, Stuttgart, Germany). Twist data were calculated by subtracting time-aligned basal data from apical data, and torsion was calculated as LV twist/LV lengthd. Speckle tracking data represent the average values for the entire myocardial region of interest, and were averaged across three cardiac cycles. Peak data represent the maximum values across the cardiac cycle.   2.3.5 Reliability of echocardiographic measurements In Chapter 3, the coefficient of variation of the sonographer (M. Stembridge) for measuring twist and untwisting velocity were 8.1% and 11%, respectively (195), which are in agreement with previously published data (154, 197). The technical error of   49 measurement of the sonographer of Chapters 4 and 5 (A. Williams) for the assessments of LV volumes, dimensions, Doppler and speckle tracking data are shown in Table 2.1 below.     Table 2.1. Technical error of measurement of the sonographer (A. Williams) for measurements of LV structure and function.  Technical Error of Measurement  Absolute Relative (%) LV mechanics Apical rotation (º) 0.9 10.7 Basal rotation (º) 0.6 14.9 Twist (º) 1.3 10.0 Longitudinal strain (%) 0.8 4.2 Circumferential strain (%) 1.3 6.7 LV volumes and hemodynamics EDV (ml) 2 1.8 ESV (ml) 2 4.5 EF (%) 3 2.5 Pulsed Doppler and tissue Doppler E (m⋅s-1) 0.04 4.8 A (m⋅s-1) 0.03 9.1 E/A 0.16 6.6 E’ (m⋅s-1) 0.01 6.5 A’ (m⋅s-1) 0.01 10.6 S’ (m⋅s-1) 0.01 9.3 LV internal diameter and wall thicknesses  LVIDd (mm) 1.0 2.3 IVST (mm) 0.4 4.2 PWT (mm) 0.5 4.9 Values are means (SD). EDV: end-diastolic volume; ESV: end-systolic volume; EF: ejection fraction; E: early diastolic inflow velocity; A: late diastolic inflow velocity; E’: early diastolic tissue velocity; A’: late diastole tissue velocity; S’: systolic tissue velocity; LVIDd: left ventricular diastolic internal diameter; IVST: interventricular septum thickness; PWT: posterior wall thickness.     50 2.4 Assessment of LV hemodynamics  During all experiments, HR was measured using conventional three-lead electrocardiography, and beat-by-beat blood pressure was monitored using finger photoplethysmography (Finometer, Amsterdam, NL). Blood pressure was additionally measured using manual sphygmomanometry immediately following echocardiographic imaging.   Cardiac output (Q) was calculated as LVSV⋅HR. Mean arterial pressure (MAP) was calculated as 1/3⋅systolic blood pressure (SBP)+2/3⋅diastolic blood pressure (DBP), and TPR was calculated as MAP/Q.     51 Chapter 3. Sex differences in LV mechanics during acute alterations to preload1  3.1 Background It is well established that sex differences exist in LV structure and function (41, 42, 91, 179). For example, compared to males, females have smaller LV wall thicknesses, volumes and mass, and these differences often remain when indexed to body size (42, 179). The disparities in structure are accompanied by differences in global LV function (i.e. systolic emptying and diastolic relaxation and filling). Despite lower absolute LVSV, females have greater EFs than males, such that they attain a lower LVESV and a greater SV for a given EDV at rest (41, 179). The differences in function may be attributed to intrinsic properties of the myocardium: the smaller female ventricle is “stiffer”, with lower diastolic compliance and a greater reliance on contractility compared to males (41, 42, 91). Whether these differences in LV structure and global function are associated with sex differences in LV mechanics remains unclear. However, recent studies have reported greater longitudinal and circumferential strain in females compared to males (9, 103, 119), providing preliminary support that sex differences in these fundamental mechanics do exist.   Left ventricular mechanics characterize myocardial deformation throughout the cardiac cycle (6, 186), and are dynamically load-dependent (59, 103, 220). Opposite rotations of the LV base and apex result in LV twist (27, 186, 199), which supports ejection during systole, and subsequent untwisting during diastole. The regional shortening and re-lengthening of the myocardium are quantified as LV strain (185) (see (145) for a more in-depth review of LV mechanics). During increases to preload, increased LVEDV and myocardial stretch increase SV via the Frank-Starling mechanism (21). Increasing preload also results in increased apical rotation, twist, diastolic untwisting velocity, circumferential strain and longitudinal strain (220). In contrast, during reductions to preload, lowered LVEDV and preload-recruitable stroke work lead to decreases in SV and longitudinal strain (46, 73, 98, 188). However, reduction of preload also results in                                                 1 A version of this chapter has been published: Williams AM, Shave RE, Stembridge M, Eves ND (2016). Females have greater left ventricular twist mechanics than males during acute reductions to preload. Am J Physiol Heart Circ Physiol 311:H76-H84.   52 increased HR and adrenergic stimulation (73, 188), as well as increased apical rotation, twist and untwisting velocity (98, 99). Although these responses of LV function to altered preload are well established, there is a paucity of data regarding whether the male and female hearts respond differently to alterations in preload.   Studies utilizing LBNP and head-up tilt have reported greater increases in HR in females, but similar reductions in LVSV and Q between the sexes (188, 224). However, females appear to have greater reductions to LVSV for a given decrease in LV filling pressure, and a steeper slope of the Frank-Starling relationship compared to males (73). These findings suggest that females may have a lower functional reserve to cope with changes to LV loading, and a potentially greater reliance on increasing contractility and chronotropy to support SV and Q during acute preload challenges.   It remains unknown whether sex differences in LV structure and global function during periods of altered preload are underpinned by sex differences in LV mechanics. Therefore, the aim of the present study was to investigate how sex affects LV mechanics in response to acute challenges to preload. It was hypothesized that females would have greater LV apical rotation and twist, and greater longitudinal strain and circumferential strain than males during decreases and increases in preload.   3.2 Methods 3.2.1 Ethical approval and study participants A total of 48 healthy individuals from the local university community, aged 18-39 years, volunteered and were enrolled in the study. Participants were excluded if they had a history of cardiovascular, metabolic or respiratory disease; were current smokers (or stopped smoking for <12 months) or had hypertension (≥140/90). Additionally, females were excluded if pregnant or nursing. The study was approved by the Institutional Clinical Research Ethics Board of the University of British Columbia. Written informed consent was obtained from all participants.     53 3.2.2 Study design Participants visited the laboratory on two occasions. Prior to each visit, participants were asked to refrain from caffeine, alcohol and exercise for a minimum of 24 hours. On day 1, participants were screened for their ability to tolerate -60 mmHg LBNP. On day 2, total blood volume was measured using the optimized carbon monoxide rebreathing technique. Participants then rested in a LBNP box, sealed at the level of the iliac crests with a velcro strap, and were angled toward the sonographer in a left lateral decubitus position. Following collection of baseline echocardiographic images, LBNP was applied at -20, -40 and -60 mmHg. Echocardiographic images were recorded at each level of LBNP, following stabilization of HR within 5 beats per minute, and MAP within 5 mmHg. Blood pressure and HR were continuously monitored using finger photoplethysmography (Finometer, Amsterdam, NL) and three-lead electrocardiography, respectively. Manual measurements of blood pressure were additionally taken at the end of each stage, using a stethoscope and sphygmomanometer.  Following LBNP, a subset of 14 individuals (8 male, 6 female) received a rapid infusion of saline. An 18- or 20-guage intravenous cannula was inserted into the antecubital vein, and warm isotonic saline (~12 ml⋅kg-1 bodyweight) was infused at a rate of ~100-200 ml⋅min-1, using a pneumatic sleeve to compress the solution (76). Following the initial rapid infusion, saline was continually infused at a rate of 10 ml⋅min-1 to maintain cardiac filling pressures (76), and echocardiographic images were recorded.   3.2.3 Specific methodology 3.2.3.1 Screening and familiarization  To screen for tolerance of LBNP, participants were exposed to -10 mmHg increments each minute up to -60 mmHg, which was then maintained for three minutes or until signs of pre-syncope. Pre-syncope was defined as a decrease in SBP below 80 mmHg, or a decrease in SBP below 90 mmHg accompanied by lightheadedness, nausea, or tunnel vision. Participants unable to tolerate up to or including -60 mmHg were excluded from the study.    54 3.2.3.2 Total blood volume Total blood volume was measured using the carbon monoxide rebreathing technique, as previously described (181). Briefly, participants were fitted with a noseclip, and a custom-made glass spirometer (Blood tec, GbR, Germany) attached to a 5 L reservoir bag of 100% O2 gas. Participants were instructed to inhale maximally, as a calculated amount of carbon monoxide (0.8 ml⋅kg-1 for males, 0.6 ml⋅kg-1 for females) was simultaneously administered to the rebreathing apparatus. Subjects held a full lung volume for 10 s, and then rebreathed from the apparatus until 2 minutes, after which they breathed room air. Venous blood was drawn from the antecubital vein at baseline and 7 minutes following the onset of rebreathing for measurement of total hemoglobin and carboxyhemoglobin (ABL 90, Radiometer, Denmark). Portable carbon monoxide analyzers (Dräger Pac 3500, Draeger Safety Inc., Texas, USA) were used to account for expired carbon monoxide at baseline, and at 4 and 7 minutes following the onset of rebreathing.  3.2.3.3 Transthoracic echocardiography and analysis of LV mechanics and hemodynamics See Chapter 2 for details.  3.2.4 Statistical analysis and power calculation Data are presented as means ± standard deviation (SD). All dependent variables were assessed using a 2 (group) x 4 (LBNP level) ANOVA for LBNP, and a 2 (group) x 2 (pre and post) ANOVA for the rapid saline infusion. When a positive effect was detected, a Fisher’s least significant difference test was used to determine pairwise differences. These statistical analyses were performed using STATISTICA (version 8.0; StatSoft, Tulsa, OK) with α set a priori to 0.05.  Linear regression analysis was used to assess the Frank-Starling relationship, ∆LVSV/∆EDV, for each participant during LBNP and saline infusion, and mean slopes were compared using an independent samples t-test (73). Linear regression was also used to assess the relationship between LV twist and untwisting velocity. Non-linear regression analysis using a second order polynomial (quadratic) least squares fit was used   55 to assess the relationships between LV twist and torsion to LV absolute and allometrically scaled volumes, respectively. The coefficients in the quadratic equation y=b0+b1x+b2x2 were calculated for each participant, and mean coefficients for male and females were compared using an independent samples t-test. Regression analyses were performed using GraphPad Prism (version 6.0f; GraphPad Software, Inc., La Jolla, CA).  No study has previously determined sex differences in cardiac mechanics during alterations in preload. However, it was determined a priori that with 20 participants per group, we were powered to detect a difference of 4.4 degrees in LV twist between the groups, utilizing a SD of 5 degrees obtained from the literature (98), an α=0.05 and a β=0.80.  3.3 Results 3.3.1 Participant characteristics Of the 48 individuals enrolled, a total of 20 males and 20 females completed the study. Seven individuals (5 females, 2 males) were unable to tolerate -60 mmHg during the familiarization, and were excluded. Additionally, one male was excluded for a previous cardiac condition. Participant characteristics and baseline cardiac and hemodynamic parameters are presented in Table 3.1. Females had smaller absolute LV dimensions compared to males, but there were no sex differences in allometrically scaled dimensions. Moreover, relative wall thickness and sphericity did not differ between groups. Total blood volume was greater in males, but normalized blood volume did not differ between the groups. There were no differences in baseline MAP or HR.    56 Table 3.1. Baseline participant characteristics and echocardiographic measurements  Males (n=20) Females (n=20) Age (yrs) 24 (6.2) 23 (3.1) Height (m) 1.79 (0.06) 1.66 (0.08)* Weight (kg) 76.9 (11.7) 62.4 (8.6)* BMI (kg⋅m-2) 23.8 (2.8) 22.6 (2.3) MAP (mmHg) 76 (6) 74 (7) HR (bpm) 56 (9) 64 (10) Total blood volume (L) 7.20 (1.15) 5.24 (0.79)* Normalized blood volume (ml⋅kg-1) 94.3 (12.0) 85.3 (16.2) LV structure and geometry Lengthd (cm) 9.14 (0.69) 8.14 (0.44)* Lengthd⋅BSA-0.5 (cm⋅m-1) 6.55 (0.40) 6.27 (0.33) LVIDd (mm) 46.7 (3.2) 41.6 (3.7)* LVIDd⋅BSA-0.5 (mm⋅m-1) 33.4 (2.0) 31.9 (2.0) IVST (mm) 11.1 (1.5) 9.7 (1.5)* IVST⋅BSA-0.5 (mm⋅m-1) 7.9 (0.9) 7.5 (1.2) PWT (mm) 9.2 (1.2) 8.3 (1.2)* PWT⋅BSA-0.5 (mm⋅m-1) 6.6 (0.8) 6.4 (1.0) Relative wall thickness 0.40 (0.05) 0.40 (0.06) Sphericity index 1.96 (0.16) 1.97 (0.14) Values are means (SD). BMI: body mass index; MAP: mean arterial pressure; HR: heart rate; Lengthd: end-diastolic length; LVIDd: left ventricular diastolic internal diameter; IVST: interventricular septum thickness; PWT: posterior wall thickness; BSA: body surface area. *p<0.05 vs males.   3.3.2 LV mechanics and structure during altered preload LV twist increased in both groups with progressive LBNP (p<0.001), and resulted predominantly from increases in apical rotation (p<0.001) (Table 3.2 and Figure 3.1). LV twist (p=0.008) and apical rotation (p=0.009) were significantly larger in females compared to males at -60 mmHg. Relative to LV length, LV torsion increased in both groups during LBNP, and was greater in females during -40 (p=0.01) and -60 mmHg (p=0.002) (Figure 3.2). Untwisting velocity was greater in females compared to males at -60 mmHg (p=0.02). There was a significant relationship between LV twist and untwisting velocity in all participants (r2=0.46, p<0.001), and slopes of the regression did not differ between the sexes. Longitudinal strain and circumferential strain declined in   57 both groups with LBNP, but longitudinal strain was greater in females at -60 mmHg (p=0.002).  LV length and LVIDd decreased in both groups with progressive LBNP but were larger in men in all conditions (p<0.001). There were no sex differences in scaled dimensions, scaled wall thicknesses or relative wall thickness at any stage. However, females had a larger sphericity index than males at -60 mmHg (p=0.007) (Table 3.2).  In the cohort receiving the rapid saline infusion, there were no sex differences in LV apical rotation, twist, torsion, untwisting velocity or sphericity index at baseline or following saline infusion. These parameters were unchanged in both groups following infusion. LV length and LVIDd were also unchanged following infusion.  Figure 3.3 illustrates the relationships between twist and LV volumes, which are presented using absolute and scaled data. A trend for greater b1 and b2 coefficients in females was observed for the relationship of twist-to-LVSV (b1: males -0.17 ± 0.44 vs. females -0.56 ± 0.60, p=0.073; b2: males 0.007 ± 0.016 vs. females 0.020 ± 0.023, p=0.074), and the relationship of torsion to allometrically scaled SV (b1: males -0.65 ± 1.50 vs. females -1.85 ± 1.96, p=0.077; b2: males 0.008 ± 0.019 vs. females 0.024 ± 0.028, p=0.073). b0, b1 and b2 coefficients did not differ between the sexes for the relationships of LV twist and torsion to LVEDV and allometrically scaled EDV, respectively.     58  Figure 3.1. Graphical representation of mean LV twist mechanics during LBNP. Blue and red lines represent data for males and females, respectively. Top panel: dotted and dashed lines represent rotations of the LV apex and base, respectively. Middle panel: solid lines represent LV twist. Lower panel: solid lines represent twist and untwisting velocities. Standard deviations are provided in Table 3.2. *p<0.05 males vs. females. -5051015200 mmHg -20 mmHg -40 mmHg -60 mmHg* -150-100-50050100150LV Twist Velocity (°sec-1 )MalesFemalesLV Twist (°)-505101520* * Rotation (°)ApexBase% Systole % Diastole % Systole % Diastole % Systole % Diastole % Systole % Diastole         50      100     150      200          50      100     150      200         50      100     150      200         50      100     150      200 59 Table 3.2. LV mechanics, structure and geometry during altered preload.   LBNP Saline Infusion   Baseline -20 mmHg -40 mmHg  -60 mmHg Pre Post Twist mechanics (peak)       Apical rot (°) M 10.6 (3.4) 9.9 (3.2) 10.7 (3.9) 13.1 (5.9)† 9.8 (3.8) 10.0 (3.5)  F 12.4 (4.5) 11.4 (5.1) 13.6 (4.8) 18.0 (6.9)*†‡§ 12.3 (5.1) 10.8 (1.2) Basal rot (°) M -3.2 (2.7) -3.0 (2.9) -3.4 (2.7) -3.6 (2.9) -3.9 (2.1) -3.3 (3.1)  F -4.1 (2.1) -3.8 (2.9) -4.2 (3.1) -4.8 (2.7) -4.1 (1.7) -2.4 (2.5) Twist (°) M 13.6 (4.6) 12.7 (3.5) 13.4 (4.2) 15.8 (5.2)‡§ 13.3 (5.3) 13.8 (4.6)  F 16.1 (5.9) 14.4 (4.8) 17.4 (5.1)‡ 21.4 (6.7)*†‡§ 15.8 (4.6) 13.6 (1.6) Untwisting velocity (°⋅s-1) M 109 (28) 105 (29) 111 (40) 118 (45) 110 (29) 95 (33)  F 126 (51) 123 (39) 140 (46) 152 (45)*‡ 117 (53) 107 (18) Strain mechanics (peak)       LS (%) M -18.5 (2.2) -16.5 (2.3)† -15.4 (2.3)† -14.4 (2.1)†‡§ -17.7 (2.7) -18.9 (1.8)  F -20.6 (1.5) -18.7 (2.3)† -17.7 (2.1)†‡ -17.0 (2.2)*†‡ -21.0 (0.8)* -21.5 (1.5)* CS, base (%) M -19.5 (3.7) -17.6 (3.7)† -16.7 (4.1)† -15.0 (3.6)†‡§ -19.8 (3.9) -17.8 (2.7)  F -20.8 (3.4) -20.2 (3.1) -17.1 (3.0)†‡ -15.9 (4.1)†‡ -21.4 (1.7) -18.4 (2.4)† CS, apex (%) M -28.2 (4.8) -27.1 (5.4) -26.1 (5.5)† -25.1 (6.6)† -27.9 (4.1) -27.6 (5.0)  F -27.6 (3.3) -25.2 (5.6) -27.0 (5.0)† -26.8 (4.9)† -27.6 (2.4) -27.5 (1.4)     60 Table 3.2 (cont). LV mechanics, structure and geometry during altered preload.  LBNP Saline Infusion  Baseline -20 mmHg -40 mmHg  -60 mmHg Pre Post LV structure and geometry       Lengthd (cm) M 9.14 (0.69) 8.96 (0.69)† 8.81 (0.69)†‡ 8.57 (0.72)†‡§ 9.10 (0.51) 9.02 (0.58)  F 8.14 (0.44)* 7.99 (0.52)*† 7.73 (0.61)*†‡ 7.58 (0.62)*†‡§ 7.90 (0.39)* 7.96 (0.32)* LVIDd (mm) M 46.7 (3.2) 45.2 (3.5)† 42.2 (4.2)†‡ 39.5 (4.3)†‡§ 46.2 (2.8) 46.3 (4.3)  F 41.6 (3.7)* 39.7 (3.0)*† 37.2 (2.9)*†‡ 32.3 (3.1)*†‡§ 39.9 (3.7)* 40.8 (4.5)* Sphericity index M 1.96 (0.16) 1.99 (0.20) 2.11 (0.24)†‡ 2.17 (0.24)†‡§ 1.98 (0.16) 1.96 (0.21)  F 1.97 (0.14) 2.03 (0.16) 2.09 (0.17)† 2.36 (0.17)*†‡§ 2.02 (0.10) 1.97 (0.19) Values are means (SD). M: males; F: females; LBNP: lower body negative pressure; Rot: rotation; LV: left ventricle; LS: longitudinal strain; CS: circumferential strain; see Table 3.1 for additional abbreviations. *p<0.05 vs males. †p<0.05 vs baseline. ‡p<0.05 vs -20 mmHg. §p<0.05 vs -40 mmHg. n=20 males, n=20 females for LBNP; n=8 males, n=6 females for rapid saline infusion.      61  Figure 3.2. LV torsion in males (blue circles) and females (red circles) during LBNP. Points represent means ± SD. *p<0.05 vs. males for given stage of LBNP.    Figure 3.3. Upper panel: the relationship of LV twist to LVSV (A) and LV torsion to SV⋅BSA-1.5 (B) Lower panel: the relationship of LV twist to LVEDV (C) and LV torsion to EDV⋅BSA-1.5 (D). Points represent group means at baseline and LBNP (closed, n=20 males, n=20 females) and following saline infusion (open, n=8 males, n=6 females). A trend toward a difference between the sexes was observed in the relationships of twist to LVSV (A) (b1, p=0.077; b2, p=0.073) and LV torsion to SV⋅BSA-1.5 (B) (b1, p=0.073; b2, p=0.074). 01234LBNPTorsion (°cm-1)FemalesMales * *0 mmHg       -20 mmHg    -40 mmHg     -60 mmHg20 30 40 50 60 70 8051015202530SV (ml)Twist (°)AFemales y=49.75-1.85x+0.024x2Males y=35.85-0.65x+0.008x240 60 80 100 120 14051015202530EDV (ml)Twist (°)CFemales y=78.51-1.65x+0.016x2Males y=59.59-1.03x+0.007x25 10 15 20 25 30 3501234SV⋅BSA-1.5 (ml⋅m-3)Torsion (°cm-1)BFemalesMalesFemales y=6.85-0.56x+0.020x2Males y=4.46-0.17x+0.007x215 20 25 30 35 40 45 50 5501234EDV⋅BSA-1.5 (ml⋅m-3)Torsion (°cm-1)DFemales y=11.08-0.47x+0.012x2Males y=7.48-0.35x+0.006x2 62 3.3.3 LV volumes and hemodynamics during altered preload LVEDV (p<0.001), ESV (p<0.001) and SV (p<0.001) gradually decreased with progressive levels of LBNP, and while absolute volumes were larger in males at all stages, there were no sex differences in allometrically scaled EDV, ESV or SV (Table 3.3). The relative decrease in LVEDV (males 34 ± 7%; females 37 ± 9%), ESV (males 25 ± 9%; females 23 ± 13%), and SV (males 41 ± 9%; females 46 ± 10%) from baseline to -60 mmHg did not differ between the sexes. However, the mean slope of the Frank-Starling relationship (∆LVSV/∆EDV) was greater in females (0.76 ± 0.09) compared to males (0.68 ± 0.09) (p=0.02). LVEF decreased in both groups with LBNP, but was higher in females at baseline (p=0.01) and at -40 mmHg (p=0.02).  HR increased in both groups with LBNP, and was higher in females at -40 (p=0.004) and -60 mmHg (p<0.001) (Table 3.3). Q was reduced with LBNP in both groups, yet MAP did not change with LBNP and was not different between the sexes. TPR increased in both groups with LBNP (p<0.001), but did not differ between the sexes.  In both groups, E decreased from baseline to -40 mmHg, but was not significantly reduced further at -60 mmHg. The reduction to E, and trend of increasing A (p=0.06) resulted in a reduced E/A ratio from baseline to -60 mmHg in both groups (males 2.09 ± 0.58 to 1.36 ± 0.28; females 2.17 ± 0.71 to 1.13 ± 0.20; p<0.001 for both). Diastolic filling velocities did not differ between the sexes during any stage. Diastolic tissue velocities decreased in both groups with LBNP (p<0.001 for both), but females had greater A’ (6.1 ± 1.4m⋅s-1 vs. 4.9 ± 0.9m⋅s-1; p=0.002) than males during -60 mmHg.  Systolic tissue velocity (S’) decreased in males from baseline to -60 mmHg (8.6 ± 1.6m⋅s-1 to 7.3 ± 1.5m⋅s-1; p<0.001), but was unchanged in females, resulting in a greater S’ in females than males at -60 mmHg (8.3 ± 1.1m⋅s-1 vs. 7.3 ± 1.5m⋅s-1; p=0.03).   Prior to and following saline infusion, absolute LV volumes were larger in males, but there were no sex differences in allometrically scaled volumes. Following saline infusion, LVEDV increased in males (p=0.003) but not in females (Table 3.3). However, scaled  63 EDV increased in both groups. Absolute and scaled LVESV were unchanged in both groups. Absolute and scaled LVSV increased in both groups (p<0.001), but EF was unchanged. E and E’ increased in both groups following infusion (p<0.01), but there were no sex differences in filling or septal tissue velocities.  64 Table 3.3. Cardiovascular responses to altered preload.   LBNP Saline Infusion   Baseline -20 mmHg -40 mmHg  -60 mmHg Pre Post HR (bpm) M 56 (9) 58 (10) 66 (11)†‡ 75 (12)†‡§ 53 (9) 56 (11)  F 64 (10) 65 (9) 75 (12)*†‡ 95 (16)*†‡§ 61 (9) 60 (10) SBP (mmHg) M 112 (5) 112 (8) 107 (11)†‡ 101 (11)†‡§ 114 (6) 120 (9)†  F 107 (6)* 102 (7)*† 99 (7)*†‡ 96 (7)*†‡ 101 (2)* 108 (7)* DBP (mmHg) M 59 (8) 61 (9) 59 (11) 62 (13) 63 (7) 70 (4)†  F 59 (9) 55 (7) 58 (10) 61 (13) 57 (6) 61 (12)* MAP (mmHg) M 76 (6) 77 (8) 74 (11) 75 (11) 79 (6) 86 (5)†  F 74 (7) 70 (6) 71 (8) 72 (10) 71 (4)* 76 (10)* EDV (ml) M 106 (18) 95 (19)† 82 (18)†‡ 70 (17)†‡§ 106 (15) 117 (19)†  F 77 (10)* 67 (9)*† 57 (8)*†‡ 48 (7)*†‡§ 76 (14)* 82 (15)* ESV (ml) M 48 (11) 44 (11)† 42 (11)†‡ 35 (9)†‡§ 47 (9) 49 (11)  F 31 (5)* 29 (4)*† 27 (3)*†‡ 24 (5)*†‡§ 29 (6)* 30 (3)* SV (ml) M 59 (9) 51 (10)† 41 (9)†‡ 35 (9)†‡§ 59 (7) 69 (10)†  F 45 (7)* 38 (7)*† 30 (6)*†‡ 24 (4)*†‡§ 46 (10)* 52 (12)*† EDV⋅BSA-1.5 (ml⋅m-3) M 39 (6) 35 (6)† 30 (6)†‡ 26 (5)†‡§ 39 (6) 43 (6)†  F 35 (5) 31 (4)† 26 (4)†‡ 22 (3)†‡§ 36 (5) 39 (4)† ESV⋅BSA-1.5 (ml⋅m-3) M 17 (3) 16 (3)† 15 (3)† 13 (3)†‡§ 17 (3) 18 (3)  F 14 (2) 13 (2)† 12 (1)†‡ 11 (2)†‡§ 14 (2) 14 (1)  65 Table 3.3 (cont). Cardiovascular responses to altered preload.   LBNP Saline Infusion   Baseline -20 mmHg -40 mmHg  -60 mmHg Pre Post SV⋅BSA-1.5 (ml⋅m-3) M 22 (4) 19 (4)† 15 (3) †‡ 13 (3) †‡§ 22 (4) 25 (4)†  F 21 (3) 17 (3)† 14 (3) †‡ 11 (2) †‡§ 22 (4) 24 (4)† EF (%) M 55 (4) 54 (4) 50 (4)†‡ 49 (6)†‡ 56 (4) 59 (4)  F 59 (3)* 56 (4)† 53 (4)*†‡ 51 (4)†‡§ 61 (4)* 63 (4) Q (L⋅min-1) M 3.24 (0.65) 2.89 (0.60)† 2.65 (0.56)†‡ 2.58 (0.71)†‡ 3.09 (0.56) 3.87 (0.89)†  F 2.92 (0.49)* 2.44 (0.38)*† 2.29 (0.37)† 2.29 (0.45)† 2.66 (0.30) 3.00 (0.43) TPR  (mmHg⋅L-1⋅min-1) M 24.3 (5.1) 27.8 (6.2)† 29.1 (8.0)† 31.3 (9.5)†‡ 23.1 (4.9) 26.1 (4.4)  F 25.9 (4.8) 29.2 (4.6)† 31.7 (6.1)† 33.0 (8.5)†‡ 25.7 (6.2) 25.9 (4.1) Values are means (SD). SBP: systolic blood pressure; DBP: diastolic blood pressure; EDV: end-diastolic volume; ESV: end-systolic volume; SV: stroke volume; EF: ejection fraction; Q: cardiac output; TPR: total peripheral resistance. See Table 3.1 for additional abbreviations. *p<0.05 vs males. †p<0.05 vs baseline. ‡p<0.05 vs -20 mmHg. §p<0.05 vs -40 mmHg. n= 20 males, n=20 females for LBNP; n=8 males, n=6 females for rapid saline infusion.     66 3.4 Discussion This is the first study to investigate sex differences in cardiac mechanics during acute alterations to preload. In support of our hypothesis, LV apical rotation, twist and longitudinal strain were all greater in females than males, but only at higher levels of LBNP. In contrast, circumferential strain was not significantly different between the sexes. The sex differences in LV twist coincided with differences in LV geometry and chronotropy, as LV sphericity index and HR were greater in females at higher levels of LBNP.  3.4.1 Sex differences in LV responses to altered preload LV twist was greater in females compared to males at -60 mmHg, and this resulted primarily from greater rotation at the apex. Due to the shorter LV length in females, LV torsion was also greater during -40 and -60 mmHg LBNP, demonstrating that females have greater twist for a given LV length compared to males. While previous reports have demonstrated similar reductions to LVEDV, ESV and SV (73), and increases to LV apical rotation and twist (98, 220) during LBNP, our data specifically demonstrate that females rely on greater apical rotation and LV twist than males during large challenges to preload.   Greater responsiveness at the apex in comparison to the base has been highlighted in previous investigations amongst male-only or mixed-sex cohorts (2, 98, 99, 198, 220), and has been suggested to help maintain the base-to-apex intraventricular pressure gradients that drive effective filling and ejection. Specifically, as LV systolic twist results in the storage of potential energy, the subsequent release of this energy in early diastole produces a rapid recoil or “suction” effect (153). In the current study, the greater apical rotation and twist in females were accompanied by a greater untwisting velocity during -60 mmHg LBNP, supporting the notion that increased apical rotation contributed to greater systolic twist and diastolic untwist mechanics in females. In accordance with previous studies, we found a strong relationship between LV twist and untwisting velocity, which supports the important role of LV twist in generating the appropriate intraventricular pressure gradients required for diastolic filling (52, 153, 198). In the  67 current study, early filling velocity was maintained in both groups between -40 and -60 mmHg; therefore, the greater untwisting velocity in females during -60 mmHg suggests that greater systolic twist and diastolic untwisting are required in the smaller female LV to generate adequate intraventricular pressure gradients, maintain passive filling and ultimately protect SV during challenges to preload.  3.4.2 Sex differences in LV adrenergic stimulation During higher levels of LBNP, HR was higher in females than in males. This elevated chronotropic response in females is commonly observed during reductions to preload (73, 188, 224), and has been proposed to reflect sex differences in sympathovagal balance. It has been proposed that compared to males, females respond with more prominent vagal withdrawal (45, 73, 188). In the current study, MAP was maintained in both groups despite reductions to Q during progressive LBNP. The higher HR during high levels of LBNP may reflect a lower SV reserve in females, requiring greater vagal withdrawal, or increased sympathetic drive, to increase HR, maintain Q and prevent reductions to MAP.  The concurrent increases to LV twist, untwisting velocity and HR have been suggested to reflect increased inotropy during reductions to preload (98, 99). Indeed, this is supported by the fact that LV twist and untwisting velocity are increased following administration of inotropic agents (2, 24, 59, 67, 153, 168). Therefore, relatively higher adrenergic stimulation or contractility may have contributed to the greater LV apical rotation, twist, and untwisting velocity in females during high levels of LBNP in this study. This is further supported by the greater systolic tissue velocity observed in females at -60 mmHg. It has also been reported that females have increased HR responsiveness to inotropic agents than males (45). Although regional adrenergic receptor densities have not been compared between the sexes, the LV apex is typically more responsive to acute stressors than the base, which likely reflects regional differences in adrenoreceptor density and sensitivity (2, 198). In the current study, relatively greater adrenergic stimulation at the apex likely contributed to the greater LV apical rotation and twist in females during high levels of LBNP.    68 3.4.3 Sex differences in LV geometry Our participants demonstrated classic sex differences in LV structure at baseline (150), with larger absolute LV dimensions and volumes in males than females. Nonetheless, relative wall thickness and sphericity index were similar, and allometrically scaled volumes and dimensions did not differ between the sexes, suggesting that LV morphology was relatively similar at baseline. With progressive LBNP, LV volumes and dimensions decreased, whereas sphericity index increased in both groups and was greater in females at -60 mmHg. In the LV wall, myocardial fibres are arranged in oblique orientations, and progressively change from a right-handed helix in the subendocardium to a left-handed helix in the subepicardium (185, 205). This continuum of helical fibre arrangement functionally underpins the generation of LV twist and shear strain (18, 19). When LV shape and helix angle are changed, the distributions of sarcomere length, passive fibre stress, and active fibre stress may be altered within the myocardium (18, 39), and subsequently impact LV twist (205, 212). In the current study, the reductions to LV volumes and increased sphericity index during LBNP likely coincided with altered LV helix angles in both males and females. However, the higher sphericity index and greater ellipsoid shape in females during -60 mmHg could reflect an altered fibre configuration that would result in larger apical rotation and twist in females.  3.4.4 Sex differences in the Frank-Starling relationship A significantly higher slope of the Frank-Starling relationship (∆LVSV/∆EDV) was observed in females compared to males. These findings agree with observations of Fu et al. (73), who reported a steeper maximal slope for ∆LVSV for any given LV pulmonary wedge pressure in females. Previous reports have suggested that the smaller female ventricle has lower diastolic compliance and greater elastance than that of males (42, 91). Accordingly, during reductions to preload, the less compliant female LV will store less elastic energy during diastole than that of males. A reduction in stored potential energy will subsequently result in a lowered capacity to utilize passive end-diastolic tension and the Frank-Starling mechanism. Therefore, females may require greater increases to LV twist and/or contractility than males to support SV.    69 The relationships between twist and LV volumes are highlighted in Figure 3.3. When plotted against absolute volumes, the curvilinear relationships of twist-to-LVEDV and twist-to-LVSV are similar for both sexes, though the female curves extend to a lower range of LV volumes and larger twist than males. However, when both twist and LV volumes are scaled, the relationships of LV torsion to allometrically scaled SV and EDV are visibly steeper in females compared to males. Thus, across a more comparable range of volumes, females appear to operate on a “steeper” portion of the twist-to-volume relationship, similar to differences in the Frank-Starling relationship. Analysis of quadratic functions for these relationships revealed a trend of greater b1 and b2 coefficients in females for the relationships of twist-to-LVSV and torsion to allometrically scaled SV, which supports the contention that the amplitudes of LV twist for a given SV differ between the sexes during reductions to preload.  The steeper slope of the curves in females may reflect a lower functional reserve, whereby if preload was challenged further, females may have a diminished reserve to further augment twist and maintain SV and MAP.  In an attempt to confirm that the differences observed in LV mechanics between the sexes were not due to differences in LV structure or body size, we normalized our twist data to LV length (i.e. torsion) and scaled LV volumes to body surface area. Furthermore, we found no association between LV length and twist (males: r2=0.002, p=0.87; females: r2=0.06, p=0.31), LV length and the change in twist from baseline to -60 mmHg (males: r2=0.12, p=0.17; females: r2=0.13, p=0.19), or LVEDV and twist (males: r2=0.06, p=0.33; females: r2=0.004, p=0.80). Combining these findings, our data support that the differences observed in LV twist during reductions to preload are a true sex difference rather than a result of variations in LV size or volume. Nonetheless, future studies might consider matching males and females for LV length or EDV to further confirm these sex differences in LV mechanics.  3.4.5 Limitations In the cohort that received the rapid saline infusion, there were no changes or sex differences in LV apical rotation, twist, and untwisting velocity. The responses of LV  70 mechanics to volume loading in prior investigations have been varied, with some reporting increased (220) or unchanged (31) apical rotation and twist, despite significant increases in EDV. These differences may be related to variations in volume loading protocols (i.e. total volume delivered, speed of infusion). Nevertheless, our findings demonstrate that changes in LVEDV within ± 10-20 ml from baseline with either LBNP or saline infusion did not have a significant effect on LV mechanics. With more substantial alterations to preload, we may have observed compensatory differences in LV mechanics between the male and female groups.   While we have accounted for large differences in LV structure using LV torsion and allometrically scaled LV volumes, we are limited in our ability to determine the physiological mechanisms responsible for the sex differences in LV mechanics observed in this study. The sex differences in LV twist and untwisting might occur due to intrinsic differences in the male and female hearts (LV size, geometry, properties of the myocardium), or differences in the adrenergic or autonomic control of the heart. It is likely that the combined influences of some or all of these factors contribute to the findings of the current study. Future work should focus on determining the independent effects of each of these factors in isolation and combination to better understand the mechanisms responsible for these sex differences in LV mechanics.  	  3.5 Summary and significance During high levels of LBNP, LV twist is greater in females compared to males, primarily as a result of greater apical rotation. In cases where passive tension and reliance on the Frank-Starling mechanism are reduced, females utilize greater LV twist and may rely on increasing contractility to a greater extent than males. While females have smaller absolute LV volumes and dimensions during high levels of LBNP, these differences in LV mechanics occur with similar relative reductions to LVEDV and SV in both sexes. Compared to males, the combination of higher LV twist and HR in females appears to protect SV and Q, and ultimately maintain MAP during reductions to preload. Overall, our data have demonstrated that females utilize greater LV mechanics than males to compensate during severe reductions to preload, and these sex differences may result  71 from differences in LV geometry, intrinsic properties of the myocardium, or adrenergic stimulation.    72 Chapter 4. The influence of adrenergic stimulation on sex differences in left ventricular twist mechanics  4.1 Background Left ventricular mechanics are fundamental to ventricular function, as LV twist supports the production of SV during ejection, and diastolic untwisting drives early filling during diastole (153, 198). Previous studies have identified sex differences in LV mechanics, where females have greater LV longitudinal and circumferential strain at rest (9, 119). Our group has also identified that females have greater LV twist and faster untwisting than males during large reductions to preload (226). It is currently unknown what structural differences or regulatory mechanisms are responsible for these sex differences in LV mechanics. However, it is feasible that differences in LV size or adrenergic stimulation may play a contributing role (153).   Females have been reported to have larger chronotropic responses to periods of acute physiological stress (73, 226), as well as having a increased high frequency power component of HRV (13, 82, 170), both of which are believed to reflect greater vagal control in females (73, 188). These findings are in contrast to males who commonly have a larger ratio of low frequency-to-high frequency power, which is believed to reflect greater sympathetic (adrenergic) control (13, 82, 116, 177). These potential sex differences in cardiac adrenergic stimulation are especially relevant to differences in LV mechanics, as altered adrenergic stimulation is reported to impact LV twist (59, 153, 168). Specifically, the administration of β1-AR agonists produces increases in SV and may even double LV twist and peak untwisting velocity (2, 144, 153). In contrast, β1-AR blockade results in reductions in LV twist, peak untwisting velocity (153) and strain (208). The changes to LV twist predominantly result from alterations to apical rotation, which is likely reflective of a greater β-AR density at the apex compared to the base (131, 146). However, given that these previous studies have involved exclusively male cohorts, it remains unknown how regional adrenergic control differs between the sexes to ultimately regulate LV twist mechanics. Therefore, the aim of this study was to investigate sex differences in LV mechanics with altered adrenergic stimulation, using  73 activation of the muscle metaboreflex with post-exercise ischemia and β1-AR blockade (bisoprolol) to augment and attenuate adrenergic stimulation, respectively. It was hypothesized that 1) during increases to adrenergic stimulation, LV twist and untwisting velocity would be lower in females than males, and 2) during reductions to adrenergic stimulation, females would have greater twist and faster untwisting than males.   4.2 Methods 4.2.1 Study participants Participants from the local university community, between the ages of 19-39 were recruited for the study. Exclusion criteria included: a history of cardiovascular, respiratory, or musculoskeletal disease; a body mass index (BMI) greater than 30 kg/m2; a resting blood pressure ≥140/90 or <110/60 mmHg and smoking (or smoking cessation <12 months).  Given the potential influence of sex-related differences in LV size on LV twist mechanics, males and females were matched for LV length. More specifically, individuals were continually enrolled until a total of 20 males and 20 females were matched for LV length. Those that could not be matched for LV length to an individual of the opposite sex were excluded.  To minimize the potential variability in LV structure (5, 218), mechanics (10, 219) and adrenergic control (137) associated with chronic endurance training, individuals performing >1 hour of moderate-intensity training five times per week, or ≥3 bouts of high intensity training per week were also excluded from the study. The study was approved by the University of British Columbia clinical research ethics board. Informed consent was obtained from all participants.  4.2.2 Study design Participants visited the laboratory on two separate occasions, and were asked to refrain from caffeine, exercise and alcohol for a minimum of 12 hours prior to the first visit, and 24 hours prior to the second visit. During visit 1, participants were assessed for resting blood pressure, adequate imaging windows and LV length. During visit 2, baseline echocardiographic images were collected following 15 minutes of quiet rest. Then, participants performed 3 minutes of isometric handgrip exercise, after which echocardiographic images were collected during the PEI period. Participants were then  74 administered bisprolol, and a final set of images were collected 2.5 hours later. To minimize differences in relative hormone levels and fluid shifts in the second visit, females who were not using combined oral contraceptives were tested in the early follicular phase of their menstrual cycles (days 3-6), and females using combined oral contraceptives were tested during the placebo or pill-free interval.  4.2.3 Specific methodology 4.2.3.1 Isometric handgrip and post-exercise ischemia  Participants performed three maximal handgrip efforts using their right hand to determine MVC, with each trial separated by at least one minute. An inflatable cuff was placed around the upper right arm, and participants performed isometric handgrip exercise at 35% MVC for 3 minutes, followed by 3-5 minutes of PEI to isolate the muscle metaboreflex (135). PEI was achieved by inflating the cuff to suprasystolic pressures (240 mmHg) ten seconds prior to handgrip release, and handgrip force was continuously recorded and displayed on a screen visible to the participant for feedback during the exercise. Collection of echocardiographic images began 30 seconds into the PEI period, and the cuff was released when imagining was complete.  4.2.3.2 β1-AR blockade Following PEI, participants rested for >15 minutes, until blood pressure and HR had returned to resting values. Participants were administered an oral 5 mg dose of bisoprolol (β1-AR antagonist), and returned to rest approximately 2.5 hours post-administration (time of peak plasma concentrations (122)) and a final set of echocardiographic images were collected after 15 minutes of quiet rest. In the time between bisoprolol administration and imaging, participants remained seated in the laboratory, and refrained from the consumption of food, but were able to drink small quantities of water ad libitum.  4.2.3.3 Transthoracic echocardiography, analysis of LV mechanics, and measurements of blood pressure and heart rate  See Chapter 2 for details.    75 4.2.3.4 Analysis of LV hemodynamics End-systolic wall stress was estimated as surrogate for LV afterload, and calculated as 0.9⋅SBP⋅(end-systolic cavity area/end-systolic myocardial area)(modified from (90)). End-systolic cavity area and myocardial area were calculated as π⋅(LVIDs/2)2 and [π⋅((PWTs+LVIDs+IVSTs)/2)2 - π⋅(LVIDs/2)2], respectively, under the assumption of a circular ventricular cavity just distal to the papillary muscles. See Chapter 2 for additional calculations.  4.2.4 Statistical analysis and sample size calculation Independent of analysis used, data are presented as mean ± standard deviation (SD) for clarity of interpretation. Normality of distribution was assessed using the Shapiro–Wilk test. Normally distributed data were assessed using an independent t-test to detect differences between the sexes in each condition. A one-way repeated measures ANOVA was used to detect within-group differences, and a Fisher’s least significant difference test was used to determine pairwise differences when a positive effect was detected. When the normality test failed, a Mann-Whitney test was used to detect sex differences in each condition for nonparametric data. A Friedman one-way repeated measures ANOVA on ranks was also used to detect within-group differences, and the Wilcoxon matched pairs test was used to determine pairwise differences. All statistical analyses were performed using STATISTICA (version 8.0; StatSoft, Tulsa, OK) with α set a priori to 0.05.  Linear least-squares regression was used to assess the relationships of LV twist mechanics with LV structure, geometry, and LV volumes in both sexes (inclusive of data from baseline, PEI and β1-AR blockade). Regression was additionally used to assess the relationship between LV twist and untwisting velocity. Pearson correlation and Spearman rank correlation were used to assess the relationships for normally distributed and nonparametric data, respectively. For clarity of interpretation, all correlation coefficients are presented as r. When a significant relationship was detected in both sexes, slopes of the regression were compared using the Extra Sum of Squares test.    76 No previous studies have investigated sex differences in LV twist with altered adrenergic stimulation, however previous work from Dedobbeleer et al. (57) reported a standard deviation (SD) of 2.3° in twist during β1-AR blockade. Utilizing this SD and an α=0.05, it was determined that 20 participants per group would allow us to detect a difference of 2.0º in LV twist between the sexes with a β=0.80.   4.3 Results 4.3.1 Baseline characteristics, LV structure and hemodynamics Of the 21 males and 26 females enrolled, 1 male and 2 females were excluded in the first visit for poor imaging windows. Four females were further excluded at the conclusion of data collection, as a male participant matched for LV length was not enrolled in the study. A total of 20 males and 20 females completed the study and were included in the analysis. Baseline characteristics are summarized in Table 4.1. MVC and thus 35% MVC were greater in males (199 ± 52N) than females (132 ± 34N; p<0.001 for both)(Table 4.1). Males had larger BMI (p=0.045) and BSA (p<0.001) than females. As per the study design, LV length was not different between the sexes (p=0.163). Despite the matching of LV length between the sexes, LVIDd was larger in males (p<0.001), resulting in sex differences in sphericity index (p=0.005). However, males had larger LV volumes (p<0.001) and SV (p=0.017) than females (Table 4.3), but allometrically scaled EDV and SV did not differ between the sexes at baseline. In contrast, scaled ESV was smaller in females at baseline (p=0.034), reflective of a greater EF in females (p=0.001). Blood pressure and HR did not differ between the sexes. There were additionally no baseline sex differences in relative wall thickness, or in scaled LVIDd, PWT, IVST. E was greater in females (females 0.94 ± 0.15m⋅s-1, males 0.82 ± 0.14m⋅s-1, p=0.01), however A (females 0.38 ± 0.07m⋅s-1, males 0.39 ± 0.11m⋅s-1), and E/A (females 2.58 ± 0.70, males 2.31 ± 0.76) did not differ between the sexes.        77 Table 4.1. Baseline characteristics, LV hemodynamics, structure and geometry  Males (n=20) Females (n=20) Participant characteristics Age (yrs) 23 (5) 22 (3) Height (m) 1.77 (0.05) 1.66 (0.07)# Weight (kg) 72.4 (6.4) 60.3 (6.4)# BMI (kg⋅m-2) 23.0 (2.0) 21.8 (1.5)* BSA (m2) 1.89 (0.10) 1.67 (0.12)# MVC (N) 571 (150) 377 (98)# Resting hemodynamics HR (bpm) 60 (10) 62 (8) SBP (mmHg) 120 (8) 115 (9) DBP (mmHg) 73 (9) 70 (8) MAP (mmHg) 88 (8) 85 (7) EF (%) 55 (3) 58 (3)# Resting LV structure and geometry Lengthd (cm) 8.45 (0.45) 8.22 (0.55) LVIDd (mm) 45.1 (3.2) 40.9 (3.1)# Sphericity index 1.88 (0.13) 2.02 (0.16)# Relative wall thickness 0.45 (0.06) 0.45 (0.07) Values are means (SD). BMI: body mass index; BSA: body surface area; MVC: maximal voluntary contraction; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; MAP: mean arterial pressure; EF: ejection fraction; Lengthd: end-diastolic length; LVIDd: left ventricular internal diameter during diastole. *p<0.05 vs. males. #p<0.01 vs. males.    4.3.2 LV mechanics in response to altered adrenergic stimulation  Table 4.2 summarizes peak LV mechanics parameters. At baseline, there were no sex differences in LV twist mechanics (twist, torsion, apical rotation, basal rotation and untwisting velocity). However, circumferential strain at the base and longitudinal strain were higher in females compared to males (p=0.025 and p=0.015, respectively).   78 4.3.2.1 LV mechanics during post exercise ischemia There were no changes from baseline in LV apical rotation, twist, untwisting velocity (Figure 4.1) or strain in either group. However, basal rotation was increased in males (p=0.037). Nonetheless, there were no sex differences in twist during PEI. Longitudinal strain remained higher in females, although circumferential strain at the base was not different between the sexes, and circumferential strain at the apex tended to be higher in females (p=0.055).  4.3.2.2 LV mechanics during β1-AR blockade In females, LV twist mechanics did not differ from baseline, although there was a trend to reduced LV twist (p=0.063). In males, LV twist and torsion were reduced (p=0.029 and p=0.032, respectively), due to a significant reduction to apical rotation (p=0.036) and a trend to reduction in basal rotation (p=0.09)(Figure 4.1). Untwisting velocity also tended to be reduced in males (p=0.075). As a result, males had lower LV apical rotation (p=0.007), twist (p=0.008) and torsion (p=0.004), and slower untwisting velocity compared to females (p=0.046). LV strain parameters were not changed from baseline, such that longitudinal strain (p<0.001) and circumferential strain at the base (p=0.02) remained higher in females. There were no sex differences in basal rotation or apical circumferential strain during β1-AR blockade.   79  Figure 4.1. Graphical representation of mean left ventricular (LV) twist mechanics at baseline, during post exercise ischemia and ß1-AR blockade (bisoprolol). Blue and red lines represent mean data for males and females, respectively. Top: dotted and dashed lines represent rotations of the LV apex and base, respectively. Middle: solid lines represent LV twist. Bottom: solid lines represent twist and untwisting velocities. SDs are provided in Table 4.2. *p<0.05 males vs. females.  -5051015Twist (°)Baseline Post Exercise Ischemia β1-AR Blockade-50050Twist Velocity (°sec-1 )MalesFemales-5051015Rotation (º)ApexBase% Systole     %Diastole% Systole     %Diastole % Systole     %Diastole         50      100     150      200         50      100     150      200          50      100     150      200* * *  80 Table 4.2. LV mechanics during altered adrenergic stimulation.   Baseline Post Exercise Ischemia β1-AR  Blockade Twist (˚) M 10.2 (2.5) 11.1 (3.2) 8.6 (1.9)†  F 11.3 (3.1) 11.3 (2.4) 10.7 (2.8)* Torsion (˚⋅cm-1) M 1.20 (0.30) 1.28 (0.36) 1.02 (0.23)†  F 1.38 (0.38) 1.37 (0.30) 1.33 (0.38)# Untwisting velocity (˚⋅s-1) M -80.3 (25.3) -77.6 (22.0) -68.2 (22.1)  F -93.5 (22.6) -79.4 (28.5) -82.0 (18.7)* Apical rot (˚) M 7.8 (1.7) 8.4 (3.3) 6.8 (2.1)†  F 8.7 (2.5) 8.9 (2.3) 8.8 (2.3)* Basal rot (˚) M -3.1 (1.8) -3.8 (1.9)† -2.5 (1.1)  F -3.3 (2.0) -3.3 (2.3) -2.4 (1.7) Longitudinal strain (%) M -17.5 (1.9) -17.0 (1.7) -17.2 (1.6)  F -19.0 (1.7)* -19.5 (1.5)# -19.0 (1.6)# Circumferential strain, base (%) M -20.3 (3.3) -20.2 (3.9) 20.2 (2.5)  F -22.3 (2.1)* -22.0 (3.0) -22.3 (3.0)* Circumferential strain, apex (%) M -26.1 (3.7) -25.5 (3.5) -25.7 (2.5)  F -25.5 (3.4) -27.6 (2.7) -26.0 (2.7) Values are means (SD). All data represent peaks across the cardiac cycle. M: males; F: females; Rot: rotation. n=20 females, 20 males for all measures but apical rotation (female n=19), basal rotation (female n=19), twist and torsion (female n=18). *p<0.05 vs. males. #p<0.01 vs. males. †p<0.05 vs. baseline.    4.3.3 Hemodynamic responses to altered adrenergic stimulation 4.3.3.1 Hemodynamics responses during post exercise ischemia Blood pressure increased from baseline in both groups (p<0.001 for both), and SBP (p=0.007), DBP (p=0.031) and MAP (p=0.006) were greater in males (Table 4.3). HR increased in males (p=0.022) and tended to increase in females (p=0.08), however HR was not different between the sexes. LVEDV increased (p<0.001 for both) but ESV was unchanged, resulting in an augmentation of both LVSV and Q in both sexes (p<0.001). There were no sex differences in scaled LVEDV or SV, but scaled ESV tended to be  81 lower in females (p=0.08). Thus, while EF was increased in males (p<0.001) and tended to increase in females (p=0.07), EF remained greater in females compared to males (p=0.025). End-systolic wall stress increased in both sexes (p<0.001), and was greater in males compared to females (p=0.013). Although TPR increased in males (p=0.014), it was unchanged in females and not different between the sexes. E increased in males (0.87 ± 0.19m⋅s-1, p=0.012), and A increased in both sexes during PEI (females 0.44 ± 0.17m⋅s-1, males 0.42 ± 0.10m⋅s-1, p<0.05). E/A, however, was unchanged and there were no sex differences in these parameters.  4.3.3.2 Hemodynamic responses during β1-AR blockade Blood pressure, HR and Q were reduced from baseline in both groups (p<0.001). SBP tended to be higher in males (p=0.08), and both DBP (p=0.012) and MAP (p=0.002) were higher in males. However, the reduction to HR was greater in females (-12 ± 6 bpm) compared to males (-8 ± 5 bpm, p=0.023). LV volumes and EF were not different from baseline in either group. Similar to baseline, scaled LVEDV and SV did not differ between the sexes, but scaled ESV was smaller (p=0.01) and EF was greater (p<0.001) in females. End-systolic wall stress was reduced in both groups (p<0.001 for both), but was not different between the sexes. TPR was unchanged and did not differ between the sexes. E was unchanged from baseline in both sexes, however A was reduced in females (0.31 ± 0.06m⋅s-1, p<0.001) but not in males (0.35 ± 0.10m⋅s-1). Thus, E/A was increased in females (2.99 ± 0.16, p<0.001) but not males (2.56 ± 0.87, p=0.072). As a result, there was a trend of higher A (p=0.09) and E/A (p=0.09) in females during this stage.            82 Table 4.3. LV hemodynamics during altered adrenergic stimulation.   Baseline Post Exercise  Ischemia β1-AR  Blockade HR (bpm) M 60 (10) 63 (10)† 52 (9)‡  F 62 (8) 65 (11)  50 (8)‡ MAP (mmHg) M 88 (8) 116 (10)‡ 81 (8)‡  F 85 (7) 107 (10)‡# 75 (11)‡# SBP (mmHg) M 120 (8) 160 (13)‡ 109 (9)‡  F 115 (9) 145 (17)‡# 104 (11)‡ DBP (mmHg) M 73 (9) 95 (11)‡ 67 (9)‡  F 70 (8) 88 (9)‡* 61 (12)‡* EF (%) M 55 (3) 58 (4)‡ 55 (3)  F 58 (3)# 60 (3)* 60 (3)# EDV (ml) M 113 (20) 120 (24)‡ 115 (19)  F 91 (14)# 96 (16)‡# 92 (13)# EDV (ml⋅m-3) M 43 (7) 46 (8)‡ 44 (7)  F 42 (6) 45 (6)‡ 43 (6) ESV (ml) M 51 (10) 51 (12) 51 (9)  F 38 (6)# 38 (7)# 37 (5)# ESV (ml⋅m-3) M 20 (3) 20 (4) 20 (3)  F 18 (3)* 18 (2) 17 (3)* SV (ml) M 62 (12) 70 (14)‡ 63 (12)  F 53 (9)* 58 (10)‡# 55 (9)# SV (ml⋅m-3) M 24 (4) 27 (5)‡ 24 (4)  F 25 (4) 27 (4)‡ 25 (4) Q (L⋅min-1) M 3.66 (0.70) 4.30 (0.62)‡ 3.26 (0.67)‡  F 3.25 (0.41)* 3.68 (0.50)‡# 2.69 (0.45)‡# Q (L⋅min-1⋅m-3) M 1.41 (0.24) 1.66 (0.26)‡ 1.26 (0.26)‡  F 1.52 (0.24) 1.73 (0.32)‡ 1.26 (0.22)‡   83 Table 4.3 (cont). LV hemodynamics during altered adrenergic stimulation.   Baseline Post Exercise  Ischemia β1-AR  Blockade TPR  (mmHg⋅L-1⋅min-1) M 25.1 (5.8) 27.6 (4.3)  26.2 (5.7)  F 26.7 (4.9) 29.5 (4.9)* 28.8 (7.1) End-systolic wall stress (kilodyne⋅cm-2) M 39.1 (5.3) 54.7 (8.4)‡ 33.5 (6.9)‡  F 37.2 (4.4) 48.5 (5.2)‡* 32.8 (6.0)‡ Values are means (SD). EDV: end-diastolic volume; ESV: end-systolic volume; SV: stroke volume; Q: cardiac output; TPR: total peripheral resistance. See Table 4.1 and 4.2 for additional abbreviations. *p<0.05 vs. males. #p<0.01 vs. males. †p<0.05 vs. baseline. ‡p<0.01 vs. baseline.     4.3.4 LV structure and geometry during altered adrenergic stimulation There were no changes from baseline in absolute or scaled wall thicknesses and LVIDd in either sex, during any stage. IVST (p<0.05) and LVIDd (p<0.001) were larger in males during all stages, and PWT was larger in males (p<0.05) except during β1-AR blockade (p=0.096). Nonetheless, scaled wall thicknesses and scaled LVIDd were not different between the sexes in any stage. Relative wall thickness and sphericity index were also unchanged in either sex, during either intervention. Relative wall thickness did not differ between the sexes, however sphericity index was greater in females at baseline (p=0.005), during PEI (p=0.007) and β1-AR blockade (p=0.003). LV length increased in males (p<0.001) and tended to increase in females (p=0.07) during PEI, but was unchanged during β1-AR blockade in either sex. LV length was not different between the sexes during either intervention, but tended to be smaller in females during β1-AR blockade (p=0.052).         84 Table 4.4. LV structure and geometry during altered adrenergic stimulation.   Baseline Post Exercise  Ischemia β1-AR  Blockade Lengthd (mm) M 84.5 (4.5) 85.7 (4.8)‡ 84.7 (4.7)  F 82.2 (5.5) 83.0 (5.3) 81.6 (5.2) LVIDd (mm) M 45.1 (3.2) 45.3 (2.7) 45.5 (3.3)  F 40.9 (3.1)# 40.8 (3.9)# 40.3 (2.8)# Sphericity index M 1.88 (0.13) 1.89 (0.12) 1.88 (0.13)  F 2.02 (0.16)# 2.05 (0.21)# 2.03 (0.17)# Relative wall thickness  M 0.45 (0.06) 0.44 (0.06) 0.45 (0.07)  F 0.45 (0.07) 0.45 (0.07) 0.48 (0.06) Values are means (SD). See Table 4.1 for abbreviations. *p<0.05 vs. males. #p<0.01 vs. males. †p<0.05 vs. baseline. ‡p<0.01 vs. baseline.     4.3.4.1 Relationships of LV mechanics with structure and geometry  There was a significant relationship between LV twist and untwisting velocity in both males (r=-0.58, p<0.001) and females (r=-0.57, p<0.001), and this was not different between the sexes. In females, there was a significant relationship for LVIDd with LV apical rotation (r=-0.30, p=0.02), and twist (r=-0.35, p=0.013)(Figure 4.2). Additionally, there were significant relationships for sphericity index with apical rotation (r=0.32, p=0.019) and twist (r=0.35, p=0.012), in females but not males. There were no relationships for LV length with twist or rotation in either group. No relationships between LVEDV or SV with LV apical rotation, basal rotation or twist were observed for either sex.   85  Figure 4.2. Relationships for LV twist mechanics with chamber structure and geometry. Data include measures during baseline, post exercise ischemia and ß1-AR blockade. Blue and red represent data for males and females, respectively. Top: closed circles represent LV twist. Bottom: open triangles and circles represent LV rotation at the apex and base, respectively. *Significant relationship (p<0.05).  20 30 40 50 600510152025LVIDd (mm)Twist (°) *30 40 50 60-1001020LVIDd (mm)Rotation (°) *1.5 2.0 2.5 3.00510152025Sphericity index*2.0 2.5 3.0-1001020Sphericity index*6 7 8 9 100510152025LV length (cm)7 8 9 10 11-1001020LV length (cm)MalesFemalesApexBase!r=-0.30p=0.02r=-0.35p=0.013r=0.32p=0.019r=0.35p=0.012 86 4.4 Discussion  This is the first study to compare LV mechanics between males and females, matched for LV length, during altered adrenergic stimulation. In support of our hypothesis, females had greater LV twist and faster untwisting velocity than males during ß1-AR blockade. However, in contrast, no sex differences in LV twist mechanics were observed with increased adrenergic stimulation during PEI.   4.4.1 Effects of post exercise ischemia on sex differences in LV mechanics In the current study, PEI was used to activate the muscle metaboreflex, and effectively increase adrenergic stimulation independently of increases to HR (151, 156). During PEI, LVSV was increased in both males and females. However, contrary to our hypothesis, LV twist was not different from baseline or between the sexes. This occurred despite a small but significant increase to basal rotation in males during PEI. The increases to LVSV in our study are in agreement with Crisafulli et al. (49, 50) who have demonstrated that SV increases to ~130% of baseline during PEI in an all male cohort. In females, Shoemaker et al. (189) have also reported a trend of elevated SV during PEI. In the current study, the elevations to LVSV resulted from increases to EDV, while ESV was unchanged, suggesting that the increases to LV contractility were enough to offset the pronounced increases in afterload as indicated by the elevated end-systolic wall stress. Likewise, both groups had increases to A filling velocity, therefore increased atrial contraction and filling potentially contributed to increasing EDV. Increases to central venous pressure also occur during PEI (136, 189), and likely increased venous return thus explaining the higher LVEDV in the current study.   Given that increases to adrenergic stimulation and LV preload can each independently increase LV twist, the concomitant increases to LVEDV and SV during PEI would be expected to accompany increases to LV twist. While LVEDV and SV were increased in this study, neither males nor females had alterations to LV apical rotation or twist during PEI.  Given that increases to afterload reduce LV twist, especially at the apex (59, 78, 221), the increased end-systolic wall stress during PEI may have countered any potential increases to apical rotation and thus LV twist in both groups. Finally, although increases  87 to LV preload are reported to augment LV twist mechanics (220), the increases to LVEDV of ~5-7 mL in the current study likely were not enough to increase LV twist. This is supported by prior investigations from our group (226) and others (31) in which small increases (~10 mL) to LVEDV and LVSV did produce significant alterations to LV twist.   While twist was not significantly altered in either sex during PEI, males did have a small but significant increase to basal rotation, but this did not result in significant sex differences in LV rotation or twist. Nonetheless, the increased basal rotation in males could provide some evidence that the responses of LV mechanics may differ between the sexes with increased adrenergic stimulation. The increases to basal rotation but not apical rotation in males may reflect greater receptor sensitivity at the base. However, this seems unlikely as greater receptor densities and augmented responsiveness to adrenergic stimulation have been demonstrated at the apex compared to the base (2, 146). Additionally, while we theorized that males would have a larger increase in apical rotation and thus twist than females, it is possible that this effect was countered by the significantly greater LV afterload (as determined by end-systolic wall stress) observed in males during PEI.  Recently, Balmain et al. (11) used PEI in an attempt to discriminate between the contributions of increased afterload and chronotropy that occur during static handgrip exercise. In contrast to our findings, they observed reductions to LV apical rotation, twist and untwisting velocity during PEI, without changes to LVEDV, ESV and SV. While the authors proposed that large increases to LV afterload attenuated LV twist, the hemodynamic data aren’t entirely consistent with increased afterload, given that increases to LVESV and reductions to SV would be expected to occur when EDV is unchanged. The reduction to LV twist is thus surprising given that increases to sympathetic activation and LV contractility occur during PEI (49, 216). In the current study, the increase to EF in males and the lack of change to ESV in both sexes suggests that an increase in LV contractility maintained LV twist and offset the increased LV afterload.    88 4.4.2 Effects of ß1-AR blockade on sex differences in LV mechanics The reduced LV twist mechanics in males compared to females during ß1-AR blockade predominantly resulted from reductions to LV apical rotation in males, whereas LV rotation and twist were unchanged in females. The lower LV apical rotation, twist and untwisting velocity in males during ß1-AR blockade provide preliminary evidence for sex-related differences in LV adrenergic control of LV twist mechanics, specifically during reductions to adrenergic stimulation. Studies using HRV consistently report greater low frequency power and low-to-high frequency ratios in males, compared to females (13, 82, 116, 177) suggesting that males are more sympathetically mediated than their female counterparts. Our data support the contention that males are more sympathetically mediated as reductions to adrenergic stimulation during ß1-AR blockade resulted in significant reductions to LV twist in males but not in females.    It has been proposed that changes to HR coincide with similar alterations to contractility and LV twist mechanics (98). However, our data do not support this mechanistic link between HR and twist in females, as they experienced a greater reduction to HR without a significant reduction to LV twist. As both HR and twist were reduced in males, this suggests that altered adrenergic stimulation may affect chronotropy and twist differently between the sexes. This postulate is partially supported by previous work that demonstrated a greater increase in HR with a ß1-AR agonist in females, but a greater increase to an index of contractility in males (45, 211). Likewise, data from Evans et al. (69) reported potentially greater reductions to HR in females than males during ß1-AR blockade with propranolol. Collectively, these data suggest that females have greater chronotropic responses to alterations in adrenergic stimulation whereas males may have greater inotropic responses. Thus, in the current study, it is possible that ß1-AR blockade reduced LV contractility in males and contributed to the attenuated LV twist mechanics compared to females, whereas females had greater reductions to HR but no alterations to LV twist.    89 4.4.3 Relationships between LV twist mechanics and chamber geometry To our knowledge, this is the first study to match LV length between the sexes, rather than scaling or indexing to LV dimensions or body size. First, we have demonstrated that for the same LV length, females have a smaller LVIDd than males, resulting in a greater sphericity index, or a greater LV ellipsoid geometry compared to males. As a result, males have greater LV volumes than females for the same LV length. Second, we did not observe any associations between LV length, EDV or SV with apical rotation, basal rotation or twist in either sex. This confirms that sex differences in LV twist mechanics are likely not fundamentally determined by differences in LV size or volume. However, there was a negative relationship between LVIDd and twist (r=-0.35, p=0.013), as well as a positive relationship for sphericity index with LV apical rotation (r=0.32, p=0.019) and twist (r=0.35, p=0.012) in females. In contrast, there were no relationships observed between LV structure or geometry and LV twist mechanics in males. Combined with the observed sex differences in LV twist during β1-AR blockade, these data suggest that LV twist may be more sensitive to LV structure and geometry in females, but more sensitive to altered adrenergic stimulation in males.   We have previously demonstrated that females have greater LV twist and sphericity index than males during significant reductions to preload utilizing LBNP, despite similar relative reductions to LV volumes in both sexes (226). In connection with the current findings, these sex differences may reflect a greater influence of LV geometry on twist in females. Given that LV deformation is primarily determined by interactions between myofibre layers (169), alterations to LV shape and thus myofibre alignment can directly alter fibre mechanics and twist in various regions of the LV wall (39). Compared to a spherical ventricle, a more ellipsoid shape favours increased active fibre stress development and ejection performance (39). To that effect, LV sphericity index has been identified as a strong independent predictor of LV rotation and twist (52). Therefore, the observed sex differences in LV sphericity index in the current and our previous study (226) suggest that sex differences in LV fibre alignment occur for a given LV length, and these intrinsic differences in myocardial structure and geometry likely play a role in  90 determining sex differences in LV twist during to differences in resting and dynamic responses of twist mechanics.   4.4.4 Limitations An important limitation to this study was that we observed no increase in LV twist in either sex during PEI and were subsequently unable to investigate whether sex differences in LV twist occur with increased adrenergic stimulation. As blood pressure, LVSV and EF increased with PEI, we are confident that this intervention augmented adrenergic stimulation and the unaltered LV twist was likely due to the concomitant increases to LV afterload. Future studies should consider administrating pharmacological ß1-AR agonists (i.e. isoproterenol, dobutamine) to effectively augment LV twist (2, 144) and to further examine whether sex differences in LV mechanics exist with increased adrenergic stimulation.  4.5 Summary and significance In males and females matched for LV length, differences in LV twist mechanics occur during reductions to adrenergic stimulation. Females have greater LV apical rotation, twist and untwisting velocity than males during ß1-AR blockade. The reductions to apical rotation and twist in males are suggestive of greater sympathetic-related adrenergic control of LV twist mechanics compared to females. Although sex differences in LV twist were not observed during increases to adrenergic stimulation with PEI, potentially greater increases to LV twist in males may have been countered by larger increases to afterload. In addition, the matching of LV length has revealed marked sex differences in LV chamber geometry, which may contribute to differences in the responses of LV twist to altered loading and adrenergic stimulation. Altogether, our data provide preliminary evidence that LV twist be more sensitive to alterations in adrenergic stimulation in males, but influenced to a greater extent by LV geometry in females.   91 Chapter 5. The influence of vagal control on sex differences in left ventricular mechanics during alterations to stroke volume  5.1 Background It has previously been established that sex differences in the autonomic control of the heart occur at rest and during periods of acute physiological stress. Vagal autonomic activity and muscarinic receptor activation are important regulators of cardiac chronotropy and myocardial inotropy, and therefore play a key role in determining LV function during challenges to preload and exercise (125, 225, 229). Studies utilizing resting heart rate variability have demonstrated greater vagal control in females, while males appear to be more sympathetically mediated (13, 66, 69, 82, 177). Additionally, during dynamic exercise and acute reductions to preload, females have augmented HR responses (likely due to increased vagal withdrawal) (73, 188, 202, 223), whereas males have greater increases in sympathetic regulators (i.e. MSNA (188) and circulating catecholamines  (45, 55, 73, 223)). However, to our knowledge, no study has investigated the specific contribution of vagal control to sex differences in the LV responses to acute physiological stress.   Further to differences in the autonomic and chronotropic responses to acute stress, the responses of LV hemodynamics and mechanics (rotation, twist and strain) also differ between the sexes (73, 188, 224, 226). During large reductions to preload and SV with LBNP, the smaller female LV has greater twist and faster untwisting than that of males, despite similar relative reductions to LV volumes (226). Yet, whether comparable changes in LV mechanics occur with increases to SV is not well known. During dynamic exercise, increases to LV twist appear to mechanically support the increases to SV up to ~50% of maximal workload, however these responses have only been examined in male cohorts (64, 198). Given that LV twist appears to support increases to SV during exercise, and defend SV during reductions to preload, it is feasible that sex differences in the relative influence of vagal versus sympathetic control, or sympathovagal balance, could contribute to differences in LV mechanics during these interventions. In Chapter 4, ß1-AR blockade reduced LV apical rotation and twist in males, but not in females,  92 providing preliminary evidence for a greater sympathetic or adrenergic control of LV twist in males. However, it remains unknown whether a potentially greater vagal control of the heart in females contributes to sex differences in LV twist during acute physiological stress.   The use of a muscarinic receptor antagonist such as glycopyrrolate allows for the selective blockade of vagal autonomic control to the heart, and thus would remove the potential influence of sex differences in vagal withdrawal during LBNP and exercise. If greater LV twist mechanics in females are predominantly determined by greater vagal control and withdrawal, vagal blockade with glycopyrrolate should eliminate the known sex differences in LV twist during reductions to SV with LBNP, and any potential sex differences during increases to SV with exercise. Therefore, the primary aim of this investigation was to determine the sex differences in LV twist mechanics during decreases and increases to SV, with and without vagal muscarinic blockade. It was hypothesized that 1) during -60 mmHg LBNP and exercise at 50% of maximal workload, females would have larger LV twist than males, and 2) sex differences in LV twist would be minimized during -60 mmHg LBNP and 50% exercise with glycopyrrolate.   5.2 Methods 5.2.1 Study participants Participants between the ages of 19-39 were recruited from the local university community at Cardiff Metropolitan University. Individuals were excluded if they had any history of cardiovascular, respiratory, or musculoskeletal disease; answered “yes” to any question in the Physical Activity Readiness Questionnaire; had a BMI greater than 30 kg/m2; had a resting SBP ≥140 or ≤100 mmHg; were current smokers (or quit <12 months). Individuals performing ≥5 hours of moderate-intensity aerobic exercise, or performing ≥3 bouts of high intensity training per week were excluded from the study to reduce any potential variability of LV structure, mechanics and autonomic regulation associated with significant endurance training (5, 10, 137, 218, 219). The study was approved by the University of British Columbia clinical research ethics board and Cardiff  93 Metropolitan University research ethics committee. Informed consent was obtained from all participants before starting the study.  5.2.2 Study design Participants visited the laboratory at the Cardiff School of Sport (Cardiff Metropolitan University, Cyncoed Campus, UK) on three occasions and were asked to refrain from caffeine, exercise and alcohol for a minimum of 24 hours prior to each visit. Potentially eligible participants were provided a copy of the consent form at least 24 hours prior to their first visit.   In the initial visit, participants signed the consent form and then were screened for adequate imaging windows, LV length and resting blood pressure. Thereafter, they were screened for their ability to tolerate up to -60 mmHg LBNP. Following 15 min rest after LBNP, participants completed an incremental exercise test to volitional fatigue on a supine cycle ergometer (Lode, Angio, Groningen, Netherlands) to determine peak power output.   During the second and third visits, participants were exposed to LBNP and performed supine cycling exercise (EX) with (GLY) or without (CON) vagal blockade using glycopyrrolate on different days (sequence of trials shown in Figure 5.1). On both days, participants first rested in a LBNP chamber, attached to a supine bed, and were sealed at the level of the iliac crests. The bed and chamber were tilted at a 45º angle toward the sonographer, positioning the participant in a left lateral position for echocardiographic imaging. Following 10 minutes of quiet rest, baseline images were collected. LBNP was then applied at -40 and -60 mmHg, and echocardiographic images were collected once HR and MAP had stabilized within 5 beats per minute and 5 mmHg, respectively. Participants rested for >20 minutes following the end of LBNP, after which they were connected to a supine cycle ergometer, rested in the supine position and tilted at a 45º angle toward the sonographer. Participants then completed exercise at 25% and 50% of peak supine power output. At each intensity, echocardiographic imaging began 2-3  94 minutes following exercise onset, when steady state HR was achieved (within 5 beats per minute).   During the visit with vagal blockade, an 18-guage catheter was inserted into the antecubital vein prior to testing. Participants then rested in the LBNP chamber as described above before baseline images were collected. Stepwise infusions of glycopyrrolate were then administered until full blockade was achieved (see Specific methodology), and a second set of baseline images were collected prior to the onset of LBNP. To ensure full vagal blockage for the entire protocol, an additional bolus of glycopyrrolate was administered when participants were rested on the supine cycle ergometer, prior to image acquisition.    Figure 5.1. Schematic of methodology for control (CON) and vagal blockade (GLY) trials. Infusion denotes administration of glycopyrrolate during GLY trials. Echo denotes collection of echocardiographic images. LBNP: lower body negative pressure; EX: submaximal exercise.    5.2.3 Specific methodology 5.2.3.1 Screening and familiarization To assess LBNP tolerance, participants were exposed to stepwise increments of -10 mmHg until -60 mmHg, which was then maintained for 3 minutes. LBNP was immediately terminated when SBP decreased below 80 mmHg, or at signs of pre-syncope (feelings of light-headedness, nausea or tunnel vision). Individuals unable to tolerate the full protocol were excluded from the study.   Pre-infusionPost-infusion / Rest-40 mmHg-60 mmHgRest25%50%!! ! ! ! ! !TimelineLBNP EXINFUSION RESTEchoEchoEchoEchoEchoEchoEcho 95 5.2.3.2 Incremental exercise testing Cycling began at 50W for females and 75W for males, and power outputs were increased in a ramp-incremental fashion, at a rate of 20W per minute for females, and 25W per minute for males. Participants were encouraged to maintain a cadence of >70 RPM, and continued to cycle until they reached volitional fatigue. Heart rate was monitored at rest and during exercise using a Polar heart rate monitor.  5.2.3.3 Vagal blockade Glycopyrrolate was administered as previously outlined (158) to achieve vagal blockade. Briefly, stepwise infusions of 0.2 mg of glycopyrrolate were administered every 2 minutes until HR was unchanged to consecutive doses of 0.2 mg. An additional 0.2 mg was administered prior to the exercise trials if HR had reduced >5 beats per minute compared to post-infusion baseline. Total doses were 1.0 ± 0.3 mg for females and 1.1 ± 0.3 mg for males, and not significantly different between the groups.  5.2.3.4 Transthoracic echocardiography, analysis of LV mechanics and hemodynamics, and measurement of blood pressure and heart rate See Chapter 2 for details.   5.2.4 Statistical analysis and power calculation For clarity of interpretation, normally distributed and nonparametric data are presented as means ± standard deviation (SD). The Shapiro-Wilk test was used to assess the normality of distribution. For normally distributed data, dependent variables were assessed using an independent t-test to detect sex differences at baseline, and during each level of LBNP and exercise. A 2 (group) x 3 (level) ANOVA was also performed to independently assess the effects of exercise and LBNP. When a positive effect was detected with the ANOVA, a Fisher’s least significant difference test was used to determine pairwise differences. For nonparametric data, dependent variables were assessed using a Mann-Whitney test to detect sex differences in each condition. Additionally, a Friedman repeated measures ANOVA on ranks was used to detect within-group differences during exercise and LBNP, and pairwise differences were assessed using the Wilcoxon matched  96 pairs test. All statistical analyses were performed using STATISTICA (version 8.0; StatSoft, Tulsa, OK) with α set a priori to 0.05.   To assess the relationship of LV torsion to SVžBSA-1.5 during LBNP and exercise, an Extra sum-of-squares F test was used to compare the linear versus second order polynomial (quadratic) fit of the data. When an appropriate model was determined, equations of the relationships were determined for each group, and the relationships were compared between the sexes using an Extra sum-of-squares F test.   Linear least-squared regression was used to assess the relationship of LV twist with untwisting velocity, as well as the relationships for LV twist mechanics with LV structure and geometry during LBNP and exercise. Pearson correlation and Spearman rank correlation were used to assess the relationships for normally distributed and nonparametric data, respectively. For clarity of interpretation, all correlation coefficients are presented as r. Linear regression of the change in LVSV (∆LVSV) for any given change in EDV (∆EDV) was performed for each participant during LBNP and mean slopes of this modified Frank-Starling relationship were compared between the sexes using an independent t-test (73).   During -60 mmHg LBNP, we have previously observed a 5.6º difference in LV twist, with a SD of 5.9º between males and females (226). With a power of 0.80 and an alpha of 0.05 we required 18 individuals in each group to detect this effect. While there are less data available to assess the effects of exercise on sex differences in LV twist, previous work from Stohr et al. (198) have reported a SD of 6.0º during exercise at 50% maximal workload. Thus, with 18 subjects per group, a SD of 6.0º and an alpha of 0.05 we had 80% power to detect a 5.8º difference in LV twist between the sexes during exercise at 50% peak power.    97 5.3 Results 5.3.1 Baseline characteristics, LV structure and hemodynamics Of 19 males and 18 females recruited and screened for the study, one male was unable to tolerate the LBNP screening and was excluded from testing. One female withdrew from the study following the screening visit. One male and one female withdrew from the study for personal reasons prior to completing the CON and GLY trials, respectively. Additionally, one male did not complete the EX trials due to a sport-related injury following screening. Thus, data are presented as means ± SD for CON-LBNP (F: n=17, M: n=17), CON-EX (F: n=17, M: n=16), GLY-LBNP (F: n=16, M: n=18), and GLY-EX (F: n=16, M: n=17).  Baseline characteristics are outlined in Table 5.1. SBP, DBP and MAP were greater in males (p<0.001 for all). HR did not differ between the sexes, however EF tended to be higher in females (p=0.074). LV length (p=0.001) and LVIDd (p<0.001) were both larger in males, although females had a larger sphericity index (p=0.026). IVST, PWT and relative wall thickness did not differ between the sexes.   98 Table 5.1. Participant demographics and baseline characteristics  Males (n=18) Females (n=17) Participant characteristics Age (yrs) 22 (2) 21 (4) Height (m) 1.79 (0.08) 1.66 (0.08)* Weight (kg) 75.8 (9.7) 65.8 (7.7)* BMI (kg⋅m-2) 23.7 (3.1) 23.8 (2.4) BSA (m2) 1.94 (0.14) 1.74 (0.13)* POpeak, supine (W) 251 (29) 201 (31)* Resting hemodynamics HR (bpm) 61 (7) 61 (9) SBP (mmHg) 124 (8) 111 (8)* DBP (mmHg) 73 (9) 62 (8)* MAP (mmHg) 89 (8) 77 (7)* EF (%) 54 (4) 56 (4) Resting LV structure Lengthd (cm) 8.8 (0.3) 8.4 (0.4)* Lengthd · BSA-0.5 (cm/m) 6.3 (0.2) 6.4 (0.2) LVIDd (mm) 49 (3) 44 (3)* LVIDd · BSA-0.5 (mm/m) 35 (3) 34 (2) Sphericity index 1.81 (0.11) 1.90 (0.11)* IVST (mm) 10 (2) 9 (1) IVST · BSA-0.5 (mm/m) 7 (1) 7 (1) PWT (mm) 10 (1) 9 (1) PWT · BSA-0.5 (mm/m) 7 (1) 7 (1) Relative wall thickness 0.39 (0.05) 0.41 (0.07) Values are means (SD). BMI: body mass index; BSA: body surface area; POpeak: peak power output; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; MAP: mean arterial pressure; EF: ejection fraction; Lengthd: end-diastolic length; LVIDd: left ventricular internal diameter during diastole; IVST, interventricular septum thickness; PWT, posterior wall thickness. *p<0.05 vs. males.    99 5.3.2 Lower body negative pressure This section will first outline the responses to LBNP in control conditions (CON-LBNP). Because glycopyrrolate was administered prior to LBNP for experiments with vagal blockade, the effects of glycopyrrolate on resting variables will be reported in the following subsection (GLY post-infusion). Finally, within-group data during LBNP with vagal blockade (GLY-LBNP) will be compared to the post-infusion (blocked) baseline.   5.3.2.1 LV mechanics during lower body negative pressure LV mechanics during LBNP without vagal blockade (CON-LBNP). Basal rotation (p<0.001), twist (p<0.001) and torsion (p<0.001) increased in both sexes with LBNP (Figure 5.2 and Table 5.2). The increases to LV twist (F: 4.8 ± 2.6º, M: 2.4 ± 2.7º; p=0.02) and torsion (F: 0.74 ± 0.34º·cm-1, M: 0.39 ± 0.33º·cm-1; p=0.008) from baseline to -60 mmHg were greater in females, resulting in greater twist (p=0.004) and torsion (p<0.001) in females compared to males during -60 mmHg. Moreover, apical rotation increased in females (p<0.001) but not males (p=0.37), resulting in larger apical rotation in females (p=0.003) during -60 mmHg. There were no sex differences in basal rotation during any stage.  Untwisting velocity increased with progressive LBNP (p=0.02), but did not differ between the sexes at any stage. Additionally, time to peak untwisting velocity increased in males (p=0.045) and tended to increase in females (p=0.07) but was not different between the sexes. There was a significant relationship between LV twist and untwisting velocity in both males (r=-0.64, p<0.001) and females (r=-0.66, p<0.001), which did not differ between the sexes (p=0.31).   Longitudinal strain (p=0.047), apical circumferential strain (p<0.001) and basal circumferential strain (p<0.001) were reduced during LBNP in both sexes. While these parameters were greater in females at rest (p<0.05 for all), they were not different between the sexes during -60 mmHg LBNP.   100 Resting LV mechanics: the effect of vagal blockade (GLY post-infusion). Compared to pre-infusion, LV basal rotation (p<0.01), twist (p<0.01), torsion (p<0.01) and untwisting velocity (p<0.01) were increased in both sexes post-infusion; however, there were no sex differences in these parameters pre- or post-infusion (Figure 5.2 and Table 5.2). By contrast, apical rotation was unchanged post-infusion, and was not different between the sexes.   Compared to pre-infusion, longitudinal strain and basal circumferential strain were reduced in both sexes post-infusion (F: p=0.036; M: p<0.001), but apical circumferential strain was unchanged. Longitudinal strain tended to be higher in females prior to infusion (p=0.074), and apical circumferential strain tended to be higher in females pre- (p=0.071) and post-infusion (p=0.069) compared to males. Otherwise, there were no significant sex differences in strain with glycopyrrolate.   LV mechanics during LBNP with vagal blockade (GLY-LBNP). In males, apical rotation, twist and torsion were not significantly altered from post-infusion baseline during GLY-LBNP, but there was a trend to increased basal rotation during -60 mmHg (p=0.061).  In females, apical rotation increased at -40 mmHg (p=0.046) and -60 mmHg (p<0.001) compared to post-infusion baseline. As a result, apical rotation was larger in females at -40 (p=0.038) and -60 mmHg (p=0.009). Likewise, twist (p=0.001) and torsion (p<0.001) increased in females with GLY-LBNP, and were greater in females compared to males at -60 mmHg (twist: p=0.035, torsion: p=0.018). Compared to post-infusion, untwisting velocity increased in males but not females at -60 mmHg, and time to peak untwisting velocity increased in both sexes with GLY-LBNP. Nonetheless, there were no sex differences in peak or time to peak untwisting velocity at any stage. A significant relationship was observed for LV twist and untwisting velocity in males (r-=0.57, p<0.001) and females (r=-0.60, p<0.001), but was not different between the sexes  Comparison of LV mechanics during LBNP with and without vagal blockade (GLY-LBNP versus CON-LBNP). The change in LV twist from baseline to -60 mmHg was not significantly different between CON-LBNP and GLY-LBNP for females (CON-LBNP  101 4.8 ± 2.6º, GLY-LBNP 4.0 ± 5.5º; p=0.93) or males (CON-LBNP 2.4 ± 2.7º, GLY-LBNP 0.3 ± 3.9º; p=0.14). The change in LV torsion from baseline to -60 mmHg also did not differ between conditions for females (CON-LBNP 0.74 ± 0.34º·cm-1, GLY-LBNP 0.87 ± 0.64º·cm-1; p=0.81) or males (CON 0.39 ± 0.33º·cm-1; GLY 0.17 ± 0.47º·cm-1, p=0.17). There were no within-group differences at -60 mmHg for LV twist (M p=0.38, F p=0.48) or torsion (M p=0.22, F p=0.36) in CON-LBNP versus GLY-LBNP.  All strain parameters were reduced with LBNP in both sexes (p<0.001 for all). Longitudinal strain was greater in females during -40 mmHg (p=0.026) but not -60 mmHg. Basal circumferential strain was greater in females at -60 mmHg (p=0.039). There were no sex differences in apical circumferential strain during LBNP.    102 Table 5.2. LV mechanics during LBNP with and without vagal blockade   CON GLY   Rest -40 mmHg -60 mmHg Pre-infusion Post-infusion -40 mmHg -60 mmHg Twist (˚) M 10.5 (2.7) 12.2 (3.1)† 12.9 (2.3)† 10.7 (3.7) 13.6 (2.6)# 13.5 (3.3) 13.9 (3.3)  F 11.2 (3.2) 12.7 (3.7)† 16.0 (3.4)†‡* 10.6 (3.4) 13.5 (3.3)# 15.2 (3.5) 17.7 (5.9)†‡* Torsion (˚cm-1)  M 1.19 (0.33) 1.43 (0.40)† 1.58 (0.31)†‡ 1.22 (0.43) 1.59 (0.35)# 1.68 (0.45) 1.75 (0.43)  F 1.34 (0.39) 1.60 (0.52)† 2.11 (0.44)†‡* 1.27 (0.42) 1.67 (0.46)# 1.97 (0.49) 2.36 (0.86)†‡* Apical rot (˚) M 7.1 (1.8) 7.7 (2.4) 7.8 (1.5) 7.0 (2.5) 8.0 (2.8) 7.7 (3.5) 7.1 (3.6)  F 8.4 (3.6) 8.7 (2.6) 11.9 (3.6)†‡* 7.5 (3.4) 8.4 (3.7) 10.4 (3.5)†* 11.6 (4.9)†* Basal rot (˚) M -3.8 (1.6) -5.3 (1.4) -5.5 (1.2)† -4.2 (1.7) -6.2 (2.2)# -6.2 (3.4) -7.5 (3.3)  F -3.1 (1.8) -4.7 (2.4)† -5.4 (2.0)† -3.4 (1.0) -5.5 (2.4)# -5.6 (2.3) -7.1 (2.5) Untwisting velocity (˚s-1) M -97 (30) -98 (35) -114 (34)† -91 (36) -134 (47)# -146 (50) -162 (38)†  F -99 (26) -113 (21) -132 (36)† -91 (28) -138 (35)# -158 (41) -162 (54) Time to peak untwisting velocity (%) M 107 (7) 126 (34)† 121 (30)† 106 (5) 126 (28)# 130 (19)† 136 (26)†  F 106 (4) 109 (6)  126 (32) 109 (5) 122 (14) 137 (29)† 134 (13) LS (%) M -17.2 (1.5) -14.7 (1.7) -13.1 (1.7)† -17.4 (1.6) -15.8 (1.7)# -13.0 (1.7)† -12.2 (1.3)†  F -18.3 (1.8)* -15.2 (1.2) -13.3 (1.7)† -18.3 (1.4) -16.6 (1.7)# -14.5 (2.0)†* -12.5 (1.9)†‡ CS, base (%) M -19.3 (2.4) -16.4 (2.2)† -15.9 (2.2)† -19.4 (2.8) -15.8 (2.5)# -11.5 (3.4)† -11.4 (3.1)†  F -22.1 (3.4)* -17.0 (3.8)† -15.3 (3.2)†‡ -20.6 (3.5) -16.9 (3.6)# -12.3 (3.7)† -14.1 (3.4)†*   103 Table 5.2 (cont). LV mechanics during LBNP with and without vagal blockade   CON GLY   Rest -40 mmHg -60 mmHg Pre-infusion Post-infusion -40 mmHg -60 mmHg CS, apex (%) M -27.0 (3.0) -23.4 (3.3)† -23.4 (4.0)† -26.1 (3.6) -24.7 (3.7) -25.3 (4.8)† -22.8 (4.6)†‡  F -29.2 (3.1)* -27.7 (3.2)* -25.7 (4.5)† -28.6 (4.1) -27.3 (4.3) -27.5 (4.3) -24.3 (3.2)‡ Values are means (SD). CON: control (n=17 females, 17 males); GLY: glycopyrrolate (n=16 females, 18 males); Rot: rotation; LS: longitudinal strain; CS: circumferential strain. †p<0.05 vs rest (CON) or post-infusion (GLY). ‡p<0.05 vs -40 mmHg. #p<0.05 vs pre-infusion in GLY. *p<0.05 vs males.   104  Figure 5.2. Graphical representation of mean left ventricular (LV) rotation and twist at rest (left), -40 mmHg (middle) and -60 mmHg (right) LBNP. Upper and lower panels depict data during CON and GLY (post-infusion). Blue and red lines represent mean data for males and females, respectively. Dotted lines represent rotations of the LV base (negative) and apex (positive). CON: control; GLY: glycopyrrolate. SD for peak data are provided in Table 5.2. *p<0.05 males vs. females. -10-505101520Rotation (Male)Twist (Male)Rotation (Female)Twist (Female)-10-505101520% Systole     %Diastole*         50      100     150      200*-10-505101520Twist and Rotation (°)-10-505101520-10-505101520Twist and Rotation (°)Rest% Systole     %Diastole % Systole     %Diastole-40 mmHg -60 mmHg**CON**         50      100     150      200         50      100     150      200-10-505101520GLY (post-infusion) 105 5.3.2.2 LV hemodynamics during lower body negative pressure  LV hemodynamics during LBNP without vagal blockade (CON-LBNP). The hemodynamic responses to LBNP are shown in Table 5.3. HR increased with LBNP (p<0.001) but did not differ between the sexes during any stage. MAP was unchanged during LBNP and was consistently higher in males at all levels of LBNP (p<0.001). TPR increased with LBNP (p<0.001), but did not differ between the sexes at any stage.  LVEDV, ESV and SV were reduced in both sexes with progressive LBNP (p<0.001 for all), and the relative reductions to EDV (F -30 ± 7%, M -26 ± 6%), ESV (F -18 ± 11%, M -17 ± 9%) and SV (F -39 ± 9%, M -34 ± 7%) did not differ between the sexes. Additionally, the slope of the Frank-Starling relationship was not different between the sexes (F 0.70 ± 0.17, M 0.71 ± 0.13). Absolute LV volumes were larger in males during all stages (p<0.01 for all), however allometrically scaled volumes were not different between the sexes. Q was reduced in both sexes from rest to -40 mmHg (p<0.001 for both), but was not further reduced from -40 to -60 mmHg. EF also declined in both sexes during LBNP (p<0.001). While EF tended to be higher in females at rest (p=0.074), there were no sex differences in EF during LBNP.   Resting LV hemodynamics: the effect of vagal blockade (GLY post-infusion). HR and MAP were increased and TPR was reduced in both sexes post-infusion (p<0.001 for all)(Table 5.3). HR and MAP were not different between the sexes post-infusion, but TPR was higher in females compared to males (p=0.043).   LVEDV and SV were reduced in both sexes post-infusion (p<0.001 for all), but ESV was unchanged, reflected in reductions to EF in both sexes (p<0.001 for both). Absolute EDV and ESV remained larger in males post-infusion (p<0.001), however SV only tended to be larger in males than females (p=0.051). There were no sex differences in allometrically scaled volumes pre- or post-infusion. Q was increased in both groups post-infusion (p<0.001 for both), and remained larger in males compared to females (p=0.01).    106 LV hemodynamics during LBNP with vagal blockade (GLY-LBNP). HR increased in both groups (p<0.001 for both) but did not differ between the sexes during GLY-LBNP.  Unlike CON-LBNP, MAP was reduced in both sexes from post-infusion to -40 mmHg (p<0.001 for both), but was not further reduced during -60 mmHg. There were no sex differences in MAP during GLY-LBNP. TPR was unchanged in both sexes, but higher in females during -40 mmHg (p=0.014) and -60 mmHg (p=0.02).  LVEDV, ESV and SV were reduced in both sexes with GLY-LBNP (p<0.001 for all), and absolute volumes were larger in males during all stages (p<0.01 for all). However, females had greater relative reductions to EDV (F: -29±4%, M: -22±4%; p<0.001) and SV (F: -38±9%, M: -27±5%; p<0.001), and as a result, allometrically scaled EDV (p=0.022) and SV (p=0.026) were reduced in females compared to males during -60 mmHg. Despite this, the slope of the Frank-Starling relationship was not different between the sexes (F: 0.71±0.13, M: 0.74±0.09).   EF was reduced in both males (p=0.007) and females (p=0.002) with LBNP, however females had greater reductions to EF than males (F: -6±7%, M: -3±2%; p=0.045). Thus, although EF was higher in females at post-infusion (p=0.005) and -40 mmHg (p=0.038), EF did not differ between the sexes during -60 mmHg. Finally, Q was reduced in males at -40mmHg (p=0.005) but not further at -60 mmHg, whereas Q was reduced in females at -40 mmHg (p<0.001) and further at -60 mmHg (p=0.006).     107 Table 5.3. LV hemodynamics during LBNP with and without vagal blockade   CON GLY   Rest -40 mmHg -60 mmHg Pre-infusion Post-infusion -40 mmHg -60 mmHg HR (bpm) M 61 (7) 73 (8)† 83 (11)†‡ 64 (9) 113 (10)# 125 (11)† 133 (13)†‡  F 61 (9) 71 (15)† 86 (17)†‡ 63 (11) 110 (11)# 122 (15)† 134 (16)†‡ MAP (mmHg)  M 89 (8) 90 (6) 89 (7) 89 (6) 97 (7)# 93 (11)† 88 (11)†‡  F 77 (7)* 80 (7)* 76 (8)* 82 (8)* 95 (7)# 92 (7)† 93 (11)†‡ EDV (ml) M 135 (16) 114 (12)† 99 (13)†‡ 133 (15) 115 (14)# 99 (13)† 90 (11)†‡  F 110 (15)* 91 (16)†* 78 (15)†‡* 113 (15)* 99 (16)#* 83 (14)†* 72 (11)†‡* EDV (ml⋅m-3) M 49 (7) 42 (5)† 36 (5)†‡ 50 (7) 43 (6)# 37 (5)† 33 (4)†‡  F 48 (5) 40 (6)† 34 (5)†‡ 49 (4) 43 (4)# 36 (4)† 30 (3)†‡* ESV (ml) M 62 (8) 57 (8)† 52 (9)†‡ 62 (8) 61 (8) 54 (8)† 50 (7)†‡  F 48 (8)* 44 (9)†* 40 (8)†‡* 52 (9)* 49 (10)* 44 (9)†* 40 (7)†‡* ESV (ml⋅m-3) M 23 (3) 21 (3)† 19 (3)†‡ 23 (3) 23 (3) 20 (3)† 19 (2)†‡  F 21 (3) 19 (3)† 17 (3)†‡ 22 (3) 21 (3) 19 (3)† 17 (3)†‡ SV (ml) M 72 (11) 57 (6)† 47 (6)†‡ 72 (9) 54 (7)# 44 (5)† 40 (5)†‡  F 62 (9)* 47 (8)†* 38 (8)†‡* 61 (8)* 49 (7)# 39 (6)†* 31 (6)†‡* SV (ml⋅m-3)  M 27 (5) 21 (3)† 17 (3)†‡ 27 (5) 20 (3)# 17 (2)† 15 (2)†‡  F 27 (4) 21 (3)† 17 (3)†‡ 27 (3) 22 (2)# 17 (2)† 13 (2)†‡* EF (%) M 54 (4) 50 (3)† 48 (4)†‡ 54 (9) 47 (7)# 45 (5) 44 (5)†  F 56 (4) 52 (5)† 49 (4)†‡ 54 (2) 50 (3)#* 48 (4)†* 44 (5)†‡  108 Table 5.3 (cont). LV hemodynamics during LBNP with and without vagal blockade   CON GLY   Rest -40 mmHg -60 mmHg Pre-infusion Post-infusion -40 mmHg -60 mmHg Q (l·min-1) M 4.40 (0.54) 4.09 (0.45)† 3.86 (0.41)† 4.56 (0.47) 6.15 (0.89)# 5.54 (0.52)† 5.27 (0.66)†  F 3.73 (0.66)* 3.31 (0.60)†* 3.17 (0.67)†* 3.84 (0.76)* 5.40 (0.70)#* 4.76 (0.69)†* 4.16 (0.66)†‡* TPR (mmHg·l-1·min-1)  M 20.5 (3.0) 22.2 (3.4) 23.2 (3.0)† 19.7 (2.5) 16.1 (2.6)# 17.0 (2.6) 17.0 (2.9)  F 21.3 (3.8) 24.8 (4.3)† 25.2 (5.4)† 22.1 (4.2) 17.8 (1.7)#* 19.3 (2.5)* 20.5 (4.9)* Values are means (SD). CON: control (n=17 females, 17 males); GLY: glycopyrrolate (n=16 females, 18 males); HR: heart rate; MAP: mean arterial pressure; EDV: end-diastolic volume; ESV: end-systolic volume; SV: stroke volume; EF: ejection fraction; Q: cardiac output; TPR: total peripheral resistance. †p<0.05 vs rest (CON) or post-infusion (GLY). ‡p<0.05 vs -40 mmHg. #p<0.05 vs pre-infusion in GLY. *p<0.05 vs males.   109 5.3.2.3 LV structure and geometry during lower body negative pressure  LV structure and geometry during LBNP without vagal blockade (CON-LBNP). LV length and LVIDd were reduced in both sexes with LBNP (p<0.001 for all) and were larger in males during all stages (p<0.01 for all)(Table 5.4). In males, sphericity index increased during -40 mmHg (p<0.001) but was not further augmented during -60 mmHg (p=0.23). In females, sphericity index increased at -40 mmHg (p=0.006) and further at -60 mmHg (p<0.001). Relative wall thickness increased in both sexes with LBNP (p<0.001 for both) but did not differ between the sexes at any stage.  LV structure and geometry at rest: the effect of vagal blockade (GLY post-infusion). Post-infusion, LV length and LVIDd were reduced in both sexes (p<0.001 for all), whereas sphericity index (p<0.01 for both) and relative wall thickness increased in both sexes (p<0.05 for both). LV length and LVIDd were larger in males pre- and post-infusion (p<0.01 for all). In contrast, sphericity index tended to be greater in females pre-infusion (p=0.07) but was not different from males post-infusion. Relative wall thickness was not different between the sexes pre- or post-infusion.   LV structure and geometry during LBNP with vagal blockade (GLY-LBNP). LV length was reduced in both sexes with GLY-LBNP (p<0.001 for both), and remained larger in males during all stages (p<0.01 for all). In females, LVIDd was reduced at -40 mmHg and further at -60 mmHg (p<0.001), however in males LVIDd was reduced at -40 mmHg (p<0.001) but not further at -60 mmHg. Accordingly, sphericity index increased in males during -40 mmHg (p<0.001) but not further during -60 mmHg, whereas sphericity index increased in females at -40 mmHg (p<0.001) and tended to further increase at -60mmHg (p=0.055). Sphericity index was therefore greater in females at -40 mmHg (p=0.019) and -60 mmHg (p=0.003) compared to males. A similar pattern was observed for relative wall thickness (p=0.017), which was increased in males during -40 mmHg (p=0.01) but not further at -60 mmHg. In females, relative wall thickness increased in at -40 mmHg (p<0.001) and tended to further increase at -60 mmHg (p=0.076). Relative wall thickness was therefore larger in females during -40 mmHg (p=0.049) and -60 mmHg (p=0.021).  110 Table 5.4. LV structure and geometry during LBNP with and without vagal blockade   CON GLY   Rest -40 mmHg -60 mmHg Pre-infusion Post-infusion -40 mmHg -60 mmHg Lengthd (mm) M 87.9 (2.8) 85.5 (3.1)† 82.1 (4.2)†‡ 87.9 (3.0) 86.0 (3.7)# 82.0 (3.8)† 79.7 (3.8)†‡  F 83.8 (3.9)* 79.6 (4.4)†* 76.9 (3.9)†‡* 83.9 (3.9)* 81.5 (5.0)#* 77.8 (4.7)†* 75.2 (4.3)†‡* LVIDd (mm) M 48.7 (3.5) 44.7 (3.3)† 42.3 (3.4)†‡ 49.0 (3.5) 45.4 (3.3)# 41.2 (3.0)† 40.5 (3.3)†  F 44.0 (2.9)* 40.6 (3.6)†* 37.0 (3.4)†‡* 44.7 (3.3)* 41.8 (3.9)#* 37.3 (3.9)†* 35.0 (3.5)†‡* Sphericity index M 1.82 (0.10) 1.92 (0.12)† 1.95 (0.14)† 1.80 (0.11) 1.90 (0.12)# 1.98 (0.09)† 1.98 (0.13)†  F 1.91 (0.11)* 1.97 (0.16)† 2.08 (0.13)†‡* 1.89 (0.15)  1.96 (0.18)# 2.10 (0.17)†* 2.17 (0.19)†* Relative wall thickness M 0.39 (0.05) 0.44 (0.05)† 0.49 (0.06)†‡ 0.40 (0.05) 0.44 (0.07)# 0.48 (0.06)† 0.51 (0.07)†  F 0.41 (0.05) 0.46 (0.06)† 0.51 (0.08)†‡ 0.40 (0.04) 0.45 (0.06)# 0.54 (0.09)†* 0.58 (0.09)†‡* Values are means (SD). See Table 5.1 for abbreviations. CON: control (n=17 females, 17 males); GLY: glycopyrrolate (n=16 females, 18 males). †p<0.05 vs rest (CON) or post-infusion (GLY). ‡p<0.05 vs -40 mmHg. #p<0.05 vs pre-infusion in GLY. *p<0.05 vs males.  111 5.3.3 Submaximal exercise 5.3.3.1 LV mechanics during exercise  LV mechanics during exercise without vagal blockade (CON-EX). Basal rotation (p<0.001), apical rotation (p<0.001), twist (p<0.001), torsion (p<0.001) and untwisting velocity (p<0.001) increased in both sexes during exercise (Figure 5.3 and Table 5.5). However, there were no sex differences in these parameters at any stage. Time to peak untwisting velocity increased in both sexes from rest to 25% peak power (p<0.01 for both), but was not further altered at 50% peak power and did not differ between the sexes at any stage. There was a significant relationship between LV twist and untwisting velocity in both males (r=-0.78, p<0.001) and females (r=-0.69, p<0.001), but did not differ between the sexes (p=0.39).  Longitudinal strain increased during exercise in both sexes (p<0.001 for both), and was greater in females during rest (p=0.002), 25% (p=0.01) and 50% peak power (p<0.001) compared to males. Basal circumferential strain increased during exercise in both males (p=0.008) and females (p=0.026), but was not different between the sexes at any stage. Apical circumferential strain increased at during exercise in both sexes (p<0.01 for both), and was greater in females at rest (p=0.046), but not different between the sexes during exercise.  LV mechanics during exercise with vagal blockade (GLY-EX). In females, LV basal rotation, apical rotation, twist and torsion increased from rest to 25% peak power (p<0.001 for all)(Figure 5.3 and Table 5.5), but were not further increased at 50%. In males, basal rotation (p=0.09), twist (p=0.05) and torsion (p=0.08) tended to increase at 25%, and were significantly augmented from rest at 50% peak power (p<0.001 for all). In contrast, apical rotation was unchanged in males during GLY-EX. Nonetheless, there were no sex differences in apical rotation, basal rotation, twist, torsion or untwisting velocity during exercise. There was a significant relationship between LV twist and untwisting velocity in both males (r=-0.69, p<0.001) and females (r=-0.79, p<0.001), but this relationship did not differ between the sexes.   112 Longitudinal strain increased during exercise in both sexes (p<0.001 for both) and was greater in females at rest (p=0.009), 25% peak power (p=0.037) and tended to be greater during 50% peak power (p=0.056). Basal circumferential strain increased at 25% peak power in females (p=0.013) but not males, but was not different between the sexes at any stage. Apical circumferential strain increased in both sexes during exercise  (p<0.001 for both), and was greater in females at 50% peak power (p=0.002).  Comparison of LV mechanics during exercise with and without vagal blockade (GLY-EX versus CON-EX). The change to LV twist from baseline to 50% peak power was not different between CON-EX and GLY-EX for females (CON-EX 5.0 ± 3.0º, GLY-EX 5.8 ± 3.8º; p=0.62) or males (CON-EX 5.4 ± 2.5º, GLY-EX 3.9 ± 3.8º; p=0.44). The change to LV torsion also did not differ between conditions for females (CON-EX 0.60 ± 0.37º·cm-1; GLY-EX 0.69 ± 0.42º·cm-1; p=0.60) or males (CON-EX 0.60 ± 0.32º·cm-1; GLY-EX 0.44 ± 0.45º·cm-1, p=0.48). There were no within-groups differences at 50% peak power for LV twist (M p=0.73, F p=0.17) or torsion (M p=0.93, F p=0.10) in CON-EX versus GLY-EX.   113 Table 5.5. LV mechanics during submaximal exercise with and without vagal blockade   CON GLY   Rest 25% 50% Rest 25% 50% Twist (˚) M 11.9 (2.7) 14.5 (3.0)† 17.7 (3.2)†‡ 13.3 (3.8) 15.7 (2.5) 17.2 (3.7)†  F 11.2 (3.3) 13.7 (3.3)† 16.4 (4.2)†‡ 12.2 (3.5) 17.2 (4.9)† 18.1 (4.9)† Torsion (˚cm-1) M 1.36 (0.33) 1.64 (0.39)† 2.01 (0.42)†‡ 1.56 (0.52) 1.81 (0.34) 2.04 (0.45)†‡  F 1.35 (0.40) 1.65 (0.41)† 1.95 (0.49)†‡ 1.50 (0.47) 2.12 (0.62)† 2.19 (0.60)† Apical rot (˚) M 8.1 (2.2) 10.0 (2.4)† 11.2 (1.8)† 8.4 (2.8) 9.25 (1.9) 9.6 (3.3)  F 8.0 (2.4) 9.9 (2.6)† 11.2 (2.8)†‡ 8.0 (3.3) 11.2 (4.4)† 11.3 (3.6)† Basal rot (˚) M -4.2 (1.3) -4.8 (1.4) -7.0 (1.9)†‡ -5.5 (1.6) -6.7 (2.3) -8.2 (2.7)†‡  F -3.6 (1.5) -4.3 (1.5) -6.2 (2.2)†‡ -4.7 (1.5) -6.8 (1.7)† -7.5 (2.6)† Untwisting velocity (˚s-1) M -107 (30) -145 (46)† -195 (50)†‡ -134 (48) -161 (47) -214 (49)†‡  F -106 (24) -135 (43)† -199 (53)†‡ -121 (40) -192 (61)† -231 (75)†‡ Time to peak untwisting velocity (%) M 108 (4) 115 (8)† 117 (9)† 125 (12) 129 (17) 125 (10)  F 108 (4) 115 (9)† 117 (7)† 129 (23) 130 (15) 126 (17) LS (%) M -17.1 (1.4) -19.9 (1.4)† -20.7 (1.4)†‡ -17.1 (1.5) -18.5 (2.3)† -19.7 (2.2)†‡  F -19.0 (1.9)* -21.3 (1.4)†* -23.0 (1.3)†‡* -18.5 (1.4)* -20.0 (1.3)†* -21.2 (1.9)†‡ CS, base (%) M -20.2 (1.9) -22.3 (3.6)† -23.0 (3.4)† -18.0 (2.0) -18.6 (2.4) -19.2 (4.0)  F -20.3 (3.9) -20.2 (3.7) -22.6 (3.6)† -16.9 (3.7) -19.2 (4.1)† -21.0 (3.5)†    114 Table 5.5 (cont). LV mechanics during submaximal exercise with and without vagal blockade   CON GLY   Rest 25% 50% Rest 25% 50% CS, apex (%) M -27.1 (3.2) -31.0 (3.4)† -33.9 (4.4)†‡ -26.6 (3.2) -29.7 (4.5)† -31.1 (4.0)†‡  F -29.5 (3.7)* -33.1 (4.3)† -36.3 (3.3)†‡ -27.7 (4.5) -31.6 (3.7)† -35.6 (3.4)†‡* Values are means (SD). See Table 5.2 for abbreviations. CON: n=17 females, 16 males. GLY: n=16 females, 17 males. †p<0.05 vs rest. ‡p<0.05 vs 25%. *p<0.05 vs males.    115  Figure 5.3. Graphical representation of mean LV rotation and twist at rest (left), 25% (middle) and 50% (right) of peak power in supine cycling exercise (EX). See Figure 5.2 for symbol legends. SD for peak data are provided in Table 5.5. *p<0.05 males vs. females.  -10-505101520-10-505101520-10-505101520Twist and Rotation (°)Rest% Systole     %Diastole % Systole     %Diastole25%CON         50      100     150      200         50      100     150      200-10-505101520-10-505101520Twist and Rotation (°)GLY -10-505101520% Systole     %Diastole50%         50      100     150      200 116 5.3.3.2 LV hemodynamics during exercise LV hemodynamics during exercise without vagal blockade (CON-EX). As would be expected, HR and MAP were increased, and TPR was reduced during exercise (Table 5.6) in both sexes (p<0.05 for all). There were no sex differences in HR or TPR, but MAP was higher in males during all stages (p<0.001).   For both sexes, LVEDV increased from rest to 25% (p<0.001 for both), but did not further increase during 50% peak power. ESV was reduced (p<0.001 for both) and SV was increased (p<0.001 for both) in both sexes during exercise. Absolute volumes were larger in males during all stages (p<0.01 for all), however there were no sex differences in allometrically scaled volumes.  EF and Q increased in both sexes with exercise (p<0.001 for all). EF was not different between the sexes at rest, but was greater in females during 25% (p=0.046) and 50% peak power (p=0.02). Q and was larger in males at rest (p<0.001), during 25% (p=0.012) and 50% peak power (p=0.003).  LV hemodynamics during exercise with vagal blockade (GLY-EX). HR increased in both sexes increased with exercise (p<0.001 for both), but was not different between the sexes during any stage. MAP was increased (p=0.002 for both) and TPR was reduced (p<0.001 for both) in both sexes with exercise. There were no sex differences in TPR at any stage. MAP was higher in males at rest (p=0.035) and during 25% and 50% peak power (p<0.01 for both).   In males, EDV was increased at 25% peak (p=0.045) but not further at 50% peak power, whereas EDV increased at 25% (p<0.001) and further at 50% in females (p=0.005). In contrast, ESV was reduced in males at 25% (p<0.001) and tended to further reduce at 50% (p=0.092), whereas ESV was unchanged in females. Absolute EDV and ESV were larger in males at rest (p<0.01 for both) and 25% (p=0.03 for both), but only tended to be larger in males at 50% peak power (p=0.08 for both). There were no sex differences in allometrically scaled EDV or ESV at any stage.  117  SV increased in both sexes during exercise (p<0.001 for both). Absolute SV tended to be larger in males at rest (p=0.064) and was larger at 25% (p=0.045), but was not different between the sexes at 50% peak power. Allometrically scaled SV was not different between the sexes during any stage. LVEF increased in both groups at 25% (p<0.001 for both) and further at 50% peak power in males (p=0.011) and females (p=0.004). EF was greater in females at rest (p<0.001) but not different between the sexes during exercise. Q increased in both groups with exercise (p<0.001 for all). Q tended to be higher in males at rest (p=0.061) and was greater in males during 25% and 50% exercise (p<0.01 for both).   118 Table 5.6. LV hemodynamics during submaximal exercise with and without vagal blockade   CON GLY   Rest 25% 50% Rest 25% 50% HR (bpm) M 63 (8) 95 (10)† 122 (16)†‡ 109 (9) 127 (13)† 151 (15)†‡  F 61 (10) 96 (8)† 122 (12)†‡ 108 (15) 126 (15)† 145 (16)†‡ MAP (mmHg)  M 89 (7) 96 (7)† 103 (8)†‡ 88 (7) 89 (9) 95 (8)†‡  F 77 (8)* 85 (7)†* 91 (9)†‡* 82 (10)* 80 (6)* 87 (9)†‡* EDV (ml) M 136 (18) 143 (20)† 144 (21)† 125 (16) 128 (19)† 130 (18)†  F 109 (15)* 118 (18)†* 119 (17)†* 106 (15)* 113 (18)†* 118 (20)†‡ EDV (ml⋅m-3)  M 49 (7) 54 (7)† 53 (8)† 46 (7) 48 (7) 49 (6)†  F 47 (4) 52 (5)† 52 (5)† 46 (5) 49 (6)† 51 (5)†‡ ESV (ml) M 63 (9) 59 (10) 57 (9) †‡ 64 (8) 58 (10)† 56 (11)†  F 49 (9)* 47 (8)†* 44 (10)†* 51 (8)* 50 (10)* 49 (10) ESV (ml⋅m-3)  M 23 (3) 22 (3)† 21 (3)† 24 (3) 22 (3)† 21 (3)†  F 21 (3) 20 (3)† 19 (3)†‡ 22 (3) 22 (3) 22 (3) SV (ml) M 73 (11) 83 (11)† 87 (14)†‡ 60 (9) 70 (10)† 74 (9)†‡  F 60 (7)* 72 (12)†* 76 (9)†‡* 55 (7) 63 (10)†* 68 (12)†‡ SV (ml⋅m-3) M 27 (5) 31 (4)† 32 (6)†‡ 23 (5) 26 (3)† 27 (3)†  F 26 (2) 31 (4)† 33 (3)†‡ 24 (4) 27 (3)† 30 (3)†‡ EF (%) M 53 (3) 58 (2)† 60 (3)†‡ 48 (2) 55 (3)† 57 (4)†‡  F 55 (4) 61 (3)†* 64 (4)†‡* 52 (3)* 56 (3)† 58 (4)†‡  119 Table 5.6 (cont). LV hemodynamics during submaximal exercise with and without vagal blockade   CON GLY   Rest 25% 50% Rest 25% 50% Q (l·min-1) M 4.47 (0.39) 7.87 (0.98)† 10.54 (1.33)†‡ 6.57 (1.00) 8.82 (0.83)† 11.11 (0.89)†‡  F 3.66 (0.58)* 6.88 (1.05)†* 9.23 (0.96)†‡* 5.92 (0.91) 7.87 (1.13)†* 9.80 (1.40)†‡* TPR (mmHg·l-1·min-1) M 20.2 (2.7) 12.4 (1.7)† 10.0 (1.8)†‡ 13.7 (2.2) 10.2 (1.4)† 8.6 (0.9)†‡  F 21.5 (3.2) 12.6 (1.9)† 10.0 (1.3)†‡ 14.1 (2.8) 10.3 (1.3)† 9.0 (1.6)†‡ Values are means (SD). See Table 5.3 for abbreviations. CON: n=17 females, 16 males. GLY: n=16 females, 17 males. †p<0.05 vs rest. ‡p<0.05 vs 25%. *p<0.05 vs males.  120 5.3.3.3 LV structure and geometry during exercise  LV structure and geometry during exercise without vagal blockade (CON-EX). LV length (rest: p=0.001; 25% and 50%: p<0.001) and LVIDd (rest: p<0.001; 25%: p=0.002; 50%: p=0.026) were larger in males than females during all stages (Table 5.7). LV length increased in males at 25% peak power (p=0.041), but was unchanged in females. In contrast, LVIDd increased in females (p=0.022) but only tended to increase in males (p=0.062) at 50% peak power. Sphericity index was larger in females at rest (p=0.03) but did not differ between the sexes during exercise. Sphericity index increased in males with exercise (p=0.021) but was unchanged in females. There were no alterations or sex differences in relative wall thickness at any stage.   LV structure and geometry during exercise with vagal blockade (GLY-EX). Significant effects of exercise were not detected for LV length, LVIDd, sphericity index or relative wall thickness. LV length (rest and 25%: p=0.003, 50%: p=0.019) and LVIDd (rest: p=0.004; 25%: p=0.007; 50%: p=0.049) were larger in males than females. There were no sex differences in sphericity index or relative wall thickness at any stage.   5.3.4 Relationship of LV torsion to SV⋅BSA-1.5 during LBNP and exercise. During LBNP, the linear relationships of LV torsion to SV⋅BSA-1.5 were not significantly different between CON-LBNP and GLY-LBNP for males (p=0.17) or females (p=0.85)(Figure 5.4). However, the relationships of LV torsion to SV⋅BSA-1.5 were significantly different between the sexes (p<0.001), with a steeper relationship observed in females (y=-0.078x+2.44) than in males (y=-0.052x+3.34).   During exercise, the linear relationships of LV torsion to SV⋅BSA-1.5 differed between GLY-EX and CON-EX in females (p=0.044) but not in males (Figure 5.4). Nonetheless, the relationships for both sexes appear to be left-shifted from CON-EX to GLY-EX conditions. There were no sex differences in these relationships in either CON-EX or GLY-EX.    121 Table 5.7. LV structure and geometry during submaximal exercise with and without vagal blockade   CON GLY   Rest 25% 50% Rest 25% 50% Lengthd (mm)  M 88.1 (2.9) 89.1 (3.1)† 89.3 (4.2)† 86.6 (4.1) 87.1 (4.5) 86.6 (4.6)  F 83.7 (3.9)* 83.1 (4.5)* 83.9 (3.9)* 81.8 (4.7)* 81.8 (4.7)* 82.6 (4.3)* LVIDd (mm) M 49.4 (3.3) 49.3 (3.9) 48.9 (3.7) 48.1 (4.2) 47.5 (3.7) 47.3 (3.7)  F 44.9 (3.0)* 44.8 (3.6)* 45.8 (3.5)†‡* 43.8 (3.6)* 43.5 (4.3)* 44.2 (4.4)* Sphericity index M 1.79 (0.09) 1.82 (0.13) 1.84 (0.15)† 1.81 (0.17) 1.84 (0.14) 1.83 (0.12)  F 1.87 (0.12)* 1.87 (0.18) 1.84 (0.15) 1.88 (0.16) 1.89 (0.17) 1.88 (0.18) Relative wall thickness  M 0.40 (0.05) 0.40 (0.07) 0.40 (0.05) 0.42 (0.07) 0.41 (0.06) 0.41 (0.05)  F 0.41 (0.05) 0.41 (0.05) 0.40 (0.05) 0.44 (0.07) 0.44 (0.06) 0.42 (0.07) Values are means (SD). See Table 5.1 for abbreviations. CON: n=17 females, 16 males. GLY: n=16 females, 17 males. †p<0.05 vs rest. ‡p<0.05 vs 25%. *p<0.05 vs males.  122  Figure 5.4. The relationships of LV torsion to SV⋅BSA-1.5 during (A) LBNP and (B) exercise. Red and blue points represent group means (SD) for females and males, respectively. Closed and open circles represent data during glycopyrrolate infusion (GLY) and control (CON) conditions. *Significantly different relationships for males vs. females (p<0.05).   5.3.5 Relationships of LV mechanics with structure and geometry during LBNP and exercise. The relationships for LV mechanics with LV structure and geometry during LBNP are shown in Figure 5.5. There were significant relationships for LVIDd with twist in both sexes (F r=-0.34, p=0.002; M r=-0.35, p<0.001). This predominantly resulted from a significant relationship of LVIDd with apical rotation in females (r=-0.50, p<0.001), but with basal rotation in males (r=0.34, p<0.001). Significant relationships were also 0 10 20 30 4001234SV⋅BSA-1.5 (ml⋅m-3)Torsion (°cm-1)LBNPp<0.001*Males y=-0.052x+3.34Females y=-0.078x+2.440 10 20 30 4001234SV⋅BSA-1.5 (ml⋅m-3)Torsion (°cm-1)CON Males y=0.10x-1.32Females y=0.07x-0.51GLY Males y=0.11x-1.14Females y=0.12x-1.32Exercise 123 observed for LV length with twist in both females (r=0.36, p<0.001) and males (r=-0.45, p<0.001), which were reflective of a significant relationship of LV length with apical rotation in females (r=-0.34, p=0.001), and basal rotation in males (r=0.44, p<0.001). In females, the relationship of sphericity index tended to be significant with LV twist (r=0.19, p=0.09), and was significant with to apical rotation (r=0.40, p<0.001), however these were not observed in males. Nonetheless, relative wall thickness was significantly related to LV twist in both females (r=0.44, p<0.001) and males (r=0.26, p=0.013). In females, this resulted from a relationship with both apical rotation (r=0.40, p<0.001) and basal rotation (r=-0.24, p=0.02), but these relationships were not significant in males.    The relationships for LV mechanics with LV structure and geometry during exercise are shown in Figure 5.6. In males, significant relationships were observed for LVIDd with LV twist (r=-0.32, p=0.002) and basal rotation (r=0.35, p<0.001). Significant relationships were also observed in males for LV length with LV twist (r=-0.36, p<0.001) and apical rotation (r=-0.31, p=0.003). These relationships were not observed in females. Relationships between LV twist with sphericity index were not observed in either sex during exercise, however males did have a significant relationship with basal rotation (r=-0.23, p=0.023). In males, relationships for relative wall thickness were significant with LV twist (r=0.22, p=0.034) and a tended to be significant with apical rotation (r=0.19, p=0.063). However, in females there was only a significant relationship with apical rotation (r=0.22, p=0.035).  124  Figure 5.5. Relationships for LV twist mechanics with LV structure and geometry during LBNP. Data include measures during CON-LBNP and GLY-LBNP. Blue and red represent data for males and females, respectively. Top: closed circles represent LV twist. Bottom: open triangles and circles represent LV rotation at the apex and base, respectively. *Significant relationship (p<0.05).    125  Figure 5.6. Relationships for LV twist mechanics with LV structure and geometry during exercise. Data include measures during CON-LBNP and GLY-LBNP. See Figure 5.5 for symbol legends. *Significant relationship (p<0.05).   126 5.4 Discussion To our knowledge, this is the first study to investigate the influence of sympathovagal balance on sex differences in LV mechanics and hemodynamic responses to periods of acute physiological stress. The key novel findings of this study are threefold. First, females have greater LV twist mechanics than males during high levels of LBNP both with and without vagal blockade. Second, sex differences in LV twist were not observed during submaximal exercise, with or without vagal blockade. Finally, the net changes to LV twist from rest to -60 mmHg and 50% peak power were not altered by vagal blockade in either of the sexes. These findings provide strong evidence that vagal control is not a key determinant of sex differences in LV twist during progressive LBNP or submaximal exercise.   5.4.1 Vagal control of LV mechanics during reductions to preload In contrast to our hypothesis, sex differences in LV twist mechanics persist at high levels of LBNP during vagal blockade with glycopyrrolate. Moreover, the change in LV twist from rest to -60 mmHg did not differ between CON-LBNP and GLY-LBNP in either sex, and remained greater in females during vagal blockade. This is illustrated in Figure 5.4, as a steeper increase in LV torsion for a given reduction to SV⋅BSA-1.5 in females compared to males. These relationships were not altered by vagal blockade, suggesting that sex differences in LV twist mechanics during large reductions to preload are not consequences of differences in vagal cardiac control. Furthermore, although LV twist was elevated post-infusion at rest, absolute LV twist and torsion were not different between CON-LBNP and GLY-LBNP during -60 mmHg. It therefore appears that increases to LV twist during LBNP are not predominantly mediated by vagal withdrawal.   During reductions to preload, females commonly have greater chronotropic responses than males, which has been suggested to reflect greater vagal withdrawal (73, 188). In Chapter 3 we also report that in females, a greater increase in HR coincided with greater LV twist compared to males during high levels of LBNP (226). Therefore, it is conceivable that differences in the chronotropic response and vagal withdrawal might be responsible for the sex differences in LV twist mechanics. However, in the current study  127 females had augmented twist mechanics despite no sex differences in HR during either CON-LBNP or GLY-LBNP. This major novel finding suggests that sex differences in LV twist are not predominantly determined by differences in HR, or by differences in the chronotropic response associated with vagal withdrawal as previously thought (73, 188).  5.4.1.1 Influence of LV volumes on LV mechanics during reductions to preload In agreement with previous data from our group (Chapter 3, (226)), the relative reductions to LVEDV and SV were not different between the sexes during CON-LBNP. In Chapter 3 and the present study, LV twist and apical rotation were greater in females than males during -60 mmHg, demonstrating that these female cohorts had augmented responses of LV twist mechanics compared to their male counterparts despite similar relative reductions to preload and SV. Although females in the present study did have greater relative reductions to LVEDV and SV than males during GLY-LBNP, the changes to LV twist and torsion from rest to -60 mmHg were not different from the CON-LBNP condition. These observations suggest that the sex differences in LV twist during LBNP are not a consequence of differences in the relative changes to LV volumes, per se.   During CON-LBNP and GLY-LBNP, the slopes of the Frank Starling relationship were not different between the sexes. In previous studies from our group (226) and others (73), this relationship was observed to be steeper in females than in males, lending to the hypothesis that females have a lower functional reserve for SV and therefore require augmented hemodynamic responses to cope during large reductions to preload (for further discussion, see section 6.2.1). However, the similar slopes in females and males in the current study do not support this contention, and demonstrate that potential differences in the Frank Starling relationship likely do not contribute to differences in LV twist mechanics during reductions to preload.    128 5.4.1.2 Influence of LV structure, geometry and myofibre orientation on LV mechanics during reductions to prelaod Previously, our group demonstrated that a greater LV twist in females coincided with a larger sphericity index compared to males during -60 mmHg (226). In the current study, sphericity index was larger in females during -60 mmHg in both CON-LBNP and GLY-LBNP conditions. Additionally, a significant sex*LBNP interaction was observed for sphericity index in both conditions, indicative of relatively greater increases to sphericity index in females. These consistent differences in LV geometry likely play a significant role in the responses and sex differences in LV twist during LBNP, as alterations to LV shape influence the helical myofibre orientations and cross-fibre interactions that determine LV twist (39, 169, 205). This interaction has been demonstrated in a human model with dramatic changes to sphericity (congestive heart failure), wherein an increased ellipsoid geometry, compared to a spherical configuration, supports increases to apical rotation and therefore is a strong independent predictor of LV twist (212). In the current study, this relationship between twist mechanics and sphericity index appears to have differed between the sexes (Figure 5.5), as sphericity index was significantly related to apical rotation in females, but was not related to basal or apical rotation in males. Therefore, it is feasible the sex differences in LV twist mechanics during large reductions to preload were influenced by differences in LV geometry. Specifically, a more favourable configuration of LV shape and myofibre orientation may have supported larger increases to apical rotation and twist in females.  Significant relationships were additionally observed between LV twist and relative wall thickness in both sexes during LBNP; however this relationship appeared to be stronger in females, in whom both apical and basal rotation were significantly related to relative wall thickness. These relationships were not observed for rotation in males, which further supports that alterations to LV structure and geometry might influence LV mechanics to a greater extent in females than in males. Although relative wall thickness did not differ between the sexes during CON-LBNP, it was greater in females during -60 mmHg in GLY-LBNP, thus the augmentation to, and differences in relative wall thickness may have contributed to the sex differences in LV twist mechanics. This contention is  129 supported by a structural model from Taber et al. (205) in which peak LV twist increases with wall thickness. Thus, the alterations to relative wall thickness during LBNP may have contributed to increasing LV twist in both sexes, but played a more significant role in augmenting twist mechanics in females than in males. Although sex differences in LV myofibre alignment and laminar structure have not been determined, it is possible that a given increase to relative wall thickness produces a more favourable reconfiguration of helical fibre layers in females, and provides a mechanical advantage for increasing apical rotation and twist to a greater extent than males.   5.4.2 Vagal control of LV mechanics during submaximal exercise Contrary to our hypothesis, we did not observe any sex differences in LV twist mechanics during submaximal exercise, both with and without vagal blockade. This can be visualized in Figure 5.4, where the alterations to LV torsion for a given increase to SV⋅BSA-1.5 were not different between the sexes in either CON-EX or GLY-EX. The increases to LV twist and torsion from rest to 50% exercise were also not different between the sexes and were not altered by vagal blockade.   Data from previous studies have indicated a greater vagal control of heart at rest in females (13, 66, 69, 82, 177), and also demonstrated augmented chronotropic responses in females compared to males during acute stress and exercise (73, 188, 223, 226). During exercise, dynamic alterations to sympathovagal balance occur wherein reductions to vagal drive are reciprocated by increasing sympathetic drive with increasing HRs (225). Thus, any sex differences in sympathovagal balance during exercise could potentially result in different alterations to LV twist mechanics; however this hypothesis was not supported by data from the current study. Firstly, the chronotropic responses during submaximal exercise did not differ between the sexes, even during vagal blockade. The alterations to sympathovagal balance therefore do not appear to have differed between our male and female cohorts during exercise. Secondly, while prior studies have provided some evidence for sex differences in the hemodynamic responses to exercise (86, 94, 223), significant sex*exercise interactions were not observed in the current study for LVEDV, ESV or SV during CON-EX. As the increases to LV rotation  130 and twist during exercise are closely related to alterations in LV volumes, it is possible the similar alterations to LV volumes during CON-EX resulted from similar responses of LV rotation and twist. In contrast, during GLY-EX the patterns of alterations to LV volumes were different between the sexes, as EDV was increased in females at both 25% and 50% but plateaued in males at 25%, and ESV was not altered in females but was reduced in males during exercise. Nonetheless, the net increases to LV twist and torsion were similar between the sexes, and were not different from CON-EX. The increases to LVSV also were similar in the sexes in both CON-EX and GLY-EX. If the alterations to SV had differed between the sexes, differences in LV mechanics may have occurred.   While the net increases to LV twist and torsion from rest to 50% peak power were not altered in either of the sexes following vagal blockade, the regional responses of LV mechanics did appear to be altered by glycopyrrolate. Specifically, during CON-EX, apical rotation, basal rotation and twist increased with exercise in both sexes; however, the increases in apical rotation were abolished in males, and plateaued in females at 25% during GLY-EX. This observation could be reflective of alterations to both sympathetic and muscarinic stimulation in those respective regions. Vagal blockade has been shown to attenuate sympathetic activity at rest (232), thus any potential reductions to SNA and adrenergic stimulation may have contributed to the blunted responses of apical rotation during GLY-EX. Moreover, there is a greater density of cholinergic nerve endings at the base compared to the apex (85, 110, 222), thus the removal of vagal stimulation during GLY-EX may have allowed for enhanced basal rotation during exercise.   The blunted responsiveness of apical rotation during GLY-EX may be reflective of a rate-dependent mechanical limitation. Previously, Stohr et al. (198) demonstrated a plateau in LV apical rotation at 50% peak power, which coincided with a mean HR of 134 bpm. In both males and females of the current study, HR was 122 bpm at 50% peak power without vagal blockade, but ~126 bpm at 25% peak power with vagal blockade. Beyond these points, apical rotation did not increase in either of the sexes supporting that a rate dependent mechanical limitation to apical rotation had occurred. Further support for apical rotation being rate limited can be gleaned from Gibbons Kroeker et al. (77),  131 who demonstrated increases to apical rotation up to a HR of 120 bpm in open-chested dogs, beyond which apical rotation and twist were actually became reduced. Additionally, in cardiac muscle, action potential duration, fibre shortening and force development can plateau and even become reduced with elevated stimulation frequencies (20, 126, 191). Collectively, these observations suggest that apical fibre dynamics may be limited at higher heart rates.   5.4.3 Effect of vagal blockade on LV mechanics at rest Although vagal blockade resulted in increases to LV basal rotation, twist and untwisting in both males and females, these parameters were not different between the sexes at rest following glycopyrrolate infusion. Vagal stimulation has a negative inotropic effect on the myocardium (58, 96, 125), and reductions to LV inotropy often result in reductions to LV rotation and twist (153). Therefore, the administration of glycopyrrolate and elimination of this negative inotropic influence likely supported the increases to LV twist mechanics in both sexes post infusion. The increases to twist primarily resulted from increases to basal rotation in both sexes, as apical rotation was not significantly augmented post-infusion. These alterations at the base, but not the apex, may be reflective of regional differences in vagal innervation (110).   Vagal blockade also resulted in reductions to LVEDV in both sexes, and presumably reductions to LV preload. These reductions to LVEDV were similar in magnitude to the reductions that occur between rest and -40 mmHg in unblocked condition (CON-LBNP). Given that -40 mmHg in CON-LBNP produced increases to LV basal rotation, and twist in both sexes, without increases to apical rotation, it is possible that similar reductions to preload occurred with the administration of glycopyrrolate and contributed to the increased LV basal rotation and twist post-infusion.   5.5 Summary and significance During large reductions to preload, females have greater LV apical rotation, twist and torsion compared to males, which persists during vagal blockade with glycopyrrolate. Given that these sex differences occurred despite similar heart rates in both groups, these  132 findings provide novel evidence that sex differences in LV twist mechanics during reductions to preload are not primarily determined by differences in vagal control or chronotropic responsiveness. During exercise, sex differences were not observed in LV twist mechanics, and the increases to LV twist from rest to 50% peak power were not altered by vagal blockade in either of the sexes. Collectively, these findings suggest that vagal cardiac control is not a key determinant of sex differences in LV twist during both reductions and increases to SV during LBNP and submaximal exercise, respectively.     133 Chapter 6. General Discussion and Conclusions  It is well established that LV twist mechanics are primarily determined by: the influences of ventricular preload on passive fibre stress at end-diastole, and the impact of afterload on active fibre shortening during systole (59, 98, 220, 221); the influence of autonomic and neurohormonal activation on chronotropy and myocardial contractility (2, 153, 168); and the alignment of myofibres, which is related to LV geometry (39, 205, 212). The series of experiments in this thesis have identified sex differences in LV mechanics in response to acute physiological stress, and specifically assessed the roles of preload, adrenergic stimulation and sympathovagal balance in determining sex differences in LV twist mechanics. The data from these studies have also demonstrated that alterations to LV rotation and twist commonly coincide with alterations to LV volumes, HR, structure and geometry.   This final chapter will compare and contrast the findings from the three experimental chapters. Additionally, to gain a stronger understanding of the relative influences of the aforementioned factors (LV volume, HR, structure and geometry) on LV twist mechanics in males and females, data from Chapters 3, 4 and 5 have been combined and reanalyzed as a single cumulate dataset. The analysis of these data will be presented and discussed to further elucidate the potential factors that determine sex differences in LV mechanics.   6.1 Sex differences in the LV twist-to-volume relationships Data from this thesis and previous studies have clearly established that LV twist is influenced by significant alterations to LV volumes. Increases to LV twist appear to defend LVSV during reductions to preload (98, 99, 226), and support increases to LVSV such as those that occur during exercise (64, 198, 220). In all three experimental chapters, LVSV was been altered to some degree, and in Chapters 3 and 5 we observed notable sex differences in the relationships of LV twist mechanics to LV volumes. To provide a more comprehensive comparison of the LV twist-to-SV relationships between the sexes, the collective data from all experimental interventions (i.e. LBNP, submaximal exercise, PEI, ß1-AR blockade and vagal blockade) were used to construct the relationships of ∆LV  134 twist and ∆torsion with ∆SV and ∆SV·BSA-1.5, respectively (Figure 6.1). In both the absolute and scaled relationships, females have a significantly steeper slope than males, reinforcing our findings in Chapters 3 and 5 that females have larger increases to LV twist and torsion for a given reduction to LV volumes compared to males. Similarly, females have a steeper slope of the polynomial relationships for ∆twist-to-∆EDV (p<0.001) and ∆torsion-to-∆EDV·BSA-1.5 (p<0.001), and the sex differences in these relationships (LVSV and EDV) are largely reflective of greater responsiveness at the apex in females.    Figure 6.1. Relationships between ∆LV twist with ∆SV (upper panel) and ∆torsion and ∆SV·BSA-1.5 (lower panel). Red and blue points represent individual data for females and males, respectively. *Significantly different relationships for males vs. females (p<0.05).   -30 -20 -10 0 10 20-2-10123∆SV⋅BSA-1.5 (ml⋅m-3)∆Torsion (°cm-1)Males y=0.14+0.0072x+0.0017x2Females y=0.12+0.0096x+0.0049x2}*p<0.001-60 -40 -20 0 20 40-20-1001020∆SV (ml)∆Twist (°)FemalesMalesMales y=1.14+0.036x+0.0020x2Females y=0.97+0.062x+0.0063x2}*p<0.001 135 Although we did not observe sex differences in LV twist mechanics following saline infusion (Chapter 3) or during submaximal exercise (Chapter 5), the quadratic functions for LV twist-to-volumes provide an indication that sex differences may occur during larger increases to SV. During LBNP, EDV was reduced by ~35-40 mL, and SV was reduced by ~20-25 mL in both sexes, whereas during supine cycling exercise EDV and SV were only increases by ~10 mL and ~15 mL in both sexes, respectively. The use of upright cycling exercise could prove more effective for greater increases to SV, as this postural change would result in a lower baseline LVEDV and SV compared to the supine position, and therefore allow for potentially greater increases to LV volumes (194).    The differences in the relationships of LV twist with LV volumes are likely reflective of sex differences in the influences of physiological mechanisms that determine LV twist mechanics during alterations to LVSV. Therefore, the potential influences of sex differences in LV preload, adrenergic stimulation and geometry will be outlined and discussed in the following sections.   6.2 Sex differences in the alterations to LV preload and afterload 6.2.1 Sex differences in reductions to preload  In Chapters 3 and 5, females had greater LV apical rotation than males during high levels of LBNP even though the relative reductions to LVEDV and SV were similar between the sexes. These findings suggest that the reductions to LV preload did not differ between the sexes, and this is further supported by Fu et al. (73) who reported similar reductions to pulmonary capillary wedge pressure (a surrogate of LVEDP and thus LVEDV) in males and females during high levels of LBNP (-40 and -60 mmHg). Additionally, although females had a steeper slope of the Frank-Starling relationship than males in Chapter 3, the slopes of this relationship did not differ between the sexes in Chapter 5. The sex differences in the Frank-Starling relationship in Chapter 3, but not Chapter 5, may be attributable to differences in the female cohorts of these studies. While the slopes of the Frank-Starling relationships were comparable between the two male cohorts (~0.68 in Chapter 3 vs. ~0.71 in Chapter 5), the females in Chapter 3 had a steeper slope of the relationship (~0.76) compared to the cohort in Chapter 5 (~0.70). It has been proposed  136 that a relatively smaller LV is characterized by greater “stiffness” (178), and LV size was substantially smaller (~30%) in the females of Chapter 3 compared to Chapter 5. Therefore, a smaller LV may have resulted in a steeper maximal slope of the LV end-diastolic pressure-volume relationship and a lower SV reserve in the females of Chapter 3. Despite these differences between cohorts, it is important to note that the sex differences in LV twist mechanics during LBNP existed in either case that the Frank-Starling relationships were or were not different between the sexes. On those accounts, it does not appear that differences in the reductions to preload or the Frank-Starling relationship contributed to the sex differences in LV twist mechanics during LBNP.    Interestingly, although apical rotation and twist increased in both sexes in Chapter 3, apical rotation did not increase in the male cohort in Chapter 5. The absolute reductions to LVEDV (~36 ml) and SV (~13 ml) in males were essentially identical between studies; however, the male participants in Chapter 3 had smaller LV volumes at baseline, and consequently experienced greater relative reductions to EDV (~34% vs. ~26%) and SV (~41% vs. ~34%) compared to the males in Chapter 5. Therefore, the males group in Chapter 3 may have experienced larger reductions to preload, and therefore required a greater compensatory mechanical response to support SV during -60 mmHg.  6.2.2 Differences in LV afterload While LV afterload has a strong influence on LV twist mechanics (59, 78, 221), the data from this research series provide little evidence for a contribution of afterload to the sex differences in LV mechanics. In Chapter 4, end-systolic wall stress was estimated as a surrogate of LV afterload, and sex differences in LV twist mechanics were observed despite no differences in end-systolic wall stress during ß1-AR blockade. This index was not determined in the other two investigations, however in Chapter 3, neither MAP nor DBP were different between the sexes, and were not altered in either of the sexes during LBNP. In Chapter 5, although MAP was elevated in males compared to females at baseline and during CON-LBNP, there were no alterations to MAP in either group which suggests that alterations to, and sex differences in, LV twist mechanics were not primarily determined by systemic afterload or blood pressure.   137 6.3 Sex differences related to sympathovagal balance and adrenergic stimulation  6.3.1 Sex differences in sympathovagal balance and the chronotropic responses to acute stress In Chapter 3, the greater LV twist mechanics in females during -60 mmHg LBNP coincided a higher HR compared to males. This elevated chronotropic response agreed with several previous reports (73, 188, 224), and it was hypothesized that differences in vagal withdrawal or sympathovagal balance might contribute to the sex differences in LV apical rotation and twist. However, in the experiments of Chapter 5, females had greater LV apical rotation and twist than males in both CON-LBNP and GLY-LBNP conditions, without any sex differences in HR during either of those conditions. It therefore does not appear that differences in HR, or the chronotropic response associated with vagal withdrawal, determine sex differences in LV twist mechanics during large reductions to preload, as previously postulated (73, 188).  The fact that vagal blockade did not alter the responses of LV twist during progressive LBNP in Chapter 5 might be explained by the relative influence of vagal control on LV contractility. While vagal stimulation has a strong impact on chronotropy, it influences myocardial inotropy to a relatively lesser extent (114). The marked chronotropic effects of vagal control are likely reflective of the dense cholinergic innervation and large numbers of muscarinic receptors in the nodal tissues (110, 222). In contrast, cholinergic innervation and muscarinic receptors are relatively sparse in the ventricles compared to the atria (58, 110, 222), therefore vagal stimulation may exert greater control in the atria and nodal tissues than in the LV.   Vagal stimulation may also exert a relatively greater influence on myocardial contractility at the LV base compared to the apex, as this region is more densely innervated by cholinergic fibers (110). This postulate is supported by observations in Chapter 5, in which GLY infusion resulted in increases to rotation and circumferential strain at the base in both sexes, but did not alter rotation or strain at the apex. Yet, in Chapters 3 and 5, basal rotation was not different between the sexes, and the sex differences in LV twist resulted from sex differences at the apex with and without vagal  138 blockade. Therefore, vagal autonomic control does not appear to contribute to the responses of LV apical rotation and twist during LBNP, or to the sex differences in LV twist mechanics during reductions to preload.   The relationships of ∆HR with ∆LV twist and ∆rotation are shown in Figure 6.2, demonstrating that increases to LV twist mechanics are associated with increases to HR. The relationship of ∆LV twist-to-∆HR is not different between the sexes, although females do have a steeper slope of the relationship for ∆apical rotation-to-∆HR (p=0.002). However, it is important to note that alterations to LV twist mechanics likely do not result from alterations to HR, per se. This is evidenced by data from Chapter 4, in which we observed a mechanistic dissociation between alterations to HR and twist in females. During ß1-AR blockade, males had significant reductions to both HR and LV twist; however, while females had significantly larger reductions to HR than males, LV twist was not altered. While the other interventions assessed during this thesis (i.e. LBNP, exercise, PEI) resulted in simultaneous alterations to LV volume and HR, the data during ß1-AR blockade provide a unique scenario in which HR was altered without concomitant alterations LV volumes or geometry. Therefore, the differential alterations to HR and LV twist in males and females during ß1-AR blockade substantiate that alterations to HR alone do not principally determine alterations to LV twist, or any sex differences in LV twist mechanics.    139  Figure 6.2. Relationships between ∆LV twist mechanics and ∆HR. Closed circles represent individual twist data (left), and open symbols represent individual peak rotation data (right) at the base (circles) and apex (triangles).    During periods of acute stress and alterations to LV volumes, the relationship between ∆HR and ∆twist are more likely reflective of alterations to autonomic or adrenergic control of the heart (baroreflex activation, altered sympathovagal balance) than of the influence of chronotropy alone. This is supported by data Gibbons Kroeker et al. (78), demonstrating in anesthetized dogs that increases to HR have a minimal effect on increasing apical rotation at lower HR ranges, beyond which further increases to HR can actually limit the magnitude of LV apical rotation and twist. This HR-related limitation to LV twist mechanics seemed to occur during exercise in Chapter 5, as the increases to apical rotation appeared to be attenuated at higher heart rates following vagal blockade (GLY-EX). Previously, increases to LV twist have been proposed to mechanically support increases to LVSV during exercise up to 50% peak power, at which LVEDV, SV and LV twist all reach a plateau (198). In this case, the plateau in twist was suggested to represent a mechanical constraint to further increasing SV. However, during GLY-EX in Chapter 5, LVEDV and SV continued to increase despite plateaus in LV apical rotation and twist. These observations suggest that limitations to LV twist are not necessarily linked with limitations to SV but may be related with a rate limitation associated with HR.   -40 -20 0 20 40 60-20-1001020∆MAP (mmHg)∆Twist (°)-50 0 50 100 150-20-1001020∆HR (bpm)∆Twist (°)-40 -20 0 20 40 60-20-1001020∆MAP (mmHg)∆Rotation (º)-50 0 50 100 150-20-1001020∆HR (bpm)∆Rotation (°)ApexBase Basal Rotation (NS)Males     r=-0.21, p<0.001Females   r=-0.14, p=0.018Basal Rotation Males     r=-0.43, p<0.001Females   r=-0.41, p<0.001Apical Rotation (M vs. F p=0.002)Males     r=0.28, p<0.001Females   r=0.45, p<0.001Apical Rotation (*p=0.038)Males     r=0.14, p=0.014Females   r=0.07, NSTwist Males     r=0.46, p<0.001Females   r=0.57,  p<0.001Twist (NS)Males     r=0.23, p<0.001Females   r=0.15, p=0.017 140 6.3.2 Sex differences in adrenergic stimulation  There is strong evidence that increases and decreases to adrenergic stimulation augment and reduce LV twist, respectively (2, 152, 168). Therefore, increases to sympathetic activation and circulating catecholamines most likely contribute to augmenting LV twist mechanics, and additionally may contribute to sex differences in LV mechanics during periods of acute physiological stress. We addressed this question in Chapter 4, and observed that ß1-AR blockade significantly reduced LV apical rotation and twist in males, but not in females, providing preliminary evidence that LV twist mechanics in males may be more sensitive to altered adrenergic stimulation compared to females. During both LBNP and exercise, males have been reported to have greater increases to circulating catecholamines compared to females (56, 223), (45, 73). Therefore, if alterations to LV twist mechanics are primarily determined by adrenergic stimulation, a greater adrenergic stimulus would be expected to produce greater increases to LV twist in males compared to females during LBNP and exercise. This was not observed in Chapters 3 or 5, as females had greater LV apical rotation and twist during LBNP, and no sex differences were observed for LV twist mechanics during exercise. As these patterns were not affected by vagal blockade, it appears unlikely that intrinsic sex differences in sympathovagal balance or the sensitivity to adrenergic stimulation are key determinants of the sex differences in LV twist mechanics during acute physiological stress.   6.4 Sex differences related to LV structure and geometry  In Chapters 3 and 5, the greater LV twist in females during LBNP always coincided with a larger sphericity index than males. This consistent observation provides an indication that differences in LV geometry could significantly contribute to sex differences in LV twist mechanics, and there is some anatomical evidence to support this hypothesis. During the transition from end-diastole to systole, myocyte shortening reduces myocardial laminar sheet angles (ß’) across the myocardial wall, and broadens the endo-to-epicardial differences in myofibre helix angles (αhelix) as those fibres become more longitudinally oriented (38, 63). Thus, these alterations to myofibre length and myolaminar sheet reorientation during systole are key determinants of LV twist and chamber shortening in the longitudinal axis (38). Moveover, as the magnitude of sheet  141 reorientation during systole is dependent on the intial configuration of LV myofibres, alterations to end-diastolic αhelix and ß’ directly impact active fibre strain, rotation and twist (18, 205). Increases to LV sphericity index are proposed to have augmented myolaminar αhelix angles (and vice versa); consequently, LV sphericity index has been identified as principal independent predictor of LV twist. In our participants, the greater sphericity index in females during large reductions to preload likely reflected more optimal myolaminar sheet and helix angles, and therefore supported the generation of greater LV apical rotation and twist compared to males.   In addition to the absolute differences in sphericity index at high levels of LBNP, sex differences also exist in the relationships for LV twist with structural and geometric parameters. In Chapter 4, we first identified significant relationships for LV twist mechanics with LVIDd and sphericity index in females but not in males. In chapter 5, similar relationships for LV twist with dimensions (LVIDd and LV length) were observed in both sexes, however these were reflective of relationships with apical rotation for females, and with basal rotation for males. Moreover, in agreement with Chapter 4, apical rotation was related to sphericity index in females, but not in males. In the cumulate dataset, ∆LV twist is significantly related to ∆LVIDd and ∆LV length in both sexes, but to a greater extent at the apex in females compared to males (Figure 6.3). Therefore, while alterations to LV internal dimensions do influence the alterations to twist mechanics in both sexes, the regional responsiveness of LV rotation differs between the sexes. These differences could be reflective of specific fibre angles at the apex in males versus females. Chen et al. have demonstrated that ß’ sheet angles vary from the base to apex, and during ejection ß’ angles become reconfigured to more transverse orientations (38, 62). It is possible that given alterations to LV dimensions produce similar alterations to ß’ angles at the base, but augment ß’ angles at the apex to a greater extent in females, and thus result in increased myofiber shortening at the apex in females.     142  Figure 6.3. Relationships for ∆LV twist mechanics with ∆Lengthd (upper panel) and ∆LVIDd (lower panel). See Figure 6.2 for figure legend.   In contrast to LV dimensions, significant relationships for ∆LV twist and rotation with ∆sphericity index and ∆relative wall thickness were exclusive to females, and were not observed in males (Figure 6.4). Thus, in addition to absolute differences in sphericity index, females appear to have a greater responsiveness of LV twist mechanics to alterations in LV chamber geometry. It is possible that a given increase to sphericity index results in more significant reconfigurations to myolaminar sheet orientations and helix angles than those in males, which further enhance the augmentations to LV apical rotation and twist. Additionally, increases to relative wall thickness have been proposed to produce a greater difference in the moment arm between the endo- and epicardial layers, and accordingly support increased LV twist (205). Similar to the relationships -1.5 -1.0 -0.5 0.0 0.5 1.0-20-1001020∆Lengthd (cm)∆Twist (°)-1.5 -1.0 -0.5 0.0 0.5 1.0-20-1001020∆Lengthd (cm)∆Rotation (º)-15 -10 -5 0 5 10-20-1001020∆LVIDd (mm)∆Twist (°)-15 -10 -5 0 5 10-20-1001020∆LVIDd (mm)∆Rotation (º)Twist Males     r=-0.13, p=0.023Females   r=-0.15, p=0.014Twist Males     r=-0.15, p=0.011Females   r=-0.21, p=0.001Apical Rotation (M vs. F p=0.014)Males     r=-0.07, NSFemales   r=-0.13, p=0.033Apical Rotation (M vs. F *p=0.001)Males     r=-0.11, NS (p=0.06)Females   r=-0.14, p=0.029Basal Rotation Males     r=0.15, p=0.008Females   r=0.16, p=0.008Basal RotationMales     r=0.15, p=0.011Females   r=0.18, p=0.003 143 with sphericity index, a given change to relative wall thickness in females could result in a more optimal reconfiguration of fibre orientations and endo-to-epicardial gradients in αhelix and ß’ angles, and ultimately support the increased LV twist mechanics compared to males.    Figure 6.4. Relationships for ∆LV twist mechanics with ∆sphericity index (upper panel) and ∆relative wall thickness (lower panel). See Figure 6.2 for figure legend.    6.5 Determinants of LV twist at rest in males and females In the separate experimental chapters, sex differences in LV twist mechanics were not observed at rest. However, to provide a more thorough comparison of LV twist between the sexes at rest, the cumulate baseline data from all three experimental chapters were assessed, and are presented in Table 6.1. In the combined dataset, females have greater -0.5 0.0 0.5 1.0-20-1001020∆Sphericity Index∆Twist (°)-0.2 0.0 0.2 0.4 0.6-20-1001020∆Relative Wall Thickness∆Twist (°)-0.5 0.0 0.5 1.0-20-1001020∆Sphericity Index∆Rotation (°)-0.2 0.0 0.2 0.4 0.6-20-1001020∆Relative Wall Thickness∆Rotation (º)Basal Rotation Males     r=-0.06, NSFemales   r=-0.13, p=0.028Basal Rotation Males     r=-0.04, NSFemales   r=-0.07, NSApical Rotation (M vs. F p<0.001)Males     r=0.03, NSFemales   r=0.13, p=0.038Apical Rotation (M vs. F p=0.005)Males     r=0.10, NSFemales   r=0.14, p=0.029Twist (M vs. F p=0.031)Males     r=0.08, NSFemales   r=0.18, p=0.006Twist (M vs. F p=0.041)Males     r=-0.01, NSFemales   r=0.13,  p=0.036 144 peak LV twist (p=0.016) and torsion (p=0.001) compared to males. Although LV rotation and untwisting velocity do not differ between the sexes, females have a trend to significantly greater apical rotation than males (p=0.096). Additionally, longitudinal strain and circumferential strain are greater in females (p<0.001 for both), but circumferential strain at the apex is not different between the sexes. Although allometrically scaled LV length is similar between the sexes, females have a smaller scaled LVIDd, (p=0.001) and therefore a greater sphericity index than males (p<0.001). Despite larger absolute LV volumes in males, allometrically scaled LVEDV, ESV and SV are not different between sexes in the combined dataset.  In an attempt to better understand the potential factors underlying these sex differences at rest, relationships for LV twist mechanics with hemodynamic, volumetric and geometric parameters were evaluated for both sexes. In both sexes, LV twist was not significantly related to EDV, LV length or LVIDd; however, LV twist and torsion had significant inverse relationships with allometrically scaled EDV in males (twist: r=-0.31, p=0.023; torsion: r=-0.36, p=0.006) and females (twist: r=-0.35, p=0.016; torsion: r=-0.41, p=0.004). These findings suggest that LV twist is not determined by LV volume at rest, per se. Rather, a relatively smaller ventricle for a given body size is related to augmented LV twist and torsion in both sexes. Allometrically scaled EDV also did not differ between the sexes, providing further support that sex differences in LV mechanics do not result from differences in absolute or relative LV volumes in this cohort.   In the combined dataset, differences in LV geometry and dimensions appear play a more significant role: LV twist mechanics were not significantly related to LV length (absolute or scaled) in either of the sexes, but significant relationships for LV torsion with allometrically scaled LVIDd do occur in males (r=-0.28, p=0.043) and females (r=-0.33, p=0.024). With a smaller LVIDd in females and a similar relative LV length in both sexes, it is feasible that a greater sphericity index in females likely contributes to the sex differences in LV twist mechanics at rest for the combined dataset. In each separate experimental chapter, there were no sex differences at rest in allometrically scaled LVEDV, scaled LV length or scaled LVIDd, which may partly explain why sex  145 differences in LV twist mechanics were not observed at baseline in those respective investigations. Nonetheless, the sex differences in the combined dataset provide further support that differences in the LV chamber geometry may play a key role in determining differences in LV twist mechanics, both at rest and during alterations to LV volumes.  Inverse relationships were also observed for resting MAP with LV twist (M r=-0.34, p=0.013; F r=-0.30, p=0.036), torsion (M r=-0.27, p=0.049; F r=-0.33, p=0.013) and apical rotation (M r=-0.53, p<0.001; F r=-0.32, p=0.025). Increases to MAP are commonly indicative of augmented LV afterload, which can attenuate active myocardial shortening during systole and therefore reduce LV twist (59, 221). Thus, the greater MAP in males may also contribute to the relatively smaller LV twist and torsion compared to females. Finally, relationships were not observed for HR with LV twist or rotation parameters in either of the sexes. This provides further support that sex differences in LV twist mechanics are not related to differences in HR, as we observed in Chapter 5 that females have augmented LV apical rotation, twist and torsion compared to males despite no differences in HR or the chronotropic response to LBNP.   146 Table 6.1. Baseline characteristics, LV hemodynamics, structure and geometry  Males (n=52) Females (n=55) p Age (yrs) 23 (5) 22 (3) 0.67 Height (c=m) 1.79 (0.07) 1.66 (0.08) <0.001 Weight (kg) 75 (10) 63 (2) <0.001 BMI (kg⋅m-2) 23.5 (2.6) 22.7 (2.3) 0.11 BSA (m2) 1.93 (0.14) 1.70 (0.13) <0.001 Resting LV mechanics (peak) Twist (˚) 11.3 (3.7) 13.3 (4.9) 0.016 Torsion (˚⋅cm-1) 1.28 (0.40) 1.62 (0.61) 0.001 Untwisting velocity (˚⋅s-1) -95 (30) -103 (31) 0.22 Apical rot (˚) 8.5 (2.9) 9.9 (4.0) 0.096 Basal rot (˚) -3.2 (1.9) -3.4 (2.2) 0.65 Longitudinal strain (%) -17.7 (1.8) -19.3 (1.8) <0.001 Circumferential strain, base (%) -19.7 (3.1) -21.9 (3.0) <0.001 Circumferential strain, apex (%) -27.0 (3.9) -27.6 (3.5) 0.40 Resting LV hemodynamics HR 63 (9) 59 (9) 0.064 MAP (mmHg) 85 (9) 80 (8) 0.004 EF (%) 54 (4) 54 (4) <0.001 EDV (ml) 119 (21) 93 (19) <0.001 EDV (ml⋅m-3) 44 (8) 42 (8) 0.13 ESV (ml) 54 (11) 39 (10) <0.001 ESV (ml⋅m-3) 27 (13) 28 (12) 0.56 SV (ml) 65 (12) 53 (11) <0.001 SV (ml⋅m-3) 24 (5) 24 (4) 0.89 Resting LV structure and geometry Lengthd (cm) 8.82 (0.57) 8.22 (0.45) <0.001 Lengthd (cm⋅m-0.5) 6.34 (0.35) 6.33 (0.29) 0.94 LVIDd (mm) 46.9 (3.5) 42.0 (3.5) <0.001 LVIDd (mm⋅m-0.5) 33.7 (2.3) 32.3 (2.2) <0.001 Sphericity index 1.89 (0.14) 1.97 (0.15) 0.001 Relative wall thickness 0.41 (0.06) 0.42 (0.06) 0.70 Values are means (SD). Rot: rotation; HR: heart rate; MAP: mean arterial pressure; EF: ejection fraction; EDV: end-diastolic volume; ESV: end-systolic volume; SV: stroke volume; Lengthd: left ventricular length at end-diastole; LVIDd: left ventricular internal diameter at end-diastole.   147 6.6 Considerations Due to the complex and integrative nature of human physiology, it is methodologically impossible to isolate the effects of alterations to a single physiological system, or the responses to a single stimulus (i.e. alterations to preload, afterload or contractility). In Chapter 3, and additional studies utilizing large reductions to LV preload (73, 98, 188, 197), the alterations to LV mechanics and hemodynamics reflect not only the responses to reduced LV volumes, but additionally involve the alterations to baroreflex activation, sympathovagal balance, reductions to LV afterload and increases to contractility (45, 142). In Chapter 4, PEI produced increases to adrenergic stimulation and LVSV, but also increased LV end-systolic wall stress in both males and females. These large increases to afterload likely countered any potential increases to LV twist, limiting our ability to distinguish any sex-related differences in the responses of LV mechanics. Finally, in Chapter 5, we examined the responses of LV mechanics during high levels of LBNP and during moderate exercise, both of which involve alterations to LVSV as well as other parameters including (but not limited to) sympathovagal balance, LV preload, afterload and contractility, baroreflex activation and even respiration. Therefore, although these experiments were specifically designed to focus on the influence of one parameter—preload, adrenergic stimulation, or vagal control—it is not possible to isolate the influences of those specific parameters or systems from other associated cardiopulmonary and neural responses during a given experimental intervention. While the inability to isolate a single effect potentially limits our interpretation of these data, the assessment of the integrated response is especially important as it obviously allows us greater external validity to translate these findings.  6.7 Relevance and implications and directions for future study 6.7.1 Influences of LV structure and geometry  A predominant theme from this experimental series surrounds the potential role of LV myofibre configuration in determining sex differences in LV mechanics. While a number of studies have previously used diffusion tensor imaging to visualize the intricate helical network of myocardial fibres and cleavage planes in human and animal hearts (38, 53, 163), these parameters have not been compared between the male and female heart. This  148 assessment could reveal key differences in the LV myocardial macro- and micro-structural elements that determine sex differences in LV rotation and twist.  Although LV length was matched between the sexes in Chapter 4, males had larger absolute LV volumes and dimensions than females, in all experiments as well as in the combined dataset. To provide additional clarity to the influences of LV size and volumes on sex differences in LV twist mechanics, future studies could aim to assess LV mechanics in cohort of males and females with matched absolute LVEDV, but significantly different sphericity indexes.  6.7.2 Influences of adrenergic stimulation While we were successful in altering adrenergic stimulation in Chapter 4, the potential role of adrenergic stimulation in sex differences in LV twist mechanics still remains relatively unclear. During elevations to adrenergic stimulation with PEI, we did not observe any alterations or sex differences in LV twist. During this intervention, any potential increases to LV twist were likely blunted by the concomitant increases to LV afterload. The use of a different experimental intervention, such as the administration of ß1-AR agonists (e.g. dobutamine infusion (2)) may prove more effective to augment LV twist mechanics, and therefore help to determine if sex differences in those responses do occur.   6.7.3 Influences of arterial and cardiopulmonary baroreflexes  Over the past decade, increasing attention has been given to sex differences in the baroreflex-mediated control of blood pressure. As LBNP unloads both arterial and cardiopulmonary baroreceptors (74), potential differences in afferent and efferent baroreflex signaling could contribute to the sex differences in LV twist mechanics during large reductions to preload. Pre-menopausal females have been proposed to have lower autonomic control of blood pressure (40, 182) and reduced vasoconstrictor responsiveness compared to males (89, 115). As a result, females may rely on the cardiac arm of the baroreflex, and increasing LV twist, to a greater extent than males (112). Nonetheless, to our knowledge, the alterations of LV twist mechanics in relation to  149 baroreflex stimulation have not been established. The administration of pharmaceutical agents such as sodium nitroprusside and phenylephrine could be used to increase and reduce blood pressure in males and females, and potentially determine any sex differences in the baroreflex-mediated responses of LV twist mechanics.   6.7.4 Relevance for clinical and sport applications   The data from this thesis have identified that females have greater twist mechanics during large reductions to preload, and the combined dataset has also provided evidence for greater apical rotation and twist in females at rest. It appears that these sex differences are likely related to differences in LV geometry, notable the ellipsoid vs. spherical shape of the ventricle. We have noted sex differences in the alterations to LV geometry during acute physiological stress, however we do not know how LV mechanics compare between males and females following chronic adaptation to prolonged physiological stress. This is relevant to both clinical populations, in which cardiac dysfunction results from chronic physiological stress and myocardial remodeling, as well as in highly-trained athletes (138, 155).   Highly trained endurance athletes have significant structural and functional cardiac remodeling (5), as well as altered adrenergic sensitivity and stimulation (32). However, the cardiovascular adaptations to chronic training differ between the sexes, whereby females have greater eccentric remodeling compared to males, as well as a larger increase to relative blood volume (102). Male athletes also appear to have greater LV adrenergic sensitivity than their female counterparts, in agreement with our findings from Chapter 4 (183). Given that LV mechanics are sensitive to alterations in LV geometry (212), preload, and adrenergic stimulation, and that sex differences exist in the physiological adaptations to chronic training, it is feasible that sex differences exist in LV mechanics in highly-trained males and females. Although we did not observe sex differences during exercise in the non-athlete participants of Chapter 5, the sex differences in cardiac adaptations to chronic endurance training may have implications for differences in LV mechanics during exercise, especially at higher intensities. Of specific interest is the influence of sex differences in chronic structural remodeling of the LV. If female athletes  150 have greater eccentric remodeling of the heart, and a potentially greater ellipsoid geometry, females may be able to augment LV twist to a greater extent than males with increasing exercise intensities. However, a potentially greater adrenergic sensitivity of LV mechanics in highly trained male could otherwise contribute to larger twist mechanics compared to their female counterparts during dynamic exercise.   Sex-related differences in LV mechanics likely also have important implications to the progression of cardiac disease. Cardiomyopathies involving both systolic and diastolic LV dysfunction commonly involve impairments to intrinsic myocardial function, as well as structural remodeling. Because the etiologies and pathologies of different forms of heart failure are varied, the alterations to LV mechanics are specific to the disease. For example, in heart failure with preserved EF, LV twist may be preserved but untwisting mechanics become impaired, which likely contributed to impaired diastolic function in these patients. Heart failure with preserved EF is more common in females compared to males, and females appear to have increased myocardial stiffness, as well as greater LV remodeling and concentric hypertrophy compared to males (80, 171, 180). The data from the current thesis support that LV twist mechanics in females may be more sensitive to altered LV structure and geometry than in males, therefore it is possible that greater LV remodeling may result in more substantial impairments to LV untwisting mechanics and diastolic function in female compared to male patients with preserved EF.   In contrast to heart failure patients with preserved EF, those with systolic dysfunction have reduced LV twist and circumferential strain compared to healthy cohorts (184). In these patients, males are reported to have lower EFs compared to females, and greater increases to LVIDd and LV mass (129). Fundamental sex-related differences in cardiac remodeling have also been reported, whereby males have augmented LV hypertrophy and greater increases to myocyte volume than females following infarct (48). The greater remodeling in males may therefore contribute to larger impairments in systolic function. While the experiments of this thesis have demonstrated greater twist mechanics in females compared to males during acute stress and at rest, future work should consider  151 examining whether sex differences in LV mechanics have implications for differences in the progression of cardiac disease and myocardial dysfunction.     6.8  Overall conclusions To our knowledge, the experiments of this thesis are the first to demonstrate sex differences in LV mechanics. During large reductions to preload, females have greater LV apical rotation, twist and torsion compared to males, which appears to be primarily due to fundamental sex differences in LV geometry, both at rest as well as during acute physiological stress. As such, potential sex differences in intrinsic LV myofibre configuration, likely play a significant role in determining sex differences in LV twist mechanics. Additionally, the data from this thesis provide support that LV twist mechanics may be more sensitive to altered adrenergic stimulation in males compared to females. However whether these sex differences occur during profound increases or decreases to adrenergic stimulation merits further investigation. Lastly, in the final experimental chapter of this thesis, it was observed the augmented LV apical rotation and twist in females during LBNP persist during vagal blockade, indicating that these sex differences in LV twist mechanics are likely not reflective of differences in vagal cardiac control. Further research is now required to elucidate the specific roles of LV structure and geometry and the adrenergic control of the heart in determining sex differences in LV twist mechanics.    152 References   1. Adams KF, Vincent LM, McAllister SM, el-Ashmawy H, Sheps DS. The influence of age and gender on left ventricular response to supine exercise in asymptomatic normal subjects. American Heart Journal 113: 732–742, 1987. 2. Akagawa E, Murata K, Tanaka N, Yamada H, Miura T, Kunichika H, Wada Y, Hadano Y, Tanaka T, Nose Y, Yasumoto K, Kono M, Matsuzaki M. 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